Unit 8 High Performance Liquid Chromatography HPLC

UNIT 8 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) Structure 8.1

Introduction Objectives

8.2 8.3

Principle Instrumentation Sample Injection System Column Packing Material or Stationary Phase Solvent Supply System Pumps Detectors

8.4 8.5 8.6 8.7

Optimization of Separation Advantages Comparison with Gas Chromatography Chromatography Applications Polyaromatic Hydrocarbons Isomeric Compounds Sugars in Popular Drinks Drug Abuse Separation of Nucleic Acids Analysis of Amino Acids Partition Chromatography Ion Chromatography Chiral Separation of Enantiomers Ion-Exclusion Chromatography Speciation Studies

8.8

Interfacing HPLC with Mass Spectrometry Thermospray Method Particle Beam Interface Atmospheric Pressure Chemical Ionization Electrospray Interface Moving Belt Interface

8.9 Summary 8.10 Terminal Questions 8.11 Answers

8.1

INTRODUCTION

During early development period of column chromatography using a 50 - 100 c m long and 1 - 5 cm diameter glass column packed with 100 - 200 µm particle size material, it was realized that column efficiency was very low taking long time for analysis. Though, it could be increased by decreasing the column length and diameter and also the particle size of t he column material. This could be made possible only after 1960 when technology for producing producing packing material with particle size of 3 to 10 µm was developed. Further, the new technology required sophisticated instruments operating at high pressure contrary to classical system where eluent flows under gravity. The first instrument of liquid chromatograph was constructed by Csaba Horvath at Yale University, USA in 1964 who describe it as high pressure liquid chromatograph (HPLC). However, he later called the technique as high performance liquid  chromatography . Thus, the new technique was named as “high pressure” or “high performance” liquid chromatography chromatography (HPLC) to distinguish it from the old procedure. Modern HPLC has emerged from the confluence of n eed, the human desire to minimize work, technological capability and the theory to guide development along rational lines. In some cases, HPLC may detect nanogram or even picogram quantities. quantities. 47

It is now the most versatile and widely used technique by chemists for the separation, qualitative identification and quantitative determination of species in a variety of organic, inorganic, biological biological and other complex materials. It is a type of elution chromatography chromatography where the sample, a mixture of solutes, is in a liquid solvent or mobile phase. The technique is also known by other synonyms such as high speed chromatography , high resolution chromatography and high efficiency chromatography  and is considered as the most sensitive method with continuous major developments. HPLC is able to separate macromolecules and ionic species, labile natural products, polymeric materials, and a wide variety of other high molecular weight polyfunctional groups. HPLC separations are based on specific interactions between sample molecules with both the stationary and mobile phases. A large variety of stationary phases available in HPLC allow a great variety of selective interactions causing better separations.

Objectives After studying this Unit, you should be able to •

explain the meaning of high performance liquid chromatography,

•

differentiate between classical liquid chromatography and HPLC,

•

discuss the basic principle and working of HPLC,

•

describe various components components of instrumentation instrumentation including stationary and mobile phases,

•

describe characteristics of stationary phases in various modes including bonded phase,

•

know the solvent delivery system, characteristics of mobile phase and elution gradient,

•

understand various detectors used in HPLC,

•

learn about versatility and advantages of HPLC,

•

know about various interfaces while using mass spectrometer as detector, and

•

learn about applications of HPLC for the analysis of a variety of solutes.

8.2

PRINCIPLE

The basic principle of separation by high performance liquid chromatography is similar to classical liquid or column chromatography (LC) though it differs with regard to the size of the column and the sample. It differs from LC in terms of speed, automation, elution time and individual manual assays of collected fractions. In case of HPLC, microgram amounts of the sample is allowed to pass through a column containing stationary solid inert phase coated with nonvolatile liquid phase by means of pressurized flow of a liquid mobile phase where components migrate at different rates due to different relative affinities. Comparison of column size, characteristics of packing material and pressure requirements to force the mobility of mobile phase in classical column chromatography and HPLC are illustrated in Fig. 8.1. According to another version, HPLC may be considered as partition chromatography where where stationary phase is a second liquid coated on an inert surface and it is immiscible with the liquid mobile phase. According to the stationary liquid phase, the technique may be subdivided into two types; liquid-liquid and liquid-bonded liquid-bonded phase chromatography. chromatography. These differ from each other in the way stationary phase is held on to the support particles of the packing. In LLC, the polar liquid i s physically adsorbed on to an inert surface where it competes with the mobile phase. However, in case of bonded phase chromatography, chromatography, liquid is chemically bonded making it more stable.

48

(a) Particle size: >150 µm Column diameter: 10-50 mm Column length: 50-200 cm Pressure: < 1 atm

(b) 40-70 µm 1 – 3 mm 50-100 cm 30-50 atm

(c) 5-10 µm 2 – 6 mm 10-50 cm 100-200 atm

Fig. 8.1: Comparison of characteristics of various forms of liquid chromatography: chromatography: (a) Classical column chromatography; chromatography; (b) HPLC with pellicular pellicular packing; (c) HPLC with microparticulate packing

