Liquid chromatography method development

When the chromatographic mode, column type, packing and dimensions have been chosen, the final stage of method development involves solvent optimisation and a choice between isocratic or gradient elution. Many separations can be achieved perfectly satisfactorily under isocratic conditions and are preferred to gradient elution techniques, as these are inconvenient due to the time required to re-equilibrate the column. A measure of the quality of separation is given by the resolution factor which can be expressed as follows  

The first term is a measure of the column efficiency, and N can be varied by changing the column length, the particle size of packing or the mobile phase velocity. 

A measure of the selectivity of the method is given by term (iii). The selectivity factor, a, may be varied by changing the temperature, or the stationary or mobile phases. However, if a 1, then no matter how long the components stay on the column, or how many theoretical plates the column is 

Thus the resolution achieved in a chromatogram is largely dependent upon values of a and k, both of which are strongly dependent on the nature of the eluant in terms of solvent strength, organic solvent, mobile phase pH (ion suppression techniques) and eluant additives (IPRs). 

In general terms, an / = 1.5 is adequate for most purposes. Values in excess of this result in over-long analysis times. In practice for isocratic elution, the following stepwise procedure is often adopted. 

---

L. van Heukelem and C.S. Thomas, Computer assisted high-performance liquid chromatography method development with applications to the isolation and analysis of phytoplankton pigments. J. Chromatogr.A 910 (2001) 31—49. 

High-pressure liquid chromatography method development of pharmaceuticals is an iterative process required to support successive phases of pharmaceutical development and clinical studies. This chapter details the approach currently in use in our laboratories, from receipt of a new chemical entity to post transfer support. 

An Expert System for High Performance Liquid Chromatography Methods Development... 

Roston, D.A. Sun, J.J. Collins, P.W. Perkins, W.E. Tremont, S.J. Supercritical fluid extraction-liquid chromatography method development for a polymeric controlled-release drug formulation. J. Pharm. Biomed. Anal. 1995, 13 (12), 1513-1520. 

Grogg-Sulser, K. Helmlin, H.-J. Clerc, J.-T. Qualitative and quantitative determination of illicit heroin street samples by reversed-phase high-performance liquid chromatography method development by CARTAGO-S. [Pg.9]

Hewitt EF, Lukulay P, Galushko S (2006) Implementation of a rapid and automated high performance liquid chromatography method development strategy for pharmaceutical drug candidates. J Chromatogr A, 1107 79-87. 

Dolan, J.W. Snyder, L.R. Saunders, D.L. Van-Heukelem, L. Simultaneous variation of temperature and gradient steepness for reversed-phase high-performance liquid chromatography method development, n. The use of further changes in conditions. J. Chromatogr. A, 1998, 803 (1-2), 33 50. 

Tswett s initial column liquid chromatography method was developed, tested, and applied in two parallel modes, liquid-solid adsorption and liquid-liquid partition. Adsorption ehromatography, based on a purely physical principle of adsorption, eonsiderably outperformed its partition counterpart with mechanically coated stationary phases to become the most important liquid chromatographic method. This remains true today in thin-layer chromatography (TLC), for which silica gel is by far the major stationary phase. In column chromatography, however, reversed-phase liquid ehromatography using chemically bonded stationary phases is the most popular method. 

Liquid Chromatography. The development of rapid, simple liquid chromatographic methods for determining the enantiomeric purity of chiral compounds is probably one of the most important developments in the study of asymmetric synthesis in the last 10 years. Several books have been published providing thorough evaluations of various enantiomeric separation techniques and their practical applications.46... 

L. Gagliardi, D. De Orsi, M.R. Del Giudice, F. Gatta, R. Porra, P. Chimenti and D. Tonelli, Development of a tandem thin-layer high-performance liquid chromatography method for the identification and determination of corticosteroids in cosmetic products. Anal. Chim. Acta 457 (2002) 187-198. 

In the last twenty years, many of the developed and validated high performance liquid chromatography methods with conventional diode array or fluorescence detectors (DAD, FLD) were improved and substituted by new hyphenation with mass spectrometric instrumentation and/or NMR, especially for the analyses of raw materials derived from Natural sources. The main goal of this coupling is achieved by improvement of selectivity and sensitivity of new instrumental configurations [7], Furthermore, with these configurations it is possible to obtain, in only one analysis, the complete chemical structure elucidation, identification and quantification of targeted compounds. 

During the last decade capillary electrophoresis (CE) has become a mature separation technique for pharmaceutical analysis. Numerous validated methods from pharmaceutical R D lahoratories and academia have been reported in literature, including identity confirmation, main component assay, purity determination, enantiomeric separation, and stoichiometry determination. In addition, CE is frequently applied as an orthogonal technique during the development of stability indicating liquid chromatography methods. As a result CE... 

Snyder, L.R. and Dolan, J.W. (1996) Initial experiments in high-performance liquid chromatographic method development. 1. Use of a starting gradient run. Journal of Chromatography. A, 721, 3-14. 

Osmotic effects play an important role mainly at high injected polymer concentrations. They may selectively affect the retention volumes of smaller polymer species contained in the polymer sample [97], Osmotic effects within porous column packings form the basis of interesting preparative liquid chromatographic method developed by Teraoka and coworkers [98-101] and denoted the high osmotic pressure liquid chromatography. 

The extracts were fractionated by a Preparative Liquid Chromatography method - PLC-8 [2], in eight distinct chemical classes FI-saturated hydrocarbons (HC), F2-monoaromatics, F3-diaromatics, F4-triaromatics, F5-polynuclear aromatics, F6-resins, F7-asphaltenes and F8-asphaltols. This method, proposed by Karam et al. as an extension of SARA method [4], was especially developed for coal-derived liquids. It combines solubility and chromatographic fractionation, affording discrete, well-defined classes of compounds which are readable for direct chromatographic and spectroscopic analysis. 

The form of the dissolved sulfur has not been characterized properly yet. While stable at ambient temperatures, a substantial amount can be converted to crystalline sulfur at elevated temperatures or by solvent separation. This observation led to the development of a rapid liquid chromatography method to determine elemental sulfur in SA binders. The procedure which has been described previously by Cassidy (17) is based on gel permeation principle and uses a Styragel column and a uv detector. Results showed that 2-14% of the elemental sulfur added reacted chemically with the asphalt. Petrossi (18) and Lee (19), who determined free sulfur by extraction with sodium sulfite followed by titration with iodine, calculated a higher percent of bonded sulfur in sulfur-asphalt compositions. The observed differences are most likely caused by variations in the asphalt composition with regard to polar aromatics and naphthene components as well as by reaction temperature and contact time. 

Varvaresou, A. et al. Development and validation of a reversed-phase ion-pair liquid chromatography method for the determination of magnesium ascorbyl phosphate and melatonin in cosmetic creams. Anal. Chim. Acta 2006, 573-574, 284—290. 

Moret, S., Hidalgo, M., and Sanchez, l.M. Development of an ion-pairing liquid chromatography method for the determination of phenoxyacetic herbicides and their main metabolites application to the analysis of soil samples. Chromatographia 2006, 63,... 

The first liquid chromatography method introduced to determine ezetimibe and its metabolites was developed by Ezzet et al. [58], and the method was used to model the pharmacokinetics of ezetimibe in humans. The liquid chromatography-mass spectrometry (LC-MS) method was used to determine imchanged ezetimibe and total ezetimibe (imchanged ezetimibe together with ezetimibe glucoronide) in human plasma samples. 

---