An HPLC Analysis of Sweeteners in Beverages

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1 CHEM 311L Quantitative Analysis Laboratory Version 1.1 An HPLC Analysis of Sweeteners in Beverages In this laboratory exercise we will perform a separation of the components of diet soft drinks using reversed-phase High Performance Liquid Chromatography (HPLC). This will allow us to confirm the presence of Caffeine, Benzoic Acid and the sweeteners Aspartame, Saccharine, and Acesulfame K in the soft drink. Once separated and identified, the amount of each sweetener present in the soft drink will be determined. HPLC is one of the most popular and widely used chromatographic techniques today. This method uses a solid or bonded stationary phase and a liquid mobile phase to separate mixture components in time. The method yields information about the identity of mixture components (qualitative information) and their concentrations (quantitative information). HPLC uses high pressure to force solvent through closed columns containing very fine particles that give high-resolution separations. The HPLC system... consists of a solvent delivery system, a sample injection valve, a high-pressure column, a detector, and a computer to control the system and display results. (Quantitative Chemical Analysis, 7 th Ed. by Daniel C. Harris; Freeman) A column packed with very small particles increases chromatographic efficiency dramatically. This is because diffusion in liquids is very slow, so small particles provide a large surface area for equilibration of the analyte between the mobile and stationary phases of the chromatographic system. However, high pressures are then needed to provide a reasonable flow rate through the column.

2 P a g e 2 A large number of possible stationary phase/mobile phase combinations exist for HPLC, each defining a specific mode of HPLC. For example, adsorption mode HPLC uses a polar, solid stationary phase such as SiO 2 or Al 2 O 3 and a nonpolar mobile phase, such as hexane or chloroform to separate compounds by their polarity. Other modes, such as ion-exchange chromatography and size exclusion chromatography separate compounds by other mechanisms. One of the most popular modes of HPLC is reversed phase, which is a type of partition chromatography. In this method, stationary phase particles (usually SiO 2 ) are coated with a chemically-bonded layer of some type of non-polar molecule. Commonly used bonded layers include 18 and 8 carbon-long straight chain alkanes (C 18 and C 8 ) and phenyl groups. The mobile phase is typically water mixed with some fraction of miscible, polar organic solvent (usually Methanol, Acetonitrile, or THF). During the separation, analyte molecules partition between the mobile phase and the bonded layer of stationary phase. Since relatively non-polar molecules will dissolve more easily into the stationary phase, they will elute last. Relatively polar compounds will not interact as strongly with the stationary phase and will therefore elute first. This mode works well for the separation of Water-soluble, non-volatile organic compounds. Since these compounds are not usually amenable to analysis by gas chromatography (GC), reversed phase HPLC is an excellent compliment to that method. When reversed-phase HPLC is used with ionized organic compounds, the ionization of the these analytes must be limited or minimized. Otherwise, these compounds are simply too polar to be retained by the non-polar stationary phase. In the case that the compound is a weak acid, the ph of the mobile phase is controlled with a buffer. Ion-pairing reagents can also be used to partially neutralize the charge of the analytes and allow them to be retained. In this experiment, the ph of the mobile phase will be buffered at ph = 4.2 using Acetic Acid to allow two of the compounds that are weak acids, Benzoic Acid and Aspartame, to be adequately retained and separated. Soft drinks frequently contain a number of additives that affect the beverage s taste and characteristics. Caffeine, one popular ingredient, is a natural xanthine alkaloid stimulant that exists in many plants as a natural insecticide, including kola nuts, coffee beans, cacao beans, and tea leaves. In some cases, it s extracted from the natural ingredients as part of the beveragemaking process (e.g. in coffee and tea and in cola beverages). In other cases, it s added by the manufacturer (e.g. in some citrus-flavored sodas). Often, even if Caffeine is naturally present, it is elevated or controlled at a consistent level by the manufacturer (e.g. in many colas). Benzoic Acid is also added to many soft drinks and other foods as a preservative against microbial growth. Diet soft drinks often contain low calorie artificial (man-made) sweeteners in place of sugar or corn syrup. We will analyze for Ceffiene, Benzoic Acid and three of these sweeteners, Aspartame, Saccharine, and Acesulfame K in a soft drifnk. The table below gives information on all five of these compounds.

3 P a g e 3 Common names IUPAC name M.W. Structure caffeine 1,3,7-trimethyl-1H-purine ,6(3H,7H)-dione benzoic acid benzoic acid aspartame (NutraSweet ) (Equal ) N-(L-α-Aspartyl)-Lphenylalanine methyl ester saccharine (Sweet'N Low ) 1,1-Dioxo-1,2-benzothiazol-3-one acesulfame potassium (Ace K) potassium 6-methyl-2,2-dioxooxathiazin-4-olate Finally, as the analytes exit the column, they must be detected by an appropriate detection system. In this experiment, a single channel UV-Visible absorbance detector will be used. The detector will be set to 254 nm, a wavelength where aromatic rings absorb light strongly, making the detection of our five compounds possible. We will use an Agilent Technologies 1200 HPLC system for our separation and quantification. This system packages together, in swappable modules, all the elements of an HPLC system discussed above. It is operated under the control of an appropriate software package supplied by the manufacturer. An autosampler module allows for processing of large numbers of samples without the need for an operator. For this experiment we will use an 80/20 Water/Methanol

4 P a g e 4 mobile phase buffered at ph = 4.2 with Acetic Acid; all being HPLC grade solvents. Flow rates and sample injection volumes will be established by your always helpful laboratory instructor. Agilent Technologies HPLC System Thus, we will separate each of our five analytes in a soft drink using a reversed-phase HPLC methodology. Each component will be detected using a UV-VIS absobance measurement. The signal response of the UV-VIS detector will allow us to determine the amount of each analyte present, when calibrated against appropriate standards.

5 P a g e 5 Pre-Lab Calculation Suppose your analysis for the Saccharine standards yields the following data: conc (M) 1.00E E E E-04 peak area 1.41E E E E+05 What is the concentration of the Saccharine in your soft drink if your analysis gives a chromatographic peak area of 3.66 x 10 5?

6 P a g e 6 Procedure 1. Due to the limited time available for this lab, you ll be provided with a solution containing a mixture of all five compounds at known concentrations. Additionally, you ll be provided with the retention times of the pure compounds and a set of calibration data (peak area vs. concentration for each compound), both determined earlier by the TA. 2. The TA and/or instructor will set-up the HPLC and start the mobile phase flow before class. Approximately 30 minutes of run time is needed to establish a stable system baseline, prior to any injections. 3. Obtain 4-6 diet beverages. Note the presence and absence of any of the compounds in Table 1 in the list of ingredients on the beverage container. 4. Bring the beverages to room temperature and (if carbonated) degas them by letting them stand for a few hours or by sonicating them. Filter ~10 ml of each using a syringe filter and syringe into a clean, labeled vial. 5. Inject each of the filtered and degassed diet drink samples and the five standard mixture into the HPLC one at a time. 6. Using the HPLC software, determine the retention times of each compound. Also, integrate each chromatogram to determine the peak areas of each compound in each standard or sample. 7. For each analyte, conduct a linear least squares analysis on the concentration and peak area data for the standard mixtures. Give the slope, y-intercept, and associated standard deviations for each analyte. (Attach a copy of a graph of the data to your assay sheet.) 8. Using these results, determine and report the concentrations of each of the five compounds in the beverage you analyzed. An error estimate for each concentration should also be provided.

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