In order to achieve the desired separation by HPLC, several operating conditions including retention time, pressure and number of plates need to be optimized. A major interest is short analysis time, or the plate count needed to accomplish a difficult separation. First of all, a proper HPLC system such a s adsorption, bonded-phase, bonded-phase, reverse phase, ion-exchange, exclusion, affinity or any other form of chromatography chromatography must be selected. Then, all the parameters in the equations as mentioned in Unit 4 that depend on the properties of the mobile and stationary phases are determined. As already described in Unit 4, these are relative retention (α), capacity factor (k’) and the plate count ( N   N ). ). It is desired that the compounds of interest should need at least ten times longer time to travel the column length than the unretained peak. Further, the viscosity of mobile phase and the diffusion coefficients of the solutes in the mobile phase are also of concern besides other characteristics of column packing. The plate height ( H   = L /   where, L is the column length and N  the  the number of plates) is  H  =  N  where, reduced by the particle diameter (d  p) and may be represented as h = H  d p   = L /   N .d  p  / d 

... (8.1)

It actually states the number of particle dia meter (d  p) that constitutes one plate height. Thus, reduced velocity may be represented as v = u.d  p /  D  D M 

… (8.2)

where, D M  is the diffusion coefficient of solute in the mobile phase. It may be considered as the ratio of  the  the time required to displace so lute molecules a distance equal to one particle diameter to the time needed for the same displacement by molecular diffusion. It expresses the balance b etween mass transport by diffusion or molecular motion across a single particle. Substituting the value of u (= L/t m), reduced velocity may be expressed as

49

v = L.d  p /tm D . D M 

… (8.3)

Thus, the complete equation for the dependence of the reduced plate height may be represented as modified van Deemter equation h = B/ v + A.v0..33 + C .v

… (8.4)

where, B = 1.2 for solid core (pellicular) packing and 2.0 for completely porous column packing. Also, A = 0 for well packed column and C  =  = 0.05 for porous particles decreasing to 0.003 for pellicular particles. No theory accurately describes the dispersion from flow in homogeneity in the mobile phase. A logarithmic plot of Eq. (8.4) is shown in Fig. 8.2. The reduced plate height has a minimum value in the range 2-3 for intermediate region of velocities where reduced velocity is 3 -5. It may be observed from Fig. 8.2 that  A term dominates all a long whereas B term arising from axial and longitudinal diffusion, dominates at law reduced velocities. This region of h / v curve is usually avoided. At high velocities, however, C  term  term responsible for increase in reduced plate height, dominates. As explained earlier, C  term  term contains the contributions from mass transfer kinetics and stagnant pockets of mobile phase. You can see that Eq. 8.4 r epresenting the reduced plate height is independent of particle diameter of the column packing. The constants A, B and C  are  are dependent on the packing of column. The number of plates in a reasonable time may be optimized while operating the

Fig. 8.2: Logarithmic plot of reduced reduced plate height, h height, h against reduced velocity,  A = 1, B set of values of constants,  A = 1, B =  = 2 and C  =  = 0.1

ν

 with a

column at the minimum in the h / v plot of Fig. 8.2. The column co lumn length and particle size of the t m and N  are  are chosen under the experimental conditions of eluent viscosity as illustrated by the following example. Assuming desired plate counts, N  =  = 5000, reduced plate height, he ight, h = 5 and a column length, L = 250 mm, required plate diameter, d  p may be calculated using Eq. (8.1). d  p =  L /   N .h = 250/5000 × 5 = 1/100 mm = 10 µm

Similarly, using viscosity parameter (η (η) and specific column resistance (ф (ф) for a fully porous packing, pressure drop (∆ ( ∆P) may be calculated using the expression. ∆P =

50

φη LvD M  3  p

d

=

N 2 h 2φη t M 

 

… (8.5)

Combinations of column lengths and particle sizes including operating pressures for different plate counts and retention times are available in literature.

SAQ 1 What are the various synonyms used for HPLC. Write each one of them. …………………………………………………………………………………………... …………………………………………………………………………………………...

8.3

INSTRUMENTATION

General instrumentation for HPLC h as following components. i)

One or more solvent reservoirs for the mobile phase.

ii)

A pump to deliver the mobile phase with varying range of pressures up to several hundred atmospheres to achieve reasonable flow rates.

iii)

Sampling valves or loops where the sample may be injected into the flowing mobile phase. Sample may be dissolved in mobile phase.

iv)

A guard column or an on-line filter to prevent contamination of the main column.

v)

A pressure gauge, inserted in front of the separation column, to measure column inlet pressure.

vi)

Separation column containing packing to accomplish desired separation. These may be modified silica gel, ion-exchange resin, gel or some other unique packing.

vii)

A detector capable enough of measuring the solute concentrations.

viii) Display and recording device for plotting time vs peak intensity. Besides, other electronic accessories for data manipulations are also required. These are schematically shown in Fig. 8.3.

Fig. 8.3: Schematic illustration of various components of HPLC instrument

51

The individual components are described below:

8.3.1

Sample Injection System

It is a limiting l imiting factor in the precision of HPLC measurement because of reproducibility with which samples may be introduced onto the column packing. Insertion of the sample into the column must be through a narrow plug so that peak broadening is minimized and the system should have no dead volume by itself. Generally, samples are dissolved in a mobile phase solvent to avoid solvent peak and 10 to 50 µL is introduced through micro sampling valves. These devices form an integral part of liquid chromatography equipment having interchangeable loops with a choice of sample size from 5 to 500 µL. The most widely used used method of introduction is based based on sampling loop as shown in Fig. 8.4. It is filled by thoroughly flushing it using a

Fig. 8.4: Schematic of injector valve with external sample loop in a microvolume microvolume sampler

sample solution by means of a microsyringe at pressures up to 7000 psi. A rotation of the valve rotor places the sample filled loop into the high pressure mobile phase stream whereby the sample is sent to the column. The system can be located within a temperature controlled oven if handling at e levated temperatures is required. Many HPLC instruments incorporate incorporate an auto sampler with an automatic injector that can inject variable volumes as per requirement. In stopped flow injection method, pump is turned off till atmospheric pressure is attained, syringe is inserted and the sample injected. The flow of sample can be brought to zero and rapidly resumed by diverting the mobile phase using a three way valve placed before the injector. This method is especially very useful for very high pressures. For best results, a two to fivefold excess of sample should be passed through the loop to ensure that previous sample has been purged thoroughly.

