What is HPLC Basic Overview_HPLC_PM_BF

What Is HPLC? Basic Principles

LAAQ-B-LC001B

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Fundamentally, chromatography is a technique used to separate the components contained in a sample. Above all, high performance liquid chromatography (HPLC) is a type of chromatography that, because of its wide application range and quantitative accuracy, is regarded as an indispensable analytical technique, particularly in the field of organic chemistry. It is also widely used as a preparation technique for the isolation and purification of target components contained in mixtures. An overview of HPLC, from the basic principles of chromatography to the characteristics of HPLC itself, is presented here.

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Invention of Chromatography by M. Tswett Ether

Chromatography

Colors

Chlorophyll CaCO 3

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The Russian-Polish botanist M. Tswett is generally recognized as the first person to establish the principles of chromatography. In a paper he presented in 1906, Tswett described how he filled a glass tube with chalk powder (CaCO3) and, by allowing an ether solution of chlorophyll to flow through the chalk, separated the chlorophyll into layers of different colors. He called this technique “chromatography”.

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Comparing Chromatography to the Flow of a River... Light leaf Heavy stone

Water flow

Base

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Chromatography can be often compared to the flow of a river. A river consists of a stationary riverbed and water that continuously moves in one direction. What happens if a leaf and a stone are thrown into the river? The relatively light leaf does not sink to the bottom, and is carried downstream by the current. On the other hand, the relatively heavy stone sinks to the bottom, and although it is gradually pulled downstream by the current, it moves much more slowly than the leaf. If you stand watch at the mouth of the river, you will eventually be able to observe the arrival of the leaf and the stone. However, although the leaf will arrive in an extremely short time, the stone will take much longer to arrive. This analogy represents the components of chromatography in the following way: •River: Separation field •Leaf and stone: Target components of sample •Standing watch at the river mouth: Detector

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Mobile Phase / Stationary Phase 

Mobile phase Strong

Weak 

Stationary phase

A site in which a moving phase (mobile phase) and a non-moving phase (stationary phase) make contact via an interface that is set up. The affinity with the mobile phase and stationary phase varies with the solute. → Separation occurs due to differences in the speed of motion.

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In chromatography, the field of separation is divided into two phases. One phase, called the “stationary phase”, does not move. The other phase, called the “mobile phase”, moves at a constant speed in one direction. The stationary phase and mobile phase make contact via an interface. They do not intermingle, and are kept in a steady state of equilibrium. In the river analogy, the riverbed corresponds to the stationary phase and the flowing water corresponds to the mobile phase. Let us suppose that some substance has been introduced into the flow of the mobile phase and led to the separation site. If this substance contains a component that is only weakly attracted by the stationary phase and a component that is strongly attracted by the stationary phase, the former component will be pulled along quickly by the flow of the mobile phase whereas the latter component will stick to the stationary phase and only move slowly. In this way, differences in the properties of the various components contained in the sample being analyzed give rise to differences in speed. This makes it possible to separate components from each other. Incidentally, in the river analogy, the interaction that determines the speed of motion is based on gravity (and buoyancy in water). In chromatography, various physical and chemical properties, such as solubility and the degree of adsorption, determine the dynamics of separation.

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ChromatoChromato-graphy / -graph / -gram / -grapher 

Chromatography:

Analytical technique

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Chromatograph:

Instrument

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Chromatogram:

Obtained “picture”

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Chromatographer:

Person

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There are many similar terms in this field and so let us clarify some of them. “Chromatography” is the name of the analytical technique itself. A “chromatograph” is an analytical instrument that is used to perform chromatography. The product names of the chromatographs given in the catalogs of analytical instrument manufacturers should all include this word. A “chromatogram” is produced by recording the results obtained with chromatography on recording paper (or some other medium). A “chromatographer” is a person who carries out a chromatography experiment.

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Three States of Matter and Chromatography Types Mobile phase Gas

Liquid

Gas chromatography

Liquid chromatography

Solid

Gas

Stationary phase

Liquid

Solid LAAQ-B-LC001B

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There are various ways of categorizing chromatography. Here, let us categorize it in terms of the three states of matter. There are generally three states of matter: gas, liquid, and solid. If we could use stationary phases and mobile phases of any state, this would give a total of nine different types of chromatography. Using a gas as the stationary phase or a solid as the mobile phase, however, is not practical (even if it is possible) and this restricts the combinations that can be used. Chromatography performed using a gas as the mobile phase and a liquid or a solid as the stationary phase is called “gas chromatography” (GC). Chromatography performed using a liquid as the mobile phase and a liquid or a solid as the stationary phase is called “liquid chromatography” (LC). Both of these techniques are indispensable, particularly in the field of organic chemistry. In addition to these, there is a technique called “supercritical fluid chromatography” (SFC), in which a supercritical fluid kept at a high temperature and high pressure is used as the mobile phase.

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Liquid Chromatography 

Chromatography in which the mobile phase is a liquid. 





The liquid used as the mobile phase is called the “eluent”.

The stationary phase is usually a solid or a liquid. In general, it is possible to analyze any substance that can be stably dissolved in the mobile phase.

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“Liquid chromatography” (LC) is chromatography in which the mobile phase is a liquid. Stationary Phase Usually solid or a regarded liquid is used the is mobile phase.bonded, (This includes the case where aasubstance as aas liquid chemically or applied, to the surface of a solid.) The most common form of stationary phase consists of fine particles of, for example, silica gel or resin packed into a cylindrical tube. These packed particles are called “packing material” or “packing” and the separation tube into which they are packed is called the “separation column” or simply the “column”. In day-to-day analysis work, “column” is sometimes used to refer to the stationary phase and “stationary phase” is sometimes used to refer to the column. Mobile Phase Various solvents are used as mobile phases. The mobile phase conveys the components of the dissolved sample through the separation field, and facilitates the repeated three-way interactions that take place between the phases and the sample, thereby leading to separation. The solvent used for the mobile phase is called the “eluent” or “eluant”. (In LC, the term “mobile phase” is also used to refer to this solvent. In this text, however, we shall use the term “eluent”.) Sample In general, it is possible to analyze any substance that can be stably dissolved in the eluent. This is one advantage that LC has over GC, which cannot be used to analyze substances that do not vaporize or that are thermally decomposed easily. The sample is generally converted to liquid form before being introduced to the system. It contains various solutes. The target substances (the analytes) are separated and detected.

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Interaction Between Solutes, Stationary Phase, and Mobile Phase 

Differences in the interactions between the solutes and stationary and mobile phases enable separation.

Solute Degree of adsorption, solubility, ionicity, etc.

Stationary phase

Mobile phase

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The solutes interact with the stationary and mobile phases. These interactions are the most important contributing factor behind separation. Representative examples of the types of interactions that take place in liquid chromatography are given below. (They are not based on strict classifications.) •Adsorption •Distribution •Hydrophobic interaction •Ion exchange •Ion pair formation •Osmosis and exclusion •Affinity

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Column Chromatography and Planar Chromatography Separation column

Paper or a substrate coated with particles

Packing material Paper Chromatography Thin Layer Chromatography (TLC)

Column Chromatography LAAQ-B-LC001B

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Liquid chromatography can be categorized by shape of separation field into column-shaped and planar types. A representative type of chromatography that uses a column-shaped field is “column chromatography”, which is performed using a separation column consisting of a cylindrical tube filled with packing material. Another type is “capillary chromatography”, which is performed using a narrow hollow tube. Unlike column chromatography, however, capillary chromatography has yet to attain general acceptance. (In the field of GC, however, capillary chromatography is a commonly used technique.) Types of chromatography that use a planar (or plate layer) field include “thin layer chromatography”, in which the stationary phase consists of a substrate of glass or some other material to which minute particles are applied, and “paper chromatography”, in which the stationary phase consists of cellulose filter paper.

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Separation Process and Chromatogram for Column Chromatography

t u tp u O

n o ti a tr n e c n o c

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Chromatogram

Time

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The separation process for column chromatography is shown in the above diagram. After the eluent is allowed to flow into the top of the column, it flows down through the spaces in the packing material due to gravity and capillary action. In this state, a sample mixture is placed at the top of the column. The solutes in the sample undergo various interactions with the solid and mobile phases, splitting up into solutes that descend quickly together with the mobile phase and solutes that adsorb to the stationary phase and descend slowly, so differences in the speed of motion emerge. At the outlet, the elution of the various solutes at different times is observed. A detector that can measure the concentrations of the solutes in the eluate is set up at the column outlet, and variations in the concentration are monitored. The graph representing the results using the horizontal axis for times and the vertical axis for solute concentrations (or more accurately, output values of detector signals proportional to solute concentrations) is called a “chromatogram”.

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Chromatogram l a n ig s r to c e t e d fo ty i s n e t In

tR tR : Retention time

Peak t0

t0 : Non-retention time

h A

A : Peak area h : Peak height

Time LAAQ-B-LC001B

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Usually, during the time period in which the sample components are not eluted, a straight line running parallel to the time axis is drawn. This is called the “baseline”. When a component is eluted, a response is obtained from the detector, and a raised section appears on the baseline. This is called a “peak”. The components in the sample are dispersed by the repeated interactions with the stationary and mobile phases, so the peaks generally take the bell-shape form of a Gaussian distribution. The time that elapses between sample injection and the appearance of the top of the peak is called the “retention time”. If the analytical conditions are the same, the same substance always gives the same retention time. Therefore, the retention time provides a means to perform the qualitative analysis of substances. The time taken for solutes in the sample to go straight through the column together with the mobile phase, without interacting with the stationary phase, and to be eluted is denoted as “t0”. There is no specific name for this parameter, but terms such as “non-retention time” and “hold-up time” seem to be commonly used. Because the eluent usually passes through the column at a constant flow rate,R tand t0 are sometimes multiplied by the eluent flow rate and handled as volumes. The volume corresponding to the retention time is called the “retention volume” and is notated as VR. The length of a straight line drawn from the top of a peak down to the baseline is called the “peak height”, and the area of the raised section above the baseline is called the “peak area”. If the intensities of the detector signals are proportional to the concentrations or absolute quantities of the peak components, then the peak areas and heights are proportional to the concentrations of the peak components. Therefore, the peak areas and heights provide a means to perform the quantitative analysis of sample components. It is generally said that using the peak areas gives greater accuracy.

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From Liquid Chromatography to High Performance Liquid Chromatography

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Higher degree of separation! → Refinement of packing material (3 to 10 µm)

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Reduction of analysis time! byequipment pump → Delivery Demand of foreluent special that can withstand high pressures

The arrival of high performance liquid chromatography!

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In order to increase the separation capability of column chromatography, in addition to increasing the surface area of the stationary phase so that the interaction efficiency is increased, it is also necessary to homogenize the separation field as much as possible so that dispersion in the mobile and stationary phases is minimized. The most effective way of achieving this is to refine the packing material. Refining the packing material, however, causes resistance to the delivery of the eluent to increase. This is similar to the way that water drains easily through sand, which has relatively large particles, whereas it does not drain easily through clay-rich soil, which has relatively fine particles. Depending on gravity and capillary action would cause analysis to take a very long time to be completed, and the idea of delivering the eluent forcibly using a high-pressure pump was proposed. This was the start of high performance liquid chromatography.

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Flow Channel Diagram for High Performance Liquid Chromatograph

Detector Column Column oven (thermostatic column chamber)

Pump

Eluent (mobile phase)

Sample injection unit (injector)

Drain Data processor

Degasser LAAQ-B-LC001B

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A high performance liquid chromatograph differs from a column chromatograph in that it is subject to the following performance requirements. Solvent Delivery Pump A solvent delivery pump that can maintain a constant, non-pulsating flow of solvent at a high pressure against the resistance of the column is required. Sample Injection Unit There is a high level of pressure between the pump and the column; a device that can inject specific amounts of sample under such conditions is required. Column The technology for filling the column evenly with refined packing material is required. Also, a material that can withstand high pressures, such as stainless steel, is required for the housing. Detector Higher degrees of separation have increased the need for high-sensitivity detection, and levels of sensitivity and stability that can respond to this need are required in the detector.

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Advantages of High Performance Liquid Chromatography 

High separation capacity, enabling the batch analysis of multiple components

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Superior quantitative capability and reproducibility

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Moderate analytical conditions

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Generally high sensitivity

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Low sample consumption

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Easy preparative separation and purification of samples



Unlike GC, the sample does not need to be vaporized.

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HPLC is a type of separation analysis, and this is the most important aspect of this analytical technique. Even if the sample consists of a mixture, it allows the target components to be separated, detected, and quantified. It also allows simultaneous analysis of multiple components. It could be said that HPLC is more suited to quantitative analysis than it is to qualitative analysis. Under the appropriate conditions, it is possible to attain a high level of reproducibility with a coefficient of variation not exceeding 1%. One advantage that HPLC has over GC is that, in general, analysis is possible for any sample that can be stably dissolved in the eluent. With GC, gas is used as the mobile phase, so substances that are difficult to vaporize or that decompose easily when heated cannot be analyzed. For this reason, particularly in the fields of pharmaceutical science and biochemistry, HPLC is used much more frequently than GC. The level of sensitivity that can be attained varies with the detector, but detection down to the µg and pg levels is usually possible and, in some cases, even smaller quantities can be detected. The amount of sample used is very small, and is usually in the range of 1 to 100 µL. The components contained in the sample are eluted from the column separately. So, if a non-destructive detector is used, the preparative separation and purification of specific components is possible. In fact, liquid chromatographs specially designed for preparative separation are commercially available.

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Fields in Which High Performance Liquid Chromatography Is Used 

Biogenic substances 

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Sugars, lipids, nucleic acids, amino acids, proteins, peptides, steroids, amines, etc.



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Medical products 

Food products

Environmental samples 

Drugs, antibiotics, etc.



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Vitamins, food additives, sugars, organic acids, amino acids, etc.

Inorganic ions Hazardous organic substances, etc.

Organic industrial products 

Synthetic polymers, additives, surfactants, etc.

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HPLC is currently being used in a broad range of fields. In particular, in the field of biochemistry, it is widely used as an indispensable analytical technique. From the perspective of an analytical instrument manufacturer, we observe that the industry that purchases the highest number of high performance liquid chromatographs is the pharmaceutical industry. It is said that the number of deliveries for this industry accounts for about 40% of the total. Although the number of deliveries to quality control departments is particularly high, it is also quite high for drug discovery and R&D departments.

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HPLC Hardware: Part 1 Solvent Delivery System, Degasser, Sample Injection Unit, Column Oven

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The analytical instrument used to perform high performance liquid chromatography is called a “high performance liquid chromatograph”. A high performance liquid chromatograph consists of several units. Here, in Part 1, I shall present all the units except the detector and data processor.

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Flow Channel Diagram for HPLC

Detector Column Column Oven (thermostatic column chamber)

Pump

Eluent (mobile phase)

Sample injection unit (injector)

Drain Data processor

Degasser LAAQ-B-LC001B

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The configuration of a high performance liquid chromatograph includes a solvent delivery pump, a sample injection unit, a column chamber, a detector, and a data processor (recorder). These units are essential. Accessories are added as necessary. The roles of these five basic units are as follows: Solvent Delivery Pump This unit delivers the eluent to the column. It incorporates features that allow it to maintain a constant, non-pulsating flow of solvent at a high pressure against the resistance of the column. Sample Injection Unit This unit introduces the sample to the column by injecting a specific quantity of sample solution. Types include a manual injector, which performs injection using a microsyringe, and an autosampler, which automatically injects a series of samples. Column Oven This unit maintains the column at a constant temperature. Temperature is an important factor that influences separation, so maintaining the column at a constant temperature makes it possible to improve the quality of separation and the reproducibility. This unit is also called a thermostatic column chamber. Detector This unit detects the components eluted from the column. There are many different types of detectors, based on various operating principles, and the detector used is selected according to the properties of the target compounds and the objective of analysis. UV-VIS absorbance detectors are the most commonly used. Data Processor (Recorder) This unit draws chromatograms by recording the signals received from the detector on charts or magnetic media. Data processors that, in addition to recording, have functions for adding peak areas and performing quantitative calculations are commonly used. Furthermore, systems that perform both instrument control and data processing using PCs are widespread.

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Solvent Delivery Pump

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Performance Requirements 

Capacity to withstand high load pressures.



Pulsations that accompany pressure



fluctuations are small. Flow rate does not fluctuate.



Solvent replacement is easy.



The flow rate setting range is wide and the flow rate is accurate.

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The solvent delivery pump is the most important part of a high performance liquid chromatograph. The basic performance requirements are as follows: 1. High-pressure discharge that is easily capable of overcoming the increase in column load pressure that results from the refinement of the packing material. 2. The pulsating flow caused by pressure fluctuations srcinating in aspiration / discharge operation only give rise to a small amount of noise in the detector. 3. The eluent flow rate does not fluctuate. 4. When replacing solution, operations such as rinsing are easy and solution consumption is relatively low. 5. The flow rate setting range is wide, and the flow rate can be set accurately.

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Solvent Delivery Pump: Representative Pumping Methods 

Syringe pump

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Plunger pump

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Diaphragm pump

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In order to satisfy performance requirements, pumps based on a wide variety of mechanisms have been devised and used. Pumps can be categorized according to the driving mechanism into a variety of types, including gas-driven pumps, motor-driven pumps, and peristaltic pumps. At present, because of their ability to maintain stable solvent delivery for long periods and to deliver solvent at high pressures, motor-driven pumps based on step motors controlled by microcomputers are widely used. Pumps can also be categorized according to the mechanism used to discharge the solution. Syringe pumps push out solvent at a constant speed using a large syringe. Plunger pumps use a reciprocating piston called a “plunger”. Diaphragm pumps push and pull an inflecting plate called a “diaphragm”. At present, plunger pumps are mainly used.

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Solvent Delivery Pump: Schematic Diagram of Plunger Pump

Motor and cam

Pump head

Check valves

Plunger

10 -100µL

Plunger seal LAAQ-B-LC001B

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In a motor-driven plunger pump, a process consisting of the aspiration, compression, and discharge of solution is repeated. The operating principle is shown in the above diagram. The operation of the step motor is converted, through a cam, to reciprocating motion of the plunger. A material such as sapphire or a special ceramic is usually used for the plunger. The eluent is aspirated and discharged by the motion of this plunger. Check valves ensure that solution flows only in one direction. The valves contain balls, typically made of ruby, which become wedged in the seats of the valves, thereby blocking the flow channels, if the solution starts to flow in the opposite direction. Some pumps use a mechanism in which the check valves are forcibly opened and closed by a magnetic force. The plunger seal prevents the solution drawn into the pump head from leaking into the drive unit. The seal is continuously being worn away by the action of the plunger, so it is a consumable item that must be replaced at regular intervals. It is typically made of fluororesin or polyethylene.

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Solvent Delivery Pump: Single Plunger Type

Check valves

Plunger head

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With a single-plunger pump, the plunger moves slowly at a constant speed during solution discharge and moves at a high speed during aspiration. This movement minimizes the reduction in pressure that occurs when the solution is aspirated, and reduces pulsation. This mechanism is called “constant displacement with quick return” (CDQR). Because of their simple structure, single-plunger pumps are easy to maintain and have come to be widely used. There is a limit, however, to the extent by which pulsation can be reduced, and increasing demands for greater sensitivity have led to a decrease in their use.

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Solvent Delivery Pump: Dual Plunger Type Check valves

Plunger heads

Type

Type

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Dual-plunger pumps are based on the idea that pulsation can be reduced by having one plunger perform aspiration while the other performs discharge. At present, this type of delivery pump is mainly used with HPLC. There are two types of dual-plunger pumps: a parallel type, in which the plungers are arranged in parallel, and a serial type, in which the plungers are arranged in series. With both types, pulsation is minimized by operating the plungers with a 180º phase difference so that they perform aspiration and discharge alternately.

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Gradient System

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Isocratic system 

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Constant eluent composition

Gradient system 

Varying eluent composition 

HPGE (High Pressure Gradient)

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LPGE (Low Pressure Gradient)

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The technique of delivering solution with a constant composition as the eluent is called “isocratic elution”. The technique of varying the eluent composition during a single analysis is called “gradient elution”.

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Aim of Gradient System (1)

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In isocratic mode CH3OH / H 2O = 6 / 4

Long analysis time!!

Poor separation!!

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CH3OH / H2O = 8 / 2

(Column: ODS type)

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In the analysis of multiple components using HPLC, attempting to clearly separate every single component results in an extremely long analysis time. On the other hand, attempting to reduce the analysis time by changing the eluent composition has an adverse effect on separation among components with relatively short retention times. Is there no way of reducing the analysis time while maintaining a good level of separation?

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Aim of Gradient System (2) 

If the eluent composition is changed gradually during analysis... t n 95% e u l e in lo n a h t e m f o n io t a 30% tr n e c n o C

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In order to separate components with short retention times, an eluent composition with a low elution strength is used immediately after sample injection. After most of these components have been eluted, the eluent composition is changed so that components with long retention times are eluted relatively quickly. This technique is called “gradient elution”. Using gradient elution in this way makes it possible to maintain good separation and reduce the analysis time. The chromatograms obtained with gradient elution contain many peaks so there is a tendency to think of gradient elution as a technique that achieves an extremely high level of separation. As can be seen above, however, the main objective of gradient elution is the batch analysis of multiple components.

