Reversed-phase HPLC carboxylic acids

For the final optimization, a modified factorial design involving three concentration levels of triethylamine and three pH levels was used. From these results, it was clear that the optimum conditions for the analysis of the carboxylic acid were so different from those required for the other compounds studied that it was not sensible to attempt to analyse all fonr together and indeed that carboxylic acids were better analysed by using conventional reversed-phase HPLC than by using ion-pairing. 

Synthetic dyes, because of their sulfonic and in some cases carboxylic acid functions, have short retention times in a reverse-phase HPLC system (168). Another problem encountered during reverse-phase HPLC is the tailing observed for compounds with sulfonic groups (216). Nevertheless, adequate pH and solvent composition have permitted the separation of some dyes in a reverse-phase system, as indicated on Table 7. 

Figure 3.20. Analysis of carboxylic acids and alcohols by reversed phase HPLC, with indirect UV detection, (a) Carboxylic acids. Chromatography conditions mobile phase, 3 X 10 4 M l-phenethyl-2-picolinium in acetate buffer (pH 4.6) column, ju-Bondapak phenyl detection, indirect UV absorbance at 254 nm. Peaks 1, acetic acid 2, propionic acid 3, butyric acid 4, valeric acid 5, caproic acid S, system peak, (b) Aliphatic alcohols. Chromatography conditions mobile phase, 4 x 10 4 M nicotinamide in water column. Ultrasphere ODS detection, indirect UV absorbance at 268 nm. Peaks 1, methanol 2, propylene glycol 3, ethanol 4, 2-propanol 5, 1-propanol 6, system peak 7, 2-butanol 8, 2-methyl-l-propanol 9, 1-butanol. (Redrawn from Refs. 23 and 24 with permission.)...

A reversed-phase HPLC post-column ion-pair extraction system was developed by Kim and Stewart [71, 72] for the analysis of carboxylic acid drugs and their salts (sodium formate, sodium acetate, 3-bromopropionic acid, 6-aminocaproic acid, 11-bromoundecanoic acid, 1-heptanesulfonic acid, / -n i t rophcny 1 acetic acid, sodium benzoate, sodium salicylate, valproic acid, probenecid, naproxen, ketoprofen, ibuprofen, mefenamic acid, flufenamic acid, and cefuroxime sodium) using a-(3,4-dimethoxy-phenyl)-4,-trimethylammoniummethylcinnamonitrile methosulfate... 

Kawamura, K. et al. Separation of aromatic carboxylic acids using quaternary ammonium salts on reversed-phase HPLC 2 application for the analysis of Loy Yang coal oxidation products. J. Sep. Sci. Technol. 2006,41, 723-732. 

The chemical scheme for C-terminal sequencing is shown in Figure 2. The first step involves treatment of the peptide or protein sample with diisopropylethylamine in order to convert the C-terminal carboxylic acid into a carboxylate salt. Derivatization of the C-terminal amino acid to a thiohydantoin is accomplished with diphenylisothiocyanatidate (liquid phase) and pyridine (gas phase). The peptide is then extensively washed with ethyl acetate and acetonitrile to remove reaction by-products. The peptide is then treated briefly with gas phase trifluoroacetic acid, followed by water vapor in case the C-terminal residue is a proline (this treatment has no effect on residues which are not proline). The derivatized amino acid is then specifically cleaved with sodium or potassium trimethylsilanolate to generate a shortened peptide or protein which is ready for continued sequencing. In the case of a C-terminal proline which was already removed by water vapor, the silanolate treatment merely converts the C-terminal carboxylic acid group on the shortened peptide to a carboxylate. The thiohydantoin amino acid is then quantitated and identified by reverse-phase HPLC. 

Analysis of Target Compounds. Matching El or Cl spectra and LC retention times to those obtained via analytical standards is exactly analogous to GC/MS methods development. Thus the major effort involves the determination of an appropriate HPLC column for any given analyte or analyte class. For example, conventional reversed phase HPLC columns are useless for extremely polar compounds such as sulfonic and certain carboxylic acids ion exchange based columns are more appropriate. 

Unstable compounds are problematic. A sample purified in the laboratory might have a short shelf-life and poor performance as a standard. Compounds altered by assays are also inconvenient. For example, substituted benzylic alcohols can dehydrate under acidic HPLC conditions, or carboxylic esters can hydrolyze in aqueous mobile phase. An impurity isolated from an active pharmaceutical ingredient as an organic salt of an organic compound poses two problems at once. The analyst must account for both the acid and the base. In the case of a toluenesulfonic acid salt of an aliphatic amine, two different methods of detection might be needed. The toluenesulfonic acid in a reverse-phase HPLC assay can by monitored by UV light, but the aliphatic amine, with no chromophore, must be measured by a different technique. 