8.3.2

Column

It is the heart of the HPLC instrument where actual separation occurs. Separation column in HPLC is usually made of heavy wall, glass lined metal or 316 grade stainless steel tubing, that can withstand high pressure and which is inert t o the chemical corrosion due to mobile phase. The interior of the tubing must be smooth with a uniform bore diameter. Straight columns that can be operated in vertical position are preferred. Some typical tubing materials used in HPLC column are listed in Table 8.1.

52

Table 8.1: Column Tubing Materials and its Uses Uses Material

Use

316 Stainless steel (SS)

General utility material, good for high pressure system

Poly (ether-ether) ketone

Inert to most organic solvents except methylene chloride, THF, DMSO and conc. Sulphuric and nitric acids. Holds pressures up to 5000 psi (34MPa).

(PEEK)

Good for metal-free biological systems. Tefzel

Inert. Common for metal-free applications.

Titanium

Withstands pressures up to 5000 psi, corrosion resistant; expensive

Fused silica Glass

Used for capillary LC.

Glass

Limited pressure range.

Glass-lined SS

Inert, withstands pressures but difficult to know when the glass is broken.

Column fittings and connectors must be so designed that void volume is zero avoiding unswept corners. Column length ranges10 ranges10 to 30 cm with inner diameter of 2 to 5 mm providing 40,000 to 60,000 plates per inch. However, shorter columns of 3 to 8 cm are also used for fast separations but in such cases, sample size will become limited. The length of the column may not only affect the resolution of a given separation –the longer the column the larger number of plates but also the speed of separation. Standard lengths vary with the manufacturer but most common values are 30, 25, 15, 12.5, 10 and 7.5 cm. It may be noted that shorter columns are described as high speed columns. The columns packed with the finer particles are more expensive than the standard 5 µm packing. Guard column: In order to increase the life of analytical column, a short guard column, also called precolumn, is placed before the main column as shown in Fig. 8.3. It removes contamination from the solvent. Guard column serves to saturate the mobile phase with the stationary phase so that losses of stationary phase in the column are minimized. However, it is essential t hat the composition of the guard column should be similar to that of the analytical column but its particle size may be larger to minimize the pressure drop.

8.3.3

Packing Material or Stationary Phase

The packing used in modern HPLC consists of small, rigid particles having a narrow particle size distribution. The most common packing material used for LC is prepared from silica (acidic) and alumina (basic) particles which are synthesized by agglomerating submicron size particles under conditions that lead to larger particle diameter. These are often coated with thin organic films which are physically or chemically bonded to the surface. For nearly all HPLC applications, chemically modified or unmodified micro particulate silicas of 3, 5 or 10 µm diameter are preferred. This form of LLC, in which both monomeric and polymeric phases have been bonded to a wide range of support materials, is called bonded phase chromatography  (BPC). Characteristics of typical packing materials used in HPLC are listed in Table 8.2. The particles used in HPLC, which are totally porous (macroporous) (macroporous) or superficially porous (pellicular) support, may be spherical or irregular in shape but it is essential that the size range is as narrow as possible to

53

Table 8.2: Characteristics of Some Commercial HPLC Column Packing Materials Type

Silica

Pellicular

Microprous

Corasil (37-50µ (37-50µm)

Lichrosorb (5,10 & 20 µm)

Vydac (30-40µ (30-40µm)

Micropak (5&10 µm) Porasil (15 & 20 µm) Spherisorb (5 µm)

Alumina

Perisorb PA

Micropak Al (5 & 10 µm) Spherisorb Al

ensure high column efficiency and permeability. These adsorbent packings retain solute molecules almost exclusively on the internal surface of the pores, thus, separating these from others. Various types of bonded phases used in HPLC are schematically shown in Fig. 8.5.

Fig. 8.5: Various shapes of stationary phase packings used in HPLC: (a) Bonded (spherical) phase; (b) Irregular large porous phase; (c) Pellicular particle beads and (d) Porous microparticle

The characteristics of various types of bonded phases are described below:

54

A.

Spherical bonded phase: These spherical packings consist of a solid, spherical nonporous nonporous core (usually a glass bead) with a layer of attached functional groups forming an outer shell containing unmodified or modified silica gel, resin, polyamide, etc. Various functional groups are used depending on the nature of solutes to be separated.

B.

Porous layer beads: A porous or pellicular layer bead type packing material consists of a solid, spherical with an a verage particle diameter 30-40 µm coated with a thin porous outer shell, typically of 1-3 µm thick. It may be a silica gel layer, a network of small spherical particles bonded to the solid core. It may also be monomeric or polymeric organic phase. Surface areas of the porous layer 2 beads range from 5 to 15 m  /g . These materials are easy to be packed because of its dense core but suffer from limited sample capacity due to small surface areas. Porous layer packings exhibit good efficiency because of improved mass transfer within the stationary phase. Longer columns are possible because the pressure drop is lower due to larger particle size of porous layer supports. Thicker coatings give rise to slower mass transfer but have increased sample capacity.

C.