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HighHigh- / LowLow-Pressure Gradient System

Low-pressure gradient unit

Mixer

Mixer

High-pressure gradient

Low-pressure gradient

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Gradient types can be categorized according to hardware configuration as either highpressure gradient or low-pressure gradient. The terms “high pressure” and “low pressure” indicate whether the location at which solutions with different compositions meet is at a high pressure or normal pressure. A high-pressure gradient system uses multiple solvent delivery pumps. The mixing ratio is regulated by the independent control of the solvent delivery flow rate for each pump. In a low-pressure gradient system, a low-pressure gradient unit that mixes the solutions is installed at a point upstream from the pump. This unit generally incorporates solenoid valves at the inlets for each solution, and the mixing ratio is regulated by controlling the opening and closing times of the valves.

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Advantages and Disadvantages of HighHigh- / LowLow-Pressure Gradient Systems 

High-pressure gradient system 

High gradient accuracy



Complex system configuration (multiple pumps required)



Low-pressure gradient system 

Simple system configuration



Degasser required

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An advantage of the high-pressure gradient system is that, because the accuracy of the mixing ratio depends on the solvent delivery performance of the pumps, using highperformance pumps makes it easy to obtain a high level of accuracy. High accuracy helps improve the reproducibility of retention times and peak area values. It also makes this system suitable for semi-micro analysis. A disadvantage is that, because one pump is required for each solution, as the number of solutions increases, so does the complexity and cost of the required HPLC system. An advantage of the low-pressure gradient system is that, because a low-pressure gradient unit can generally handle four solutions, the equipment cost per solution is relatively low. A disadvantage is that, because different solutions are mixed under normal pressure, bubbles are easily formed and a degasser is therefore required.

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Degasser



Problems caused by dissolved air in the eluent 

Unstable delivery by pump



More noise and large baseline drift in detector cell

In order to avoid these problems, the eluent must be degassed.

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Since analysis is performed in air at a pressure of one atmosphere, air bubbles are always dissolved in the eluent. If the eluent is passed through the HPLC system in this state, problems srcinating in the dissolved air may occur. The most serious problems occur if the bubbles enter the solvent delivery pump. The compression and expansion of the bubbles by the plunger is enough to disrupt the delivery of the all-important eluent. The flow rate may drop, pulsation may occur, or solvent delivery may stop completely. Problems also occur if bubbles enter the flow cell in the detector. Noise may be produced when the bubbles pass through, and baseline fluctuations may occur if bubbles accumulate in the cell. Furthermore, even if there are no bubbles, depending on the quantity of dissolved gas, the detected background level may be affected, and the peak response itself may even change. In order to prevent these problems, the eluent is degassed beforehand. Degassing methods consist of “offline degassing”, in which the eluent is degassed before it is set in the instrument, and “online degassing”, in which the eluent is continuously maintained in a degassed state after being set in the instrument. The device used to perform continuous online degassing is called a “degasser”. A method in which the pressure is reduced with an aspirator while the eluent is either subjected to ultrasound or agitated with a magnetic stirrer is commonly used to perform offline degassing. Although this method is simple and relatively inexpensive, air is gradually dissolved back into the eluent afterwards, and not only is the effect of degassing diminished during analysis, baseline fluctuations may also occur. It is recommended that the following online degasser is used.

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Online Degasser Regulator Helium cylinder

Polymeric film tube

Vacuum chamber

To pump

To pump To draft

Drain valve

Eluent container

Helium purge method

Eluent container

Gas-liquid separation membrane method

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An online degasser is used to degas the eluent continuously, even during analysis. It is not absolutely essential for HPLC, but it is required to fully attain the basic performance specifications of an HPLC system. There are two types of online degasser: those that use the helium purge method and those that use the gas-liquid separation membrane method. Helium Purge Method With this method, helium gas is bubbled into the eluent, and air is thereby replaced with helium. The solubility of helium in liquids is generally low, and does not cause the formation of bubbles when water and organic solvents are mixed. The greatest advantage of this method is that, in comparison with other methods, it has a large degassing effect. It does, however, have some disadvantages. For example, a helium gas cylinder is required, and when using mixtures of solvents, highly volatile solvents may vaporize, causing a change in the composition. Gas-Liquid Separation Membrane Method (Vacuum Degassing Method) With this method, the eluent is passed through a tube made of polymeric film, and the outside of the tube is decompressed (i.e., in a vacuum). Because the tube is made of a material that allows the permeation of gases but not liquids, by the time the eluent reaches the tube outlet, some degree of degassing has been achieved. All this method requires is the electricity to run a vacuum pump. Hardly any maintenance is necessary. For this reason, it is currently the most commonly used method. One disadvantage of this method, though, is that sufficient degassing may not be achieved for high eluent flow rates.

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Sample Injection Unit (Injector)

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Performance Requirements 

No sample remaining in unit

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Minimal broadening of sample band

 

Free adjustment of injection volume Minimal loss



Superior durability and pressure resistance

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The performance specifications required of the sample injection unit used in HPLC are as follows: 1. It must have a structure that does not allow the sample to remain in the unit. 2. It must have a structure that minimizes spread of the sample band. 3. It must be possible to freely set the sample injection volume. 4. Sample loss must be minimal. 5. It must have superior durability and pressure resistance. In order to satisfy these requirements, nearly all commercially available sample injection units for HPLC, whether they are manual injectors or autosamplers, are based on mechanisms that allow flow channel selection using 6-port valves.

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Manual Injector

From pump

To column

LOAD position

From pump

To column

INJECT position LAAQ-B-LC001B

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With a manual injector, the sample is injected manually using a microsyringe. Regardless of the manufacturer of the HPLC system itself, a large number of currently used products incorporate Rheodyne 6-port valves.

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Manual Injector: Operating Principle of Sample Injection

From pump

From pump

p o o L

p o o L

To column

To column

LOAD

INJECT

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The injection principle is as follows: First, the sample is injected in the LOAD state with the microsyringe. The injected sample solution is stored in the sample loop. Next, the knob is turned to switch to the INJECT state. This causes the eluent delivered from the pump to flow through the loop, thereby conveying the sample to the column. The injection procedure can be summarized as follows: 1. Insert the microsyringe in the INJECT position. 2. Turn the knob to switch to the LOAD position. 3. Push the microsyringe plunger to inject the sample. 4. Turn the knob to switch to the INJECT position. (Start data collection at the same time.) 5. Remove the microsyringe. 6. Rinse the injection port. If the column load pressure is high, or if the loop volume is large, turn the knob to the LOAD position before inserting the microsyringe.

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Manual Injector: Injection Method 

Syringe measurement method 



It is desirable that no more than half the loop volume is injected.

Loop measurement method 

It is desirable that at least 3 times the loop volume is injected.

LAAQ-B-LC001B

33

Syringe Measurement Method With this method, the volume is measured with the microsyringe, and this volume is injected into the manual injector. An important advantage of this method is that the injection volume can be changed freely as long as it is within the range of the microsyringe’s measuring capacity. A disadvantage is that it is easy for inconsistencies in the volume to occur due to variations in the skill level and personal style of the measurer. The speed of the injected sample band is higher near the center of the tube and lower near the internal wall of the tube. For this reason, if a volume almost equal to the loop volume is injected, there is a possibility that some of it may leave the loop. If possible, do not inject more than half the loop volume. Loop Measurement Method With this method, using the way that the sample leaves the loop if more than the loop volume is injected, an amount equal to the loop volume is conveyed to the column by deliberately injecting more than the loop volume. An advantage of this method is that there is little chance of inconsistencies occurring between analysts, so the injection reproducibility is high. A disadvantage is that the loop must be replaced in order to change the sample injection volume. As previously mentioned, the injected sample band does not flow through the loop evenly, so if an amount only slightly larger than the loop volume is injected, eluent may remain on the inner wall. Therefore, inject at least 3 times the loop volume.

33

Autosampler (Pressure Injection Method) To column

Frompump

Frompump

Tocolumn

Sample Loop

LAAQ-B-LC001B

LOAD

INJECT

34

Injection by an autosampler consists of the automatic execution of the operations performed by a manual injector using a microsyringe based on control by a computer program. Pressure Injection Method With this method, after a specific amount of sample is aspirated from the sample vial and conveyed to the sample loop attached to a 6-port valve, the valve is switched so that eluent is delivered into the loop and the sample is consequently conveyed to the column. In other words, some of the operations performed by an analyst using a manual injector are performed by a machine. An advantage of this method is that, as with a manual injector, injection based on the loop measurement method is possible, and a large-volume injection can be facilitated by replacement of the sample loop.

34

Autosampler (Total(Total-Volume Injection Method) To column

From pump

From pump

To column

Needle

Sample vial Measuring pump

LOAD

INJECT

LAAQ-B-LC001B

35

Total-Volume Injection Method With this method, a specific amount of sample is measured from the sample vial into a tube, and eluent is delivered directly into this tube, thereby conveying the sample to the column. The most important advantage of this method is that, because eluent flows around inside the needle at all times except during injection operation, sample carryover is unlikely to occur. Another advantage is that, because it is not necessary to aspirate a volume greater than that conveyed to the column, there is little sample loss. With this method, even if there are only very small bubbles in the section of tube leading from the measuring pump to the tip of the needle, it can become impossible to aspirate accurate amounts. For this reason, the rinsing fluid that fills the autosampler interior must also be degassed.

35

Column Oven



Air circulation heating type



Block heating type 



Aluminum block heater

Insulated column jacket type 

Water bath

LAAQ-B-LC001B

36

In HPLC, particularly in the widely used techniques of reversed phase chromatography, normal phase chromatography, and ion exchange chromatography, temperature control is an extremely important consideration. For example, the following are generally observed if the column temperature increases. (There are some exceptions.) • The retention time becomes shorter. • The load pressure decreases due to a decrease in the viscosity of the eluent. • The number of theoretical plates improves due to an increase in the diffusion coefficient. For this reason, it can be said that within a range in which possible deterioration of the column can be ignored, analysis is often performed at as high a temperature as possible (40°C to 60°C). A wide variety of heating mechanisms, such as air circulation heating and block heating, are employed in column chambers. In general, air circulation heating is most commonly used, although the use of block heating, which requires relatively little space, has increased in recent years.

36

Tubing and Preparation for Solvent Delivery Prior to Analysis

LAAQ-B-LC001B

37

When first setting up an HPLC system or when changing the flow-channel configuration in accordance with the type of analysis, the units and columns must be connected with tubing. When connecting the units with tubing, in order to prevent the spread of sample inside the tubing, short tubes with narrow diameters must be used to the extent possible without hampering execution of the experiment. When the tubing is connected, rinsing fluid and eluent are delivered from the pump. At this time, care is required to ensure that the flow channels are not blocked. Care is also required regarding the purity of the solvents delivered.

37

Tubing



Material 

Stainless steel (SUS)



PEEK (polyether ether ketone)



Fluororesin



O.D. (outer diameter) 



I.D. (inner diameter) 

LAAQ-B-LC001B

1.6 mm



0.1 mm 0.3 mm



0.5 mm



0.8 mm etc.

38

Materials used for tubing include stainless steel (SUS316) and PEEK (polyether ether ketone). Stainless steel can withstand pressures of 100 MPa or more, making it particularly suitable for tubing in places subject to high load pressures (e.g., flow channels upstream from the separation column). A disadvantage is that it is prone to corrosion by acids or halogens. PEEK is a type of engineering plastic, and despite being a resin, it can withstand pressures of up to around 25 MPa. It can be used across the entire pH range (i.e., pH 1 to 14), but it does not allow the use of organic solvents with a high solvency, such as chloroform and/or THF. Various types of fluororesin tubes are also used for tubing. In general, they have a high resistance to organic solvents and are easy to handle but there seem to be many types that have a relatively low pressure resistance. They are suitable for the tubing situated downstream from the column and drain tubes. The outer diameter of the tubing used for HPLC is 1.6 mm (1/16 inches). This seems to be an accepted standard almost everywhere in the world. The inner diameter of the tubing is determined in accordance with the purpose of use. In general, tubing with an inner diameter of 0.25 to 0.3 mm is used in standard HPLC. Tubing with an inner diameter of 0.1 mm is used for semi-micro HPLC. Although resistance occurs at the standard flow rate (approx. 1 mL/min), this property is utilized in resistance tubes. Tubing with an inner diameter of 0.5 mm or more is used in preparative LC. It is also used for sample loops and post-column reaction tubes in cases where a significant degree of volume is required.

38

Connectors



Male nut (SUS) Ferrule (SUS) 



Ferrule

Sealing possible up to 40 MPa

Male nut

Male nut (PEEK) 

Can be connected without any tools



Resists pressures of up to approx. 25 MPa

Male nut (PEEK)

LAAQ-B-LC001B

39

The tubing is attached to connection ports using connectors. A stainless steel connector consists of two parts: a ferrule and a male nut. When these are threaded onto a stainless steel tube and the male nut is tightened, the ferrule is pressed into the tube and thereby secured. A spanner is required for tightening. Tighten the nut as far as possible by hand, and then turn it about half a rotation using the spanner. (Tighten it approx. 45° from the point where the ferrule is secured.) Excessive tightening may result in damage to the thread of the nut. Although stainless steel connectors have a high pressure resistance, they require a spanner, and for the connection of columns that are frequently detached and reattached, something easier to use is more suitable. The PEEK male nut was developed in response to this need. No tools are required for this connector. It can withstand pressures of up to 25 MPa when tightened with fingers. Also, because the ferrule is not secured to the tube, connection is always in a position that suits the connection port. Although the type of connector shown in the above diagram consists of a ferrule and nut combined into one, types, like stainless steel connectors, that consist of separable ferrules and nuts are also widely used.

39

Dead Volume (Extra-column volume) 

Dead volume can cause peaks broadening. Male nut

Dead volume

Tube

Poor connection

Excellent connection LAAQ-B-LC001B

40

The volume of the space outside the column that has no direct relationship with separation is called the “dead volume”. If the dead volume is large, it can cause peaks to spread. Therefore, care is required to ensure that the dead volume is minimized, especially with respect to parts of the flow channel that the sample passes through (i.e., between the injector, column, and detector). As much as possible, use injectors and detector flow cells of structures that have minimal dead volume. Also, for the tubing that connects these parts, use tubes that are as short and have as small an inner diameter as possible without creating an undue level of resistance or causing handling problems. Care is also required when connecting the tubing. As shown in the above diagram, if the tube is inserted to the inside end of the connection port, there is no problem. If, however, it is not inserted to the end, dead volume is created. In this case, the peaks may be broadened or they may have shoulders.

40

Mobile Phase



Water



Organic Solvent



“Ultrapure water” can be used with confidence.



HPLC-grade solvent can be used with confidence.



Commercial “distilled



Special-grade solvent is

water for HPLC” is also acceptable.

acceptable depending on the detection conditions. 

LAAQ-B-LC001B

Care is required regarding solvents containing stabilizers (e.g., tetrahydrofuran and chloroform)

41

Sometimes, powdered cuttings and organic dirts are present on new tubing. After connecting new tubing, be sure to rinse the flow channels. It is generally advisable to use alcohol-based solvents (e.g., 2-propanol) for rinsing. It is not necessary to use solvents of a particularly high purity for this purpose. After rinsing the flow channels, prepare eluent and deliver it. For this, use solvent of as high a purity as possible. The water prepared by an “ultrapure water system” can be used with confidence. In general, however, this level of purity is not always necessary. Purified water that has undergone a purification process consisting of at least two stages, such as reverse osmosis and ion exchange or ion exchange and distillation, is usually acceptable. Of course, commercial purified water specifically intended for HPLC may also be used. HPLC-grade organic solvents can be used with confidence. In general, however, HPLCgrade solvents are a little expensive, and depending on the analytical conditions, it may be acceptable to use special-grade solvent. Solvents such as tetrahydrofuran and chloroform contain additives, and this may cause problems in detection or separation. Of course, solvents containing additives are more stable, so, in some cases, it may be better to use such solvents as long as there are no problems with analysis. Therefore, decide whether or not to use solvents containing additives in accordance with the specific details of the analytical process.

41

Replacement of Eluent 

Mutually insoluble solvents must not be exchanged directly.



Water

Aqueous solutions containing salt and organic solvents must not be exchanged directly.

Buffer solution

2-Propanol

Water

Hexane

Water-soluble organic solvent

LAAQ-B-LC001B

42

When replacing the solution in the flow channels, exercise care regarding the mutual solubility of the pre- and post-replacement solutions. When exchanging solvents that do not mix together, such as water and hexane, do not exchange them directly. First replace one with a solvent that dissolves in both (e.g., 2propanol). Some inorganic salts dissolve easily in water but do not dissolve easily in organic solvents. Therefore, when replacing a buffer solution with an organic solvent, first deliver water through the flow channels to rid them of salt and organic solvent. Also, care is required to ensure that none of the pre-replacement solution is mixed with the post-replacement solution. Pour some of the solution about to be delivered into a small beaker and rinse the suction filters and tubes in this solution before setting the solution vial.

42

Mixing, Filtration, and Offline Degassing of the Eluent

Decompression by aspirator

Membrane filter with pore size of approx. 0.45 µm

Decompression by aspirator Ultrasonic cleaning unit LAAQ-B-LC001B

43

After preparing the eluent, mix it well. This is to homogenize the solution and to prevent problems related to bubbles occurring during delivery by expelling supersaturated dissolved air. Even if an online degasser is used, in some cases it is easy for bubbles to be produced immediately after the start of delivery. In order to start delivery smoothly, it is recommended that a moderate amount of degassing is performed beforehand. More specifically, connect the inlet of an aspirator to the mouth of the bottle and, while applying ultrasonic waves to the solution, decompress the bottle. If an ultrasonic cleaning unit is not available, perform decompression while agitating the solution intensely with a magnetic stirrer. This operation need not be performed for a long time. A few tens of seconds is sufficient. As long as the production of bubbles at the start of delivery is prevented, the online degasser will handle degassing in subsequent operation. In the case of an eluent that contains a relatively high concentration of salt, such as a buffer solution, it is recommended that the technique of filtration under reduced pressure is used. Use a membrane filter with a pore size of approx. 0.45 µm. Filtration under reduced pressure takes care of both filtration and degassing at the same time.

43

Reversed Phase Chromatography Part 1 Basic Principles

LAAQ-B-LC001B

44

A large number of separation modes are used in high performance liquid chromatography, but the most widely used mode by far is “reversed phase (distribution) chromatography”. The principle and characteristics of this separation mode are described here.

44

Polarity of Substances



Polarity





Property of a substance whereby the positions of the electrons give rise to positive and negative poles



Water: Polar





Methane: Nonpolar –

H H LAAQ-B-LC001B

H

H

Methane

H

+

Solvents of similar polarities can be easily dissolved together. Polar and nonpolar molecules have a similar relationship to that of water and oil.

H

O

C

Miscibility of solvents

H

Water

O

H C C H Acetic acid

O

–

45

Two atoms share an electron cloud to form a “covalent bond”, and this whole structure constitutes a molecule. However, even though the electron cloud is “shared”, it is not necessarily evenly distributed between the bonded atoms, and the electrons may be located more closely to the atom that exerts greater pull on them. Electrons are negatively charged, so the atom to which the electrons are pulled becomes a negative pole, and the other atom becomes a positive pole. This type of bonded state is described as “polar”. The strength with which a bonded atom pulls electrons is called “electronegativity”. Comparing the electronegativities of some commonly encountered atoms gives the following: F > O > Cl, N > Br > C, H If the center of the negative charge and the center of the positive charge in a molecule do not coincide, that molecule is polar. Water molecules are typical polar molecules. In methane, however, although there is polarity in the individual C-H bonds, overall the molecule has a regular tetrahedral structure, so there is no polarity. In general, it is said that substances that are either both polar or both nonpolar have a high mutual solubility. On the other hand, polar and nonpolar substances have a low mutual solubility. Using water to represent polar solvents and oil to represent nonpolar solvents, the relationship between a polar and nonpolar solvent can be likened to the relationship between oil and water.

45

Nonpolar (Hydrophobic) Functional Groups and Polar (Hydrophilic) Functional Groups



Nonpolar Functional Groups 

-(CH2)nCH3 







-COOH



-NH2



-OH

Alkyl groups



-C6H5 

Polar Functional Groups

Phenyl groups





LAAQ-B-LC001B

Carboxyl groups Amino groups Hydroxyl groups

46

In some cases, molecules with complex structures contain both nonpolar and polar parts. The overall polarity of such a molecule is determined by the functional groups that are bonded. Representative examples of nonpolar functional groups include alkyl groups and phenyl groups, which are composed entirely of weakly electronegative carbon and hydrogen atoms. The longer the alkyl group chain, the lower the polarity. Polar functional groups include molecules composed of strongly electronegative halogen and nitrogen atoms. Representative examples include carboxyl groups, amino groups, and hydroxyl groups.

46

Partition Chromatography



A liquid (or a substance regarded as a liquid) is used as the stationary phase, and the solute is separated according to whether it dissolves more readily in the stationary or mobile phase.



Liquid-liquid chromatography

LAAQ-B-LC001B

47

Depending on how readily a solute dissolves in two solvents that are not mutually soluble, a difference may emerge between the concentration of solute in each solvent. The technique of using this property to transfer a component dissolved in one solvent to another solvent, or to concentrate or clean up the component, is called “solvent extraction”. The type of chromatography that directly applies the principle of solvent extraction is called “partition chromatography”. In partition chromatography, the stationary and mobile phases are both thought of as liquids, and the strength of retention of a solute is determined according to whether the solute dissolves more readily in the stationary or mobile phase. Of course, the liquids used for the stationary and mobile phases must not be mutually soluble.

