14- Lab 14- R-HPLC for Detn of Caffeine

Chem 223:

Experiment 14

Determination of caffeine in soft drinks d rinks by High Performance Liquid Chromatography (HPLC). References: th

1. Harris, 7 ed. Chap. 25 2. Harvey: 578-589 3. Kealey: pp. 155-164 4. Experimental procedure. The University of Ad elaine. Australia. Department of Chemistry. 5. Experiment 5. University Kuala Lumpur. Malaysian Institute of Chemical And BioEngineering Technology 6. Experiment 5. Chem 426. Washington State University. 7. Expriment . Chem 427. Portland State University

I. Purpose of the experiment

The traditional method for the determination of caffeine is via extraction with spectrophotometric quantification. Use of the liquid chromatography permits a fast and easy separation of caffeine from other sub stances such as tannic acid, caffeine acid, and sucrose found in these beverages. The amount present in soft drinks is controlled by the manufacturer, and can be obtained upon request. The following experiment illustrates the utility of high performance liquid chromatography chromatography (HPLC) as an analytical tool for the determination the amounts of caffeine in various commercially available soft drinks. From the resulting chromatograms, measurements of retention time t R  and peak areas are made. If the flow rate and pump pressure are held constant throughout the entire experiment, t R  may be used as qualitative measure and the peak area as a quantitative measure. A calibration curve for peak area vs. concentration of the caffeine standards can then be employed to determine the concentration of caffeine in the beverages. II. Introduction

Chromatographic met hods are based on differential affinities of solutes for two phases, a stationary phase and a mobile phase. Molecular interactions leading to these affinities are  polar forces (dipole-dipole and H-bonding) and dispersion forces (induced dipoles). Thus, in a chromatographic system, solute molecules will be attracted to the phase of similar   polarity. Mobile phases in chromatography are either eith er liquid or gaseous. Gas chromatography (GC) is the only technique that uses a gas as a mobile phase, while many techniques employ liquids (liquid chromatography, LC). High Performance Liquid Chromatography (HPLC) is a modern (late 60's) modification of the classical open column techniques that established chromatography as the ulti mate separation technique. The name is derived from the fact that much higher column efficiencies are possible when wh en the particle size of the stationary phase is small (3-10 mm in HPLC versus 40 mm conventional open column LC). As a result of o f these smaller 

 particles, large back-pressures require the mobile phase to be pumped through the column under high pressures. HPLC has several advantages over GC: 1. HPLC is not limited to volatile compounds. 2. A greater control and wider selection of stationary and mobile phases. 3. Many detectors are non-destructive, allowing further analysis of eluted compounds. The basic liquid chromatograph consists of four elements. These are: a pump to move the mobile phase, and injector to deliver the sample onto the column, a column to separate the sample, and a detector to visualize the eluted compounds. The basic HPLC system is illuminated in fig. 1

http://www.uams.edu/metabolic/ Fig. 1 : The basic HPLC system Columns

In HPLC, narrow columns with internal diameters 2-80 mm are used. These columns are  packed with particles having an average diameter of less than 50 microns (50 x 10 -6m). Such columns require high pressures (1000-6000 psi) to maintain a convenient flow o f the eluting solvent, usually in the range 0.1-10 mL/min, but occasionally higher. Resolution is considerably superior to that achieved with an ordinary column, in part because of the tight  packing of the stationary phase, which reduces lateral diffusion, and because of the large surface area of the packing. Compared with classical column chromatography, where the columns are gravity fed and a separation can take hours or even days, HPLC can offer analysis times of 5-30 min. There are two main classes of column: "normal" and "reversed" phase. Normal phase columns are most usually packed with silica gel; they work i n the  partition/adsorption mode in the same manner as a normal silica gel column in conventional chromatography.

Reverse phase chromatography, which is the most common form of HPLC, is a type of   partition chromatography. Frequently, reversed phase columns are packed with a chemically  bonded octadecylsilyl coated silica; such columns are referred to as C-18 and are very non polar. Other popular bonded phase co lumns have octasilyl, cyanopropyl, or phenylsilyl  packings.