Porous particles: Totally porous particles have a large surface area in the range 100 to 860 m 2 /g with average average being 400 m2 /g. The mean pore pore diameter is inversely related to the specific surface area where small molecules enter the pores. The particles can be packed into the HPLC column of efficiencies up to 800 theoretical plates per centimeter if 5 µm particle sizes are used. However,

larger particles give proportionately small number of theoretical plates whence the efficiency of separation goes down. D.

Macroporous particles: These are recently introduced graphitized carbon and styrene-divinylbenzene styrene-divinylbenzene polymers having large channels b esides micropores. The rigid, porous polymeric macroporous macroporous beads do not swell or shrink with changes in the ionic strength of the mobile phase (can be used over an extended pH range of 1 and 13) or deform at high velocities and are most suited for separations in nonaqeous media. These materials have increased the choice of stationary phases and the scope of HPLC, particularly for highly polar and basic substances.

An illustration of various types of bonded phases used in HPLC is shown in Fig. 8.6 where different topographies are obtained depending on the nature of the ligand . It may be noted that different packing materials are used in different type of techniques of adsorption, partition, ion-exchange, ion-exchange, size exclusion chromatography. chromatography.

(a)

(b)

(c)

Fig. 8.6: Various shapes shapes of bonded HPLC column packing materials (a) Types Types of bonded bonded phases, (b) Topography Topography of ligands and (c) Size of ligands ligands

SAQ 2 Explain why small particle size is required in HPLC? How is it important in attaining higher efficiency? …………………………………………………………………………………………... …………………………………………………………………………………………... …………………………………………………………………………………………...

SAQ 3 Choose the correct answer from the choices given. i)

Which one of the following is the most appropriate particle size (in µ m) for packing material in HPLC? a) 1-5

ii)

b) 3-5

c) 10-20

d) 20-50

Which one of the following column length (in cm) should be used for faster HPLC separation? a) 2-5

b) 5-10

c) 10-15

d) 20-30

55

iii)

Which of the following materials meet the requirements to fabricate HPLC column? a) Glass lined metal

iv)

c) Stainless steel

d) Steel

What is the average surface area ( in m2 /g) of porous porous particles in HPLC column? column? a) 100

v)

b) Quartz

b) 300

c) 800

d) 400

Which one of the following ranges of flow rates (in mL/min) should be adequate for analytical HPLC? a) 0.02 – 1.0 b) 0.05 - 2.0

c)

1.0-2.0

d) 0.5 – 2.0

Let us now study about the s tationary phases used in various chromatographic chromatographic modes. i)

Adsorption Chromatography

In majority of the cases of adsorption chromatography, chromatography, silica column packings are used where main mechanism is the interaction of its OH groups with the polar or unsaturated functional groups of a solute/solvent molecule by hydrogen bonding or dipole interaction. The slightly acidic silanol (Si-OH) groups in silica gel are at the surface and extend out from the surface in the internal channels of the pore structure. The number and topographical arrangement of the several types of OH groups, as shown in Fig. 8.7, determine the activity of the adsorbent and thereby the retention of the solutes. These OH groups can be divided into three types: •

silanol (free OH),

•

siloxane bond (Si-O-Si) and

•

hydrogen bond (Si-OH…O).

Fig. 8.7: Structure of silica gel depicting the various types of hydroxyl groups groups that interact with the functional groups of solute/solvent molecules

Each of these groups has different activity that increases in the following order: Bound < free < H-bond. According to current models of adsorption process, it is assumed that adsorption sites are completely covered by either of solute or solvent molecules that are adsorbed depending on their relative strength in this competitive interaction. The 56

competition between the solute and the mobile phase molecules for an active site provides the driving force and selectivity in separations. Interaction between a solute molecule and the adsorbent surface is best when functional groups overlap adsorption sites. Adsorption chromatography is less influenced by difference in molecular weight but certainly more by functional groups. For compounds of low to moderate polarity, adsorption chromatography chromatography often makes possible the separation of complex mixtures into classes of compounds with similar chemical functionality. Typical examples of group separations are polynuclear aromatics from a petroleum sample and the triglycerides from a liquid extract. ii)

Partition Chromatography

It can be subdivided into liquid-liquid chromatography chromatography (LLC) and bonded phase chromatography (BPC), the difference being in the method by which stationary phase is held on the support particles of the packing. In case of LLC, a liquid stationary phase is retained on the surface of the p acking by physical adsorption. With bonded phase, the stationary phase is bonded chemically to the surface of inert support. Of late bonded phase has become predominant over liquid phase because of certain disadvantages. The packings for bonded phase are prepared from rigid silica or silica based compositions. These are formed as uniform, porous, mechanically sturdy particles commonly having diameters 3, 5 or 10 µm. The surface of fully hydrolysed silica is made up of chemically silanol groups. The most useful bonded phase coatings are siloxanes formed by the reaction of hydrolysed surface with an organochlorosilane as shown below: CH3 Si

OH + Cl

Si

CH3 R

Si

O

CH3

Si

R + HCl

CH3 CH3

CH3 Si Si

OH + Cl

Si

Cl

O

H2O

CH3 Si

OH

Si

O

Si

O

Si

CH3

CH3

CH3

CH3

Si CH3

O

Si

Cl

Cl

CH3

Surface coverage by silanization is limited to 4 µmol/m2 or less because of steric effects. The unreacted SiOH groups impart an undesirable polarity to the surface, which may lead to chromatographic chromatographic tailing of the peaks. In order to avoid this effect, siloxane packings are often capped by further reaction with chloromethylsilane chloromethylsilane that can react with many of the unreacted silanol groups. Two types of partition chromatography have been recognized based on relative polarities of stationary phase and mobile phase. In normal phase LC or HPLC, stationary phase consists of highly polar water or triethyleneglycol supported on silica or alumina particles and a nonpolar mobile phase solvent solvent such as hexane is used. In contrast, in the reversed phase chromatography, chromatography, the stationary phase is nonpolar, often a hydrocarbon and the mobile phase is polar such as water, methanol or acetonitrile where most the polar component appears first. Perhaps three quarters of all the HPLC is currently being carried out in columns with reversed phase.