47

Normal Phase / Reversed Phase

Stationary phase

Mobile phase

Normal phase

High polarity

Low polarity

(hydrophilic)

(hydrophobic)

Reversed phase

Low polarity

High polarity

(hydrophobic)

(hydrophilic)

LAAQ-B-LC001B

48

Partition chromatography can be performed in one of two modes: normal phase and reversed phase. The combination of a stationary phase with a high polarity and a mobile phase with a low polarity is called “normal phase” and the opposite combination is called “reversed phase”. Reversed phase chromatography is described here. The term “reversed phase” gives the impression that this technique is somewhat unorthodox. In fact, most people that use HPLC perform separation with reversed phase chromatography, and it can fairly be described as a standard separation mode.

48

Reversed Phase Chromatography



Stationary phase: Low polarity 



Octadecyl group-bonded silical gel (ODS)

Mobile phase: High polarity 

Water, methanol, acetonitrile



Salt is sometimes added.

LAAQ-B-LC001B

49

Stationary Phase Compounds with a low polarity, such as those composed of aliphatic chains without localized electrons, are used. However, to be used as packing material for HPLC, the substance used must be chemically stable and capable of withstanding high pressures, so it is not true to say that any substance with a low polarity is sufficient. The most commonly used substance is produced by chemically bonding an octadecyl group (-C18H37) to the surface of silica gel. This type of packing material is commonly known as “ODS”, and an “ODS column”, into which ODS is packed, is almost synonymous with a “column for reversed phase chromatography”. Mobile Phase The most commonly used solvents are water, methanol, and acetonitrile. Water is the solvent with the highest polarity, and by mixing it with methanol or acetonitrile, which have lower polarities, the overall polarity of the solution can be adjusted. Salts and acids are also added sometimes in order, for example, to adjust the pH value or form ion pairs.

49

Separation Column for Reversed Phase Chromatography 

C18 (ODS) type



Phenyl type



C8 (octyl) type



TMS type



C4 (butyl) type



Cyano type

Si

-O-Si

CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3

C18 (ODS) LAAQ-B-LC001B

50

In general, packing material produced by chemically bonding hydrophobic (low-polarity) functional groups to a silica gel substrate is used as the stationary phase. The most widespread of such packing materials is a type called “ODS”, which is formed by bonding octadecyl groups (-C18H37) to the surface of silica gel. The structure of this material is illustrated above. In addition to ODS, packing materials produced by bonding octyl groups, which have a short aliphatic chain, phenyl groups, and cyanopropyl groups are commercially available, and are used in cases where a different separation selectivity from that of ODS is required. Also, the support material is not limited to silica gel. For example, materials formed by bonding octadecyl groups to the surface of a resin are also available.

50

Effect of Chain Length of Stationary Phase C8 Medium

C18 (ODS) Strong

C4 Weak

LAAQ-B-LC001B

51

If a stationary phase produced by chemically bonding an aliphatic chain to silica gel is used, the length of the aliphatic chain influences the retention strength for the solute. It is said that, in general, longer chains have a greater retention strength. Beyond a certain length, however, the retention strength does not change significantly. To effect an overall increase or decrease in the speed with which a component is eluted, rather than replacing the stationary phase, changing the composition of the mobile phase, as described later, is significantly simpler and cheaper. Therefore, as long as an ODS column is used as the separation column, there is unlikely to be any problem deciding on the separation conditions. The analysis of a protein is an example of a situation necessitating the use of a stationary phase produced by bonding octyl groups or groups with shorter aliphatic chains. In general, proteins are denatured and precipitated in organic solvents, so there cannot be a high concentration of organic solvent in the mobile phase. Therefore, a stationary phase produced by bonding a short aliphatic chain is used, thereby decreasing the overall retention strength, and the amount of organic solvent added to the mobile phase is decreased.

51

Hydrophobic Interaction H2O H2O H2O

H2O

H2O

H2O

H2O

Nonpolar solute

H2O H2O

If a nonpolar H2O substance is added...

H2O H2O

H2O H2O

H2O H2O

Nonpolar solute

LAAQ-B-LC001B

H2O

H2O H2O

…the network is broken and...

Network of hydrogen bonds

H2O

H2O

Nonpolar stationary phase

…the nonpolar substance is pushed to a nonpolar location.

52

Although reversed phase chromatography is regarded as a type of partition mode, it is said that the retention mechanism is difficult to explain in terms of partition. On the other hand, because the force that acts between the nonpolar solute and the nonpolar stationary phase is only a weak dispersion force (van der Waals force), it is impossible to explain the mechanism simply in terms of the theory of adsorption. Therefore, the concept of “hydrophobic interaction” is used as a model to explain the retention mechanism of reversed phase chromatography. The polar mobile phase molecules are formed by a network of hydrogen bonds. Although polar and ionic solutes can participate in this network, a nonpolar solute cannot form hydrogen bonds easily. So, in order to dissolve, it must break the network, consequently creating an energy imbalance. A simple way for the entire solution to regain a stable energy balance would be to push out the nonpolar solute. If the solution is in contact with a nonpolar stationary phase, pushing the solute toward this stationary phase would reduce the number of breaks in the network, and improve the energy balance. The retention mechanism of reversed phase chromatography, then, can be understood by considering a model in which the solute is repelled by the mobile phase and pushed onto the stationary phase, rather than one in which the solute and stationary phase are positively attracting each other.

52

Relationship Between Retention Time and Polarity

OH

C18 (ODS) Weak Strong CH3

LAAQ-B-LC001B

53

In reversed phase chromatography, strongly hydrophobic substances (i.e., substances with a relatively low polarity) are strongly retained by the stationary phase, and therefore have relatively long retention times. Therefore, in a chromatogram containing multiple peaks, the substances are eluted, broadly speaking, in descending order of polarity.

53

Basic Settings for Eluent Used in Reversed Phase Mode 

Water (buffer solution) + water-soluble organic solvent Water-soluble organic solvent: Methanol Acetonitrile Tetrahydrofuran etc.  The mixing ratio of the water (buffer solution) and organic solvent has the greatest influence on separation. 



If a buffer solution is used, its pH value is an important separation parameter.

LAAQ-B-LC001B

54

In general, a solution of the following composition is used as the eluent in reversed phase mode: Water (buffer solution) + water-soluble organic solvent In many cases, separation adjustment is performed by changing the composition of this eluent. In gas chromatography, the composition of the carrier gas, which acts as the mobile phase, is hardly ever changed. In liquid chromatography, however, the composition of the mobile phase is a key aspect of separation adjustment. The most commonly used water-soluble organic solvents are methanol and acetonitrile. Other solvents, such as tetrahydrofuran (THF) are also used. The factor that has the greatest influence over the retention and separation of the solute is the ratio with which the water (buffer solution) and water-soluble organic solvent are mixed. In many cases, the mixing ratio of the organic solvent with respect to water has a greater influence on solute retention than the type of organic solvent used. Instead of just using water, sometimes salt or another substance is added in order to create a pH buffer solution. In this case, the pH has a great influence over separation. Although the type of buffer salt used and its concentration influence separation, it is the pH that needs foremost consideration.

54

Difference in Solute Retention Strengths for Water and Water-Soluble Organic Solvents Tightly packed network

H2O

H2O

H2O H2O

H2O

Loose network

CH3OH

CH3OH

H2O H2O

CH3OH CH3OH

CH3OH

Nonpolar solute

CH3OH CH3OH

Nonpolar solute

Nonpolar stationary phase LAAQ-B-LC001B

55

Let us review some of the points made about hydrophobic interaction. Polar mobile phase molecules are formed by a network of hydrogen bonds. If a nonpolar solute enters this network, hydrogen bonds are broken, and this creates an energy imbalance. In order to minimize this imbalance, the solute is pushed onto the nonpolar stationary phase. This is the basic principle behind solute retention due to hydrophobic interaction. Because water has a very high polarity, its network of hydrogen bonds is believed to be extremely tightly packed. Solvents such as methanol and acetonitrile, however, despite having some level of polarity, are not as polar as water, so their hydrogen bonds are believed to be much weaker. In solvents that form loose networks like this, the force with which a nonpolar solute is pushed onto the nonpolar stationary phase is not that strong. The above gives rise to the following basic rule concerning reversed phase mode: The greater the proportion of water in the eluent, the greater the solute retention strength. Or The greater the polarity of the eluent, the greater the solute retention strength.

55

Relationship between Polarity of Eluent and Retention Time in Reversed Phase Mode

Eluent: Methanol / Water

60/40

70/30

80/20 LAAQ-B-LC001B

56

In practice, using single solvents such as water or methanol as the eluent is quite rare. Usually, mixtures of these solvents are used. This makes it possible to control the overall solute retention strength. The above diagram illustrates how differences in the eluent affect the chromatogram. As the polarity of the eluent decreases (i.e., as the proportion of methanol increases), the overall retention time decreases.

56

Chromatogram Parameters Methods for Expressing Separation and Column Performance

LAAQ-B-LC001B

57

The parameters that can be obtained from chromatograms are explained here. Retention Factor, k Sometimes called theretention “capacitystrength factor” or this parameter expresses the solute of the the “capacity stationaryratio”, phase. Theoretical Plate Number, N This parameter is an indicator of the performance of the separation column. Separation Factor, a This parameter is equal to the ratio of the retention factors for two peaks. Resolution, RS This parameter expresses the degree of separation between two peaks.

57

Retention Factor, k l a n ig s r o t c e t e d fo th g n rte S

tR

k

= t R − t0 t0

t0

tR: Retention time t0: Non-retention time Time

LAAQ-B-LC001B

58

If we hypothesize that the solute does not interact with the stationary phase at all, and remains within the eluent the whole time, then the corresponding peak would appear at t0. This means that the time obtained by subtracting t0 from the retention time, tR, can be regarded as time for which the solute stayed in the stationary phase. If a solute remains in the stationary phase for a relatively long time, it indicates that the retention strength for that solute is relatively high. It is therefore possible to express the strength with which a solute is retained by calculating the ratio of times that the solute remains in the stationary and mobile phases. This is called the retention factor. If the retention factor (k) is 1, it indicates that the solute remains in the stationary and mobile phases for the same time. If it is less than 1, it indicates that the solute is not retained to a significant degree before elution. If it is 3 or greater, it indicates that the solute undergoes significant interaction with the stationary phase before elution. One problem is the calculation of t0. In theory, the volume of the eluent inside the column can be calculated by multiplying the internal volume of the column (i.e., the volume of the cylinder) by the porosity of the packing material. Dividing this by the eluent flow rate gives t0. For example, if the inner diameter and length of the column are 0.46 cm and 15 cm respectively, the porosity is 0.6, and the eluent flow rate is 0.8 mL/min, then t0 can be calculated as follows: 2

t0 = (0.23 × π × 15) × 0.6 ÷ 0.8 = 1.87 [min] In practice, however, because the porosity inside the column is hardly ever known, and the volume of other parts, such as tubing, affects the calculation, it is difficult to obtain an accurate value for t0. A working value can be obtained, however, by actually measuring the retention time for a solute that is known not to be retained by the stationary phase. With the reversed phase mode, substances such as nitrite ion and urea are often used. 58

Theoretical Plate Number, N

N

= 16

2

tR W

2

= 5.54

H W1/2 H1/2

= 2π

W LAAQ-B-LC001B

tR W1 / 2

tR

•H

2

Area 59

One theoretical way of handling chromatography is the “plate theory” model. This is based on the concept of handling the process of chromatography as repeated solvent extraction in a flask. The solute that enters the separation site is partitioned between the stationary and mobile phases according to a specific ratio. The mobile phase moves, so the solute partitioned in the mobile phase also moves, and is partitioned again. As this behavior is repeated again and again, substances with different partition coefficients are separated in a way that can be thought of as repeated solvent extraction performed to increase the degree of refinement. In this model, if one occurrence of solvent extraction is denoted as one “plate”, then the “theoretical plate number” is the number of plates corresponding to the extraction performed by the separation column. If the theoretical plate number is large, this means that extraction is performed a correspondingly large number of times, and indicates a relatively high level of separation performance. Although the formulas defining the theoretical plate number are given above, the reason why these formulas are used is not given here. (In fact, textbooks on the fundamentals and practical application of HPLC usually do not give the derivation of these formulas.) For more details, refer to specialized literature on the subject. For the purposes of this text, remember that the theoretical plate number is an indicator of the efficiency (performance) of the separation column.

59

Evaluation of Column Efficiency Based on Theoretical Plate Number 

If the retention times are the same, the peak width is smaller for the one with the larger theoretical plate



number.

If the peak width is the same, the retention time is longer for the one with the larger theoretical plate number.

N: Large

N: Small

N: Large

N: Small

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60

In one of the formulas given for the theoretical plate number, the retention time appears in the numerator and the peak width appears in the denominator. This shows that these quantities are important factors in the evaluation of column performance. If peaks are sharp, they can be completely separated from nearby peaks. Therefore, a highperformance column can be thought of as one that gives small peak widths. The sample band is diffused inside the column, so peaks with a short retention time are relatively sharp, whereas peaks with a long retention time are relatively broad (in the case of an isocratic system). Therefore, if two columns give the same peak width for a given solute, the column that gives a longer retention time can be evaluated as having a higher lever of performance. If a separation column is used repeatedly, the peaks gradually become broader and the retention times gradually become shorter. In other words, the column performance deteriorates. Appropriate management can be performed by regularly obtaining the theoretical plate number. There is no specific value, however, for the theoretical plate number below which the column must be replaced. Each case must be evaluated independently according to whether or not the desired separation can be achieved and whether or not the decreases in sensitivity and area reproducibility caused by the broadening of peaks are within acceptable limits.

60

Separation Factor, a



Separation factor: Ratio of k’s of two peaks k1

k2

k2

α

= (k2

LAAQ-B-LC001B

k1

> k1 )

61

The separation factor for two peaks is the ratio of their retention factors. The relationship between the elution positions of two peaks is expressed using this parameter. It can also be said that the separation factor expresses the separation selectivity. This is because the size of α indicates whether the two peaks are in closely neighboring positions or separated positions. The separation factor only expresses the positional relationship between two peaks. It provides no information about peak separation (i.e., the degree of overlap). Even if the separation factor is large, if the peaks are broad, they may not be well separated. Even if the separation factor is small, if the theoretical plate number for the column is high and the peaks are sharp, they may be sufficiently separated.

61

Resolution, RS

tR1

tR2 RS

=

tR

− tR

2

2 (W

1

= 1.18 × W1/2h,1

W1/2h,2 W1

1

1

h1/2

+ W2 ) − tR ,1 + W1 / 2

tR W1/ 2 h

2

1

h,2

W2

LAAQ-B-LC001B

62

The resolution indicates the extent to which two peaks are separated or, from a different perspective, the extent to which they overlap. While the separation factor indicates only the positional relationship between two peaks, and does not indicate the degree of overlap, the resolution does, to a certain extent, indicate the degree of separation. The formulas used to obtain the resolution are given above. It can be seen that this parameter is equal to the ratio of the difference between the retention times of the two peaks and the average value of the two peak widths. If the distance between the peaks is large compared to the peak widths, they are well separated, whereas if the opposite is true, they are overlapping.

62

Resolution Required for Complete Separation (tR2 - tR1)

(tR2 - tR1)

W1 W2

W1 W2

tR2 - tR1 = W1 = W2

tR2 - tR1 = W1 = W2

RS = 1 LAAQ-B-LC001B

RS = 1 If the peaks are Gaussian distributions, RS > 1.5 is necessary for complete separation.

If the peaks are isosceles triangles, they are completely separated.

63

What level of resolution is required for two peaks to be completely separated? Let us suppose that the two peaks are isosceles triangles. If the two triangles are standing alongside each other with their bases making contact, the difference between the retention times and the average value of the peak widths are equal, and the resolution equals 1. This means that if the resolution is greater than 1, the bases of the two triangles do not make contact, and complete separation is attained. This does not apply, however, to peaks shaped like Gaussian distributions. At a resolution of 1, the peak skirts overlap. To be able to say that complete separation is attained, a resolution of at least 1.5 is probably required. The above reasoning is based on the assumption that the two peaks are almost the same height. If the heights are different, sufficient separation may not be attained at the levels of resolution specified above. In particular, the smaller peak may be partially covered by the larger peak, making it impossible to identify the peak top.

63

Relationship Between Resolution and Other Parameters 

The resolution is a function of the separation factor, the theoretical plate

RS

number, and the retention factor. 

=

tR 2

1 (W1 + W2 ) 2

=1

The separation can be improved by improving these 3 parameters!

LAAQ-B-LC001B

− t R1

4

N

α −1 α

k’2 k’2

+1

64

By manipulating the formula previously given for the resolution in the way shown above, it can be demonstrated that this parameter is a function of the theoretical plate number (N), the separation factor (α), and the capacity factor (k’). The above formula indicates that an increase in the theoretical plate number, an increase in the separation factor, or an increase in the retention factor translates to an increase in the resolution. Therefore, the separation can be improved by improving these 3 parameters.

64

Contribution of Capacity Factor to Resolution 



Increasing the capacity factor improves separation! A capacity around 3 tofactor 10 is of appropriate. Exceeding this just increases the analysis time.

n 1.0 o ti lu o 0.8 s re r fo 0.6 o i tr a 0.4 n o ti u 0.2 ib tr n o C0.0

0

5

10

15

20

Capacity factor

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65

If retention of the target substance is weak, the separation can be improved by taking steps, such as changing the eluent composition, that will strengthen overall retention. The effect is particularly noticeable in cases where the k’ value does not exceed 10. If the k’ value does exceed 10, however, increasing the retention strength even further will probably not improve separation. In such a case, it would be more effective to improve the theoretical plate number or the separation factor.

65

Contribution of Theoretical Plate Number to Resolution 

The resolution increases in proportion to the square root of the theoretical plate number.

n 2.0 o ti u l o s e r r o f r o t c 1.0 a f n io t u irb t n o C0.0

0

10000 2 0000 30000 Theoretical plate number

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66

Whereas the capacity factor and separation factor can be adjusted using the chemical interaction between the stationary and mobile phases, the theoretical plate number depends on the performance of the separation column. Consequently, in order to improve it, the column length must be increased or the column must be replaced with one capable of superior performance. The theoretical plate number is almost proportional to the column length, so lengthening the column is an effective way of improving the resolution. This, however, causes the analysis time and the load pressure at the column inlet to increase. As such, in terms of convenience, it may not necessarily be the best approach. Therefore, it is necessary to strike a balance between the desired separation performance and practicalities.

66

To Improve Separation...

Before adjustment

k’ increased

Eluent replaced with one of lower elution strength.

N increased

Column replaced with one of superior performance. Column lengthened.

α

Column (packing material) replaced. Eluent composition changed. Column temperature changed.

increased

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67

The specific measures that can be used to improve the resolution can be summarized as follows: Increase the Capacity Factor ( k’) Change the eluent composition so that elution is generally slower. In reversed phase mode, reduce the proportion of organic solvent in the eluent. There are also methods that involve changing the stationary phase. For example, in ion exchange mode, replace the column with one filled with packing material that has a large exchange capacity. Increase the Theoretical Plate Number ( N) In general, replace the column with one of superior performance. For example, use a column filled with packing material of a smaller pore size. It is also effective to either use a longer column or multiple connected columns. In some cases, the theoretical plate number can be improved by increasing the column temperature or changing the solvent composition so that eluent viscosity decreases. Increase the Separation Factor (α ) Change separation conditions such as the column used, the eluent composition, and the temperature.

67

pH Buffer Solution Used for Eluent Selection and Preparation of Buffer Solution

LAAQ-B-LC001B

68

Sometimes, for reasons related to separation or detection, a pH buffer solution must be used as the eluent. Here, the basic concepts behind the pH buffer solution used for the eluent are explained.

68

Acid Dissociation Equilibrium H+

If an acid is added... ...the equilibrium shifts to the left to offset the increase in H +.

A- + H+

HA If an alkali is added... …the equilibrium shifts to the right to offset the decrease in H +.

OH-

The equilibrium always shifts in a way that offsets changes.

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69

A typical type of acid dissociation equilibrium is shown above. Let us suppose that an acid, HA, which is in an undissociated state, and its A– ions, which are in a dissociated state, are in equilibrium at a certain ratio. If a small quantity of another acid or an alkali is added to this solution, although the H+ concentration temporarily increases or decreases, the above equilibrium shifts in a way that offsets this change, so the H+ concentration does not change significantly. A solution like this, whose pH value only changes slightly when a small quantity of an acid or alkali is added, is called a “pH buffer solution”. The pH buffering power is first exhibited when HA and A– are in a complementary state. If the equilibrium shifts greatly to the left or right, there is unlikely to be a significant level of buffering action. In an aqueous solution, strong acids, such as hydrochloric acid, nitric acid, and sulfuric acid, are always in an almost completely dissociated state. In other words, the equilibrium is shifted greatly to the right as shown above. Aqueous solutions of these strong acids have almost no pH buffering power. On the other hand, with weak acids, such as phosphoric acid, acetic acid, and citric acid, it is possible, depending on the pH value, to maintain an equilibrium between the undissociated and dissociated states at a certain ratio. These weak acids can be used for buffer solutions.

