Benzoic acids HPLC separation

In this experiment a theoretical model is used to optimize the HPLC separation of substituted benzoic acids by adjusting the pH of the mobile phase. An empirical model is then used... 

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. 

In reversed-phase HPLC separation of polyphenolics on the basis of polarity, the elution order of polyphenolics may be predicted. The more-polar polyphenolics are generally eluted first under reversed-phase conditions. Glycosylation in flavonoids increases their polarity and therefore their mobility in the re-versed-phase system. The elution order of benzoic acids, hydroxycinnamic acids, and agly-cones of flavonoids can normally be determined on the basis of the number of polar hydroxyl groups and lipophilic methoxyl groups. For additional information about elution order for various classes of polyphenolics, see Background Information. 

This part of the chapter describes HPLC techniques for the separation and determination of preservatives, as follows S02, postharvest preservatives (PPs), benzoic acid (BA), sorbic acid (SA), and the ethyl, methyl, propyl esters of 4-hydroxybenzoic acid (EsHBA) and 5-nitrofury-lacrylic acid (5-NFA). The propionic, lactic, acetic acids are discussed in the chapter on organic acids. 

Flak and Schaber (5,62) used reverse-phase HPLC for the quantitative and simultaneous determination of benzoic acid and sorbic acid, as well as 4-hydroxybenzoic acid, salicylic, 5-nitrofurylacrylic, and p-chlorobenzoic acid and the EsHBA (methyl, ethyl, propyl) in wines and beverages. The first five compounds can be determined by isocratic elution from a Clg column using 0.12 M acetate, pH 3.8 acetonitrile (85 15), and all may be separated with gradient elution (increasing acetonitrile from 10 to 60%, with a simultaneous decrease of the pH of the acetate buffer from 3.9 initially to 3.3). 

Mannio and Cosio (78) described a sensitive, specific, and rapid chromatographic procedure to determine BA and SA in different food products. Benzoic acid and SA were extracted from foods by a microdialysis probe connected online to an HPLC column that allows separation of BA and SA. Detection was done at 228 and 260 nm for BA and SA, respectively. The procedure was linear from 1 to 80 ppm, with a detection limit of 1 ppm for SA and 2 ppm for BA. The assay was successfully applied to soft drinks, fruit juices, and dairy products (cheese, yogurt, and cream). 

There are a large number of other published procedures for the separation of a number of sweeteners and preservatives at one time these are all based on reverse-phase HPLC. Perhaps one of the most startling is the method published by Williams (1986). This uses a small particle size (3 xm) C8 column and allows the separation of a range of colours, sweeteners and preservatives in less than 5 min. The materials separated were amaranth, quinoline yellow, quinine sulphate, sunset yellow, caffeine, aspartame, saccharin, vanillin, sorbic acid, benzoic acid and green S. 

One of the older methods used to detect the presence of preservatives in soft drinks and juices is thin-layer chromatography (Woidich el al., 1967). This provides a useful method to detect benzoic and sorbic acids as well as the substituted benzoic acids. The first stage involves the extraction of the preservatives with diethyl ether prior to their chromatographic separation on polyamide plates. Although it is difficult to use this procedure to quantify the level of these preservatives in a sample, it is not impossible. This approach can still be used today by a laboratory that does not have access to HPLC. 

Djakovic-Sekulic et al. " separated eight anilides of 2,2-dimethylpropanoic acid, nine anilides of benzoic acid, and nine anilides of ot-phenyl acetic acid by RP-18 HPLC using mixtures containing methanol and water in volume proportions 6h-4, 6.5h-3.5, and 7-1-3 as the mobile phases. The log k values of investigated anilides have been correlated with the dipole moments ( raph) or permittivities (Emph) of the mobile phases applied, with numerical values of one topological index from among those based on the distance matrix R, W, A, B, B) or on the adjacency matrix (M, M, x with... 

The most common analytical method for detection of benzoic acid or sorbic acid has been reversed-phase HPLC (Saad et al., 2005). Organic acids in wine vinegars have also been detected by reversed-phase HPLC (RP-HPLC) using two C18 columns, UV detection at 210 nm, and sample filtration through Sep-Pack C18. However, complete separation of organic acids has not been achieved with this procedure (Morales, Gonzalez, and Troncoso, 1998). 

Data obtained from Joseph, Seema M. and Palasota, John A. 2001. The combined effects of pH and percent methanol on the HPLC separation of benzoic acid and phenol. Journal of Chemical Education 78 1381. 

Some new phenacyl labels [104] for carboxylic acids have been synthesized recently, and l-(4-hydroxy-phenyl)-2-bromoethanone (4-HBE) was found to be a useful UV derivatizing agent. 4-HBE reacted with aliphatic and aromatic adds. A 4 2 1 molar ratio of 4-HBE, triethylamine and organic add was used. The reaction was carried out at 80 °C for 1 h with 76% conversion. HPLC separation was carried out on an Ultrasphere RP-18 column with methanol/water (60 40 v/v) as eluent. Detection was at 289 nm, which avoided most of the interference found at lower wavelengths. The detection limit was 1 ng for the derivative of benzoic acid. 

Polymers developed specifically for HPLC include the macroporous crosslinked vinylpyridinium type, which has proved very effective in HILIC of sugars in aqueous acetonitrile. These polymers can be used at column temperatures up to 70°C, under which conditions, with the resin in the phosphate or sulfate form, baseline resolution of mixtures of the common monosaccharides and some disaccharides, such as maltose and lactose, can be achieved. Stationary phases in which a polymer replaces silica as the support for the aminopropyl bonded phase much used in HILIC of carbohydrates have also been successfully applied in separations of mono- and disaccharides in this case precolumn derivatization with 4-amino-benzoic acid ethyl ether (ABEE) has been recommended to overcome the problem of glycosylamine formation that occurs with imderivatized sugars. 

Radioiododebromination f ljiodide is evaporated under vacuum in a 1 ml glass ampoule made from a Pasteur Pipette. 10 pi (5 mM) of the bromo precursor and 40 pi 0.2 M benzoic acid are added, both dissolved in EtOH. The solution is shaken in order to dissolve the 1 and evaporated slowly ( ) until the precipitating solid appears dry. The ampoule is sealed under vacuum with a gas torch and heated at 200-230°C for 10 min. Then the cold ampoule is opened and the reaction mixture dissolved in 60-pl ethanol. For separation, this solution is injected completely in an analytical RP18-HPLC column. Seventy percent RCY is obtained for [ IjPHIPA 3-10. 

To illustrate the hyphenation between FIA and HPLC, a system for food additives (acesulfame-k, saccharin, caffeine, benzoic acid and sorbic acid) determination [40] is shown in Figure 3.14. The system includes a dialysis cell in order to eliminate the sample matrix. Thus, a peristaltic pump dispenses the sample, which is loaded into a holding coil of an injection valve (IVl) and later injected into the donor stream. Both, donor and acceptor streams are also propelled by the peristaltic pump. Once the dialysis has been performed, analytes are loaded into a holding coil of a second injection valve (IV2). At this point, the sample passes to the HPLC system. The sample is injected into a mobile phase stream dispensed by the HPLC pump. So, the pretreated sample passes through the precolumn and column, and the analytes are separated and detected. Thus, the treatment, separation and detection steps are carried out in an efficient and high automated way. 

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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