HPLC method development

Remcho, V. T. McNair, H. M. Rasmussen, H. T. HPLC Method Development with the Photodiode Array Detector, /. Chem. Educ. 1992, 69, A117-A119. 

Snyder, L. R. Glajch, J. L. Kirkland, J. J. Practical HPLC Method Development. Wiley-Interscience New York, 1988. 

L.R. Snyder, J. Kirkland and J. Glaich, Practical HPLC Method Development, 2nd Edn., Wiley-Interscience, New York, 1997. ISBN 047100703X. 

Snyder L.R., Dolan J.W., Molnar I., and Djordjevic, N.M., Selectivity control in reversed-phase HPLC methods development varying temperature and solvent strength to optimize separations, LC-GC, 15 (2), 136, 1997. 

Parameters that should be tested in HPLC method development are flow rate, column temperature, batch and supplier of the column, injection volume, mobile phase composition and buffer pH, and detection wavelength [2], For GC/GLC methods, one should investigate the effects of column temperature, mobile phase flow rate, and column lots or suppliers [38], For capillary electrophoresis, changes in temperature, buffer pH, ionic strength, buffer concentrations, detector wavelength, rinse times, and capillaries lots and supplier should be studied [35, 36], Typical variation such as extraction time, and stability of the analytical solution should be also evaluated [37],... 

Figure 4.13 from Snyder, Kirkland and Glajch, Practical Hplc Method Development, 2nd ed. (1997) by... 

Snyder L.R., Kirkland J.J, and Glajch J.L. Practical HPLC Method Development, 2nd ed., John Wiley Sons New York, 1997, Chap. 4. 

The actual Amb a 1 concentration of the extract can be quantitated using a reversed-phase HPLC method developed at Dynavax. This is a custom two-step method that employs chromatography to separate the Amb a 1 from the other extracted proteins. The Amb a 1 concentration is then determined from the resolved Amb a 1 peak area and a standard curve of purified Amb a 1. This is the only step at which the Amb a 1 concentration of the process material is measured by a two-step process. Following the extraction step, the Amb a 1 rapidly becomes enriched over two purification steps, and the Bradford assay adequately reflects Amb a 1 concentration through the remainder of the process. 

The first consideration when investigating HPLC method development protocols is the chemical structure of the analyte, in particular, the presence of functional groups capable of interacting with the stationary phase and containing or in the vicinity of the stereogenic elements [79]. Since the natural target of macrocyclic antibiotics is the A-acyl-D-alanyl-D-alanine terminus (see Section 2.1), the early choice of suitable substrates for this kind of CSPs was that of amino acids [45]. However, it turned out that the macrocyclic CSPs were very successful not only in amino acids enantioresolution, but also in the separation of a wide variety of different structures. 

The mobile phase controls HPLC separation While the HPLC stationary phase provides retention and influences the separation mechanism, it is the mobile phase which controls the overall separation. HPLC method development efforts focus on finding a set of mobile phase conditions that provide adequate separation of the analyte peak(s) from other components in the sample. 

Introduction to HPLC, CLC-10 and HPLC Method Development, CLC-20, (Computer-based Instruction), Savant, Eullerton, CA, http //www.savant4training.com/savant2.htm. 

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