Experiment UPHPLC: Separation and Quantification of Components in Diet Soft Drinks

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1 Experiment UPHPLC: Separation and Quantification of Components in Diet Soft Drinks bjective: The purpose of this experiment is to quantify the caffeine content of a diet soda sample using Ultra-High Performance Liquid Chromatography (UHPLC). In order to quantify the caffeine, it must be isolated from the other components in the mixture. In this experiment, you will determine a set of HPLC conditions suitable for separating caffeine, benzoic acid, and aspartame and then quantify the caffeine content of your soda sample using a standard calibration curve. At the end of this experiment you should understand the mechanisms by which components in a mixture are separated, identified, and quantified using HPLC and understand how to vary experimental parameters to optimize a separation. Equipment Chemicals ThermoScientific UHPLC Diet soda sample (Degas for 20 min followed by filtration with 0.22mm syringe filter Hamilton Syringe Caffeine standards 100% (0.045g/250mL) 250 ml Vol flask Additional dilutions as necessary to bracket soda sample HPLC-grade methanol 20mM phosphate buffer, ph 3 Standard samples mixture containing caffeine, aspartame, benzoic acid and uracil Safety and Waste Disposal The chemicals used in this experiment should pose no significant safety hazards. Good laboratory procedure should be followed at all times. Discussion The fundamentals of chromatographic separations and a detailed discussion of the application of HPLC is covered in the appropriate chapter in your text. The theory of HPLC and UHPLC are similar, the former using ultra high pressure. The following discussion summarizes important concepts from these chapters, but the student is encouraged to read the full text. Introduction to HPLC and Instrument Components. High performance liquid chromatography (HPLC) and Ultra-High Performance Liquid Chromatography (UHPLC) are actually the same techniques. UHPLC uses twice the pressure from conventional HPLC, up to 22,000 psi to obtain rapid flow rate and improve resolution. This is done by using columns that uses particle sizes of < 2 um. HPLC and UPHPLC are important analytical tools for separating and quantifying components in complex liquid mixtures. By choosing the appropriate equipment (i.e. column and detector), this method is applicable to samples with components ranging from small organic and inorganic molecules and ions to polymers and proteins with high molecular weights. The various types of liquid chromatography and their characteristics are summarized in the table below. In this experiment, we will use reversed-phase partition chromatography. TYPE Adsorption Partition (reversedphase) Partition (normalphase) Ion Exchange Size- Exclusion SAMPLE PLARITY non-polar to somewhat polar non-polar to somewhat polar somewhat polar to highly polar highly polar to ionic non-polar to ionic Table 1. Various Types and Applications of HPLC MLECULAR WEIGHT RAGE STATIARY PHASE MBILE PHASE silica or alumina non-polar to polar non-polar liquid adsorbed or chemically bonded to the packing material highly polar liquid adsorbed or chemically bonded to the packing material ion-exchange resins made of insoluble, highmolecular weight solids functionalized typically with sulfonic acid (cationic exchange) or amine (anionic exchange) groups relatively polar relatively non-polar aqueous buffers with added organic solvents to moderate solvent strength small, porous, silica or polymeric particles polar to non-polar

2 Experiment. HPLC: Separation and Quantification of Components in Diet Soft Drinks Modified 11/2017 Figure 1. Shows the components of our ThermoScientific Ultimate 3000 UHPLC.: Figure 1. Control of the above components and data acquisition and analysis are performed on a Dell personal computer. Figure 2 The workup is through ThermoScientiic Chromeleon Chromatography Data System Software. updated UHPLC of Diet Soda

