Analytical Biochemistry, ( ISI ), Volume (383), Year (2008-12) , Pages (93-102)

Title : ( Application of Statistical Design for the Optimization of Amino Acid Separation by reverse-phase HPLC )

Authors: REZA GHeshlaghi , Jeno Scharer , Murray Moo-Young , Peter Douglas ,

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Modified resolution and overall separation factors used to quantify the separation of complex chromatography systems are described. These factors were proven to be applicable to the optimization of amino acid resolution in reverse-phase (RP) HPLC chromatograms. To optimize precolumn derivatization with phenylisothiocyanate, a 25-1 fractional factorial design in triplicate was employed. The five independent variables for optimizing the overall separation factor were triethylamine content of the aqueous buffer, pH of the aqueous buffer, separation temperature, methanol/acetonitrile concentration ratio in the organic eluent, and mobile phase flow rate. Of these, triethylamine concentration and methanol/acetonitrile ratio were the most important. The methodology captured the interaction between variables. Temperature appeared in the interaction terms; consequently, it was included in the hierarchic model. The preliminary model based on the factorial experiments was not able to explain the response curvature in the design space; therefore, a central composite design was used to provide a quadratic model. Constrained nonlinear programming was used for optimization purposes. The quadratic model predicted the optimal levels of the variables. In this study, the best levels of the five independent variables that provide the maximum modified resolution for each pair of consecutive amino acids appearing in the chromatograph were determined. These results are of outmost importance for accurate analysis of a subset of the amino acids.

Keywords

, Experimental design, Central composite design, Amino acid analysis optimization, Precolumn derivatization using phenylisothiocyanate, Modified resolution, Overall separation
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@article{paperid:1010384,
author = {GHeshlaghi, REZA and Jeno Scharer and Murray Moo-Young and Peter Douglas},
title = {Application of Statistical Design for the Optimization of Amino Acid Separation by reverse-phase HPLC},
journal = {Analytical Biochemistry},
year = {2008},
volume = {383},
month = {December},
issn = {0003-2697},
pages = {93--102},
numpages = {9},
keywords = {Experimental design; Central composite design; Amino acid analysis optimization; Precolumn derivatization using phenylisothiocyanate; Modified resolution; Overall separation factor},
}

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%0 Journal Article
%T Application of Statistical Design for the Optimization of Amino Acid Separation by reverse-phase HPLC
%A GHeshlaghi, REZA
%A Jeno Scharer
%A Murray Moo-Young
%A Peter Douglas
%J Analytical Biochemistry
%@ 0003-2697
%D 2008

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