The eluent used with reversed phase columns is relatively po lar, e.g. MeOH/H 2O. Unlike normal phase chromatography, the more polar components of a mixture elute first, since these  partition is relatively unfavorable on the highly non-polar packing. Increasing the polarity of  the solvent increases the retention time of a particular component. The situation is the reverse of normal adsorption chromatography: Normal vs. Reverse Phase Normal

Reverse

Packing polarity

High

Low

Solvent polarity

Low

High

Elution order

Non-polar first, then  polar 

Polar first, then non polar 

Effect of increasing solvent  polarity

Decreases retention time

Increases retention time

The other major improvement over column chromatography concerns the detection methods which can be used. These methods are highly automated and extremely sensitive. Important parameters associated with the chromatographic process are measured directly from the chromatogram for example, resolution, R, describes the degree of separation of  successive solute peaks. A value of 1.5 indicates complete (baseline) resolution, while an R value of 1.0 is considered adequate for analytical purposes. The separation factor  describes the relative retention of two components for separation to occur, a must be greater than 1. The capacity factor, k', gives an indication of the relative amount of a  particular solute in each phase (stationary and mobile). It is of the utmost importance, since the observed retention characteristics of a solute depend on its distribution between these phases, and, therefore, its relative affinity towards each. The capacity factor is  proportional to the volumes of liquid and mobile phases, and is an indication of the increase in retention volume (or time) for a given solute relative to that of th e mobile  phase.

The number of theoretical plates, N, is a measure of the efficiency of the chromatographic  process. Obviously, if the individual molecules of a particular solute spread out over a wide as they travel through the column, separation will be difficult, and the separation  process inefficient. Peak width is proportional to this spreading, and is a reflection of  the number of theoretical equilibrium steps of t he solute between the two phases that have occurred within the column. The higher the value of N (or the narrower the peaks), the more efficient the chromatographic process.

III.

Reagents and instrument.

- Solutions provided: - Chemicals: Caffeine reagent grade for standards.

Fig. 2: Molecular structure of caffeine (1,3,7- trimetylxanthine) -

Solutions available: + Solvent for mobile phase: 20% Methanol:80% water (pH 3.50) + Caffeine stock Standard (1000ppm): Accurately weigh out 0.10 gram (to 0.0001g) of caffeine, quantitatively transfer into a 100 mL volumetric flask, dissolve in about 50 mL methanol, fill to the mark with distilled water, and mix thoroughly. + Internal standard stock solution (1000ppm): Accurately weigh out 0.10 gram ( to 0.0001g) of m-methoxybenzioc acid (MW=152.15), quantitatively transfer into a 100 mL volu metric flask, dissolve in about 50 mL of methanol fill to the mark with distilled water, and mix thoroughly.

-Solution to be prepared:

+ Preparing the working standard solution Place 0; 0.5; 1.0; 1.5; 2.0 and 2.5 mL of the caffeine stock solution into a series of six clean and dry 25-mL volumetric flasks. Add 2.5 mL of the internal standard stock solution to each of the six flasks. Dilute to the mark with the  previously prepared solution of 20% methanol: 80% H 2O solvent, adjusted to about pH 3.50 with glacial acetic acid. This is the same solvent to be used as the mobile phase.  Preparing the unknowns : For carbonated beverages, you must first remove the carbonation. You can leave the cap o ff for a couple of days or put a small amount of the beverage in a beaker a nd carefully bring it to a boil on a hotplate. Bring the unknown back to room temperature before you proceed with the filtering. Remove all suspended particulates (solids) before you inject into the LC system. +

Pour 10 to 15 mL of each soft drink samples into a small clean, dry beaker. To decarbonate the beverage, transfer into another dry beaker back and forth until no

more the bubbling is observed. The soda is now adequately decarbonated. Into a clean, dry 25-mL volumetric flask pipette 8 mL of decarbonated Pepsi-Cola, and into a second 25-mL flask, pipette 8 mL of decarbonated Coca-Cola. Dilute each volumetric flask to the mark with 20 methanol: 80% water solvent adjusted to pH 3.50. -Apparatus and equipment:

+ Volumetric flasks, five 100 mL, four 20 mL, Pipettes, 2 mL, 4 mL, 8 mL (one of  each). + The Shimadzu HPLC instrument comprises four main components: the injector LC10Advp, the solvent delivery system , column furnace CTO 10AS and the PDA (Photodiode array) SPD-M10A ( D2 and W lamp). Cartridge: Supelco LiChrospher  RP-18 (6.1 x 250 mm) (a reversed phase column). IV. Experimental Procedure

Prior to injection of the standards into the column run the mobile phase (20% methanol: 80% H2O, pH 3.50) to equilibrate the column for 5 to 10 minutes. Simultaneously monitor the detector response to insure that there are no substances left on the column from previous experiments. Shake the five caffeine solutions adequately for proper dissolution and then degas each for 5 min before injection into the chromatograph. Set the pump flow rate at 2.3 mL/min. A. Operation of the HPLC - General Procedure