69

Acid Dissociation Constant and pHpH-Based Abundance Ratio A- + H+

HA

The acid dissociation constant, Ka, is defined as follows: − +

[ A ][H ]

Ka

= [HA]

pH = pK a

+ log

[A − ] [HA ]

1.0

0.8

CH3COO -

CH3COOH

o ti ra

e0.6 c n a d0.4 n u b A

0.2

0.0 123456789

⎛ pH = − log[H + ] ⎞ ⎜⎜ ⎟⎟ ⎝ pK a = − log K a ⎠

pH

pKa Relationship Between Abundance Ratio and pH Value of Acetic Acid and Ac etic Acid Ions

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70

The “acid dissociation constant”, Ka, is a constant that expresses the level of the dissociation equilibrium of an acid. Ka is a constant that is specific to the acid type. Stronger acids have a large value whereas weaker acids have a smaller value. Note that -log Ka is notated as “pKa”. In general, this “pKa” notation is used more often. As mentioned previously, pH buffering power is exhibited when HA and A– are in a complementary state. If the concentration of the two is the same, from the formula given above, it can be seen that pH and p Ka must be equal. Conversely, if pH and p Ka are equal, the concentrations of HA and A – must be almost the same, and the pH buffering power must be high. Therefore, to prepare a buffer solution of a certain pH value, it is advisable to use an acid that has a pKa value that is close to the desired pH value.

70

Preparing pH Buffer Solution



Use a weak acid with a p Ka value close to the desired pH value. 



Example: Preparing a buffer solution for a pH value of around 4.8. → Use acetic acid, which has a pKa value of 4.8.

Make the concentrations of HA and A- roughly equal. → Mix an acid with its salt. 

Example: Mix acetic acid and sodium acetate so that they have the same molar concentration.

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71

The acid selection and preparations methods used to prepare a pH buffer solution are described here. Selection of Acid Type Use an acid with a pKa value close to the desired pH value. The p Ka values of compounds are given in physics and chemical dictionaries and in books that refer to acid-base equilibrium. Preparation Procedure Prepare the solution by mixing the acid with its salt. For example, to prepare acetate buffer solution, mix acetic acid in an undissociated state with its salt, sodium acetate. Sodium ions are strongly basic, and because they almost completely dissociate in aqueous solutions, adding sodium acetate is essentially equivalent to adding dissociated acetate ions. To adjust the pH value of a buffer solution so that it is close to the p Ka value, mix the acid with its salt so that they have the same molar concentration. To separate the pH value somewhat from the pKa value, change the mixing ratio of the acid and its salt. If possible, keep the difference between the solution’s pH value and the pKa value less than 1.

71

Buffer Solutions Used for HPLC Eluent



Requirements 







LAAQ-B-LC001B



Commonly Used Acids

High buffering power at prescribed pH.



Phosphoric acid

Does not adversely affect detection.



Acetic acid



Citric acid

Does not damage column or equipment. Inexpensive.









pKa 2.1, 7.2, 12.3 pKa 4.8 pKa 3.1, 4.8, 6.4

Concentration 

If only to adjust pH, 10 mmol/L is sufficient. 72

One obvious requirement of a buffer solution is that it has a high buffering power in a neighborhood of the desired pH. If it is to be used as an HPLC eluent, several other properties are required. First, it is important that it does not adversely affect detection. With UV detection, it must have no UV absorption. With fluorescence detection, it must have no fluorescence. With electrical conductivity detection, it must either have a low equivalent conductivity or it must be difficult to ionize. It is important to use a buffer solution that does not damage the column or any other equipment. In general, the wetted parts of an HPLC system are composed of stainless steel, so it is advisable to avoid halogens or other substances that may corrode the steel. With LCMS, nonvolatile salts that may precipitate at the interface must not be used. Phosphate buffer solution satisfies the above conditions, is relatively inexpensive, and is the most commonly used substance. Acetate and citrate buffer solutions are also commonly used. All of these solutions are used in the acidic region, for pH values in a range of approximately 2 to 7. Because the ODS column, which is the most representative separation column for HPLC, is not suited to alkalis, the use of buffer solutions with pH values in the alkaline range is relatively uncommon. The buffering capacity increases with the concentration of the buffer salt. Increases in the salt concentration of the eluent, however, increase the risk of clogging and hasten the deterioration of the pump’s plunger seal. Therefore, it is advisable to prepare the buffer solution with as low a concentration as possible within the range in which an appropriate pH buffering capacity is attained. In general, when using a buffer solution for the sole purpose of maintaining the pH of the eluent at a constant level, use a concentration of approximately 10 mmol/L. This may not be appropriate, however, if the salt concentration exerts a large influence on separation (e.g., in 72 ion exchange mode).

Characteristics of Phosphate Buffer Solution 

Advantages 



Three dissociation states (pKa 2.1, 7.2, 12.3) 



No UV absorption



Inexpensive

No volatility 

Possible to prepare buffer solutions of various pH values.



Disadvantages Difficult to use for LCMS or evaporative light scattering detection.

LAAQ-B-LC001B

73

The most commonly used buffer solution in HPLC is phosphate buffer solution. Phosphoric acid dissociates in three stages, so there are three pKa values, and it has a buffering power in the neighborhoods of the corresponding pH values. In particular, it is often used as an acidic buffer solution in the range of pH 2 to 3 and as a neutral buffer solution in a neighborhood of pH 7. Also, because it has almost no UV absorption, there is almost no background signal in UV detection, which is the most representative HPLC detection method. This is another reason why this buffer solution is used so widely. Although phosphate buffer solution is used so widely, because it has no volatility, it is difficult to use in LCMS and evaporative light scattering detection. The lack of volatility also makes post-processing difficult in the preparative separation and purification of peak components. In this case, use another acid that has volatility, even if it has inferior buffering capacity.

73

Reversed Phase Chromatography Part 2 Consideration of Analytical Conditions

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74

Here, following on from Part 1, the methods used to set the analytical conditions in reversed phase chromatography are described. Reversed phase ion pair chromatography is also briefly mentioned.

74

Guidelines for Setting Mobile Phase Conditions (1) Neutral (Nonionic) Substances 



Eluent Composition 

Water / acetonitrile



Water / methanol

Separation Adjustment 

Changing the mixing ratio of the water and organic solvent



Changing the type of organic solvent

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75

Drawing on the points made so far, let us summarize the basic guidelines for setting separation conditions in reversed phase chromatography. Regarding the stationary phase, it is generally appropriate to use an ODS column. Situations where another type of column must be used are quite rare. Regarding the eluent composition, if the target components are substances that do not ionize, use a mixture of water and acetonitrile or a mixture of water and methanol. Choose between acetonitrile or methanol on the basis of which better facilitates separation and which is more convenient generally. Adjust overall retention by changing the mixing ratio of the water and these organic solvents.

75

pH of Eluent and Retention of Ionic Solutes COOH Acidic

Increased hydrophobicity pH of eluent

COO Alkaline

Increased hydrophilicity

+ H LAAQ-B-LC001B

76

In the analysis of ionic solute, the pH value of the eluent greatly influences the retention strength. The above diagram shows how the retention strength for benzoic acid changes. The acid dissociation constant (pKa) of benzoic acid is 4.8. In eluents of greater acidity, it exists as undissociated benzoic acid, whereas in eluents of greater alkalinity, it exists as benzoic acid ions. In general, because ionic substances have more affinity towards networks of hydrogen bonds, the ionized form of a substance dissolves more readily in polar eluents, and is less easily retained by the stationary phase. Therefore, for an acidic (anionic) substance such as benzoic acid, if the pH of the eluent is more acidic than the acid dissociation constant, the retention strength increases, whereas if it is more alkaline than the acid dissociation constant, the retention strength decreases. Naturally, the opposite occurs with basic (cationic) substances. With neutral substances that do not ionize, the pH of the eluent has almost no influence on the retention strength.

76

Guidelines for Setting Mobile Phase Conditions (2) Acidic (Anionic) Substances 

Eluent Composition 

Acidic buffer solution / acetonitrile



Acidic buffer solution / methanol

Increase retention strength by making the eluent acidic and suppressing ionization!

LAAQ-B-LC001B

77

With substances (e.g., organic acids) for which the target components are anionized, prepare the eluent by mixing an acidic buffer solution with either acetonitrile or methanol. The eluent is rendered acidic in order to suppress the ionization of the target components and to make it easier for them to be retained by the stationary phase. A phosphate is used as the buffer salt. This is because it has a high pH buffering power in a neighborhood of pH 2 to 3, it has almost no UV absorption, and it is inexpensive. The concentration of the buffer salt is usually in the range of 5 to 100 mmol/L, and is often set to approximately 10 mmol/L.

77

Analysis of Basic Substances (1) Problems Encountered with Alkaline Eluents

N+ H

With alkaline eluents, although the ionization of basic substances is suppressed, and the retention strength increases...

N

OH

Si O

OH

Si

OH

…silica gel dissolves in alkalis, so the packing material deteriorates rapidly.

OH OH

OH

LAAQ-B-LC001B

78

Although we have guidelines for setting the mobile phase conditions for neutral and acidic substances, how should we handle basic (cationic) substances? One possible method is to use a mixture of an alkali buffer solution and either acetonitrile or methanol in order to suppress ionization. This method is certainly effective with regard to separation, but it creates another problem and is not often used. The problem is that the silica gel dissolves in alkalis; consequently, the packing material deteriorates rapidly. The usable pH range for a standard ODS column is approximately 2 to 8, and it is said that staying within a range of approximately 2 to 5 helps prolong the service life of the column. In other words, the pH of the eluent must be set in the acidic-to-neutral range. However, packing material in which the surface of the silica gel has been specially treated in order to protect it from the influence of alkalis, thereby improving the alkali resistance, has recently been developed. In addition to silica gel, there is also packing material that was produced by bonding octadecyl groups with a polymer substrate. Therefore, when using an alkali as the eluent, it is necessary to use a separation column filled with a packing material that can withstand it.

78

Analysis of Basic Substances (2) Influence of Residual Silanol Groups

Basic substances interact with the residual silanol groups, causing delayed elution and tailing.

O

Si

Si

-O-Si-O Residual silanol group

O Si

N+ H

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79

Another possible method that can be used for basic substances, as with acidic substances, is to use a mixture of an acidic buffer solution and an organic solvent. In this case, although the target component is cationized, the decrease in retention strength is compensated for by lowering the proportion of organic solvent in the eluent. It is appropriate to set the pH of the buffer solution in the neighborhood of 2 to 3. This is to suppress the influence of residual silanol groups for which the octadecyl groups have not bonded to the surface of the stationary phase. Although octadecyl groups are chemically bonded to silanol groups on the surface of the silica gel, because of their bulky structure, it is impossible to bond to all silanol groups. The residual silanol groups are weakly acidic and therefore attract basic substances, which causes tailing of the peaks. Using an acidic buffer solution makes it possible to suppress dissociation of the residual silanol groups and reduce their influence to some extent. In order to suppress the influence of these residual silanol groups, nearly all ODS columns that are currently available have been subjected to “end-capping”. With this treatment, bonding relatively compact trimethylsilyl groups and other substances with the residual silanol groups makes it possible to reduce the influence of the silanol groups on separation. Even if the eluent is made acidic, with packing material containing a large number of residual silanol groups, it is impossible to suppress the tailing that occurs with basic substances. When analyzing basic substances, be sure to use a column that has been subjected to endcapping. Note that residual silanol groups are not the only cause of tailing. For example, tailing is prone to occur if packing material containing metallic impurities is used for compounds that easily form complexes with metals. Separation columns with reduced metal content are commercially available. So, if necessary, try using one of these columns instead.

79

Analysis of Basic Substances (3) Addition of Sodium Perchlorate

ClO4

N+ H

Ion pair

Si O

Si

Basic substances form ion pairs with perchlorate ions, thereby balancing the charge and increasing the retention strength.

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80

If tailing still occurs even if the eluent is made acidic, adding anions with a low charge density, such as perchlorate ions, to the eluent may have some effect. Anions and cations with low charge densities form ion pairs in aqueous solutions, thereby balancing the charge. This causes the hydrophobicity and the strength of retention by the stationary phase to increase. It also suppresses tailing. Sodium perchlorate is widely used for this purpose. For the eluent, use a solution produced by adding 50 to 200 mmol/L of sodium perchlorate to an acidic buffer solution and mix this with an organic solvent. To further increase the retention strength, add sodium alkylsulfonate to the eluent. Further details are provided in the section on reversed phase ion pair chromatography.

80

Guidelines for Setting Mobile Phase Conditions (3) Basic Substances (Cationic Substances) 

Eluent Composition 

Acidic buffer solution containing anions with a low charge density (e.g., perchlorate ions) / acetonitrile



As above / methanol

Making eluent acidic → Suppresses dissociation of residual silanol groups → Prevents tailing! Adding perchlorate ions → Forms ion pairs → Increases retention strength! → Suppresses tailing! LAAQ-B-LC001B

81

The guidelines for setting the mobile phase conditions in the analysis of basic substances can be summarized as follows: (1) Alkaline Buffer Solution / Acetonitrile (Methanol) Packing material with a high alkali resistance must be used. (2) Acidic Buffer Solution / Acetonitrile (Methanol) End-capped packing material must be used. Depending on the properties of the target substances, it may not be possible to suppress tailing. (3) Acidic Buffer Solution Containing Sodium Perchlorate / Acetonitrile (Methanol) Concentration of perchlorate: approximately 50 to 200 mmol/L. End-capped packing material must be used. Tailing suppression effect is relatively high. (4) Acidic Buffer Solution Containing Alkyl Sulfonic Acid / Acetonitrile (Methanol) Concentration of alkyl sulfonic acid: approximately 5 to 50 mmol/L. The concentration and pH of the alkyl sulfonic acid and the concentration of the organic solvent must be optimized. Of the above, method (3) is relatively easy to implement and trouble-free. Therefore, first try method (3), and if there seems to be a problem, try method 4 or try using a different separation column or separation mode.

81

Reversed Phase Ion Pair Chromatography 

Increase the retention strength by adding an ion pair reagent with the opposite charge to the target substance into the eluent. Ion pair formation

Ion pair formation

Ion exchangeexchange-like effect LAAQ-B-LC001B

BasicSubstance

Ion exchangeexchange-like effect AcidicSubstance

82

If the target substance is highly soluble in water, and has either cationic or anionic properties, it is usually appropriate to use cation or anion exchange chromatography. If, however, you want to simultaneously analyze the uncharged solutes in the sample, use reversed phase ion pair chromatography. Ions that have the opposite charge to the target substance, and that contain hydrophobic functional groups, are dissolved in the eluent. The added ions are referred to as an “ion pair reagent”. The target substance and the ion pair reagent form ion pairs in the eluent. As a result, the overall charge is balanced out, the hydrophobicity increases, and the retention strength of the stationary phase increases. Furthermore, if an ion pair reagent that has hydrophobic functional groups, such as alkyl groups, is used, the ion pair reagent itself is retained in the stationary phase, and due to a type of ion exchange-like interaction, solutes with the opposite charge are also retained. This method makes it possible for highly water-soluble ions that are not retained at all with an eluent that does not contain an ion pair reagent to be retained with an ODS column.

82

Representative Ion Pair Reagents



Anionic Compounds 



Tetra-n-butylammonium hydroxide (TBA)

Cationic Compounds 

Pentanesulfonic acid sodium salt (C5)



Hexanesulfonic acid sodium salt (C6)



Heptanesulfonic acid sodium salt (C7)



Octanesulfonic acid sodium salt (C8)

LAAQ-B-LC001B

83

UV detection is often used in HPLC; so, for ion pair reagents, it is appropriate to use compounds with as little UV absorption as possible. Tetrabutylammonium salts are often used in the analysis of anionic substances. Alkylamines are also used for this purpose. Alkyl sulfonate salts are used in the analysis of cationic substances. They come in a variety of chain lengths and use a type that is suitable for the retention and separation conditions. Prepare the eluent by adding one of the above to a pH buffer solution at an appropriate concentration. It is common to use a concentration in a range of approximately 5 to 50 mmol/L. In general, increasing the concentration increases the retention strength. Beyond a certain point, however, ion pair reagent molecules combine with each other, causing micellization, and the retention strength stops increasing.

83

Points to Note Concerning the Use of Ion Pairs 

Selection of Ion Pair Reagent 



In general, the retention strength increases with the length of the alkyl chain.

pH of Eluent 

The retention strength changes according to whether or not ionization takes place.



Concentration of Ion Pair Reagent 



In general, the retention strength increases with the ion pair concentration, but there is an upper limit.

Proportion of Organic Solvent in Eluent 

Optimize the separation conditions by considering the type and concentration of the ion pair reagent.

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84

In ion pair chromatography, there are many parameters, such as the type and concentration of the ion pair reagent, the pH of the eluent, and the concentration of the organic solvent, involved in the setting of the mobile phase conditions. Also, in the simultaneous analysis of ionic substances and nonionic substances, because of differences in the behavior of substances are retained by the ion pair effect and substances that are not, setting conditions isthat even more complex. Therefore, in cases where the addition of an ion pair reagent may not be absolutely necessary, it may be faster to first consider the condition setting guidelines previously given for standard reversed phase chromatography. If this is not successful, consider the use of ion pairs.

84

HPLC Separation Modes Separation Modes Other Than Reversed Phase Chromatography

LAAQ-B-LC001B

85

An overview of some representative separation modes used in HPLC is given here.

85

HPLC Separation Modes



Adsorption (liquid-solid) chromatography



Partition (liquid-liquid) chromatography 

Normal phase partition chromatography



Reversed phase partition chromatography



Ion exchange chromatography



Size exclusion chromatography

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86

Representative HPLC separation modes can be categorized into four types: “adsorption”, “partition”, “ion exchange”, and “size exclusion”. In fact, there are other separation modes, and in some cases, multiple separation modes are used simultaneously. It would be impossible to exhaustively categorize every type. For now, just remember the four types mentioned above.

86

Adsorption Chromatography



 

A solid such as silica gel is used as the stationary phase, and differences, mainly in the degree of adsorption to its surface, are used to separate the solutes. Liquid-solid chromatography The retention strength increases with the hydrophilicity of the solute.

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“Adsorption chromatography” refers to the separation mechanism in which a solid such as silica gel or alumina is used as the stationary phase, and solutes are retained by the stationary phase mainly because of adsorption to its surface. Chemically unmodified silica gel is often used as the stationary phase. Because the silanol groups (Si-OH) on its surface are hydrophilic, polar solutes are retained by the stationary phase due to hydrogen bonding and hydrophilic interactions, such as dipolar interactions. Substances with higher degrees of hydrophilicity undergo stronger interactions with the stationary phase; consequently, they are retained with greater strength in the column. Because the force that acts between the solutes and the stationary phase in this mode is almost the same as the one that acts in normal phase partition chromatography, which is described later, adsorption chromatography and normal phase partition chromatography are not distinguished in some cases.

87

Partition Chromatography



A liquid (or a substance regarded as a liquid) is used as the stationary phase, and the solute is separated according to whether it dissolves more readily in the stationary or mobile phase.



Liquid-liquid chromatography

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In adsorption chromatography, the strength of solute retention is mainly determined by the degree of adsorption to a solid surface. In partition chromatography, the retention strength is determined by whether the solute dissolves more easily in the stationary phase, which can be thought of as a liquid, or the mobile phase. Of course, the liquids used for the stationary phase and mobile phase must not be mutually soluble. Apparently, in early partition chromatography, the stationary phase was actually impregnated with a liquid. The impregnated liquid would gradually flow out, however, so materials created by chemically bonding functional groups to the surface of the stationary phase came into use.

88

Normal Phase and Reversed Phase

Solid phase

Mobile phase

Normal phase

High polarity (hydrophilic)

Low polarity (hydrophobic)

Reversed phase

Low polarity

High polarity

(hydrophobic)

(hydrophilic)

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In partition chromatography, the stationary phase and mobile phase must not be mutually soluble. As a general rule, similar substances tend to be mutually soluble; so, in order to ensure mutual insolubility, substances with contrasting properties are set as the stationary and mobile phases. Therefore, use a combination of stationary phase and mobile phase in which one has a high polarity (hydrophilic) and one has a low polarity (hydrophobic). The combination of a stationary phase with a high polarity and a mobile phase with a low polarity is called “normal phase” and the opposite combination is called “reversed phase”.

89

Normal Phase (Partition) Chromatography 



Partition chromatography in which the stationary phase has a high polarity (hydrophilic) and the mobile phase has a low polarity (hydrophobic) Essentially based on the same separation mechanism as adsorption chromatography in which the stationary phase has a hydrophilic base, such as silica gel

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Normal phase chromatography is a kind of partition chromatography in which the stationary phase has a high polarity and the mobile phase has a low polarity. Materials created by bonding polar functional groups, such as aminopropyl groups and cyanopropyl groups, to the surface of silica gel are often used as the stationary phase. Nonpolar solvents, such as hexane and chloroform, are used as the mobile phase. As with adsorption chromatography based on silica gel, the force that acts between the solutes and the stationary phase is based on hydrophilic interactions, such as hydrogen bonding and dipolar interactions. Therefore, it has become common to not distinguish between adsorption chromatography and normal phase partition chromatography. In this textbook, then, the principles of both of these techniques are explained as “normal phase chromatography”.

90

Invention of Chromatography by M. Tswett Ether

Chromatography

Colors

Chlorophyll CaCO 3

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In a paper he presented in 1906, Tswett described how he separated the chlorophyll contained in plant constituents using chalk powder (CaCO 3) as an adsorbent. He called this technique “chromatography”. This is said to be the start of chromatography. This technique could be categorized according to the separation mode as adsorption / normal phase chromatography.

91

Stationary Phase and Mobile Phase Used in Normal Phase Mode



Stationary Phase 

Silica gel: -Si-OH



Cyano type: -Si-CH2CH2CH2CN



Amino type: -Si-CH CH CH NH



Diol type: -Si-CH2CH2CH2OCH(OH)-CH2OH

2



2

2

2

Mobile Phase 

Basic solvents: Aliphatic hydrocarbons, aromatic hydrocarbons, etc.



Additional solvents: Alcohols, ethers, etc.

