Chapter 13 Amino acids and peptides

of mobile-phase (B %) does not change during the separation. Isocratic elution is suitable for analysis of compounds with a relatively narrow range of polarity and hydrophobicity. In gradient elution, mobile phase strength (B %) increases during the separation. This means that sample retention is measured by k value decrease for each band as it migrates through the column [1-3]. Chromatographers are cautioned to avoid gradient elution when isocratic elution is possible to use. It is common knowledge that development and optimization of HPLC gradient methods can be time consuming and require many experiments [1,2]. Moreover, the transfer of a gradient elution method between columns, instruments and laboratories is notoriously more difficult than the transfer of an isocratic elution method [4,5]. Despite that gradient elution is a powerful method for separation and peak identification of many branches of chromatography provided that the optimum gradient profile, the profile that yields the best separation of the chromatographic peaks of a mixture of analytes can be easily determined [1-3, 6] The principal reason for using a gradient elution approach is a necessity to analyze samples containing solutes with a wide range of hydrophobicity [1-3, 6].As we mentioned before, optimization of gradient elution is a substantially more difficult task than optimization of isocratic conditions and can require many experiments, especially for multi-component samples [7][8][9][10]. Prediction of retention in gradient elution from structural formulae of analytes can potentially save time in method development, however, only a limited number of studies in this field have been published [11][12][13]. An approach described earlier requires many preliminary experiments to "train a column" and values of molecular descriptors which are mainly not known. Such an approach can be considered as theoretically interesting but without practical value [14][15][16]. The solvatic Abstract Application of the solvatic retention model of reversed-phase liquid chromatography was studied to predict retention of phenylisothiocyanate derivatives of amino acids from structural formulae and stationary and mobile phase properties. The gradient elution mode with methanol and acetonitrile aqueous mobile phases was used. It was shown that practically acceptable prediction or retention time values can be achieved after the first approximation step when experimental data of one run are used. The zero approximation level predictions-from structural formulae, column and mobile phase properties can be used as a "first guess" method from which further optimization can begin.

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“…For experimental work amino acid sample was prepared by pre-column phenylisothiocyanate derivatization [20][21][22][23].…”

Section: Materials and Suppliesmentioning

confidence: 99%

Application of the Solvatic Model for Prediction of Retention in RP-LC for Multi-Step Gradient Profiles

Vorslova

Golushko²,

Galushko³

et al. 2014

Chromatographia

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“…This implies that an efficient chromatographic separation prior to the detection of CML is essential. Two chromatographic approaches for amino acid analysis have been widely employed: (i) pre-column derivatisation to fluorescent and more hydrophobic derivatives, which are separated by reversed-phase LC and (ii) separation of the native amino acids by ion-exchange LC, followed by their post-column derivatisation [13]. Both approaches have their advantages and disadvantages, but for the present application pre-column derivatisation was selected, its main benefits being technical simplicity, speed and the relative ease to manipulate the separation of the amino acids by tuning the mobile phase composition.…”

Section: Separation and Detectionmentioning

confidence: 99%

“…Pre-column derivatisation of amino acids has been achieved with a wide variety of reagents [13]. For the sake of robustness and sensitivity, FMOC chloride was employed in the present work, because this reagent forms highly fluorescent and very stable derivatives with both primary and secondary amines in a matter of seconds.…”

Section: Separation and Detectionmentioning

confidence: 99%

Liquid chromatographic method for the quantitative determination of N?-carboxymethyllysine in human plasma proteins

VANDEMERBEL¹

2004

Journal of Chromatography B

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“…Derivatization with phenylisothiocyanate (PITC) was chosen because this method has several advantages -possibilities for pre-column derivatization, stability of derivatives for up to 48 hours (at 5 -8 o C), and also the compatibility with using HPLC with a UV detector [24][25][26].…”

Section: Introductionmentioning

confidence: 99%

Prediction of Reversed-Phase Liquid Chromatography Retention Parameters for Phenylisothiocyanate Derivatives of Amino Acids

Vorslova¹,

Golushko²,

Galushko³

et al. 2014

Latvian Journal of Chemistry

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We report our experience with highly polar and charged analyte retention parameter prediction for a reversed-phase high-performance liquid chromatographic method. The solvatic retention model has been used to predict retention of phenylisothiocyanate derivatives of 25 natural amino acids under gradient elution conditions. Retention factors have been calculated from molecular parameters of analyte structures and from the column and eluent characteristics. A step-by-step method which includes the first guess prediction of initial conditions from structural formula and fine tuning of the retention model parameters using data from successive runs can substantially save method development time.

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Chapter 13 Amino acids and peptides

Cited by 11 publications

References 336 publications

Application of the Solvatic Model for Prediction of Retention in RP-LC for Multi-Step Gradient Profiles

Application of the Solvatic Model for Prediction of Retention in RP-LC for Multi-Step Gradient Profiles

Liquid chromatographic method for the quantitative determination of N?-carboxymethyllysine in human plasma proteins

Prediction of Reversed-Phase Liquid Chromatography Retention Parameters for Phenylisothiocyanate Derivatives of Amino Acids

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