HPTLC Determination of Organic Acid Preservatives in Beverages

In a high performance TLC (HPTLC) method sorbic acid (2,4-hexadienoic acid) and benzoic acid were determined without preliminary extraction or cleanup by the chromatography of aliquots of samples and of standards on preadsorbent silica gel or C18-bonded silica gel plates containing fluorescent indicator.

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Figure 2 RF values of aromatic carboxylic acids. 1, Benzoic; 2, 2-chloro; 3, 3-chloro; 4, 4-chloro; 5, 2-hydroxy; 6, 3-hydroxy; 7, 4-hydroxy; 8, 2-nitro; 9, 3-nitro; 10, 4-nitro; 11, 2-amino; 12, 4-aminobenzoic acid in benzene containing formic acid and diethylene glycol monoethyl ether. (Reproduced with premission from Guinchard etal., 1976.)

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Figure 2 RF values of aromatic carboxylic acids. 1, Benzoic; 2, 2-chloro; 3, 3-chloro; 4, 4-chloro; 5, 2-hydroxy; 6, 3-hydroxy; 7, 4-hydroxy; 8, 2-nitro; 9, 3-nitro; 10, 4-nitro; 11, 2-amino; 12, 4-aminobenzoic acid in benzene containing formic acid and diethylene glycol monoethyl ether. (Reproduced with premission from Guinchard etal., 1976.)

The zones which quenched fluorescence upon UV irradiation at 254 nm were compared by scanning densitometry. This procedure was preferred to measurement of densitometry based on UV absorption.

Preadsorbent high-performance LHPKDF silica gel (Whatman) plates (20 x 10 mm) with 19 lanes were used for normal-phase experiments with the solvent «-pentyl formate-chloroform-formic acid (2 : 7:1) in which the hRF values for sorbic acid and benzoic acid were 61 and 58. For reversed-phase TLC on (Whatman) C18 LKC18F plates (20 x 20 mm) with meth-anol-0.5 mol L_1 sodium chloride (1:1), the respective hRF values for these two acids were 44 and 59. It was found necessary to apply a stream of warm air during spotting of samples with a 10 |L Drummond digital microdispenser and, after this stage, to dry the plates. Development was then effected in a Camag twin-trough chamber to 7 cm beyond the sorbent-preadsorbent interface with normal-phase plates and to 10 cm for C18 plates. The plates were then dried and the areas of the dark quenched zones against a fluorescent background were scanned at the predetermined maximum absorption (between 200 and 370 nm) with a Shimadzu Model 930 densitometer operated in the reflectance mode. From the chromatography of 0.50, 1.00, 2.00, 4.00, 6.00 and 8.00 |L of standards for sorbic and benzoic acids containing 125-2000 ng and 1.00-16.0 |g respectively, linear calibration plots of scan area/weight were obtained. For quantification, the sample scan area was compared with that of a closely matching standard within the linear calibration range and the corresponding weight found. Recovery analyses were carried out with beverage samples spiked with sorbic and benzoic acids, which were compared with the corresponding unfortified samples. They averaged at 98.0% for all analyses.

By the HPTLC method, sorbic and benzoic acids present separately in a variety of beverages have been directly quantified. The analysis of standards on the same TLC plate eliminates the requirement for an internal standard, as in high performance liquid chromatography (HPLC). By contrast with the HPTLC and HPLC methods, spectrophotometric

Figure 3 RF values of 2-substituted benzoic acids in different solvents. The solvent composition, organic component-water (40:60 v/v) with addition of 0.1 mol L~1 tetramethylammonium bromide (pK value in parantheses): open circles, benzoic acid (4.19); filled triangles, 2-hydroxy (2.97); open squares, 2-acetoxy (3.5); filled squares, 2-carboxy (2.91/5.59); filled circles, 2-nitro (2.16); open squares, 2-methyl (3.91); open triangles, 2-amino (6.97); filled/inverted triangles, 2-chloro (2.92). (Reproduced with permission from Jost etal., 1984.)

Figure 3 RF values of 2-substituted benzoic acids in different solvents. The solvent composition, organic component-water (40:60 v/v) with addition of 0.1 mol L~1 tetramethylammonium bromide (pK value in parantheses): open circles, benzoic acid (4.19); filled triangles, 2-hydroxy (2.97); open squares, 2-acetoxy (3.5); filled squares, 2-carboxy (2.91/5.59); filled circles, 2-nitro (2.16); open squares, 2-methyl (3.91); open triangles, 2-amino (6.97); filled/inverted triangles, 2-chloro (2.92). (Reproduced with permission from Jost etal., 1984.)

analysis requires a preliminary sample preparation by steam distillation. However, very low concentrations of benzoic acid are more amenable to HPLC analysis and when sorbic and benzoic acids are present together the method is less satisfactory due to sample streaking, even on a C18-bonded silica gel layer (particularly at higher loads).

In view of these limitations, a modified method was adopted, involving solid-phase extraction (SPE) on a C18 cartridge followed by the preceding quantification method established on preadsorbent C18 plates.

The extraction procedure was validated by spiking commercial samples with known amounts of the acids in turn and demonstrating the satisfactory recovery of each. With this total method, sample interferences were eliminated and samples too low for analysis by direct spotting could be analysed. The whole TLC methodology is considered to be applicable to a wide range of solid and syrupy-type samples containing either or both of the two preservatives at concentrations as low as those measurable by HPLC.

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