Saccharin, HPLC analysis

Di Pietra, A. M., V. Cavrini, D. Bonazzi, and L. Benfenati. 1990. HPLC analysis of aspartame and saccharin in pharmaceutical and dietary formulations. Chromatographia 30 215-218. 

For the analysis and separation of benzoic acid, caffeine, aspartame, and saccharin in dietetic soft drinks, a HPLC system consisting of a Varian MCH-5N-CAP 150 x 4.6 mm column and a variable wave length UV/VIS detector was recommended [32]. The mobile phase is a gradient, beginning with 90% 0.01 M KH2PO4 (pH = 2) and methanol, and ending in 25 minutes with 60 % buffer / 40 % methanol. 

Sample preparation for saccharin analysis by HPLC can be performed as indicated in Section I.C. Methods for extraction have also been described for desserts and sweets containing food thickeners (38) soy sauce, orange juice, and yogurt (60) chewing gum (17,39), and different food samples (42). Aminobenzoic acid, theophyllin, sodium fumarate, and adenine sulfate have been used as internal standards (17,31,39,44). 

A Hannisdal. Analysis of acesulfame-K, saccharin and preservatives in beverages and jams by HPLC. Z Lebensm Unters Forsch 194(6) 517-519, 1992. 

Veerabhadrarao et al. (76) used reverse-phase HPLC for the determination of some food additives (acesulfame, saccharine, BA, p-hydroxybenzoic acid). The samples (beverages, tomato sauce) were diluted and then separated on a /rBondapak CJg column with methanol/acetic acid/water (20 5 75) or (35 5 60) as mobile phases. The determination was done at 254 nm. Recoveries varied from 98 to 106% for direct analysis and from 91.6 to 101.8% for extraction of samples (76). 

Synergism occurs with fructose (Hyvonen el al., 1978), aspartame, cycla-mate (Bakal, 1987) and sucralose (Tate Lyle Pic, 1986). Negative synergy (i.e. suppression) occurs with acesulfame K blends. Analysis of saccharin is usually done using HPLC (Halm Gilikson, 1987) or spectrophotometric methods (Ramappa Nayak, 1983). 

Three spectrophotometric procedures are given in the AOAC compendium of methods (960.22, 962.13 and 967.11) for the analysis of caffeine, all of which have an extraction stage followed by a quantification procedure. There is also an HPLC method, discussed earlier, which was designed to measure saccharin, benzoic acid and caffeine at the same time (AOAC, 978.08). Again, the HPLC method, EN 12856 1999 (Anon, 1999a), can be used for the analysis of caffeine, but this analyte was not included in the collaborative study. 

While RPLC and RPIPC techniques applied with UV detection have already been elaborated for analyzing saccharin and acesulfam-K [67,68], the liquid chromatographic analysis of sodium cyclamate is barely substantiated in the literature because of the non-chromophoric structure of this compound. The only exception is the HPLC method introduced by Hermann et al. with direct photometric detection [69],... 

When you examine the chart obtained from the analysis, you may find that the peak corresponding to aspartame appears to be rather small. The peak is small because aspartame absorbs ultraviolet radiation most efficiently at 220 nm, whereas the detector is set to measure the absorption of light at 254 nm. Nevertheless, the observed retention time of aspartame will not depend upon the setting of the detector, and therefore the interpretation of the results should not be affected. The expected order of elution is saccharine (first), caffeine, aspartame, and benzoic acid. Another interesting point is that although the caffeine peak appears to be quite large in this analysis, it is nevertheless quite small when compared with the peak that would be obtained if you injected coffee into the HPLC. For a caffeine peak from coffee to fit onto your graph, you would have to dilute the coffee at least 10-fold. Even decaffeinated coffee usually has more caffeine in it than most sodas (decaffeinated coffee is required to be only 95-96% decaffeinated). 

Sweeteners used in the food industry typically are limited to the bulk sweeteners, sucrose, fructose, glucose, and com syrups, or the high potency sweeteners, saccharin, aspartame, sucralose, and acesulfame k. While various enzymatic and colorimetric methods may be used, high performance liquid chromatography is the most commonly used technique in this analysis [101]. HPLC offers speed, sensitivity, accuracy, and precision to the analyst. Several types of HPLC columns (anion and cation... 

HPLC has also found substantial application in the analysis of the high potency sweeteners. In one methodology, equilibrium dialysis of a food against water (15 h, 12,000-14,000 dalton cutofO was used to eliminate interference from lipids, carbohydrates, and proteins. Direct chromatography of the dialysate on a reverse phase column (UV detection 220 r m) gave very good results for acesulfame-k, saccharin, and aspartame. 

Kritsunankul et al. [76] proposed flow injection online dialysis for sample pretreatment prior to the simultaneous determination of some food additives by HPLC and UV detection (FID-HPLC). For this, a liquid sample or mixed standard solution (900 pL) was injected into a donor stream (5%, w/v, sucrose) of a FID system and was pushed further through a dialysis cell, while an acceptor solution (0.025 mol/L phosphate buffer, pH 3.75) was held on the opposite side of the dialysis membrane. The dialysate was then flowed to an injection loop of the HPLC valve, where it was further injected into the HPLC system and analyzed under isocratic reversed-phase HPLC conditions and UV detection (230 nm) (Figure 24.6). The order of elution of five food additives was acesulfame-K, saccharin, caffeine, benzoic acid, and sorbic acid, with an analysis time of 14 min. This system has advantages of high degrees of automation for sample pretreatment, that is, online sample separation and dilution and low consumption of chemicals and materials. 

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