57

Most commonly, the R group of the siloxane in these coatings is a n-octyl (C-8 chain) or n-octadecyl (C-18 chain). With such preparations, the long chain hydrocarbon hydrocarbon groups are aligned parallel to one another and perpendicular to the particle surface, giving a brush or bristle-like structure as illustrated in Fig. 8.6. The relationship the between polarity of the sample with that of the column packing material and mobile phase is illustrated in Fig. 8.8. Retention increases with the hydrophobic character of the solute samples. Generally, the lower the polarity of the mobile phase, the higher is i ts eluent strength. The effect of chain length of the alkyl group upon column performance performance is illustrated in Fig. 8.8 where it is observed that longer chains produce packings that are more retentive. For example, maximum sample size for a C18 packing is roughly double that for a C4 preparation under similar experimental conditions.

Fig. 8.8: Relationship between the polarity polarity of the sample with that of the packing material and the mobile phase in reverse phase HPLC

In commercial normal-phase bonded packings, the R in the siloxane structure is a polar functional group such as cyano (−C2H4CN), diol (–C3H6OCH2CHOHCH 2OH), amino (−C3H6NH2), and dimethylamino d imethylamino (C3H6N(CH3)2). The polarities of these packing materials vary over a considerable range with the cyano type being the last polar and the amino types the most. Diol packings are intermediate in polarity. With normal phase packings, elution is carried with relatively non-polar solvents such as ethyl ether, chloroform and n-hexane. iii)

Ion-exchange Chromatography

In this case, column packings have charge bearing functional groups attached to a polymer matrix. The functional groups are permanently bonded ionic groups associated with counterions of the opposite charge. Some ion-exchange ion-exchange packings bear negatively charged groups and are used for exchanging cationic species whereas others are designed for exchanging anionic species. Similarly, some functional groups such as –COOH or -PO32– have weak acidic or basic properties whereas some others have considerable affinity for heavy metal cations. Several structural types of packings, as shown in Fig. 8.9, have been used in ion-exchange HPLC. Of these, the pellicular type consists of a resin coating, about about 1-2 µm thick, on a µ glass bead of 30-40 m diameter. Superficially porous resins are obtained by coating glass beads with a thin layer of silica microspheres on which ion exchanger is bonded. This increases the interface between the resin and mobile phase. Either type of these packings have low exchange capacity, 0.01 – 0.1 meq/g. The exchanger may also be bonded to silica microparticles by means of silylation reactions or polymerized into pores of a superficially porous silica gel. 58

(a)

(b)

(c)

(d)

Fig. 8.9: Various structural types of ion-exchange packings: packings: (a) pellicular with ion-exchange film; (b) exchanger beads coated superficially with porous resin; (c) macroreticular resin bead and (d) anion exchanger surface sulfonated and bonded electrostatically

During preparation of ion exchanger by silylation, a vinyl group is chosen for R 3 in -SiOSiR1R2R3 leading to a vinylated silica which is then polymerized with styrene. CH = CH2 + CH = CH2

CH

CH2

C6H5

CH

CH2

C6H5

Afterwards, the bonded phase is treated with chloromethyl ether and subsequently trimethylamine trimethylamine or hydroxyethyldimethylamine hydroxyethyldimethylamine to prepare the quaternary amine exchanger as is shown below: CH2

CH2

CH

CH2

+ ClCH2OCH3

C6H5

CH2

CH2

CH

CH2 + CH3OH

C6H5CH2Cl

RNH2

N(CH3)2CH2CH2OH CH2

CH2 CH

CH2

+ C6H5CH2NH2-R Weak anion exchanger

CH2

CH2

CH

CH2

+ C6H5CH2N(CH3)3 Strong anion exchanger

Hydrophilic Hydrophilic polymers allow the separation of proteins, nucleic acids and other large ionic molecules. The microporosity of these ion exchangers minimizes possible exclusion effects. iv)

Size Exclusion Chromatography

In this case, column packings are either semi-rigid, cross-linked macromolecular polymers or rigid, controlled pore size glasses or silicas. The semi-rigid materials swell and care must be taken to their use limited to a maximum pressure of 300 psi due to bed compressibility. The styrene-divinyl benzene polymers allow fractionation within a molecular weight range of 100 to 5000 million. Partially sulphonated polystyrene beads are compatible with aqueous systems and non-sulphonated ones with non-aqueous systems with bead diameters ~5 µm. Another class of hydrophilic porous packing is prepared by suspension polymerization of 2-hydroxyethyl 2-hydroxyethyl methacrylate with ethylene dimethacrylate. These packings can withstand pressures up to 3000 psi and are usable with aqueous systems and with a variety of polar organic solvents. Porous glasses and silicas cover a wide range of pore size diameters. For example, a series of particle size diameters and operating ranges of molecular weights are listed in Table 8.3.