The greatest enemy of HPLC is fine particu late matter, which can damage the pu mping system and irreversibly block the column. Therefore, all solvents have been filtered through fine membranes (0.4-0.5 micron) and all solutions to be injected MUST be  prepared either with filtered solvent, or filtered as specified later in these notes. B. Start-up Procedure

First, ensure that there is sufficient filtered solvent in the reservoir for your run. Using the screw on the right hand side of the instrument, pressure the column to 800 psi (beginning of the yellow region). Switch the solvent selector on the inlet manifold at the front of the pump to the running solvent. Switch on the power to the pump and slowly increase the flow rate to 3 mL/min. Switch on the U.V. detector and once the absorbance reading has settled (10 min), set the zero to 0.01 AU. C. To inject a sample: Switch the injector lever (top) to "load". Switch the lower lever to vertical and remove the plug (store in hole in injector switch). Wipe the needle with a clean tissue and insert into the injector. Inject the sample into the loop with even  pressure (excess of solvent in the loop will b e pushed out of the vent tube on the right of  the injector). Replace the injector plug and move the low lever to the horizontal position. Smoothly switch the injector lever to "inject", and at the same time press "inject " on the data module to start the data collection. The data module plots a real time c hromatogram, and at the end of the run time (15 min) replots the chromatogram with details of  retention time (R T), peak area (A or H) and relative areas of the peaks (conc.). Although

the integration is not affected if a plotted peak is off scale, the chromatogram can be replotted at a different attenuation by resetting the ATN (powers of 2, the bigger the attenuation setting the smaller the peaks will look, normally set at 30) and then recalling the plot (Recall). The plot is stored until the next injection. "Feed" moves the p aper  forward for the next plot. D. Shut down procedure

After the last run, flush the column with 50 mL of 20% methanol: 80% water  (not at pH 3.50). Increase the flow to 3mL/min slowly and flush the column with solvent for 10 min. S top the flow and switch off the pu mp. Remove the plots from the Data module and switch off at the front of the unit. Switch off the U.V. detector. Depressure the column by unscrewing the pressure screw on the right of the instrument until 4 threads are showing.  Notes: Normal running pressure for this experiment is between 1500-2200 psi. If a high  pressure shutdown occurs (>2500 psi), consult a de monstrator. Look for leaks at connections through the system; if there are any, consult a demonstrator. Listen to the pump during the experiment; if there are any unusual noises, consult a demonstrator. Look at the outlet flow; it should be a thin stream. If there is no flow, consult a demonstrator. Analysis of data

i.

Standard caffeine samples are used to identify the caffeine peak and the retention time of caffeine is recorded. From 20 ppm to the 100 ppm, the peak is increased.

ii.

The retention time has been used to determine the present of caffeine in the soda sample.

iii.

The peak ratios between the standard and internal standard are measured to quantitatively determine the amount of caffeine in the sample. Then the standard curve is constructed.

iv.

The peak ratios between the standard and internal standard in the soda sample chromatograph has been measured. The concentration and peak ratio area has been used to determine the concentration of the caffeine in t he soda sample.

v.

Express this concentration also in grams/liter.

FOR YOUR REPORT

(a) Data should be presented in the form o f a Table which is easily read and which clearly shows how the values were obtained. (b) Calculations should be clearly shown with an explanation for each step. (c) Treatment of Data: The t R  can be used as a diagnostic tool to determine qualitatively the presence of a substance in a chromatographic mixture. In a tabular 

form, record for the caffeine standards, the concentration, retention times, and peak  areas. Also, calculate the mean and standard deviation of the retention times. Plot the peak area vs. the concentration in mg/mL for the caffeine standards. From the average retention time for the caffeine standard identify the caffeine peak in the coffee, tea, and beverages. (d) Calculate the average and standard deviation of the peak areas and the concentration of caffeine in mg/mL in the samples. Include corrections for dilution in the calculations. More Questions 1. Explain the rationale for using a reverse phase column for the determination of 

caffeine 2. Would the construction of a calibration curve based on peak high (rather than area) give accurate results for the determination of caffeine in this experiment?

3. Could an ion-exchange column be used for the determination of caffeine? Explain. 4. Choose a caffeine peak and determine: (i) Capacity factor, (ii) Number of theoretical plates, (iii) Asymmetric factor