3 ptimization of Resolution and Column Performance The goal of any liquid chromatography experiment is to achieve the desired separation in the shortest possible time. With the UHPLC, an experiment that used to run min now takes 2-4 minutes. Still the instrument must be optimized to improve resolution. ptimization of the experiment usually involves manipulation of column and mobile phase parameters to alter the relative migration rates of the components in the mixture and to reduce zone broadening. These can generally be optimized fairly independently. Migration Rates The length of time it takes for a given component/solute to travel through the column and be detected is determined by the flow rate of the mobile phase, m, and the partitioning of the solute between the mobile and stationary phases. Since the solute molecules can only travel when they are dissolved in the mobile phase, the greater their concentration in the mobile phase, the faster they will elute. The partition coefficient, K, is defined in equation 1 K = C S C M (1) where C S is the concentration of the solute dissolved in or adsorbed to the stationary phase, and C M is the concentration of the solute in the mobile phase. The quantities C S and C M, however, are rarely determined in chromatographic experiments. Instead, a quantity called the retention factor, k, is determined. The retention factor for a component A is defined as k À = t R t M t M (2) where t R is the retention time of component A and t M is the retention time of an un-retained species (uracil), which is also called the dead time. The average rate of linear migration of component A is related to both the flow rate of the mobile phase and the retention factor. 1 v = m ( 1 + k À ) The retention factors should normally lie in a range of 2-5, but for complex mixtures a larger range may be required to separate all the components. The value of the retention factor for a given component depends on the chemical identity of the component and the following experimental variables: mobile phase flow rate mobile phase composition column temperature column composition Zone Broadening: The extent to which the component bands spread as they travel down the column affects the efficiency of the separation. The theoretical plate height, H, is defined in equation 4 and is based on a Gaussian analysis of the peak width, σ, as it exits the column at point L. H = σ2 L (4) where σ = LW 4t R (5) and W is the width of the peak at the base. The data analysis program on our HPLC actually reports the width at half maximum, W 1/2, for each peak rather than the width at the base. Assuming a Gaussian peak shape, W = ( )W 1/2 (6) so, ( ) 2 L W 1/2 H = t R ( ) 2 (7)

4 The number of theoretical plates in the column,, is = L H (8) Efficient columns have small H and large for a given component. The theoretical plate height is affected by the following experimental parameters: mobile phase flow rate diffusion coefficient of the solute in the mobile phase diffusion coefficient in the stationary phase (depends on temperature and viscosity) retention factor diameter of the particles packing the column thickness of the liquid coating on the stationary phase Resolution The resolution of two adjacent peaks, R S, is determined by their separation and their widths. [ ] W A +W B = 2 (t R ) B (t R ) A R S = 2 [(t R ) B (t R ) A ] # ( ) ( W 1/2) + W $ % 1/2 A ( ) B & '( (9) In other words, R S depends on both migration rates and zone broadening. A resolution of 1.5 means that the overlap of the peaks is about 0.3%, so conditions should be optimized to achieve at least this resolution if possible. In this experiment, you will adjust only the composition of the mobile phase to optimize the retention factors and resolution. We will not attempt to optimize the zone broadening independently by changing the column or the flow rate. Solution Preparation. (Subject to modifications) Safety and Waste Disposal. Check all glassware for stress, stars, or fatigue before performing this experiment. Standard solutions of benzoic acid and aspartame will be provided for you. You will need to prepare a 1000ppm standard solution of caffeine. Individual diet soda samples will be provided to you. The identity of the vials will be written on the label. I. Preparation individual solutions of Caffeine, Benzoic Acid and Aspartame Prepare individual, 200ppm solutions of Caffeine, Benzoic Acid and Aspartame. Spike each with saturated solution of uracil (provided). II. Preparation of Standard solutions Prepare a 1000ppm standard solution of caffeine. The exact concentration of Benzoic acid and Aspartame will be provided later. For your pre-calculations, assume it will also be about 1000ppm Using the standard solutions of aspartame, benzoic acid and caffeine, prepare the following solutions. Table 2. Recommended stock solution and serial dilution for trace elements to be analyzed. Components Concentration range Stock Solution necessary * Standard Solution Range (10ml Total Volume per set) ppm Set 1 (Blank) Set 2 Set 3 Set 4 Set 5 1 Caffeine ppm 1000 ppm 0 ppm 25 ppm 100 ppm 200 ppm 300 ppm 2 Benzoic Acid ppm 1000 ppm 0 ppm 25 ppm 100 ppm 200 ppm 300 ppm 3 Aspartame ppm 1000 ppm 0 ppm 25 ppm 100 ppm 200 ppm 300 ppm 4 Uracil Saturated Use the procedure that you followed in experiment 5, Atomic Spectroscopy of Hair to determine the volumes necessary to prepare these standards III. Preparation of your unknown. For your unknown sample, prepare three or 4 vial solutions so you can carry out the chromatograms in triplicate or quartet. For your soda sample, prepare three or 4 vial solutions so you can carry out the chromatograms in triplicate or quartet. Components in Diet Soft Drinks The ingredient list for most diet soft-drinks includes caffeine, benzoic acid, and aspartame (utrasweet ). The structures of these compounds are shown below along with their UV-Vis spectra.