59

Table 8.3: Correlation of Pore Size Diameter and Operating Range of Mol. Wt Pore-size diameter (µm)

Operating range

4

1000-8000

10

1000-30000

25

2500-125000

55

11000-350000

150

100000-1000000

250

200000-1500000

(Daltons)

These packings are chemically resistant at pH 10) × 106

125

(0.2 – 5) × 10

300

(0.03 – 1) × 105

500

(0.05 – 5) × 105

1000

(5 – 20) × 105

10

2

4

Ion Chromatography

It differs from ion-exchange chromatography in the nature of exchange resins. The technique involves an ion-exchange column and a means of suppressing (removing) ionic species other than the sample ions in the eluting mobile phase to facilitate detection of the sample by a conductivity monitor as schematically illustrated in Fig. 8.10.

60

Electric inte inte rator rator

Fig. 8.10: Schematic diagram of ion chromatograph chromatograph with separation separation column

The column packing consists of a neutral polymer core of ~ 10 µm diameter depending on whether the packing will be used for the separation of cations or anions. Contrary to the conventional ion-exchange chromatography chromatography where core is sulphonated or aminated leading to the formation of sulfonic acid or quaternary amine groups, in ion chromatography, a monolayer of aminated or sulphonated polymeric anion exchange beads is used. Similarly, for a cation exchanger, there would be an intermediate layer of aminated groups covered by a thin layer of sulphonated resin beads. Due to the proximity of all the active sites t o the eluent-resin interface, this type of exchanger has favorable favorable mass transfer characteristics. It has low exchange capacity, about 0.020 meq/g of copolymer. In most applications, silica based materials are inappropriate due to their degradation in the presence of aqueous eluents and their poor selectivity for some ionic species. The eluent passes through a suppressor column where the eluting or background electrolyte is effectively removed by converting it into water or, water and carbon dioxide i.e., sodium ions are replaced by hydronium ions or methylsulfonate methylsulfonate ions with hydroxyl ions. A miniaturized ‘self-regenerating’ ‘self-regenerating’ suppressor cartridge c artridge incorporating an + electrolysis cell is also available a vailable where H3O  and O2 are continually formed by the electrolysis of a stream of deionized water passing through an anode compartment and similarly, OH¯ and H2 are formed in a cathode compartment. Both compartments are separated from the eluent either by cation or anionexchange membranes depending on whether anionic or cationic analytes are to be separated. vi)

Chiral Chromatography

Quite often only one enantiomer possesses the desired therapeutic activity whereas the other may be inactive or even harmful. The separation of enantiomers by HPLC using chiral stationary phase (CSP) is based on the formation of transient diastereoisomeric diastereoisomeric compounds between between the

61

enantiomorphs of the solute and the chiral selector which is an integral part of the stationary phase. The difference in stability between these complexes results in difference in their retention times, the enantiomer forming the less stable complex being eluted first. A large number of chiral phases are commercially available. All of these are coated on silica gel support. The coating itself is a polymeric material to which an optically active isomer is bonded. For example, the l form of the amino acid, proline has been bonded to polystyrene- p-divinylbenzene, -divinylbenzene, a cross linked copolymer to give an optically active stationary phase for the separation of racemic mixtures of amino acids. In this case, Cu2+ ions are introduced into the solution of the analyte enantiomers to be separated whereby a t ernary complex, as shown in Fig. 8.11. is formed between the stationary phase, amino acid anion and Cu2+. The formation constant for this complex differs for d  and  and l forms of the analyte amino acid; thus, making their separation possible.

Fig. 8.11: Illustration of a t ernary complex formed between an L-proline bonded phase, 2+ an analyte amino acid and a Cu  ion

Cyclodextrin-bonded Cyclodextrin-bonded stationary phases have been demonstrated to be be particularly efficient in resolving structural isomers. Some examples areprostaglandin A1, A2 and B1B2, α- and β  and  β -naphthols, -naphthols, o,o′ and p, p′-biphenyls and the ortho-, meta- and para- isomers of nitrophenol, nitroaniline, xylene, cresol and aminobenzoic acid. Recently introduced graphitized carbon and new generation of rigid porous polymeric microbeads based on styrene/divinyl benzene as alternatives to silica can be used over a wide range of pH between 1 to 13. Some examples of column packings used in HPLC and their applications are listed in Table 8.5. Table 8.5: Some Typical Column Packings Used in HPLC HPLC Packing

Mode of HPLC

Applications

Microparticulate silicas; spherical or irregular particles; mean particle size 3, 5 and 10µm chemically modified versions of the above (bonded-phase packings)

LSC (adsorption)

Non-polar to moderately polar mixtures, e.g., polyaromatics, fats, oils, mixtures of isomers

Octadecyl (ODS or C18)

BP (bonded phase) and Ion Pair Chromatography (IPC)

Wide range of moderately polar mixtures, e.g., pharmaceuticals and drugs, amino acids

Octyl (C8)

BPC, IPC

More polar mixtures, e.g., pesticides, herbicides, peptides, metabolites in body fluids Table continued on next page

62

Short chain (C3 or less)

BPC, IPC

IPC applications of above three packings include bases, dyestuffs and other multiply charged species; used instead of IEC

Diol

BPC

Very polar and water-soluble compounds, e.g., food and drink additives

Nitrile

Normal phase and BPC

Alternative to silica, can give better results

Aminoakyl

BPC

Carbohydrates including sugars

Anion and cation exchangers (tertiary amine or sulphonic acid)

IEC (Ion-exchange chromatography)

Ionic and ionizable compounds, e.g., vitamins, water-soluble drugs, amino acids, food and drink additives

Controlled porosity silicas (chemically modified to reduce adsorption effects)

Size exclusion Chromatography

Polymer mixtures, screening of unknown samples. Increasing use for separating mixtures of smaller molecules before other modes of HPLC

Chiral amino acids bound to aminopropyl

Chiral Chromatography (CC)

Chiral peptides

CC

Cyclodextrins

CC

Mixtures of enantiomers especially of drugs

It may be mentioned that besides various modes of HPLC discussed above, thin layer chromatography chromatography is another mode which is already discussed in Unit 6. Hence, it is not included in the discussion here.

