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 dif-
ficult until the development of ion chromatography (IC)
(1). IC is an HPLC version of ion exchange that has be-
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come the method of choice for routine anion analysis (2).
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In IC, ions are usually detected by measuring the con-
ductivity of eluate (3). The two techniques for conduc-
tivity detection are “single column” or “nonsuppressed”
ion chromatography (NSIC) and “suppressed” ion chro-
matography (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 conduc-
tivity detector, any standard HPLC system can perform
NSIC. Moreover, NSIC presents several analytical ad-
vantages: calibration from borate–gluconate is more lin-
ear 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 per-
formed only by NSIC (5).
Because of the importance of IC, we felt a need for
developing a series of IC experiments that could be in-
tegrated 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 phosphovanadomolyb-
date 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® , Pepsi-
Cola® , 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 pre-
column. Samples are injected by using a Rheodyne-type
injector with a 100-µL loop, and detected using a Wa-
ters 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 Journal of Chemical Education • Vol. 73 No. 12 December 1996
The concentrate solution A is prepared by dissolv-
ing 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 n-
butanol, 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 con-
ductivity of the eluent should be about 180 µS.
Reagents
Potassium dihydrogen phosphate, boric acid, sodium
gluconate, sodium tetraborate decahydrate, glycerin, n-
butanol, and acetonitrile must be of analytical grade or
better. The use of Milli-Q treated water is strongly rec-
ommended.
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 phos-
phate are prepared by dilution.
About 100 mL of cola is boiled for 20 min in a bea-
ker 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 pre-
vent retention of neutral organics via hydrophobic in-
teractions with the column packing (11). Each solution
is filtered through a 0.45-µm filter unit before injection.
Figures 1 and 2 depict chromatograms correspond-
ing 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 co-
las. After the session, students calculate the concentra-
tion of phosphate in the cola samples and also its rela-
tive 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
 In the Laboratory

same cola brands.

Table 1. Phosphate Contents of the Analyzed
In our case (10 students, 2 instructors, and one ses-
Cola Beverages
sion of 3 hours), the repeatability RSD ranged from 0.5
to 1.5%. We calculate the reproducibility RSD to lie Phosphatea Reproducibility
Cola brand
within 2.8–5.6%. Table 1 shows the average concentra- (mg/L) (% RSD)
tion and the reproducibility of phosphate found in sev- Coca-Cola 530 2.8
eral cola beverages by the students in one session. Based Coca-Cola Light 500 4.0
on the reproducibilities, the mean values were appro- Pepsi-Cola 540 2.7
priately rounded.
As a first insight, a relative difference among brands Pepsi Diet 480 4.2
is detectable. The application of one-way ANOVA to the Crystal Pepsi 450 5.6
collected results (at least 4 data for each analyzed cola Schweppes Black Cola 570 3.5
brand) confirmed that the brands differ significantly.
Moreover, from the estimate of the within-sample vari- Tab 440 3.4
ance, the least significant difference (12) among means a
Expressed as orthophosphoric acid.
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, Literature Cited
and Coca-Cola/Pepsi-Cola do not differ significantly in
the content of phosphate. 1. Walton, H. F.; Rockling, R. D. Ion Exchange in Analytical Chemistry; CRC:
Boston, 1990; p 59.
The pH of degassed undiluted colas ranged between 2. Harris, D. C. Quantitative Chemical Analysis; W. H. Freeman: New York,
2.4 and 2.5, in good agreement with the observations of 1995; p 706.
3. Small, H.; Stevens, T. S.; Bauman, W. C. Anal. Chem. 1975, 47, 1801–1809.
Murphy (10). Using the first ionization constant for phos- 4. Schmuckler, G.; Jagoe, A. L.; Girard, J. E.; Buell, P. E. J. Chromatogr. 1986,
phoric acid, Ka = 7.5 × 10{3, the total concentration of 356, 413–419.
5. Okada, T.; Kuwamoto, T. Anal. Chem. 1985, 57, 829–833.
phosphoric acid is calculated. The results range within 6. Williams, W. J. Handbook of Anion Determination; Butterworths: London,
6.09 × 10 {3 and 4.50 × 10{3 M, which correspond to 597– 1979; pp 435, 487.
441 mg/L. This suggests that phosphoric acid is the 7. Ryder, D. S. J. Chromatogr. 1986, 354, 438–441.
8. Schmuckler, G.; Brenman, L. LC-GC Int. 1992, 5, 36–38.
strongest acid present in cola, and accordingly the sul- 9. Tanaka, T.; Hiiro, K.; Kawahara, A.; Wakida, S. Bunseki Kagaku 1983, 32,
fate levels found in the analysis could be attributed to 771–773.
10. Murphy, J. J. Chem. Educ. 1983, 60, 420–421.
the water used in the cola formulation. 11. Water Ion Chromatography Cookbook; Manual no. 20195; Millipore Corpo-
ration, Water Chromatography Division: Mildford, 1989.
12. Miller, J. C.; Miller, J. N. Statistics for Analytical Chemistry; Ellis Horwood:
Chichester, 1993; pp 66–69.

Figure 1. Ion chromatogram for 1:50 diluted Coca-Cola ® (I, phos- Figure 2. Ion chromatogram for 1:50 diluted Pepsi-Cola ® (I, phos-
phate; II, sulfate). phate; II, sulfate).

Vol. 73 No. 12 December 1996 • Journal of Chemical Education 1175