In the Laboratory
Determination of Phosphate in Cola Beverages Using Nonsuppressed Ion Chromatography An Experiment Introducing Ion Chromatography for Quantitative Analysis M. A. Bello and A. Gustavo González Departamento de Química Analítica, Universidad de Sevilla, 41012 Sevilla, Spain The determination of inorganic anions was very difficult until the development of ion chromatography (IC) (1). IC is an HPLC version of ion exchange that has become the method of choice for routine anion analysis (2). In IC, ions are usually detected by measuring the conductivity of eluate (3). The two techniques for conductivity detection are “single column” or “nonsuppressed” ion chromatography (NSIC) and “suppressed” ion chromatography (SIC). Although SIC presents the advantage of a signal-to-noise ratio about one order of magnitude better than NSIC, NSIC is often selected because of its simple instrumentation. With the addition of a conductivity detector, any standard HPLC system can perform NSIC. Moreover, NSIC presents several analytical advantages: calibration from borate–gluconate is more linear than with eluents commonly used in SIC (4); and anion exchange and conductivity detection of very weak acids (e.g., borate, cyanide, sulfide, silicate) can be performed only by NSIC (5). Because of the importance of IC, we felt a need for developing a series of IC experiments that could be integrated into existing laboratory courses in analytical chemistry. In the present paper we report one of these experiments: The determination of phosphate in cola beverages by NSIC. Phosphate, when present at minor or trace levels, is currently determined spectrophotometrically, preferably as molybdenum blue or using the phosphovanadomolybdate method (6). These standard procedures are tiresome compared with recent procedures based in IC (7–9). Colas are actually relatively rich in phosphoric acid, having concentrations of about 5 × 10{3 M (10), and may be considered as very suitable laboratory samples for the experiments (Coca-Cola® , Coca-Cola Light® , PepsiCola® , Pepsi Diet® , Crystal Pepsi®, Schweppes Black Cola®, Tab®, etc.). Experimental Procedure
Apparatus A Waters 501 HPLC pump is used with a Waters IC-Pak A HR column and a Waters Guard Pak precolumn. Samples are injected by using a Rheodyne-type injector with a 100-µL loop, and detected using a Waters 341 conductivity detector. Peak evaluation is made with an Hewlett Packard HP3395 integrator. The detector sensitivity selected for experiments was 1 µS and the eluent flow rate was 1 mL/min. Eluent Preparation
1174
The concentrate solution A is prepared by dissolving 16 g of sodium gluconate, 18 g of boric acid, and 25 g of sodium tetraborate in a mixture 25:75 v/v of glycerin:water to 1 L. The working eluent is prepared by mixing 12 mL of concentrate solution A, 20 mL of nbutanol, 120 mL of acetonitrile, and water up to 1 L (pH 8.4). This eluent is degassed with a flow of helium and microfiltered (0.45 µm) before use. The background conductivity of the eluent should be about 180 µS.