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Stationary Phase Materials created by chemically modifying silica gel with cyanopropyl groups or aminopropyl groups are typically used as the stationary phase in normal phase chromatography. While chemically unmodified silica gel and silica gel modified with cyanopropyl groups are widely used in general applications, silica gel modified with aminopropyl groups is often used in the analysis of sugars. Mobile Phase The mobile phase is prepared by adding an additive solvent to a basic solvent. The following are often used as the basic solvent: •Hydrocarbons (pentane, hexane, heptane, octane) •Aromatic hydrocarbons (benzene, toluene, xylene) •Organochloride compounds (chloroform, dichlormethane) The following are used as the additive solvent: •Alcohols (2-propanol, ethanol, methanol) •Ethers (methyl-t-butyl ether, diethyl ether) •Tetrahydrofuran, dioxane, pyridine, ethyl acetate, acetonitrile, acetone In general, the basic solvent is selected first, and the additive solvent is added to adjust the retention time and separation. Selecting solvents with little UV absorption is beneficial for UV detection. A small amount of acid is sometimes added to the mobile phase in order to suppress the ionization of solutes.

92

Relationship between Hydrogen Bonding and Retention Time in Normal Phase Mode

SiOH

Strong

HO

SiOH Weak Very weak

OH

Steric hindrance LAAQ-B-LC001B

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In normal phase mode, the solutes are retained by the stationary phase due to hydrophilic interactions, such as hydrogen bonding, that take place between the solutes and the stationary phase. For this reason, elution tends to take longer if the sample contains any of the following functional groups: • -COOH: Carboxyl groups • -NH2: Amino groups • -OH: Hydroxyl groups On the other hand, if the sample is a hydrocarbon and there are no hydrogen bonding sites, or if there are large hydrocarbon groups around the hydrogen bonding sites that act as steric hindrances, elution tends to occur sooner.

93

Relationship Between Eluent Polarity and Retention Time in Normal Phase Mode

Eluent: Hexane/methanol

100/0

98/2

95/5 LAAQ-B-LC001B

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Since normal phase mode is the opposite of reversed phase mode, the relationship between the eluent polarity and the solute retention time is reversed. The above diagram shows how differences in the eluent affect the chromatogram. As the polarity of the eluent increases (i.e., as the proportion of methanol increases), the overall retention time decreases.

94

Comparison of Normal Phase and Reversed Phase 

Normal Phase 





Effective for separation of structural isomers





Wide range of applications



Effective for separation of homologs



Stationary phase has long service life



Stabilizes quickly



Eluents are inexpensive and easy to use

Offers separation selectivity not available with reversed phase



Reversed Phase

Stabilizes slowly and is prone to fluctuations in retention time Eluents are expensive

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Normal phase and reversed phase are, in a sense, opposites and have their own distinct characteristics. In general, normal phase mode is said to be effective for the separation of structural isomers that differ in terms of the connection points of functional groups. On the other hand, it is not effective for the separation of homologs that differ only in the length of aliphatic chain. Reversed phase mode is more effective for this application. Regarding the time taken for the stationary phase and mobile phase to reach an equilibrium, reversed phase mode is significantly faster than normal phase mode. Reversed phase mode is also superior in terms of the stability of the stationary phase. Many of the columns used for normal phase mode have shorter service lives. In actual analysis work, the cost and safety of the eluent used cannot be ignored. Water, methanol, and acetonitrile, which are solvents mainly used in reversed phase mode, are inexpensive and relatively easy to handle. The fact that these solvents have little UV absorption is an important advantage in HPLC, in which absorption detectors are often used. On the other hand, many of the nonaqueous solvents that are mainly used in normal phase mode are expensive and somewhat difficult to handle due, for example, to adverse effects on health and the danger of explosions resulting from contact with flames or static electricity. In addition to the above points, reversed phase mode can be used for a wide range of target substances so, at present, the most common approach in situations where either mode can be used is to first try using reversed phase mode.

95

Ion Exchange Chromatography

R Anion exchange

N+ R R

Cation exchange

SO3-

+

++++ + ++++

Electrostatic interaction (Coulomb force) LAAQ-B-LC001B

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Ion exchange chromatography is a separation mode in which, if the solutes and stationary phase have the opposite charge, they are attracted to each other by electrostatic interaction, and the solutes are consequently retained. Therefore, the force that acts between the solutes and the stationary phase probably consists mainly of Coulomb forces. Ion exchange chromatography can be divided into two types according to the charge on the target solutes: cation exchange chromatography and anion exchange chromatography. Naturally, ionic substances are the target of ion exchange chromatography. Proteins, peptides, and amino acids are all ionic substances, so they can be analyzed using ion exchange chromatography. Therefore, this separation mode is used widely in the field of biochemistry. This separation mode is also used for inorganic ions, such as chloride ions (Cl–) and sodium ions (Na+). HPLC that is used to analyze such inorganic ions is referred to as “ion chromatography”. Whereas “ion exchange chromatography” refers to a separation mode used in HPLC, “ion chromatography” refers to the branch of HPLC in which mainly inorganic ions are analyzed using dedicated hardware (although the definition is somewhat vague). Although these terms names are similar, try to use them correctly.

96

Stationary Phase Used in Ion Exchange Mode 

Base Material  



Cation Exchange Column  



Resin is often used. Silica gel is also used.

Strong cation exchange Week cation exchange

(SCX) (WCX)

-SO3-COO-

Anion Exchange Column  

Strong anion exchange Week anion exchange

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(SAX) (WAX)

-NR3+ -NHR2+ 97

Base Material Although various substances are used for the base material of the ion exchanger, most of them are resinous. Many different types of resin, including polystyrene resin and methacrylic resin, are used. Silica is an inorganic material that is also often used, and there are many other types of materials that are being developed and sold. Exchange Groups Sulfonic acid groups (-SO3–) and carboxyl groups (-COO–) are representative examples of cation exchange groups. Quaternary ammonium groups (-NR3+) are a representative example of anion exchange groups.

97

Dependence of Exchange Capacity of Ion Exchanger on pH of Eluent Strongly acidic cation exchanger

Strongly basic anion exchanger

y it c a p

tiy c a p

c a e g n a h c x E

c a e g n a h c Weakly basic x E

0

Weakly acidic cation exchanger 7 pH

anion exchanger

14

0

Cation exchange mode

7 pH

14

Anion exchange mode

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Ion exchangers can be categorized according to whether they perform “cation exchange” or “anion exchange” or according to whether they perform “strong ion exchange” or “weak ion exchange”. Strong ion exchangers have functional groups whose exchange capacity does not depend greatly on the pH of the eluent and are continuously ionizing. More specifically, exchangers created by chemically bonding sulfonic acid groups and quaternary ammonium groups are often used. Weak ion exchangers have functional groups whose dissociation is suppressed and whose exchange capacity decreases to an extent that depends on the pH of the eluent. More specifically, exchangers created by chemically bonding carboxyl groups and tertiary ammonium groups are often used. Use an exchanger that is appropriate for the substance to be separated and the pH characteristics of that substance.

98

Relationship between Retention Time and Salt Concentration of Eluent in Ion Exchange Mode

Resin The exchange groups are in equilibrium with anions in the eluent.

Resin An eluent ion is driven away and a solute ion is adsorbed.

Resin The solute ion is driven away by an eluent ion and is adsorbed by the next exchange group.

Solute ions and eluent ions compete for ion exchange groups. If the salt concentration of the eluent increases, the solutes are eluted sooner. 99

In ion exchange chromatography, the solutes and the stationary phase have the opposite charge and attract each other electrostatically. Why, then, are the solutes eluted from the column? Why are they not continuously held by the stationary phase? This is because ions with a charge of the same sign as the solutes are added to the eluent, and these ions compete with the solute ions for exchange groups. Therefore, the overall elution rate can be increased without greatly changing the separation selectivity by increasing the overall salt concentration of the eluent. In other words, the effect that is achieved in reversed phase and normal phase chromatography by changing the mixing ratio of the solvents comprising the eluent in order to adjust the polarity and thereby increase or decrease the overall elution time is achieved in ion exchange chromatography by adjusting the salt concentration of the eluent.

99

Ion Exclusion Chromatography

H+

H+

H+

Depending on the level of dissociation, some weak acid ions can enter the pore.

Strong acid ions are repelled by charge and cannot enter the pore. LAAQ-B-LC001B

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Ion exclusion chromatography is a separation mode that is like ion exchange chromatography in that it uses an ion exchanger. The mechanism of operation, however, is completely different. If the solutes and the stationary phase have a charge of the same sign, a Coulomb repulsive force acts. It is considered that the strength of this repulsive force increases with the ease with which the solutes ionize. On the other hand, ions that do not dissociate readily and substances with no charge are not subject to the same level of repulsion, so they reach the interior of the pores in the packing material. They are consequently retained by the stationary phase due to size exclusion and reversed phase interactions. Ion exclusion chromatography, then, is a separation mode in which the retention strength is determined mainly by the degree of dissociation of the solute ions. In practice, this mode is often used for the separation of short-chain fatty acids.

100

Size Exclusion Chromatography 



Separation is based on the size (bulkiness) of molecules. The name varies with the application field!  

Size Exclusion Chromatography Gel Permeation Chromatography(SEC) (GPC) 



Chemical industry fields, synthetic polymers, nonaqueous systems

Gel Filtration Chromatography (GFC) 

Biochemical fields, biological macromolecules, aqueous systems

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Whereas all of the previously described separation modes use chemical or electrostatic interactions to produce retention strength, in size exclusion chromatography, there are (ideally) no interactions between the solute molecules and the stationary phase. Gel particles with a gigantic network structure are used as the stationary phase, and separation takes according to the degree to which the solute molecules penetrate the pores of these place particles. Although this mode is called “size exclusion chromatography”, for a while now, it has been referred to by different names that vary with the industry field. In chemical industry fields, the name “gel permeation chromatography (GPC)” is used even today. In general, it is used in the quality control and property evaluation of chemically synthesized resins. Hydrophobic resins, such as polystyrene, are used as the stationary phase and substances such as tetrahydrofuran, chloroform, and dimethylformamide are used as the eluent. “Gel filtration chromatography (GFC)” is a name often heard in biochemical fields. It is mainly used in relation to the analysis of biological macromolecules, such as proteins, peptides, nucleic acids, and polysaccharides. Because all of these macromolecules are soluble in water, highly hydrophilic resins are often used as the stationary phase, and aqueous solutions are often used as the eluent.

In addition, because the separation mechanism used in this mode is also called “molecular sieving”, this mode is also sometimes referred to as “molecular sieving chromatography”.

101

Principle of Size Exclusion Mode

The size of the solute molecules determines whether or not they can enter the pores.

Packing material

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When solutes are carried by the flow of the eluent into the stationary phase, which consists of macromolecules with a gigantic network structure, relatively small molecules penetrate the interior of the network whereas relatively large molecules cannot enter the network and are excluded. In other words, the range in which the solute molecules can move around inside the column (i.e.,move the effective volume) size. Solutes with a slowly, low molecular weight can around in a largevaries rangewith and their consequently are eluted whereas solutes with a high molecular weight are restricted and consequently are eluted quickly.

102

Relationship Between Molecular Weight and Retention Time in Size Exclusion Mode Exclusion limit t h g i e w r a l u

) s i x a ic m th ir lc a e o g o M (l

Permeability limit

Elution capacity

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Solute molecules larger than a certain size are not able to enter the network structure of the stationary phase at all, and are all excluded. This size is called the “exclusion limit”. Solute molecules smaller than a certain size completely permeate the interior of the stationary phase and are consequently all eluted at almost the same position. This size is called the “permeability limit”. In size exclusion mode, separation takes place between the exclusion limit and the permeability limit. These limits are determined by the packing material used, so it is necessary to estimate the molecular weight of the target substances and select an appropriate separation column.

103

Creating a Molecular Weight Calibration Curve For separation of large molecular weights t h ig e w r la u c le o M

) s i x a ic

For wide-range separation (mix gel)

m h itr a g o (l

Elution capacity For separation of small molecular weights LAAQ-B-LC001B

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The kind of curve shown above, which expresses the relationship between the elution capacity (retention time) of a solute and its molecular weight, is called the “calibration curve” of the size exclusion column. Molecular weight markers, for which the molecular weight is already known, are commercially available, so obtain elution capacities (retention times) for these markers and use the results to create a calibration curve. It is difficult to cover a wide range of molecular weights with the same packing material (i.e., column) so, in general, size exclusion columns are sold as series consisting of multiple types of packing material that have the same properties but differing degrees of crosslinkage. Select a column that is appropriate for the desired molecular weight range. If the molecular weight distribution of the sample covers a wide range, select multiple columns from the same series that collectively cover this range, and connect them in series. A possible alternative approach is to use a column that has been filled with packing materials that have differing degrees of cross-linkage.

104

Calculating Molecular Weights

Chromatogram



Various Average Molecular Weights 



Calibration curve 



Retention time LAAQ-B-LC001B

Mn: Number-average molecular weight Mw: molecular Weight-average weight Mz: Z-average molecular weight, etc.

Molecular weights and molecular weight distributions are calculated using special calculation software. 105

Special calculation software is required to calculate various average molecular weights and molecular weight distributions. Usually, a chromatogram is sampled with a chromatogram data processor (software), and special software performs molecular weight calculations using the data file obtained. Basically, the chromatogram is sliced at regular intervals, and the respective area values are obtained. At the same time, the molecular weights for the slices are calculated from a previously created calibration curve. Then, calculating the area at the position of each molecular weight makes it possible to obtain the average molecular weights. There are various types of average molecular weight, such as the “number-average molecular weight”, which is based on the numbers of molecules, and the “weight-average molecular weight”, which is based on the weights. It is said that these correlate with the various properties of macromolecular compounds, so use these values as necessary and evaluate the results.

105

Guidelines for Selecting Separation Mode (1) Required Information



Soluble solvent



Molecular weight



Structural formula and chemical properties 

Do the substances ionize?



Is there UV absorption or fluorescence?



Is derivatization possible? etc.

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There are many different separation modes in HPLC, so it is necessary to select a mode that is appropriate for the target substances and samples. The following kind of information is required for the selection of the separation mode: Soluble Solvent In principle, HPLC can be used for any substance that can be dissolved in a liquid. Conversely, however, if the liquids in which the substance dissolves are not known, then appropriate analytical conditions cannot be established. First and foremost, then, it is essential to know which solvents the substance dissolves in. Molecular Weight Size exclusion mode is used for substances of more than a certain molecular weight, whereas other separation modes are used for substances of less than this molecular weight. Size exclusion mode is the first choice for molecular weights of more than a few thousand. Even in this case, however, in order to select a column with an appropriate exclusion limit, information on the approximate molecular weight range is required.

In addition, it is desirable to have as much information as possible on the chemical properties, including the structural formulas, of the substances and samples to be analyzed. In particular, knowing if the substances ionize makes it possible to know whether or not ion exchange mode or reversed phase ion pair mode can be used, and the existence of UV absorption or fluorescence has a bearing on detector selection.

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Guidelines for Selecting Separation Mode (2) Basic Policy

Reversed phase mode using an ODS column is the first choice! Exceptions Large molecular weight (> 2,000) Optical isomers Chiral column Stereoisomers, positional isomers

Size exclusion

Normal phase / adsorption Inorganic ions Ion chromatography Sugars, amino acids, short-chain fatty acids Special column LAAQ-B-LC001B

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The most commonly used HPLC separation mode is reversed phase chromatography. The main reason for this is its wide application range. Therefore, except in a few situations, reversed phase mode is the first choice. Some exceptions are described below: For compounds with a molecular weight of more than a few thousand, size exclusion mode is used. In the separation of optical isomers that have the same chemical properties, a special optical isomer separation column called a “chiral column” is often used. For the separation of multiple stereoisomers or positional isomers, normal phase/adsorption mode is more suitable than reversed phase mode. Also, regarding the analysis of lipophilic substances that do not dissolve in mixtures of water and organic solvents, this mode is the first choice in many cases. In the analysis of inorganic ions, the technique called “ion chromatography”, which combines ion exchange mode and electrical conductivity detection, is used. Although ion chromatography is, in principle, a branch of HPLC, dedicated equipment is commercially available that is unlike standard HPLC, which is mainly used for the analysis of organic substances, so it is distinguished by the use of a different name. In addition, for sugars, amino acids, and short-chain fatty acids, the special separation columns listed below are commercially available. It is advisable to select one these columns in accordance with the application. • Monosaccharides, oligosaccharides: Reversed phase mode using column with amino group-bonded packing material Size exclusion-ligand exchange Anion exchange, etc. • Amino acids:

Cation exchange (pre-column derivatization-) reversed phase mode, etc.

• Short-chain fatty acids: Ion exclusion Ion exchange, etc. 107

HPLC Hardware: Part 2 Detectors and Their Ranges of Application

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Representative detectors that are used in HPLC and their ranges of application are described here.

108

Detection Condition Requirements



Sensitivity 



The detector must have the appropriate level of sensitivity.

Selectivity 

The detector must be able to detect the target substance without, if possible, detecting other substances.



Adaptability to separation conditions



Operability, etc.

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HPLC analytical conditions can be divided broadly into separation conditions and detection conditions. (There is also the category of pretreatment conditions but this is omitted here.) The most important considerations with regard to the detection conditions are probably sensitivity and selectivity. Sensitivity Although high sensitivity is generally required, this may not apply to all situations. If peaks are clipped or if the linearity of the calibration curve is poor, it may be better to use a detector of lower sensitivity. Selectivity It must be possible to detect the target substances, ideally without detecting other substances. Even if other substances are detected, this is not a problem as long as they are separated in the column. In fact, if no other substances are detected, separation may not be necessary. (In this case, the name of the analytical technique used is “flow injection analysis (FIA)”, which is distinct from HPLC.) As with sensitivity, a high level of selectivity may not necessarily be suitable in every situation. For example, when investigating the impurity content of samples, a low level of selectivity may be preferable. Adaptability to Separation Conditions With some detectors, there are restrictions with regard to the setting range of separation conditions. For example, some detectors cannot be used for gradient analysis and some detectors cannot handle nonvolatile salts. Also, it is usually not permissible to add substances to the eluent that are detected by the detector. It is necessary to confirm that these restrictions do not present problems before setting the analytical conditions.

109

Representative HPLC Detectors  

     

UV-VIS absorbance detector Photodiode array-type UV-VIS absorbance detector Fluorescence detector Refractive index detector Evaporative light scattering detector Electrical conductivity detector Electrochemical detector Mass spectrometer

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The following performance specifications are demanded from HPLC detectors: • Easy operability and high reliability • Quick response and high sensitivity • Low noise level and little drift • No dispersion of separated sample bands • Minimally influenced by changes in the type of solvent, flow rate, and temperature. The representative detectors used in HPLC are listed above. Here, the principles and ranges of application of these detectors are described in order.

110

UV-VIS Absorbance Detector C:

Detection cell

Ein

Concentration

Eout

A

l A

=

·C·l

= –log (Eout / Ein)

C

( A: absorbance, E: absorption coefficient) LAAQ-B-LC001B

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A UV-VIS absorbance detector is created by installing a flow cell in a “UV-VIS spectrophotometer”. It directs light of a certain wavelength through a solution, and observes the decrease of intensity in the light that passes through. Because, in accordance with the Lambert-Beer law, the absorbance is proportional to the concentration of the absorbing substance, measuring changes in the absorbance of the eluent makes it possible to calculate the concentrations of peak components. These detectors can be used for substances that have absorption in the ultraviolet region (approx. 190 to 370 nm) or the visible wavelength region (approx. 370 to 600 nm; up to approx. 900 nm for some detectors). Not only do many organic compounds, which are the main target substances of HPLC, have this absorption, the detectors have a relatively high sensitivity and are not easily influenced by factors such as temperature and pulsations. For this reason, they are the most representative HPLC detectors and are widely used.

111

Optical System of UVUV-VIS Absorbance Detector Grating Sample cell

Ein

Eout

Ein

Ein

Photodiode

Photodiode

Reference cell D2 / W lamp

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The principle behind the optical system of this detector is illustrated above. A deuterium lamp (ultraviolet light) or a tungsten lamp (visible light) is mainly used as the light source. Monochromatic light produced by a diffraction grating passes through a cell, and when it enters a silicon photodiode or some other detector, it is converted to an electrical signal. Usually, before the light reaches the cell, it is split into two beams, with one beam passing through a sample cell and the other beam passing through a reference cell (which is usually just an empty space) for reference. Measurement data is obtained from the ratio of the intensities of these two beams.

112

Spectrum and Selection of Detection Wavelength

The longer wavelength is more selective.

200

250

300 Wavelength [nm]

350

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Whether or not an organic compound has absorption in the ultraviolet or visible regions depends on its molecular structure. In general, organic compounds with double bonds have ultraviolet absorption. This detector is particularly suited, however, to substances with conjugated structures, such as conjugated dienes and benzene skeletons. There is a tendency for compounds with large numbers of conjugated double bonds to have absorption at longer wavelengths. The ultraviolet spectrum for caffeine is shown above. All compounds have unique spectra like this. Usually, high sensitivity is desirable; in this case, set a wavelength that corresponds to the absorption maximum as the detection wavelength. However, in terms of selectivity, it is advantageous to set a longer wavelength, so, if there are many co-existing substances in the sample, set a long wavelength as the detection wavelength, even if sensitivity is somewhat sacrificed.

113

Optical System of Photodiode Array Detector Sample cell

Grating

A single photodiode measures the absorbance for the corresponding wavelength at a resolution of approx. 1 nm.

