Reversed phase liquid chromatography buffers

Figure 8.43 Separation of enantiomers using complexation chromatography. A, Separation of alkyloxiranes on a 42 m x 0.2S mm I.O. open tubular column coated with 0.06 M Mn(II) bis-3-(pentafluoro-propionyl)-IR-camphorate in OV-ioi at 40 C. B, Separation of D,L-amino acids by reversed-phase liquid chromatography using a mobile phase containing 0.005 M L-histidine methyl ester and 0.0025 M copper sulfate in an ammonium acetate buffer at pH 5.5. A stepwise gradient using increasing amounts of acetonitrile was used for this separation.

Gritti, F. and Guiochon, G, Effect of the pH, the concentration and the nature of the buffer on the adsorption mechanism of an ionic compound in reversed-phase liquid chromatography, ii. Analytical and overload band profiles on Symmetry-C-18 and Xterra-C-18, J. Chromatogr. A, 1041, 63, 2004. 

A. P. Schellinger and P. W. Carr, Solubility of Buffers in Aqueous-Organic Eluents for Reversed-Phase Liquid Chromatography, LCGC 2004,22, 544 ... 

Application of Prep-HPLC to PGS Analysis. Reverse phase liquid chromatography has proven to be well suited for cleanup of plant extracts by prep-HPLC (4, 46, 47, 48). When the mobile phase is initially an aqueous buffer at pH 2.8, all but the highly charged (e.g., zeatin ribotide with 5 AMP used as a representative compound for zeatin ribotide) plant hormones are retained at the head of the column (Fig. 1). Since the PGS are retained, samples can be injected onto the column in a dilute form. In-... 

Acetylcholineesterase and choline oxidase The detector fabrication of glutaral-dehyde co-crosslinking of AChE and ChO with bovine serum albumin on the Pt working electrode of a conventional thin layer electrochemical flow cell. A mobile phase of phosphate buffer 0.1 M pH 6.5 containing 5 mM-sodium hexane sulfonate and 10 mM-tetra-methylammonium phosphate was used in the ion-pair, reversed phase liquid chromatography. 

S. Heinisch and J. L. Rocca, Effect of mobile phase composition, pH and buffer type on the retention of ionizable compounds in reversed-phase liquid chromatography Application to method development,/. Chromatogr. A 1048 (2004), 183-193. 

Electrochemical detectors are based upon the volta-metric oxidation or reduction of separated analytes at a micro- or thin-film electrode. A number of pharmacologically active compounds that are aldehydes, ketones, or quinones (such as doxorubicin), or nitro compounds (such as nitrofurantoin) are amenable to reduction at a mercury or platinum electrode electron-rich indole derivatives and catecholamines can be oxidized at these electrodes. An important condition that must be fulfilled for electrochemical detection to be practicable is that the mobile phase must be capable of conducting an electrical current. This makes electrochemical detection particularly useful in reversed-phase liquid chromatography, where buffered water mixed with one or more organic cosolvents is usually the mobile phase. 

Micelles and cyclodextrins are the most common reagents used for this technique. Micellar electrokinetic capillary chromatography (MECC or MEKC) is generally used for the separation of small molecules [6], Sodium dodecyl sulfate at concentrations from 20 to 150 mM in conjunction with 20 mM borate buffer (pH 9.3) or phosphate buffer (pH 7.0) represent the most common operating conditions. The mechanism of separation is related to reversed-phase liquid chromatography, at least for neutral solutes. Organic solvents such as 5-20% methanol or acetonitrile are useful to modify selectivity when there is too much retention in the system. Alternative surfactants such as bile salts (sodium cholate), cationic surfactants (cetyltrimethy-lammonium bromide), nonionic surfactants (poly-oxyethylene-23-lauryl ether), and alkyl glucosides can be used as well. 

In addition to the three classical separation methods mentioned above, reversed-phase liquid chromatography (RPLC) is becoming increasingly popular for the separation of highly polar and ionic species, respectively. Long-chain fatty acids, for example, are separated on a chemically bonded octadecyl phase after protonation in the mobile phase with a suitable aqueous buffer solution. This separation mode is known as ion suppression [18]. 

The use of positive-ion ESI with reverse-phase liquid chromatography is common for most methods. Mobile phases are often a combination of either methanol or acetonitrile combined with water that contains additives such as acids or volatile ammonium buffers. Optimization between increased sensitivity with a higher percent of organic solvent in the mobile phase and adequate retention on the column is important for LC-MS/MS detection. 

Reversed phase liquid chromatography on C-18 bonded phase has often been used for separation of amino acid derivatives. As these columns can be different from one supplier to another, it is impossible to recommend the conditions for the mobile phase. Lindroth and Mopper (1979) and Hill et al. (1979) used phosphate buffer at different pH values and methanol in the mobile phase. 

Diastereomeric amides from L-leucinamide and arylpropionic acids have been separated by reversed phase liquid chromatography using acetonitrile/phosphate buffer (pH 6.5) as the mobile phase [65], Some workers also found that the inclusion of triethylamine (0.02%) improved the separation [71]. Rossetti et al. [72] used HI LC to separate the diastereomeric phenylethyl-amides of several NSAIDs the solvent systems used were benzene/methanol (93 7) and chloroform/ethyl acetate (15 1), and separation was improved by multiple development in the same solvent system. 

Reversed-Phase Liquid Chromatography (RPLC) is an important tool in protein chemistry. Examination of sorption isotherms revealed that alcohohc buffers did not desorb proteins near physiological pH in RPLC systems, while buffers containing a poly(ethoxy alcohol) surfactant did not desorb protein at pH 2, but they did at pH 7 with concentrations of surfactant apparently well above the critical micellar concentration (cmc) [2]. It has been proposed that a necessary condition for the desorption of a protein from a surface is that the surface tension of the solvent falls between that of the protein and the surface [6]. This condition is fiilfilled for many proteins with surfactant solutions near conditions of physiological pH and ionic strength. Therefore, it was expected that separations of proteins could be thieved in these conditions. 

Schellinger, A.P. Stoll, D.R. Carr, P.W. High-speed gradient elution reversed-phase liquid chromatography of bases in buffered eluents Part I. Retention repeatability and column re-equilibration. J. Chromatogr. A, 2008, 1192, 41-53. 

Figure 10.270 Separation of the nine most abundant steviol glycosides by reversed-phase liquid chromatography. Separator column Capcell Pak CIS MG II, 5 pm column dimensions 250mmx4.6mm i.d. column temperature 40°C eluent MeCN/phosphate buffer, pH 2.6 (32 68 v/v) flow rate 1 mlVmin ...

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