Reagents Potassium dihydrogen phosphate, boric acid, sodium gluconate, sodium tetraborate decahydrate, glycerin, nbutanol, and acetonitrile must be of analytical grade or better. The use of Milli-Q treated water is strongly recommended. A standard stock solution of 1000 mg/L phosphate is prepared for calibration. Procedures From the standard phosphate stock solution, at least five standards covering the range 1–10 mg/L of phosphate are prepared by dilution. About 100 mL of cola is boiled for 20 min in a beaker covered by a watch glass and then sonicated for 10 min or treated with a flow of nitrogen to remove carbon dioxide. Samples are prepared for analysis by diluting the degassed cola 1:50 with water and passing it through a Waters C18 SEP-Pak Plus cartridge (short body) to prevent retention of neutral organics via hydrophobic interactions with the column packing (11). Each solution is filtered through a 0.45-µm filter unit before injection. Figures 1 and 2 depict chromatograms corresponding to Coca-Cola and Pepsi-Cola, respectively. Phosphate, which appears as peak I, elutes in approximately 11.30 min. Peak II corresponds to sulfate, whose presence will not be discussed here. Quantitation of phosphate in the cola samples is achieved through a calibration curve. Using the peak height as analytical signal, a calibration line covering the range of 1–10 mg/L phosphate proves suitable for all the colas studied. Discussion Each student should obtain at least 3 different colas. After the session, students calculate the concentration of phosphate in the cola samples and also its relative standard deviation (RSD) from replicate injections of the same cola brand (repeatability). From the students’ reports, the instructor may evaluate the reproducibility using the results obtained by different students for the
Journal of Chemical Education • Vol. 73 No. 12 December 1996
In the Laboratory
same cola brands. In our case (10 students, 2 instructors, and one session of 3 hours), the repeatability RSD ranged from 0.5 to 1.5%. We calculate the reproducibility RSD to lie within 2.8–5.6%. Table 1 shows the average concentration and the reproducibility of phosphate found in several cola beverages by the students in one session. Based on the reproducibilities, the mean values were appropriately rounded. As a first insight, a relative difference among brands is detectable. The application of one-way ANOVA to the collected results (at least 4 data for each analyzed cola brand) confirmed that the brands differ significantly. Moreover, from the estimate of the within-sample variance, the least significant difference (12) among means was calculated, giving 27. Comparing this value with the differences between the means one can see that only the couples Crystal Pepsi/Tab, Coca-Cola Light/Pepsi Diet, and Coca-Cola/Pepsi-Cola do not differ significantly in the content of phosphate. The pH of degassed undiluted colas ranged between 2.4 and 2.5, in good agreement with the observations of Murphy (10). Using the first ionization constant for phosphoric acid, Ka = 7.5 × 10{3, the total concentration of phosphoric acid is calculated. The results range within 6.09 × 10 {3 and 4.50 × 10{3 M, which correspond to 597– 441 mg/L. This suggests that phosphoric acid is the strongest acid present in cola, and accordingly the sulfate levels found in the analysis could be attributed to the water used in the cola formulation.
Figure 1. Ion chromatogram for 1:50 diluted Coca-Cola ® (I, phosphate; II, sulfate).
Table 1. Phosphate Contents of the Analyzed Cola Beverages Phosphatea Reproducibility Cola brand (mg/L) (% RSD) Coca-Cola
530
2.8
Coca-Cola Light
500
4.0
Pepsi-Cola
540
2.7
Pepsi Diet
480
4.2
Crystal
Pepsi
450
5.6
Schweppes Black Cola
570
3.5
Tab
440
3.4
a
Expressed as orthophosphoric acid.
Literature Cited 1. Walton, H. F.; Rockling, R. D. Ion Exchange in Analytical Chemistry; CRC: Boston, 1990; p 59. 2. Harris, D. C. Quantitative Chemical Analysis; W. H. Freeman: New York, 1995; p 706. 3. Small, H.; Stevens, T. S.; Bauman, W. C. Anal. Chem. 1975, 47, 1801–1809. 4. Schmuckler, G.; Jagoe, A. L.; Girard, J. E.; Buell, P. E. J. Chromatogr. 1986, 356, 413–419. 5. Okada, T.; Kuwamoto, T. Anal. Chem. 1985, 57, 829–833. 6. Williams, W. J. Handbook of Anion Determination; Butterworths: London, 1979; pp 435, 487. 7. Ryder, D. S. J. Chromatogr. 1986, 354, 438–441. 8. Schmuckler, G.; Brenman, L. LC-GC Int. 1992, 5, 36–38. 9. Tanaka, T.; Hiiro, K.; Kawahara, A.; Wakida, S. Bunseki Kagaku 1983, 32, 771–773. 10. Murphy, J. J. Chem. Educ. 1983, 60, 420–421. 11. Water Ion Chromatography Cookbook; Manual no. 20195; Millipore Corporation, Water Chromatography Division: Mildford, 1989. 12. Miller, J. C.; Miller, J. N. Statistics for Analytical Chemistry; Ellis Horwood: Chichester, 1993; pp 66–69.
Figure 2. Ion chromatogram for 1:50 diluted Pepsi-Cola ® (I, phosphate; II, sulfate).
Vol. 73 No. 12 December 1996 • Journal of Chemical Education
1175