D2 / W lamp

Photodiode array

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Photodiode array-type absorbance detectors can be classified, in terms of the measurement principle, together with the other absorbance detectors previously mentioned. With standard absorbance detectors, however, the light is divided by a diffraction grating before it reaches the cell and monochromatic light is used, whereas with photodiode array detectors, polychromatic light is passed the advantage cell and guided to the detector. This givesin real photodiode array detectors thethrough important of being able to obtain spectra time. The principle behind the optical system of this type of detector is illustrated above. After polychromatic light passes through the cell, it is divided by a diffraction grating, and guided to photodiode array elements. The photodiode array consists of a large number of elements (512 in many cases), and these elements simultaneously convert light components corresponding to a continuous range of distinct wavelengths (separated by intervals of 1 nm in many cases) into electrical signals. This allows real-time spectrum measurement.

114

Data Obtained with a Photodiode Array Detector Spectrum Chromatogram

e c n a b r o s b

A

W LAAQ-B-LC001B

Retention time

en el av

gt

h

115

The data obtained with a photodiode array detector is not handled as the usual timeabsorbance 2D data, but rather as 3D data incorporating a wavelength axis. For this reason, the data is processed using special analysis software instead of the standard type of chromatograph data processor. This software makes it possible to view the spectra corresponding wavelengths. to specified times and the chromatograms corresponding to specific

115

Advantages of Photodiode Array Detectors 

Peak Identification Using Spectra 





Complementation of identification based on retention time Library searches

Evaluation of Peak Purity 

Purity evaluation performed by comparison of the shape of spectra from the peak detection start point to the peak detection end point

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The ability to obtain 3D data is not the only advantage offered by a photodiode array detector. It can obtain chromatograms simultaneously at multiple wavelengths so, in addition to being able to perform the role of multiple single-wavelength absorbance detectors as a single unit, it offers the following advantages: Peak Identification Based on UV Spectra Basically, retention time is the only means of identification available in chromatography; so, in terms of qualitative capability, chromatography is a poorer analytical technique than techniques such as NMR, MS, and FTIR. For this reason, attempts are made to offset this weakness using the selectivity and qualitative capability of the detector. With a photodiode array detector, a UV spectrum can be obtained for each peak, so these spectra can be used as a means of identification. Furthermore, many types of analysis software incorporate a library search function, making it possible to search the library for the UV spectra that most closely resemble the ones obtained. Evaluation of Peak Purity From the detection start point to the detection end point of a pure peak, the shape of the spectrum should not change simply due to changes in absorbance. Therefore, by obtaining and comparing the spectra obtained at different times for the same peak, it is possible to evaluate the optical purity of that peak. Many software products incorporate a function that obtains and compares the spectra for three points of a given peak: the top, a point on the up-slope, and a point on the down-slope. This is called the “three-point spectrum method”. Furthermore, software products incorporating a function that compares spectra for all points from the detection start point to the detection end point and displays the evaluation results graphically as a “peak purity curve” are available.

116

Fluorescence Detector Excitation wavelength

+

*

*

hv1 h v2 + Excited state

Fluorescence wavelength

Quasi-excited state

h v1

h v2

Fluorescence Ground state

LAAQ-B-LC001B

117

“Fluorescence” is a phenomenon whereby certain substances emit light when they themselves are irradiated with light. If such a substance is irradiated with light of a certain wavelength, light of a longer wavelength is emitted. The molecules in organic compounds are connected via shared electrons. When irradiated with light, the kinetic energy of such an electron changes, and the electron is transferred from the ground state to an excited state. Usually, this electron simply returns to the ground state, but in some cases, it moves to a state with a slightly lower energy level, the “preexcited state”, before finally returning to the ground state. In this case, light with a wavelength longer than that of the irradiating light is emitted. This is called “fluorescence”. The number of substances that emit fluorescence is relatively small compared to the number of substances that have absorption in the ultraviolet region, so the selectivity of fluorescence detection can be described as high. In general, fluorescence detection is more sensitive than absorbance detection by 2 or 3 digits.

117

Optical System of Fluorescence Detector

Xenon lamp Fluorescence grating Photomultiplier tube

Fluorescence

Excitation grating

Excitation light

Sample cell

LAAQ-B-LC001B

118

There are two types of fluorescence detectors: filter types and spectral types. Recently, spectral fluorescence detectors have come to be widely used. The principle behind the optical system of this type of detector is illustrated above. A xenon lamp is used as the light source. The light is divided by the excitation grating and monochromatic light is directed to the sample cell. The fluorescence emitted by the fluorescent substance in the cell is itself divided by the fluorescence grating, and the intensity of this fluorescence is converted to electric signals by the photomultiplier tube that acts as the detector.

118

Fluorescence Derivatization Reagents



OPA Reagent (Reacts with Primary Amines) S-R’

CHO + R-NH2 CHO

R’-SH

N-R

o-phthalaldhyde (OPA) 

ADAM Reagent (Reacts with Fatty Acids) + R-COOH CHN2

LAAQ-B-LC001B

CH2OCOR

9-anthryldiazomethane (ADAM) 119

Even target components that do not naturally emit fluorescence can be analyzed using a fluorescence detector by converting them to derivatives of fluorescent substances with either pre-column or post-column derivatization. A wide variety of fluorescence derivatization reagents are commercially available. Using a reagent that is suitable for the purpose makes it possible to improve the sensitivity and selectivity.

119

Differential Refractive Index Detector (Deflection(Deflection-Type)

Light-receiving unit Reference cell

Light Sample cell

LAAQ-B-LC001B

120

A differential refractive index detector measures the difference in refractive index between the sample cell and reference cell, so it has almost no specificity for substances. It can therefore be described as a universal detector that can be used for many different substances. On the other hand, it does have some disadvantages requiring care. For example, it is less sensitive than other it has no substance selectivity, which makes it susceptible to the influence ofdetectors, foreign substances, and it is also susceptible to the influence of temperature and pulsation. In addition, it does not allow the eluent composition to be changed during analysis, meaning it cannot be used for the gradient elution method. For application examples, this detector is often used for the detection of substances that have hardly any UV absorption, such as sugars. Because its response is constant and does not depend on the substance, it is also used as a detector for the measurement of molecular weight distributions. Furthermore, it is sometimes used for preparative separation.

120

Optical System of Differential Refractive Index Detector (Deflection(Deflection-Type)

Slit

W lamp

Reference cell Sample cell The slit image moves if the refractive index inside the flow cell changes.

Photodiode

LAAQ-B-LC001B

121

Differential refractive index detectors can be broadly categorized into three types: Deflectiontype, Fresnel-type, and Interfero-type. Deflection-Type This type of detector measures the overall change in the refraction angle that occurs due to the difference in refractive index when light passes through a sample cell and reference cell that have been arranged in series. In the actual instrument, the image that is created in the detector unit moves a distance that is proportional to the refraction angle, so the difference in light intensity that arises due to this is detected. Fresnel-Type This type of detector uses the way that the transmittance of light at the boundary surface between two media changes in accordance with the difference of refractive index between the two media. When the light from the light source enters the cell underneath a prism, the apparent intensity of light transmitted from the cell varies due to the difference in refractive index between the prism and the liquid in the cell. Therefore, detecting the transmitted light after reflection from below makes it possible to detect the difference in the refractive index. Interfero-Type This type of detector splits the light emitted from the light source into two parts with a beam splitter, passes it through a sample cell and a reference cell, refocuses it, and measures the changes in light intensity that occur due to the interference caused by the difference in refractive index.

121

Evaporative Light Scattering Detector

Light-receiving unit Drift tube Nebulizer Column eluate Nebulizer gas

Assist gas

Drain

Light source

The column eluate is evaporated and the light scattered by the particles of nonvolatile substances is detected. LAAQ-B-LC001B

122

An evaporative light scattering detector removes volatile eluent by nebulizing and heating the column eluate, irradiates the microscopic particles of the remaining nonvolatile substances, and detects the scattered light that is created. The detection can be broadly divided into three stages: (1) nebulization of the column eluate, (2) evaporation of the eluent, and (3) detection of the scattered light created by the nonvolatile components. An outline of the flow channels in an evaporative light scattering detector is shown above. Except for some nonvolatile compounds, this detector can detect nearly all compounds, and like the differential refractive index detector, it is highly versatile. The greatest advantage that this detector has over the differential refractive index detector is that it allows gradient elution to be used.

122

Electrical Conductivity Detector

NaCl aqueous solution

Pure water

Cl-

The bulb does not light with water.

Na+

The bulb lights if there are ions.

LAAQ-B-LC001B

123

Electrical conductivity is the inverse of resistance. Therefore, it can be said that an electrical conductivity detector detects how easily electricity flows. The electrical resistance of pure water is large, but if ions are dissolved in it, electric current flows more easily. Utilizing this behavior, electrical conductivity detection is used to selectively detect ions. This detector is mainly used in the field generally referred to as “ion chromatography”, which involves the analysis of inorganic ions, alkanoic acid, and amines. However, because this detector also detects the ions in the eluent, it is sometimes used together with an ion exchanger called a “suppressor”, which helps reduce the background level.

123

Principle of Electrical Conductivity Detector

V

K

=

I

=

A

L

Electrode

LAAQ-B-LC001B

E

=

L

k A

I

K: I: E: A: L: k:

A L

•k

K

A•

Electrical conductivity [S] Electric current [A] Voltage [V] Electrode surface area [cm 2] Distance between electrodes [cm] Specific electrical conductivity [S•cm -1] 124

The electrical conductivity, K, is obtained by measuring the current,I, that flows when a voltage, E, is applied between two electrodes that face each other. The unit used for K is [S] (siemens) or [mho], which is the inverse of [ Ω] (ohm). The electrical conductivity is proportional to the electrode surface area, A, and inversely proportional to the distance between the electrodes, L. The quantity k, which is equal to the electrical conductivity for A=1 and L=1, is introduced and analyzed in general. This quantity is called the “specific electrical conductivity”. Its unit is [S•cm -1]. The quantity L/A is a constant determined by the shape of the cell, and is called the “cell constant”. With an actual electrical conductivity detector, an altering-current voltage is applied to the cell in order to prevent polarization around the electrodes, thereby ensuring that the electrical conductivity of the solution is measured correctly. The electrical conductivity varies greatly with the temperature. More specifically, the electrical conductivity changes by approx. 1.7% when the temperature changes by 1ºC. For this reason, with some electrical conductivity detectors, the cell unit is housed in the column oven and is also equipped with its own temperature control function in order to ensure stable detection.

124

Limiting Equivalent Ion Conductance, λ [S• [S•cm2/mol], in Aqueous Solution (25º (25ºC)

Cation H+ Li+ Na+ K+ NH4+ (CH3)3NH+ Mg2+ Ca2+

Anion

λ

OH – F – Cl – Br – NO 3– CH 3COO– C 6H5COO– SO 42–

349.8 38.6 50.1 73.5 73.5 47.2 53.0 59.5

LAAQ-B-LC001B

λ

198.3 55.4 76.3 78.1 71.4 40.9 32.3 80.0

125

In the same way that the absorbance varies with the type of substance, the electrical conductivity varies with the type of ion. Regarding absorbance, each substance is assigned a “molar absorbance coefficient”, which indicates the size of the absorbance. The electrical conductivity equivalent of this is the “equivalent ion conductance”. The equivalent ion conductances of some representative ions are given in the above table. In general, the smaller the ion, the larger the equivalent ion conductance. This means that, with an electrical conductivity detector, smaller ions can be detected with a high level of sensitivity. Because of the electroneutrality principle, it is impossible for the concentration of a specific cation or anion to change at a certain peak position. If the concentration of a certain ion increases, in order to balance the total amount of positive and negative charge in the solution, either the concentration of another ion with a charge of the same sign decreases or the concentration of an ion with a charge of the opposite sign increases. By utilizing this principle, it is possible to give a response at the peak of a target ion that is equal to or greater than the ion’s equivalent conductance. For example, in the measurement of Cl–, if the eluent buffer solution is prepared so that the concentration of H + increases with increases in the concentration of Cl –, the response is dramatically improved by the contribution of H+, which has a large equivalent conductance.

125

Electrochemical Detector Electrode HO

R

HO 2eO

R + 2H+

O LAAQ-B-LC001B

126

Simply put, an electrochemical detector is an oxidation-reduction electrode that is used as a detector. It has the advantage of enabling the high-sensitivity, selective detection of substances that are easily oxidized or reduced. Electrochemical detectors can be categorized into two types: amperometric and coulometric. An amperometric detector is based on the measurement of electric current, and generally has an electrolysis efficiency of no more than 10%. A coulometric detector is like an amperometric detector in that it uses a fixed-potential electrode reaction, but it increases the electrolysis efficiency to 100%. While the coulometric detector, for which the reaction proceeds at a level of 100%, offers advantages such as not being greatly influenced by decreases in sensitivity due to dirt on the electrode and fluctuations in the eluent flow rate, it also has disadvantages, such as requiring a long time for initial stabilization because of the large electrode surface area and having a large cell capacity. In terms of sensitivity, there is not a great difference between the two types of detector. This is because an increase in the electrolysis capacity results in an increase in the size of signals corresponding to foreign substances as well as those corresponding to the target substances. Materials such as glassy carbon, platinum, silver, and gold are often used for the electrodes. In particular, glassy carbon is used in a variety of applications, including the analysis of phenol compounds.

126

Cell Structure of Electrochemical Detector (Amperometric Type)

Reference electrode

Working electrode

(Ag/AgCl)

(glassy carbon)

Eluent

Electrode couple LAAQ-B-LC001B

127

An example of the cell structure used in an amperometric detector is shown above. If there is an oxidizing / reducing substance on the surface of the working electrode, to which a constant voltage is applied, that substance exchanges electrons with the electrode. For example, if silver or silver oxide on saturated potassium chloride is used as the reference electrode, glassy carbon is used as the working electrode, and a potential difference of approx. +0.8 V is applied, substances such as catechol are oxidized and electrons are discharged. In the case of catechol, the number of discharged electrons is proportional to the quantity of catechol, so measuring the size of the flowing current makes it possible to ascertain the quantity of catechol present. It is said that the handling of electrochemical detectors is somewhat troublesome. This is because they are susceptible to baseline fluctuations due to impurities in the eluent and changes in the ambient temperature, and to changes in sensitivity due to dirt on the electrodes. It is important to be familiar with the characteristics, operating methods, and maintenance methods of this detector and, in particular, to maintain the flow cell in a clean state and prevent contamination of the eluent, column, or any other piece of equipment.

127

LCMS instruments are the most promising analytical instruments. Using a mass spectrometer as an LC detector not only enables detection with a high level of selectivity, it can also help facilitate structural analysis. Although it has been some time since LCMS emerged, it is only quite recently that it has become possible to use it in routine analysis work. It is said that this is because of the difficulty in developing an interface for linking the LC and MS sections. When a liquid is decompressed and vaporized, its volume increases by a factor ranging from several thousands to several tens of thousands. The MS section must be maintained at a high vacuum, so it is not acceptable for a liquid to enter the MS section in this state. Also, if the liquid contains nonvolatile salts, these salts crystallize without vaporizing, and can cause clogging at the inlet to the MS section. Therefore, an interface that can effectively remove excess eluent from the system and convey the target components efficiently to the MS section is required.

128

Atmospheric Pressure Ionization Electrospray Ionization (ESI)

Liquid Sample Liquid Samples Neburaizing Nebulizing Gas Gas

-+ ++- + -+ +- + - + 2)

+ + +- -+++

+ + +-+-++

+

+ 11)) + 33) E 2 Io ) CC + CCh So vap) E n oou haa v u E o o High Voltage lv f r a Io v lolo rgrg High Voltage e ap n Eavp o mnbEEx eeddD nSt olvtioonla Dr ap ra xc l e otio orpo nt f n or t io cluus lpel at n siio te onn io t n

Atmospheric Pressure Chemical Ionization (APCI) Molecular ion reaction Liquid Sample Liquid Samples Nebulizing Gas Neburaizing Gas

Heater Heater

LAAQ-B-LC001B

Corona ColonaDischarge Discharge Needle Nnndle

129

At present, the most widely used LCMS interface mechanisms are electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). In ESI, which utilizes the phenomenon of electrostatic atomization, the ions in a solution are extracted as ions in a gas phase in their srcinal state. This technique is said to be particularly suitable for the analysis of substances with relatively high polarities. In APCI, after the solution is vaporized by heating, it is ionized using corona discharge, and through chemical reactions between these ions and solutes, the solutes are ionized.

129

Advantages of LCMS (1) Quantitative analysis with excellent selectivity m/z=100

A TIC

B A:100 B:100 C:150

D:150 m/z=150

C

D

130

With LCMS, it is possible to selectively detect substances of specific molecular weights. It therefore enables detection with an extremely high level of selectivity. For example, even if two substances are eluted at the same retention time, as long as their molecular weights are different, they can be individually quantified using a chromatogram obtained by detecting at their own mass-to-charge ratios. This means that special measures to separate the substances are not required.

130

Advantages of LCMS (2) Peaks can be identified with MS spectra.

M/Z

M/Z

M/Z

131

Obtaining MS spectra for individual peaks helps facilitate peak identification and structural determination. Although traditional LC is capable of a high level of quantitative analysis, its performance in qualitative analysis has been described as rather poor. LCMS has the potential to fully overcome this disadvantage. With LCMS, however, fragment ions do not appear in large numbers, so LCMS is inferior to GCMS as a technique that can help facilitate structural determination. If the main objective of analysis is structural determination, LC/MS/MS is more suitable than LC/MS.

131

Comparison of Detectors

Absorbance Fluorescence

LAAQ-B-LC001B

Possibility of Gradient System

Light-absorbing substances

ng

Possible

None

Evaporative light scattering

Electrochemical

Sensitivity

Fluorescentsubstances

Differential refractive index

Electrical conductivity

Selectivity

pg µg

Possible Impossible

Nonvolatilesubstances

µg

Possible

Ionic substances

ng

Partially possible

Oxidizing / reducing substances

pg

Partially possible

Note: The above table indicates general characteristics. There are exceptions.

132

Finally, let us compare the different detectors. UV-VIS absorbance detectors are the most versatile, and are widely used. In comparison, fluorescence detectors and electrochemical detectors can be described as offering high sensitivity and selectivity. In particular, when combined with derivatization, fluorescence detectors can be used in a wide variety of applications. Differential refractive index detectors have hardly any selectivity and a low level of sensitivity, so they are not suitable for high-sensitivity analysis. Because of their lack of selectivity, however, they are widely used in the measurement of molecular weight distributions and preparative applications. Electrical conductivity detectors can be described as special detectors for “ion chromatography”, which is used to analyze inorganic ions and alkanoic acids.

132

Post-Column Derivatization

Reaction chamber

Pump Reaction solution LAAQ-B-LC001B

133

If the sensitivity or selectivity for the target substance is poor, it may be possible to make an improvement by derivatizing the substance to another substance using a chemical reaction. The derivatization method in which a reaction solution delivered by a separate pump is mixed with the eluate from the separation column is called “post-column derivatization”. The method which“pre-column the derivatization reaction is performed is introduced to HPLC isin called derivatization”. (Refer to the before sectionthe on sample pretreatment for details on pre-column derivatization: P149.) Post-column derivatization is performed after separation in order to improve the sensitivity and selectivity of detection, so, in a general sense, it is regarded as part of the detection system. The chemical reaction used in post-column derivatization must satisfy the following requirements: • The reaction must be quantitative. (Even if there are multiple reaction products by reaction, a detection response that is proportional to the concentration of the srcinal substance is sufficient.) • The reaction speed must be reasonably high. (It does not have to be complete.) • Mixing and heating are sufficient to cause the reaction. • The detection sensitivity for un-reacted derivatization reagent is low (or, if possible, undetectable). • The composition or components of the eluent do not inhibit the reaction. With this method, because the reaction time and temperature can be controlled accurately and because the target components meet the reagent after being separated from the sample matrix and dissolved in the eluent, it is easy to maintain the reaction conditions at a constant level, and there is a high level of quantitative performance. It is also simple to automate the system. 133

Application Examples of PostPostColumn Methods 



Amino Acids

Bromate Ions 

Orthophthalic acid, OPA (fluorescence)



Ninhydrin (visible absorption)

Reducing Sugars 











Arginine (fluorescence)

Cyanide Ions 

Carbamate Pesticides 



Alkaline hydrolysis - OPA (fluorescence)

Chlorination - pyrazolone (visible absorption)

Transition Metal Ions 

LAAQ-B-LC001B

Tribromide ionization (ultraviolet absorption) o-Dianisidine (visible absorption)

4-(2-Pyridylazo) resorcinol, PAR (visible absorption) 134

Some representative examples of ways in which post-column derivatization is used in practice are given above. Because the improved sensitivity and selectivity are the main objectives, fluorescence and visible absorption are often used for detection. Instead of causing a chemical reaction involving the target substances, it is also possible to improve the detection sensitivity and selectivity by changing the eluent composition. This can, in a general sense, also be referred to as a “post-column method”. A representative example of this is the use of a suppressor in ion chromatography. In order to reduce the background level of the eluent attained in electrical conductivity detection and improve the sensitivity for the target ions, the suppressor exchanges specific ions in the eluent with other ions. The suppressor does not, however, exert any influence over the target ions themselves. The detection sensitivity can also be improved by mixing with buffer solutions, acids, or alkalis after passage through the separation column. This is called “post-column pH buffering”. A representative example of this is the technique of separating short-chain fatty acids with ion exclusion mode, mixing with a buffer solution, and then performing electrical conductivity detection.