5 CH 3 H 3 C CH nm Caffeine C 8 H MW = pk a = 10.4 H nm Benzoic Acid C 7 H 6 2 MW = pk a = 4.2 H 2 H H H nm Aspartame (L-Aspartyl-L-phenylalanine methyl ester) C 14 H MW =

6 Instrument Setup and Analysis. otes on Use of UHPLC: Your instructor or the lab tech will show you how to use the equipment and the software. Ask the LabTech is to be used for this experiment. See Figure 2 Sequence of Analysis: 1. Make sure that the recommended chromatography method (instrument parameters) is optimized for separation of caffeine, benzoic acid and aspartame. Using this experimental method for all the chromatogram runs in this experiment. The first analysis will be that of the pure components, caffeine, benzoic acid and aspartame, separately. Determine the retention time of each component. 2. The next series of chromatograms are for the standard samples (1-5) containing caffeine, benzoic acid and aspartame, also known as the standard mix. Uracil has been added to the standard mixture to provide a dead time marker (i.e. uracil is un-retained). Use the UV-Vis spectra of the components shown above to choose appropriate wavelength(s) to monitor the chromatograms. Use the chromatogram from the standard mix to calculate the retention factors for each component and verify the retention time with that of the chromatograms in step 1 above. Calculate the resolution between each pair of adjacent peaks, and the values for H and for caffeine (only) under the conditions used. The instrument will generate a calibration curve for caffeine, benzoic acid and aspartame to determine the concentration of these compounds for your unknown. You many have to determine W 1/2 by measuring the base of the signal with a ruler. 3. ext run the chromatography of your unknown, in triplicate (or quartet). The unknown contains, aspartame, benzoic acid and caffeine. Determine concentration caffeine and one other components in your unknown. (If Aspartame is not detected, then this is the component not to analyze.) 4. ext run the four diet cola samples, labeled A, B, C & D. These will be in vials and will be provided to you. You will be assigned two cola samples. Carry out the chromatography, in triplicate (or quartet) of these two unknown samples and identify the cola for your unknown. 5. ptional: Calculate the concentration of caffeine in these two unknown sample and compare it to the cola standards. (10% bonus) Talking points: How would the choice of monitored wavelength affects the sensitivity of the caffeine analysis. Would you choose the same wavelength if you are to quantify benzoic acid and aspartame in the presence of caffeine? If significant zone broadening had resulted in unsatisfactory resolution in the chromatograms, what recommendations would improve the resolution? Comment on the feasibility of your suggestion. Show sample calculations and attach all relevant chromatograms and spreadsheet printouts. Reference: Principles of Instrumental Analysis, 7 th Edition, Douglas Skoog, F. James Holler, Timothy ieman, Saunders College Publishing, Philadelphia, The Analysis of Artificial Sweeteners and Additives in Beverages by HPLC, Journal of Chemical Education, vol. 68(8), August 1991, p A195-A200.

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