SAQ 4 Complete the following sentences with suitable words. i)

Silylation is a process where...................................................................................

ii)

While using normal phase packing, elution is carried out using............................. …………………………………………………………………………………….

iii)

Functional groups such as ......................................have weakly acidic properties.

iv)

Styrene-divinyl Styrene-divinyl benzene polymers a llow fractionation of substances having molecular weight in the range range of.............................................. ...............................

v)

Background electrolyte is effectively removed in.....................................where it is converted into .....................................................................................................

vi)

Chiral stationary phase is used for the separation of ............................. ................ and is based on the formation of ............................................................................

8.3.4

Solvent Supply System

Nature and composition of mobile phase in LC plays an important role as i t provides a dimension in terms of retention time for experimental manipulation. In case of HPLC, high purity solvents without any dissolved gases should be used because any impurity may affect the retention time and hence separation of the constituents. The eluent system consists of reservoirs of reservoirs from which one or more solvents can be selected. 63

Essential features of a modern HPLC system includes flow control and inlet filter through a Millipore filter under vacuum. Also degassing facility such as a supply of an inert gas is a must. It helps in removing dissolved gases that may have adverse effect on the column performance. The general criteria for the selection of a mobile phase are: •

It should dissolve the sample.

•

It should keep the column stable.

•

It should be compatible with the detector.

•

It should be immiscible with the stationary phase.

•

Its viscosity should not be high.

•

Active fluorides should be avoided when using glass co lumn.

The eluting power of a solvent is determined by its overall polarity, the polarity of the stationary phase, and the nature of sample components. The capacity factor, k ′, is controlled by the strength of solvent which can be easily predicted in adsorption chromatography. chromatography. Snyder has defined solvent strength parameter, εo, as the adsorption energy per unit area of adsorbent . Some common solvents used in adsorption chromatography are listed in Table 8.6 in the order of increasing solvent solvent strength. It also includes adsorption strength strength of the various functional groups groups of solute molecules. Such a list is also called eluotropic series of solvents. It has been observed observed that log k ′ for a given solute varies linearly l inearly o with ε . Other properties of solvents which must be taken into account include boiling point and viscosity, detector compatibility, flammability and toxicity. Generally, the lower boiling and hence, the low viscosity solvents give higher chromatographic efficiency and lower back pressure. Table 8.6: Solvent Strength Parameter, ε o and the Physical Properties of Selected Solvents Used in HPLC Solvent

Pentane Hexane Cyclohexane Carbon disulphide Carbon tetrachloride 1-Chlorobutane  Di-isopropyl ether 2-Chloropropane Benzene Diethyl ether Chloroform Methylene dichloride Tetrahydrofuran Acetone 1,4-Dioxane Ethyl acetate 1-Pentanol Acetonitrile Methanol Water

64

o

ε 

(SiO2)

0.00 —0.05 0.14 0.14

0.25 0.38 0.26

0.47 0.49 0.38 0.50

o

ε 

(Al2O3)

Refractive index, 20ºC

0.00 0.00 0.04 0.15 0.18 0.26 0.28 0.29 0.32 0.38 0.40 0.42 0.45 0.56 0.56 0.58 0.61 0.65

Viscosity, 20ºC (mN  ─ 2 sec m ) 0.23 0.313 0.980 0.363 0.965 0.47 0.379 0.335 0.65 0.23 0.57 0.44 0.55 0.32 1.54 0.45 4.1 0.375

0.95 Large

0.60 1.00

1.329 1.333

1.358 1.375 1.426 1.628 1.460 1.402 1.368 1.378 1.501 1.353 1.443 1.425 1.407 1.359 1.422 1.370 1.410 1.344

As the column flow rate is proportional to the product of the linear velocity and the cross sectional area of the column, the solvent consumption is considerably reduced as illustrated in Table 8.7. Table 8.7: Solvent Consumption for Different Diameter HPLC Columns ID (mm)

Flow rate for linear velocity of 0.14 cm/sec (mL/min)

Volume in a 8 hr day (mL)

0.51

0.02

6.9

0.71

0.027

13

1.02

0.044

24

1.29

0.079

38

1.59

0.12

57

4.6

1.00

480

When two more solvents with a fixed composition are used, it is called isocratic elution. This, however, is a very cumbersome process and instead gradient elution is used. continuous change in the composition of the mobile Gradient Elution: It involves continuous phase by allowing a more polar solvent to flow into the r eservoir containing a less polar one, whence the mixture flows to the column as illustrated in Fig. 8.12. Thus, a complex mixture of solutes that cannot be separated by isocratic separation can be separated by gradient elution. It is especially useful for separating components that differ widely in polarity. For gradient elution using a low pressure mixing system, the solvents from different reservoirs are fed to a mixing chamber and then mixed solvent is pumped into the column.

Fig. 8.12: Schematic illustration of low pressure pressure gradient using three three solvents of different polarity

Time proportionating electrovalves used in modern instruments are regulated by a microprocessor; thus, thus, the resolution for each chromatogram. chromatogram. It can reduce the run time and increase the sensitivity. As the gradient develops, tailings are made to elute quicker. The commercial liquid chromatographs are designed designed to mix two or more solvents in a progressive manner from 0 to 100% of one component. If one of the solvents gives an appreciable response at the detector, then the generation of a solvent gradient gradient will also introduce a baseline drift in response. In such a case, column will also need time to 65

regenerate the starting solvent composition each time a fresh gradient run is started and ideally, a blank gradient is run between samples to prevent prevent the occurrence of artifact peaks which can be observed. This can make gradient elution seem slower than literature values. It may be noted that gradient elution produces effects similar to temperature programming in gas chromatography.