134

Quantitative Analysis Absolute Calibration Curve Method and Internal Standard Method

LAAQ-B-LC001B

135

When the analytical conditions have been decided, let us analyze an actual sample. First, let us analyze a standard sample, and obtain the retention time for the peak of the target substance. When analyzing an actual sample, if a peak appears at the same retention time, this is probably the target substance. This identification by retention time, then, is the qualitative tool used in chromatography. Next, let us analyze a standard sample with a known concentration, obtain the peak area value, and obtain the relationship between the concentration and the area value. This relationship is expressed using a “calibration curve”. The quantitation of the target substance in an actual sample is performed using this calibration curve.

135

Qualitative Analysis



Identification based on retention time



Acquisition of spectra with detector





UV spectra



MS spectra

Transfer to other analytical instruments after preparative separation

LAAQ-B-LC001B

136

With chromatography, there is hardly any information that helps ascertain the structure of target substances (unless a detector such as a mass spectrometer is used). In this sense, then, it must be conceded that chromatography is an analytical technique with a low level of qualitative capability compared to techniques such as NMR and FTIR. Basically, qualitative analysis in chromatography is performed according to whether or not the retention time coincides with that obtained for the peak of the standard sample. This means that identification based on the retention time is essentially the only qualitative tool available. The retention time is determined by the conditions listed below. When performing identification (or quantitative analysis), these conditions must be kept constant throughout the analysis of the standard sample and the actual sample. •Column (packing material, column size) •Eluent composition •Eluent flow rate •Temperature (column, solvent delivery system) There is always a possibility that other components contained in the sample coincidentally give peaks at the same retention time. In order to prevent the incorrect identification of these peaks, it is effective to connect a detector that can obtain spectra and use it to assist in identification. Two representative detectors that are used for this purpose are the photodiode array-type absorbance detector and the mass spectrometer (LCMS). For each peak that appears, the former can obtain a UV spectrum and the latter can obtain an MS spectrum. Another method used is to prepare (i.e., collect) the eluate corresponding to the time period during which the peaks appear and subject it to structural analysis with another analytical technique with superior qualitative capability. 136

Quantitative Analysis



Quantitation performed with peak area or height.



Calibration curve created beforehand using a standard. 

Absolute calibration curve method



Internal standard method



Standard addition method

LAAQ-B-LC001B

137

HPLC is generally regarded as an analytical technique that, as long as the appropriate conditions are maintained, can produce highly accurate quantitative values. In order to achieve a high level of quantitative accuracy, the equipment used must be capable of the following level of performance: • The solvent delivery pump must have excellent constant flow performance. • The components to be quantified must be sufficiently separated from other components. • The sample introduction section must have excellent reproducibility. • Variation of the column temperature must be minimal. • The detector must have a good response, and its linearity must be wide. • An excellent data processor must be used. If the above conditions are satisfied, analysis in which the coefficient of variation of quantitative values does not exceed 1% can be achieved with little difficulty. In some cases, peak area values are used to perform quantitative calculations, whereas in other cases, peak heights are used. It is said that, in general, using peak area values gives greater because the recent advancement in data processing hasis made it accuracy. possible toFurthermore, obtain area values easily, performing quantitation using area values currently considered to be common. Before analyzing an actual sample, it is necessary to analyze a standard sample with a known concentration, and obtain a graphical representation of the relationship between the concentration and the peak area values. This representation is called a “calibration curve”.

137

Calibration Curve for Absolute Calibration Curve Method Concentration

Area A1

C1

A C2

Calibration curve A4

2

a raA3 e k a eA P 2

A3 C3

A1 A4

C1

C4 LAAQ-B-LC001B

C2 C3 Concentration

C4 138

The procedure for the absolute calibration curve method, which is the most commonly used method, is described below. Prepare 3 or 4 standard solutions of the target substance with different concentrations, introduce the same quantities of these solutions to HPLC, obtain chromatograms, and measure the peak area values. By representing the concentrations of the target substance in the standard solutions on a horizontal axis and representing the corresponding peak areas on a vertical axis, create the kind of calibration curve shown above. Next, introduce an actual sample solution under the same conditions, obtain a chromatogram, and measure the peak area value. Calculate the concentration of the target substance in the actual sample by obtaining the concentration (horizontal axis) that corresponds to the measured peak area value (vertical axis) in the calibration curve. With this method, all measurements must be performed under exactly the same conditions.

138

Calibration Curve for Internal Standard Method Concentration Target Internal substance standard

C1

CIS

C2

CIS

C3

CIS

C4

CIS

Area

A1

AIS

A

A

2

IS

A3

AIS

A4

AIS

d r a d n a t s l a rn A4 /AIS e t n i r o f a re A3 /AIS A / e c n a t A2 /AIS s b u s t e g r A1/AIS a t r o f a re A

Calibration curve

C1/CIS C2 /CIS C3 /CIS C4 /CIS Concentration of target substance / Concentration of internal standard

LAAQ-B-LC001B

139

The procedure for the internal standard method, which is the second most commonly used method after the absolute calibration curve method, is described below. First, select a substance (the “internal standard”) other than the target substance. This substance must be stable, it must have similar chemical properties to the target substance, and its peak must appear near that of the target substance and be completely separated from the peaks of other sample components. Prepare 3 or 4 standard solutions with different concentrations of the standard substance (hereafter referred to as “X”) and the same concentrations of the internal standard (hereafter referred to as “IS”), introduce the same quantities of these solutions to the HPLC, obtain chromatograms, and measure the peak area values. Representing the value [Concentration of X / Concentration of IS] for the standard solutions on a horizontal axis and representing the value [Peak area of X / Peak area of IS] for the standard solutions on a vertical axis, create the kind of calibration curve shown above. Next, prepare a sample solution for measurement by adding IS to an actual sample at roughly the same concentration used with the standard solutions, introduce this solution under the same conditions, obtain a chromatogram, and measure the peak area value. Calculate the concentration of the target substance in the actual sample by obtaining the concentration ratio of X and IS (horizontal axis) that corresponds to the peak area ratio of X and IS (vertical axis), and multiplying this by the concentration of the added IS.

139

Advantages of Internal Standard Method (1) 

Not affected by inconsistencies in injection volume. X

IS

AX / AIS

10 µL injected Same area ratio IS X 9 µL injected CX / CIS LAAQ-B-LC001B

140

Not only is the procedure for the internal standard method more troublesome than that for the absolute calibration curve method, an appropriate internal standard must be selected. What, then, are the advantages of using this method? One advantage is that it is not affected by inconsistencies in the injection volume. With the internal standard method, quantitation for both standard samples and actual samples is always based on the ratio with respect to the internal standard. If the injection volume decreases, although the peak area value for the target substance decreases by a corresponding amount, the peak area value for the internal standard also decreases by the same proportion. Therefore, the area ratio for the two remains constant, regardless of the injection volume, and the correct concentration can be obtained from the calibration curve.

140

Advantages of Internal Standard Method (2) 

Not affected by the pretreatment recovery rate. X

IS

IS

A /

100% recovery rate

X

A

Same area ratio IS 90% recovery rate

X

CX / CIS LAAQ-B-LC001B

141

The quantitative values obtained with the internal standard method are not affected by the pretreatment recovery rate for exactly the same reason why they are not affected by inconsistencies in the injection volume. Note that this depends on a certain condition. The recovery rates for the target substance and the internal standard must be exactly the same for each sample. This is why they must have similar chemical properties. In cases where samples are injected in the HPLC system after undergoing complex pretreatment, it is better to use the internal standard method. Although it is not directly related to quantitation, the additive recovery rate can be calculated from the peak area values for the internal standard.

141

Selection Criteria for Internal Standard



It must have similar chemical properties to the target substance.



Its peak must appear relatively near that of the



target substance. It must not already be contained in the actual samples.



Its peak must be completely separated from those of other sample components.



It must be chemically stable.

LAAQ-B-LC001B

142

The selection criteria for the internal standard are shown above. The severity of these criteria discourages the adoption of the internal standard method. In cases where the internal standard method is simply being used in order to prevent errors resulting from inconsistencies in injection volume, the first criterion, namely, that the internal standard must have similar chemical properties to the target substance, can be ignored. Also, although it is not specified above, it is better to use a safe, inexpensive compound as the internal standard.

142

Sample Pretreatment Tasks Performed Before Injection

LAAQ-B-LC001B

143

It is ideal if sample pretreatment is not necessary. This is because it requires effort and can cause errors in quantitative values. In HPLC, however, the number of cases where pretreatment is not necessary is relatively small, and some form of pretreatment is nearly always required. There are countless pretreatment methods, the suitability of which vary with the target substance and the sample properties, and it would be almost impossible to describe every single one. Some representative pretreatment methods used in HPLC are described here.

143

Objectives of Pretreatment



To improve the accuracy of quantitative values



To improve sensitivity and selectivity



To protect and prevent the deterioration of columns and analytical instruments



To simplify measurement operations and procedures



To stabilize target substances

LAAQ-B-LC001B

144

The objective of quantitative analysis is to obtain correct quantitative values, so the ultimate objective of pretreatment is the same. If the samples cannot be injected directly into the HPLC, or if they can be injected but the desired separation or detection cannot be attained, pretreatment is performed. At the same time, the practical importance of performing pretreatment in order to protect the separation column cannot be ignored. Of all the units that make up an HPLC system, the separation column is the most prone to deterioration, and this deterioration has a significant effect on analysis results.

144

Substances That Must Not Be Injected into the Column 

Insoluble substances (e.g., microscopic particles and precipitation)



Substances that are precipitated in the



eluent Substances that irreversibly adsorb to the packing material



Substances that dissolve, or chemically react, with the packing material

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145

With respect to protection of the separation column, the following substances must not be injected into the column. Remove these substances in pretreatment. Insoluble Substances Insoluble substances can cause clogging in the column; remove them as a first priority. They can be removed simply by performing filtration or centrifugal separation. Substances That are Precipitated in the Eluent When an organic solvent is used as the eluent, if a sample consisting of an aqueous solution that contains a high concentration of inorganic salt is injected, the salt may not dissolve fully in the organic solvent and may be precipitated. Check beforehand that precipitation does not occur when the sample solution and eluent are mixed. Also, if the sample consists of an aqueous solution that contains a protein, it may be denatured and precipitated immediately after being mixed with the eluent. Perform deproteinization. Substances That Irreversibly Adsorb to the Packing Material For example, if a silica gel-based packing material is used, basic proteins adsorb strongly. Also, ions of transition metals such as iron sometimes adsorb to the surface of the packing material, forming oxides that remain on the surface. If possible, remove such substances in pretreatment. Substances That Dissolve, or Chemically React, with the Packing Material As much as possible, do not inject strong acids or alkalis or highly corrosive substances. In particular, there are many restrictions on the solvents that can be injected when using resin-based packing materials; in such cases, follow the guidance given in instruction manuals. 145

Filtration and Centrifugal Separation



In general, filter every sample before injection!



It is convenient to use a disposable filter with a pore diameter of approx. 0.45 µm.



Centrifugal separation is applicable for samples that are difficult to filter.

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Filter

Syringe

146

In general, in order to prevent clogging of the column and the flow channels, filter all types of sample. In general, a membrane filter with a pore diameter of approx. 0.45 µm is used. Disposable types that can be attached to the tip of the syringe are commercially available. A filter made of a material that is suitable for the solvent must be used so, when purchasing a filter, select one that is suitable for the sample solvent to be filtered. If there is a large amount of suspended matter in the sample, or the sample volume is small, centrifugal separation may be more suitable than filtration. The volume of sample injected into an HPLC system does not exceed the order of a few hundred µL, so it should be sufficient to prepare a centrifuge tube with a capacity of approx. 1.5 mL together with a compatible centrifugal separator.

146

Deproteinization Precipitation Addition of organic solvent (e.g., acetonitrile) Addition of acid (e.g., trichloroacetic acid, perchloric acid) Addition of heavy metal or neutral salt

Ultrafiltration

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147

Proteins may be denatured and precipitated when they are mixed with the eluent and, consequently, cause clogging. They may also adsorb strongly to the surface of the packing material, and adversely affect the performance of the separation column. Unless proteins are themselves the target substances, it is better to remove them before injection.

Precipitation If an organic solvent or acid is added to a solution containing a protein, the protein is denatured and precipitated. This property can be used to remove proteins by applying centrifugal separation. One commonly used organic solvent is acetonitrile. In the analysis of blood serum and blood plasma, add at least twice the volume of acetonitrile to the sample, mix the two, perform centrifugal separation on the mixture, and inject the supernatant into the HPLC system. Because this method makes it possible to extract the target components at the same time, it is widely used. Two commonly used acids are 10% trichloroacetic acid and 6% perchloric acid. The procedure is the same as that for organic solvent. In the analysis of blood serum or blood plasma, however, only roughly half of the volume of the sample needs to be added. In addition, copper sulfate, zinc sulfate, and saturated ammonium sulfate are used as precipitants.

Ultrafiltration The method of using an ultrafilter membrane to perform deproteinization in accordance with molecular size is relatively simple and is suitable for processing multiple samples. However, tt does have disadvantages, such as a low recovery rate for substances that adsorb to proteins easily and high running costs. As with standard filtration, various types of disposable ultrafilters are commercially available. There are types that apply air pressure and types that are set in centrifugal separators and subjected to centrifugal force. 147

Solid Phase Extraction (1)

(2)

Conditioning

Sample addition

(3) Rinsing

(4) Elution Solvent with low elution strength

Solvent with high elution strength Target component Unwanted components

148

Solvent extraction has been widely used as an HPLC pretreatment method for some time. This method is used to separate and concentrate solutes using partition across a liquid-liquid interface. There is also a method used to perform separation and concentration using interactions such as adsorption and partition across a solid-liquid interface. This method is called “solid phase extraction” (SPE), and can be described as a pretreatment technique that directly applies the separation principles of liquid chromatography. Various types of SPE cartridges, which consist of 100 to 500 mg of the same type of packing material used in liquid chromatography packed into a small housing, are commercially available. Sample solution is passed through one of these cartridges, the target component is held, and then another solution is passed through the cartridge, causing the elution of the target component. The principle is the same as that used in liquid chromatography, so the points that apply to the various separation modes used in liquid chromatography can be applied to the setting of the pretreatment conditions. It is said that this method generally gives a better recovery rate than solvent elution, and is also superior in terms of reproducibility. The amount of organic solvent required is relatively small, so there is no need to be especially concerned about the discharge of harmful substances into the environment. Also, using suction and pressurization devices enables the batch processing of multiple samples. This method has traditionally been used for “purification”, which involves the extraction of target components from samples with complex compositions and the removal of unwanted components. In recent years, however, it has received attention for its capability as a “concentration” technique, with which large numbers of samples can be delivered and eluted using a relatively small amount of solvent. In the analysis of trace amounts of harmful organic substances (environmental pollutants) that are present in the environment, because the required level of sensitivity cannot be attained directly even with GCMS, let alone HPLC, concentration is required as pretreatment. With solvent extraction, the procedure is troublesome and harmful organic solvents must be used. With solid phase extraction, however, the procedure is simple and solvent consumption is low, making it suitable for the processing of multiple samples. This is why this method is now widely used. 148

Pre-Column Derivatization 

OPA Reagent (Reacts with Primary Amines) S-R’

CHO + R-NH2 CHO

N-R

R’-SH

o-phthalaldhyde (OPA) 

2,4-DNPH (Reacts with Aldehydes and Ketones) NHNH2 +

O 2N

NO2

R R’

NHN=C C=O

H+

O2N

R R’

NO2

2,4-dinitrophenylhydrazine (2,4-DNPH) LAAQ-B-LC001B

149

One technique used for sample pretreatment is “pre-column derivatization”. In cases where the target substance is not retained as it is by the column and cannot be separated from other components, or in cases where the detection sensitivity or selectivity is poor, this technique is used to convert the substance to another compound via a chemical reaction before introducing it into the HPLC system. The chemical reaction used in pre-column derivatization must satisfy the following requirements: • A single derivative is produced from one target substance. • The reaction must be quantitative, and not easily influenced by the sample matrix. • The reaction products must be stable. • The reaction products must separate from the unreacted derivatization reagent. On the other hand, because the chemical reaction takes place before introduction to the HPLC system, there are no restrictions on the reaction procedures (e.g., heating, filtration, or evaporative concentration) or time, and by setting a small scale for the volume of sample and reaction reagent, even expensive reagent can be used economically.

149

Evaluation of the Reliability of Analysis Validation of Analytical Methods

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150

If the procedures are followed faithfully, quantitative values are obtained. How far, though, can these values be trusted? Furthermore, how far can the analytical methods themselves be trusted? A very basic explanation of the “validation of analytical methods”, which is performed to demonstrate the validity of the analytical methods used for analysis, is given here. Using analytical methods that have been properly validated ensures the reliability of the measurements obtained with these methods.

150

What Is “Validation of Analytical Methods” Methods”? 



Scientifically demonstrating that the analytical methods concur with the intended (i.e., that errorspurpose are within a permissible range) Evaluating required items from the validation characteristics

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

Validation characteristics 

Accuracy / trueness



Precision

 

Specificity Detection limit



Quantitation limit



Linearity



Range



(Robustness)

151

“Validation of analytical methods” consists of scientifically demonstrating that the analytical methods used in a test concur with the intended purpose. It is extremely important for ensuring the reliability of test results. Although this may seem like a rather difficult concept, for example, comparing with the quantitative values obtained with other analytical methods, checking reproducibility by performing repeated injections, and checking linearity by creating a calibration curve are all forms of validation of analytical methods. In other words, it would be reasonable to think of it as the checks that are usually performed when adopting new analytical methods. In the past, there were no established rules for the implementation of validation, and the methods used depended greatly on the developers of analytical methods. For this reason, it was difficult to say whether or not the results of such methods could be described as valid from an official standpoint. Now, however, guidelines for validation are specified, for example, in the related resources for the Japanese Pharmacopoeia (13th edition (1996) onwards) so, in general, it can be considered acceptable to perform the validation of analytical methods by following these guidelines. More specifically, items from the list of validation characteristics shown above on the right that must be verified for the analytical methods concerned are selected and tested. (It is not always necessary to verify all of the characteristics.)

In general, evaluation of the validation characteristics is performed using statistical techniques. As evaluation is based on test results, there is always some degree of error. Therefore, before establishing an implementation plan for the validation of analytical methods, the statistical techniques used for evaluation must be thoroughly understood.

151

Accuracy / Trueness 

Definition





Degree of bias in measurements obtained with analytical procedures



Difference between true value

Evaluation Method 

Comparison with theoretical values (or authenticated values)



Comparison with results obtained using other analytical procedures for which the accuracy (trueness) is known



Recovery test

and grand mean of measurements

True value

Measurement

Average

95% confidence interval

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152

“Accuracy” (or “trueness”) is the degree of bias from the “true value”, which some say only God knows. If the true value is thought of as the center of a target, then if the places that are actually hit describe a distribution that centers on the center of the target, the accuracy is high, and if the center of this distribution is on the left or the right of the center of the target, the accuracy is low. With a relative analytical technique like chromatography, however, the true value cannot be defined, so the accuracy effectively refers to the recovery rate based on a standard sample. If the theoretical value is known, or if there is a value that has been authenticated or agreed on as a true value, the accuracy can be evaluated by comparing this value with the average of the measurements. If there is another analytical technique for which the accuracy is already known, the accuracy can be evaluated by comparison with the value obtained with this technique. The accuracy can also be evaluated by performing a recovery test. A certain amount of the target substance is added to a sample, this is analyzed together with a sample containing none of the target substance, and the difference in the quantitative values obtained is checked to see if it corresponds to the amount of target substance added. In each case, measurement is performed several times, and both the average value and the 95% confidence interval are calculated. It is checked that the true value (or the value assumed to be the true value) is contained within this confidence interval.

152

Precision



Definition 



Degree of coincidence of series of measurements obtained by repeatedly analyzing multiple samples taken from a homogenous test substance



Repeatability / IntraAssay Precision 

Precision of measurements taken over a short time period under the same conditions



Intermediate Precision



Reproducibility

Variance, standard deviation, or relative standard deviation of measurements

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153

“Precision” is the degree of inconsistency, or random error, in measurements. Regardless of the distance from the center of the target (true value), if the places hit are closely packed, the precision is high; if they are distributed over a wide range, the precision is low. The precision is expressed at three levels, which differ in terms of the repetition conditions. “Repeatability” (or “intra-assay precision”) is the precision of measurements taken over a short time period under the same conditions. In other words, it is the precision based only on random error. However, with instrumental analysis techniques, such as HPLC, that require an analysis cycle of some length, time delay is also a variable factor. “Intermediate precision” is the precision obtained when analysis is performed by multiple analysts, using multiple instruments, on multiple days, in one laboratory, and “reproducibility” is the precision obtained when analysis is performed in multiple laboratories. In other words, these are the levels of precision obtained when, in addition to random error, the influence of other variable factors is included. In order to make a detailed evaluation of intermediate precision and reproducibility that includes an examination of variable factors, multi-way analysis of variance based on the number of factors is used to obtain the confidence intervals for the standard deviation and total variance. (For details on the “analysis of variance”, refer to reference documentation on statistics.) In this case, if there is a large number of variable factors, a large number of experiments and troublesome calculations must be performed, so the experiments should be performed in accordance with an appropriate experimental design.

