Saccharinic acids chromatography

In the carbohydrate series, the acids encountered are aldonic, aldaric, uronic, or saccharinic acids. Often, these acids are readily transformed into their lactones or methyl esters, and it is as derivatives of these that they are commonly studied. Table VIII (see p. 136) records examples in which derivatives of such acids and lactones have been subjected to gas-liquid chromatography. 

Ishizu et al.29H found that D-xylose and D-fructose react with aqueous calcium hydroxide to produce 13 lactonizable saccharinic and other acids. These were identified after separation by cellulose column and gas-liquid chromatography, and the Cs-saccharinic acids, 2-C-methyl-D-threonic acid (117) and 2-C-methyl-D-erythronic acid (118), were among those isolated. These authors299 later reported that L-sorbose reacts similarly, to generate 14 lactones, including the 2-C-methyl-L-xr/o o-l,4-lactone and 2-C-methyl-L-lyxono-1,4-lactone, which were also prepared from 1-deoxy-L-threo-pentulose via the cyanohydrin reaction. 

As would be expected from the above considerations, substitution at C6 of a hexose has, at most, only a minor effect on the course of saccha-rinic acid formation. Thus, 6-0-(a-D-galactosyl)-D-glucose (melibiose) plus lime-water gives lactic acid and a mixture of the corresponding metasac-charinic, isosaccharinic, and saccharinic acids.The qualitative experiments with melibiose and with 6-0-methyl-D-glucose, based mainly on paper chromatography, suggest that the metasaccharinic acids may be the principal products from hexoses substituted at C6. 

Reverse-phase chromatography has been used extensively for the determination of saccharin. Smyly et al. (30) and Eng et al. (39) used /rBondapak Cl 8 and 5% acetic acid for the determination of saccharin. Based on this work, an Association of Official Analytical Chemists (AOAC) collaborative study was conducted, and the method using a mobile phase buffered to pH 3 with sodium acetate and modified with 3% isopropanol was adopted. Webb and Beckman (61) used this method successfully for the separation of saccharin from aspartame, caffeine, sodium benzoate, and artificial colors and flavors. Veerabhadrarao et al. (27) added methanol to the mobile phase (methanol acetic acid water, 4 1 1, v/v) for improved separation of saccharin from caffeine, benzoic and p-hydroxybenzoic acids, vanillin, aspartame, acesulfame-K, and dulcin. Saccharin was also determined using LiChrosorb Cl8 and 4 6 v/v methanol phosphate buffer,... 

High-performance ion chromatography was used by Biemer (17) to separate acesulfame-K from saccharin and cyclamate. A Dionex AS4A anion separator column and a mobile phase of 300 mg Na2C03/L were used for the separation of the sweeteners. The regenerate solution consisted of 25 mM sulfuric acid solution. Wu et al. (63) determined acesulfame-K on Dionex AS4A-SC and 0.5 mM Na2C03 mobile phase. 

Reverse-phase chromatography has been used for the determination of dulcin. Veerabhadrarao et al. (27) described a method for the separation of dulcin, acesulfame-K, and saccharin on pBondapak Cl8 using methanol acetic acid water (7 1 12, v/v). Prodolliet and Bruelhart (33) used the same column, but a mobile phase of 12.5 mM KH2P04 (pH 3.5) acetonitrile (9 1, v/v) for the separation of dulcin from saccharin, acesulfame-K, alitame, aspartame and its degradation products, other food additives, and natural constituents. Lawrence and Charbonneau (16) used a gradient of 0-100% mobile phase B (20 mM KH2P04, pH 3.5 acetonitrile, 8 2, v/v) in A (20 mM KH2P04, pH 5.0 acetonitrile, 97 3, v/v) on Supelcosil LC-18 for the separation of dulcin from saccharin, aspartame, acesulfame-K, cyclamate, sucralose, and alitame. 

Ion-pair chromatography has also been used for the determination of dulcin. Wu et al. (47) added the ion pair tetraethylammonium hydroxide to the mobile-phase methanol 85% phosphoric acid, pH 6 (2 8), for the separation of dulcin from saccharin, cyclamate, aspartame, and acesulfame-K on /xBondapak 08. Herrmann et al. (24) separated dulcin from saccharin, cyclamate, and aspartame on Hypersil MOS 3 using a mobile phase consisting of 5 mM tetrabutylam-... 

Fig. 1. Elution diagram of the chromatography on DEAE cellulose of urine from a rat which received [3 l C] saccharin at 50 mg/kg. Each fraction contained 10 ml. Peak I carbonate Peak II sulfamoylbenzoic acid Peak III saccharin.

Polarographic methods of analysis have been applied to samples of foods containing saccharin (1+1-1+1+ ). In a procedure (1+1+) saccharin is extracted into organic solvents in an acidic medium. Further purification is achieved by column chromatography. The residue obtained is dissolved in 0.1 N NaOH and an aliquot is polarographed in a supporting electrolyte of 0.1 N HC1, 0.1 N KC1 and 0.1 Bu N Br. 

Saccharin has been detected and estimated after extraction from food samples on Whatman No. 1 filter paper (61,62). The sample in solution form, such as carbonated water can be used for paper chromatography. The spotted paper is developed in BuOH-ACOH-HpO (k0 l0 22) for l8 hours and sprayed with a solution of phthalic acid and aniline for spot development. Saccharin with an Rp 0.17 can be estimated colorimetrically after elution of the spot with 60 AcOH (62). 

Gas chromatography Acesulfame-K, aspartame, cyclamate, saccharin, and stevioside are determined by gas chromatography, but the main drawback of this technique is that a derivatization is required. Acesulfame-K is methylated with ethereal diazomethane, aspartame is converted into its N- 2-methylpropoxycarbonyl) methyl ester derivative, menthol and isobutyl chloroformate are used to convert aspartame to 3-[(isobutoxycarbonyl)amino]-4-[[a-(methoxycarbonyl)phenethyl]amino]-4-oxobutyric acid, cyclamate is determined as cyclohexene resulting from the reaction with nitrite, saccharin is converted to N-methylsaccharin, and stevioside is hydrolyzed. Detection is carried out utilizing flame-ionization, flame-photometric electron-capture detectors or nitrogen-phosphorus detection. 

Figure 10.173 Determination of saccharin in a nickel/iron bath by ion-exclusion chromatography. Separator column Metrosep Organic Acids eluent 0.5 mmol/L H2S04/acetone (85 15 v/v) flow rate 0.5 mL/min detection ...

A FIA indirect method was described for saccharin determination by Yebra et al. [88]. This method was based on the precipitation of silver saccharinate in an acetic acid medium. The precipitate was dissolved in ammonia, and the dissolved silver was continuously measured by FA AS. The FIA precipitation-dissolution manifold used is similar to that used for cyclamate determination [83] however, in this work, a Scientific System 05-105 column fitted with a removable screen-type stainless-steel filter (pore size 0.5 pm, chamber inner volume 580 pL, filtration area 3 cm ), originally designed as a cleaning device for liquid chromatography, was employed for filtration purposes. 

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