8.3.5

Pumps

A variety of pumps are used to maintain flow rate and pressure of the mobile phase. Also a degasser is needed to remove dissolved air and other gases from the solvent. A desirable feature of the delivery system is the capability of generating solvent gradient. A pump should be able to operate up to a p ressure of 100 atm (1500 psi) though in some cases 400 atm (6000 psi) is desired. For most analytical columns, only moderate flow rates of 0.5 – 2 mL/min may be required. However, for microbore columns, low flow rates of only a few microlitres/min may be sufficient. Also, a pump should have a small hold up volume. Some typical pumps are described below: i)

Reciprocating piston pump: It is the most popular type of pump as it is inexpensive and can permit a wide range of flow rates. It consists o f a small motor driven piston moving rapidly back and forth in a hydraulic chamber that may vary from 35 to 400 µL. The piston sucks in solvent from the mobile phase reservoir by means of check valves. Usually, a hydraulic fluid transmits the pumping action to the solvent via a flexible diaphragm; thus, minimizing solvent contamination and corrosion problems with pump parts.

A wide range of flow rates may be obtained by varying either the stroke volume during each cycle of the pump or the stroke frequency. Delivery of solvent through reciprocating pump is continuous without any restrictions on the reservoir or operating time. These have very small initial volume and accurate elution gradient. Its advantages include small internal volume (35 to 400 µL), their high output pressures (up to 10,000 psi), their ready adaptability to gradient elution and their constant flow rates which are largely independent of column back pressure and solvent viscosity.

66

ii)

Syringe type displacement pump:  These pumps work through positive solvent displacement by a mechanically driven piston at a constant flow rate. The piston is actuated by a screw feed drive through a gear box usually run by a digital stepping motor. The rate of solvent delivery is controlled by changing the voltage of the motor. The solvent chamber has finite capacity of 250-500 mL which may be refilled if need be. Pulse less flow is achieved along with high pressure capability of 200-475 atm.

iii)

Constant pressure pump: In this type t ype of pump, pressure delivered through a large piston drives the mobile phase. Since the pressure on the solvent is proportional to the ratio of the area of the two pistons, usually between 30:1 and 50:1, a low pressure gas source of 1-10 atm can be used to generate high liquid pressures (1-400 atm). A valving arrangement permits the rapid refill of the solvent chamber whose capacity is about 70 mL. This system provides pulse less and continuous pumping, including high flow rates for preparative applications. This type of pump is useful for pumping columns but inconvenient for solvent gradient columns.

iv)

Pneumatic pump: These types of pumps are simple, inexpensive and pulse free but suffer from limited capacity a nd pressure output. In this case, mobile phase is contained in a collapsible container housed in a vessel that can be pressurized by a compressed gas. These are not amenable to gradient elution and are limited to pressures less than 2000 psi.

Most commercial instruments are equipped with computer controlled devices for measuring the flow rate by determining the pressure drop across a restrictor located at the pump outlet. Any difference in signal from a preset value is then used to decrease or increase the speed of the pump motor. Composition of solvents may be continuously varied or in a stepwise fashion.

8.3.6

Detectors

A detector is an important part of the HPLC instrument and should be chosen very carefully for selective separation and accurate determination. The single most crucial factor is continuous detection based on the progress of separation of a component which may be immediately displayed and then recorded. However, a good detector must have following characteristics: •

It should have linear response to solute concentration in the range 0.1 µg/mL to 1 ng/mL.

•

It should respond to solute only and not to the solvent or change in solute to solvent ratio.

•

It should be insensitive to change in temperature, pressure and flow rate.

Though highly sensitive detectors have been developed for HPLC but there is no universal detector which could be used for all kinds of samples and for all concentration ranges. The choice of a detector depends on the problem a t hand though sometimes more than one detector may be used. These are of two basic types. i)

Bulk Property Detectors

These types of detectors measure on overall change in a physical property of the mobile phase with and without solute e.g. refractive index, dielectric constant, density, electrical and thermal conductivity, conductivity, vapour pressure etc.. These types of detectors are somewhat insensitive and require good temperature control. ii)

Solute Property Detectors

These respond to a physical property of the solute that is not exhibited by the pure mobile phase. These are highly sensitive with a detection signal for a few ng or even lesser amount of sample. For example absorbance, fluorescence, fluorescence, diffusion current and electrochemical detectors are considered in this category. Besides low detection limit, a HPLC detector must meet following requirements: •

Selective response towards one or more c lasses of solutes.

•

It must be small and compatible with liquid flow.

•

It should have good stability and reproducibility. reproducibility.

•

It must have low dead volume to minimize e xtra-column band broadening. broadening.

•

It must have high reliability and ease of use.

•

It must have small response time, at least 10 times less than the peak width of a solute.

•

It should have a linear response to solute that e xtends over several orders of magnitude.

Some characteristics of commonly used detectors in HPLC systems are listed in Table 8.8.

67

Table 8.8: Performance of some HPLC Detectors Detector property Absorbance Fluorescence Electrochemical Conductivity Refractive Index Mass spectrometry FTIR Light scattering Optical activity Photo ionization Element selective

*

Typical LOD (mass) 10 pg 10 fg 100 pg 100 pg-1 ng 1 ng