153

Specificity 

Definition





The ability to accurately analyze the target substance in the presence of other expected substances



The discrimination capability of the analytical methods



Multiple analytical procedures may be combined in order to attain the required level of discrimination

LAAQ-B-LC001B

Evaluation Method 



Confirmation that the target substance can be discriminated (separated) from co-existing components, related substances, decomposition products, etc. If reference standards for impurities cannot be obtained, the measurement results for samples thought to contain the impurities are compared. 154

“Specificity” is the ability to accurately analyze the target substance in the presence of other substances that are thought to co-exist in the sample. In the context of chromatography, if the target substance is well separated and the detection sensitivity is high, the specificity is high. If the co-existing components contained in the sample are known, and their reference standards are available, the specificity can be evaluated by preparing a solution containing only the target substance, a solution containing only the co-existing components, and a solution containing both the target substance and the co-existing components, and comparing the analysis results. If reference standards for impurities are not available, the same kind of comparison can be performed using samples thought to contain impurities, such as samples that have been allowed to stand for a long time and samples that have been exposed to extreme conditions. In chromatography, the specificity can be improved by improving separation or increasing the detection selectivity. Evaluation of the specificity in relation to separation is usually performed using the resolution (RS). As technical guidelines for liquid chromatography, the Japanese Pharmacopoeia Technical Information (1996) specifies that the resolution for the target substance with respect to neighboring components must, in general, be at least 1.5, and that the resolution for related substances under investigation with respect to neighboring related substances must be at least 1.2. In order to improve the specificity in relation to detection, it is considered appropriate to use a detection method with as high a selectivity as possible. Also, using a photodiode array-type UV-VIS absorbance detector or a mass spectrometer makes it possible to increase the qualitative capability for peaks and to prevent the mistaken identification of peaks.

154

Detection Limit



Definition





The minimum quantity of a target substance that can be detected.



Quantitation is not absolutely necessary.

Evaluation Method 

Calculated from the standard deviation of measurements and the slope of the calibration curve.  DL = 3.3 σ/slope (σ: Standard deviation of measurements) (Slope: Slope of calibration curve)



Calculated from the signal-to-noise ratio. 

Concentration for which S/N = 3 or 2

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155

The “detection limit” is the minimum quantity or concentration of a target substance that can be detected. As long as the presence of the target substance can be confirmed, it is not necessary for quantitation to be possible with the permissible level of accuracy and precision. In chromatography, the detection limit is the minimum quantity for which a peak can be recognized. One method for obtaining the detection limit is based on the standard deviation of the responses (peak areas) and the slope of the calibration curve. The detection limit, DL, is calculated using the following formula:

DL = 3.3 σ/slope

σ: Standard deviation of responses (peak areas) Slope: Slope of calibration curve The standard deviation of the responses obtained with a blank sample and a sample containing the target substance at a concentration near the detection limit is used as the standard deviation, σ. In chromatography, the method of obtaining the detection limit using the signal-to-noise ratio is often used. A chromatogram is obtained for a sample containing the target substance at a concentration near the detection limit, and the ratio (S/N) of the signal height to the average noise width is calculated. From the result, the concentration corresponding to an S/N value of 3 or 2 is calculated, and this is used as the detection limit.

155

Quantitation Limit 

Definition





The minimum quantity of a target substance that can be quantified



Quantitation with an appropriate level of accuracy and precision must be possible. (In general, the relative standard deviation must not exceed 10%.)

Evaluation Method 

Calculated from the standard deviation of measurements and the slope of the calibration curve. 



QL = 10 σ/slope (σ: Standard deviation of measurements) (Slope: Slope of calibration curve)

Calculated from the signal-tonoise ratio. 

Concentration for which S/N = 10

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156

The “quantitation limit” is the minimum quantity or concentration of a target substance that can be quantified. Naturally, the ability to obtain quantitative values with an appropriate level of accuracy or precision is a prerequisite. In general, it is required that the relative standard deviation does not exceed 10%. The method used to obtain the quantitation limit is similar to the method used to obtain the detection limit. In terms of the standard deviation of the responses (peak areas) and the slope of the calibration curve, the quantitation limit, QL, is calculated using the following formula:

QL = 10 σ/slope

σ: Standard deviation of responses (peak areas) Slope: Slope of calibration curve The standard deviation of the responses obtained with a blank sample and a sample containing the target substance at a concentration near the quantitation limit is used as the standard deviation, σ. In terms of the signal-to-noise ratio, the concentration corresponding to an S/N value of 10 is calculated, and this is used as the quantitation limit.

156

Linearity 

Definition 





The ability of the analytical method to produce measurements for the quantity of a target substance that satisfy a linear relationship. Values produced by converting quantities or measurements of the target substance using a precisely defined formula may be used.

Evaluation Method 



LAAQ-B-LC001B

Samples containing different quantities of the target substance (usually 5 concentrations) are analyzed regressionrepeatedly, equations and and correlation coefficients are obtained. Residuals obtained from the regression equations of the measurements are plotted, and it is confirmed that there is no specific slope. 157

“Linearity” is the ability to produce measurement results that are directly proportional to the quantity (concentration) of the target substance. Naturally, it is necessary for there to be linearity throughout the entire measurable concentration range. The evaluation of linearity starts with the visual observation of a graph obtained by plotting the response (peak area) as a function of the quantity (concentration) of the target substance. Even if the correlation coefficient is near 1, in many cases, the points do not describe a good linear relationship when they are actually plotted. At least 5 different concentrations are usually used. If a linear relationship is confirmed, regression analysis is performed using the least squares method, and quantities such as the correlation coefficient, the y-intercept, and the slope of the regression line are calculated. If a linear relationship cannot be confirmed, it is expressed as an appropriate function. If necessary, the residuals from the regression equation of the measurements are obtained and plotted against the quantity (concentration) of the target substance, and it is confirmed that no identifiable slope is observed.

157

Range



Definition 



The region between the lower and upper limits of the quantity of a target substance that gives appropriate levels of accuracy and precision

LAAQ-B-LC001B

Evaluation Method 

The accuracy, precision, and linearity are investigated for samples containing quantities of a target substance that correspond to the lower limit, upper limit, and approximate center of the range. 158

The “range” refers to the region between the lower and upper limits of the quantity (concentration) of target substance that give appropriate levels of accuracy and precision. With a linear analytical procedure, linearity must be maintained throughout this range. In general, the accuracy and precision are highest at the center of the range in which linearity holds, and decrease as the concentration approaches the lower and upper limits. Therefore, if the required levels of accuracy and precision are attained near the lower and upper limits of the measured concentration range, in general, accuracy and precision are assured throughout the entire range.

158

Robustness



Definition 



The ability of an analytical procedure to remain unaffected

Evaluation Method 

by small changes in analytical conditions.

Some or all of the variable factors (i.e., the analytical conditions) are changed and the effects are evaluated.

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159

The “robustness” is the ability of an analytical method to resist changes in measurements when the analytical conditions are deliberately changed within a small range. It is evaluated by changing variable factors (i.e., analytical conditions) such as the separation column used, the composition and pH of the eluent, the temperature, and the detection parameters, and investigating the stability of the measurements. If the measurements fluctuate greatly when small changes are made to the analytical conditions, the analytical method is modified so that stable measurements are obtained. The results of robustness evaluation are reflected in the significant figures of a value that indicates the analytical conditions for the ultimate analytical method as well as points to note. Using an analytical method with a low level of robustness can create major problems afterwards. There are cases of the separation changing significantly when a separation column of a different lot is used, and cases in which separation cannot be reproduced unless the eluent pH is unified to two decimal places. Make a list of all conceivable variable factors, and conduct an investigation for each one.

159

Maintenance of Separation Column Extending the Column’s Service Life

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160

A separation column is basically a consumable item. However carefully it is used, it will eventually deteriorate. Even so, columns are too expensive to discard each time they are used, so it is best to try and ensure as long a service life as possible. Some general points regarding the use of separation columns are given here.

160

SilicaSilica-Based Packing Materials and ResinResin-Based Packing Materials Silica-Based pHrange

Resin-Based Generally a wide range

2- 7.5

Organic solvent

No restrictions

Significant restrictions

Pressure resistance

25 MPa max.

Low pressure resistance

Temperature

60ºC max.

Depends on packing material

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161

The types of packing material used in HPLC can broadly be categorized into silica-based types and resin-based types. Some general points regarding the use of these types of columns are given here. Silica-Based Columns Because silica-based columns are superior in terms of theoretical plate number, pressure resistance, and durability, and are relatively inexpensive, they are currently the most commonly used HPLC columns. In particular, the ODS columns used in reversed phase chromatography are employed in a wide variety of analytical applications. The parameter requiring the most care when using a silica-based column is the eluent pH. The pH must be in the range of 2 to 8, and it is desirable for it to be in the range of 3 to 5. If the pH is outside this range, the silica gel that forms the support material dissolves, and deterioration of the performance is accelerated. Resin-Based Columns One advantage of resin-based columns is that the eluent pH range that can be used is significantly wider than that of silica-based columns (although there are exceptions). They are also stable when modified with basic functional groups (e.g., quaternary ammonium). In the use of resin-based columns, however, greater consideration of detailed points is required than for silica-based columns. Pressure resistance: In general, the pressure resistance of resin-based columns is low, and some are broken at pressures no greater than a few MPa. Also, sudden changes in pressure must be avoided when, for example, replacing valves. Swell and contraction: Resin swells or contracts according to the polarity of the solvent so, when a resin-based column is used, the eluent composition cannot be changed very much. In principle, the solvent used when packing is performed is used as the eluent in analysis. In particular, the amount of organic solvent that can be added is subject to significant restrictions. Be sure to refer to the instruction manual of the column used, and use a permissible amount of solvent.

161

General Handling of Columns 





Observe restrictions related to solvents and the pH range. Never allow the packing material to dry. Do not allow solids or microscopic particles to enter the column. 





Filter samples.

LAAQ-B-LC001B

Use as low a load pressure as possible. 

Do not exceed the upper pressure limit.



Do not subject the column to sudden pressure changes.

Do not subject the column to intense shocks. 162

Some handling precautions that generally apply to HPLC separation columns are given above. In all cases, carefully read the instruction manual provided with the separation column, and try as much as possible to observe the specifications given (e.g., for solvent type, pH range, load pressure/flow rate, and temperature). Usage precautions are also given; refer to these as necessary. If you do not understand the contents of the instruction manual or the required information is not given, it may be better to contact the manufacturer. Recently, useful information is often given on manufacturers’ websites.

162

Typical Problems (1) Column Clogging 

Preventive Measures 

Filter samples.



Check that samples dissolve in the eluent.



Get in the habit of observing pressure values.



Corrective Action 







Check for clogging in parts other than the column. Rinse with an appropriate solvent. Connect the column in reverse and flush out the insoluble substances at a low flow rate. Open the column end and perform ultrasonic cleaning of the filter.

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163

Some examples of problems that can srcinate in the column are described here. Problems related to pressure increases caused by clogging in the column are relatively frequent. Preventive measures include filtering the samples and checking the solubility of the samples with respect to the eluent. Also, in anticipation of such a problem, it is advisable to get into a habit of checking the pumps’ pressure gauges. Even if clogging does occur, it would be a mistake to immediately assume that it has occurred in the column. It is possible that some insoluble substance is blocking a flow channel in a place other than the column, so remove the tubing joints in order from the downstream end, and try to identify the location of the clogging. If the clogging is in a tube, this can often be remedied simply by pumping eluent in the reverse direction. If the column is identified as the cause, think carefully about what is obstructing the flow, and try the following corrective actions. • Dissolve the substance that is causing the clogging, and rinse out the column with a solvent that will hardly damage the packing material. • Connect the column in reverse, and flush out the insoluble substances by pumping eluent at a low flow rate. • Open the column inlet, remove the inline filter, and either subject it to ultrasonic cleaning or replace it.

163

Typical Problems (2) Peak Deformation Cause

CorrectiveAction

Sample overload

Reduce the sample injection volume or concentration.

Inappropriate sample

Replace the sample solvent with one of a

solvent Dirt

low elution capacity. Rinsethecolumn.

Gap in column inlet

Repair the column by supplementing it with packing material.

Influence of secondary retention effects

Rinse the column. Replace the column with one that is only minimally influenced.

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164

There are many possible causes of changes in the shapes of peaks, so implement the corrective action that corresponds to the cause. If the concentration of the target substance in the sample is so high that it is not retained in the stationary phase, tailing occurs. A typical example theconcentration way that peakistailing occurs for nitric acid ions andpeak sulfuric acid ions when a sample withofa this highissalt injected in ion chromatography. Either dilute the sample or reduce the injection volume. If the sample solvent has a higher elution capacity than the eluent (in reversed phase mode, this corresponds to the situation where there is a large proportion of organic solvent), as the injection volume is increased, a portion of the component is eluted before the actual retention time. This is because the sample solvent temporarily acts as the eluent, and some of the solutes travel through the column quickly together with the sample solvent. Either replace the sample solvent with one of a low elution capacity or reduce the injection volume. If all of the peaks that appear are broad, or if only some of the peaks are deformed, it is possible that dirt in the column may be the cause. Rinse the column using the procedure described later. If all of the peaks have “shoulders”, or if the peak for a single component splits into two, there may be a gap in the packing material near the column inlet. This may be a depression caused by pressure or it may be a result of the packing material gradually dissolving or being crushed into small particles. If it is an ODS column, it may be possible to repair the column by supplementing with packing material. In addition, peaks are sometimes deformed by the influence of forces other than the principal interactions involved in peaks retention, namely, secondary retention effects. For example, in reversedthe phase chromatography, if the for basic substances and complexing substances are deformed, influence of residual silanol groups and metal impurities is possible. The problem may be resolved by rinsing the column, but in some cases, it may be better to replace the column with one thought to be only minimally influenced by secondary retention effects.

164

Typical Problems (3) Decrease in Retention Time 

Check whether the cause of the problem is not the column.



If the column is identified as the cause...



Eluent composition



Rinsing



Eluent flow rate



Replacement

 Column temperature

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165

Many of the causes of a change in retention time are not related to the column. In particular, sudden increases in retention time may be caused by a temporary drop in the eluent flow rate, which may occur due to air bubbles in a pump, liquid leakage, or clogging in a suction filter. Therefore, first check that the eluent flow rate and eluent composition are correct. It is rare for the column to be the cause of an increase in retention time. However, it may well be the cause of a decrease in retention time. This is usually because of a decrease in the retention strength of the column. Decreases in retention strength are caused by dirt on the surface of the stationary phase or a decrease in the number of retention sites (e.g., functional groups) of the stationary phase. In the former case, the problem may be resolved by rinsing the column. In the latter case, the column must be replaced with a new column.

165

Typical Problems (4) Baseline Drift 

Check whether the cause of the problem is not the column. 

If the problem persists when the column is removed, it is caused by the eluent, the solvent delivery system (pump or degasser), or the detector.

LAAQ-B-LC001B



If the column is identified as the cause... 

Rinsing



Review of temperature control



Replacement

166

If the baseline is unstable, it is possible that the cause is not related to the column. Remove the column, replace it with a resistance tube (tubing with an inner diameter of 0.1 mm and a length of 2 to 4 m), and try delivering solvent in the same way. If the drift does not stop, the cause is probably related to the eluent, the solvent delivery system (e.g., pump or degasser), or the detector. If the column is identified as the cause, the most likely problem is that dirt is being eluted from the column. Rinse the column. Very rarely, small fluctuations in the column temperature disrupt the state of equilibrium that exists between the stationary phase and eluent, and this is picked up by the detector and results in baseline drift. This phenomenon occurs only when the eluent itself is detected. For example, it may occur when using a differential refractive index detector or an electrical conductivity detector. It is possible that analysis is being influenced by changes in the ambient temperature. Investigate whether or not the problem is resolved by maintaining a constant temperature in the laboratory.

166

Guard Column and Pre-column

Guard column

PrePre-column

LAAQ-B-LC001B

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Separation columns are consumable items. No matter how carefully they are used, they eventually deteriorate. An expensive column, however, is not easy to replace, so it is desirable to extend the service life as much as possible. For this reason, it is recommended that a guard column is attached immediately before the main column. In general, the guard column is filled with the same packing material as the main column, and is no more than a tenth of the size. Guard columns are nearly always provided for particularly expensive main columns (e.g., those used in ion chromatography and size exclusion chromatography). On the other hand, with relatively inexpensive columns such as ODS columns, there is little benefit in using guard columns, so they are not provided in many cases. If one of the previously described problems occurs when a guard column is used, the cause may be the guard column rather than the main column. Therefore, remove the guard column and investigate whether or not the same problem occurs with only the main column. It may be possible to resolve the problem simply by rinsing or replacing the guard column. Separately from the guard column, another column is sometimes attached between the pump and the injector. Here, in order to distinguish it from the guard column, this column is called the “pre-column”. A pre-column may be attached for one of the following two reasons: • To remove or temporarily trap impurities contained in the eluent (e.g., an ammonia trap column used to analyze amino acids) • To delay deterioration (dissolution) of the column due to the eluent (e.g., a presaturation column employed when using an alkaline eluent in reversed phase mode)

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Column Rinsing

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Use an eluent with a high elution capacity 

Reversed phase mode: Solution with a high proportion of organic solvent

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Ion exchange mode: Solution with a high salt concentration

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Consider secondary retention effects 

To remove basic substances from a reversed phase column → Use an acidic solution and add an ion pair reagent.

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To remove hydrophobic substances from an ion exchange column → Add an organic solvent.

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The first rule that applies to the rinsing fluid used for a separation column is to use a solution that would have a high elution capacity if it were used as an eluent. There are some people who think that rinsing simply consists of washing with water, but in some cases, this degree of rinsing is completely meaningless. For example, with reversed phase mode, because the overall elution capacity increases (i.e., the retention strength decreases) as the eluent polarity decreases, it is better to use a solution with a large proportion of organic solvent. In many cases, effective rinsing of an ODS column can be expected with methanol or acetonitrile. However, if a buffer solution is used as the eluent, measures, such as rinsing the buffer salt beforehand with water, must be taken to ensure that salt is not precipitated. The second rule is to use a solution that can elute substances that have adsorbed to the packing material due to secondary retention effects. In reversed phase mode, basic substances sometimes adsorb to the packing material due to the influence of, for example, residual silanol groups, and these substances cannot be rinsed away with an organic solvent. It is effective to use an aqueous solution of sodium perchlorate with a concentration of approx. 100 mmol/L and a pH adjusted to lie in the range of 2 to 3 using phosphoric acid, mixed together with methanol or acetonitrile. In ion exchange mode, if a hydrophobic substance adsorbs to the substrate of the packing material, rinsing may be possible by adding a small amount of organic solvent to the eluent. In ion chromatography, metal ions contained in the sample sometimes remain in the packing material, causing peaks for phosphoric acid ions to be smaller or to not appear at all. This problem may be resolved by pumping an aqueous solution of disodium ethylenediamine tetraacetate (EDTA-2Na) with a concentration of approx. 10 mmol/L for a while. The appropriate rinsing method varies with the type of column. In many cases, the relevant information can be found in the column’s instruction manual, in technical data produced by the manufacturer, or on the manufacturer’s website.

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Checking Column Performance

N

= 16

2

tR W

2

H W1/2

H1/2

= 5.54 = 2π

W LAAQ-B-LC001B

tR W1 / 2

tR

•H

2

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The performance of a separation column is checked by calculating the theoretical plate number using a standard substance. Shipment inspection data should be provided with all commercial separation columns. The degree of deterioration that has occurred since shipment can be ascertained by setting up the same analytical conditions and injecting the same substance specified in the inspection data and calculating the theoretical plate number. Another method is to use the analytical conditions used in daily analysis work as the inspection conditions, and to calculate the theoretical plate number when creating a calibration curve. Currently, many HPLC workstation software packages incorporate functions for calculating theoretical plate numbers and outputting analysis reports. This software makes it possible to check columns without having to perform any extra operations. There are no clear criteria regarding the decrease in theoretical plate number that necessitates column replacement. In general, the column can probably be used as long as the target component is fully separated and any decrease in sensitivity caused by broadening of the peaks is within permissible limits. Therefore, it is reasonable to specify the theoretical plate number that, through experience, is identified as being the point at which these conditions fail to be met as the criterion for replacement.

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Column Storage

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Storage Solution 





It is generally safe to use the same storage solution as used at shipment.

Storage Conditions 

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In order to prevent putrefaction, alcohol or some other preservative substance may be added.



LAAQ-B-LC001B

Insert an airtight stopper in the column end. Never allow the packing material to dry. Make a record of the storage solution and final usage conditions and store it together with the column. Store the column in a location not subject to shocks or sudden temperature changes. 170

If the separation column is stored separately from the instrument, it is filled with a solution. In most cases, the storage solution used at shipment is specified in the separation column’s instruction manual. It is safe to fill the column with a solution of a similar composition. With an ODS column, it is advisable to fill the column with methanol or acetonitrile after rinsing the buffer salt out. A mixture of one of these solvents with water is also acceptable. When storing the column for a long time, possible putrefaction of the storage solution must be considered. If the column is filled with an organic solvent, there is no danger of microbial activity. However, if the column is filled with an aqueous solution (in particular, an approximately neutral buffer solution), putrefaction may occur. If the packing material can withstand it, it is advisable to add alcohol to a concentration of 10% or greater or to add a preservative substance. If this is not possible, the storage solution should be periodically replaced, even if this is troublesome. The most important point regarding column storage is to be sure not to allow the packing material to dry. In particular, with resin-based packing material, it is reasonable to assume that if it does dry, it will never be restored to its srcinal condition. Be sure to insert an airtight stopper in the column end. When storing a column, for the benefit of the next person using it, make a record of the storage solution used and the final usage conditions, and store it together with the column. Instruments and software packages that record the state of the column have recently become commercially available, so it is convenient to use one of these products. Regarding the storage location, there is no particular need to refrigerate the column; it is sufficient to put it back in the box that it came in and store it in a drawer or some other appropriate place.

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