Use of the reverse-phase HPLC system is highly flexible since it can also be applied to ionizable compounds such as carboxylic acids, phenols, and amines. The partition coefficients relate to the unionized compounds that are generally assumed to be the principal forms in which these compounds are transported into biota, even though their concentration may be low in comparison with the dissociated states at physiological pH values acidic compounds such as highly chlorinated phenols or many carboxylic acids have... 

Deacylated quillaja saponins were modified by incorporation of a Cn alkyl chain at the carboxyl group of the glucuronic acid residue to yield a family of saponin analogs referred to as GPI-OIOO. These stable derivatives, when compared by reverse phase HPLC to the acylated or deacylated forms of quillaja saponins, are more hydrophobic. The presence of the alkyl chain in GPI-OIOO is expected to increase the selfassociating properties of these derivatives in water, as well as their capacity to form complexes with proteins that are held by hydrophobic interactions. These analogs are similar to the deacylated quillaja saponins in that they appear to be devoid of toxicity in mice at a dose up to 1 mg. 

Pfizer reported the discovery of a new class of structurally complex alkyl carboxylic acids, as exemplified by CP-225917 and CP-263114 (Figure 20), that are weak and non-selective inhibitors of squalene synthase and FPTase. These compounds were isolated by acid base liquid-liquid partition, Sephadex LH20 chromatography and reverse phase HPLC from ethyl acetate extracts of an unidentified fungus that also produced zaragozic acid A [97]. 

The primary structure was assessed by peptide mapping and N- and C-terminal sequencing. N-terminal sequence analysis showed that a single sequence was detected, MKAIFVLNAA, which corresponds exactly to the first 10 amino acids at the N terminus of P40 as predicted from the DNA sequence. Reverse phase HPLC analysis of a digestion of P40 with a lysine-specific endopeptidase, i.e. endoproteinase Lys-C, was used for identification and primary structure confirmation (Fig. 9). Endoproteinase Lys-C hydolyzes peptide bonds at the carboxylic side of lysine residues. The seventeen peaks resolved were characterized by mass spectrometry, allowing the confirmation of 99 % of the primary sequence. 

The use of small spherical particles for NPC results in plate numbers in excess of 10,000 which compares favourably with the efficiencies of stationary phases currently in use for reversed phase HPLC. A list of the common functional groups used in NPC are shown in Table 6.1. Most of the popular supports used in NPC are silica based since alumina undergoes undesirable side reactions (e.g. irreversible reaction with carboxylic acid). The introduction of bonded phase supports has led to the availability of many other polar stationary phases, some of which can also be used in the reversed phase mode (Lochmuller et al., 1979). A bonded diamino phase was found to be optimal for aromatic hydrocarbons (Chmielowiec and George, 1980). 

Recently, Bayliss et al. [132] described the synthesis of anthracene-9-carbonyl chloride and its aplications as a label for fluorescence and UV absorbance detection of hydroxy compounds. The preparation and properties of esters of short-chain alcohols, diols, trichothecene mycotoxins and sterols were investigated. Anthracene-9-carbonyl chloride was prepared from commercial anthracene-9-carboxylic acid. Derivatization was carried out in acetonitrile free from water or active hydrogen compounds. The reaction rate was dejjendent on the structure of the alcohol. The derivatization of diethylene glycol was complete at ambient temp>erature within 10 min (0.25 M reagent) or 30 min (0.1 M reagent) without a catalyst, but required 1 h for diolesteroL testosterone and the trichothecene T-2 toxin. For sterically hindered alcohols such as t-butanol and 17a-methyl-testosterone, more than 10 h, or refluxing for 1 h, was needed to complete the reaction. The derivatives had absorption maxima at 250 run. Both normal and reversed phase HPLC were applied to the separation of the derivatives. 

Valeur et al (1994) elucidated the principal cleavage pathways of sphingomyelin in positive ion PSP. Sphingomyelin from bovine milk, bovine brain and chicken egg yolk were analysed by reversed-phase HPLC-PSP-MS. Spectra of individual molecular species showed prominent ceramide-derived fragment ions as well as ions of long-chain base (LCB) and carboxylic acid origin of the ceramide unit. 

It is used as a derivatization reagent for aliphatic carboxylic acids that lack strong UV-chromaphores to improve detectability and retention on a reversed-phase HPLC column (14). It is used to determine extraction efficiencies of individual carboxylic acids. 

For the library samples that contain very polar by-products, excess reagents, or impurities, an ion-pair reagent may be added to the mobile phase to improve retention of the polar components on the reversed-phase HPLC column. For small polar basic components such as amines, 0.1-1% of heptafluorobutyric acid can be added into the mobile phase containing acetonitrile or methanol in water. For small polar acidic components such as carboxylic acids, 5-20 mM of tetrabutyl-ammonium dihydrogenphosphate can be added into the mobile phase containing acetonitrile or methanol in 50 mM phosphate buffer (pH 7.4). 

Fluorescent labelling reagent used for the reversed phase hplc sepn. and detn. of carboxylic acids. Mp 214-216°. 

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