Retention time in reversed-phase chromatography

Figure 4.1 Correlation of predicted and observed retention times in reversed-phase chromatography. The predicted retention times for 58 peptides of 2 to 16 residues in length were obtained by summation of retention coefficients for each residue in the peptide. Retention coefficients were determined from the retention of model synthetic peptides with the structure Ac-Gly-XX-(Leu)3-(Lys)2-amide, where X was substituted by the 20 protein amino acids. (Reproduced from D. Guo, C.T. Mant, A.K. Taneja, and R.S. Hodges, J. Chromatogr., 359 519 [1986]. With permission from Elsevier Science.)...

In a series of experiments designed to explore further the role of polarity in affecting retention time in reversed-phase chromatography, we developed chemical procedures for the condensation of molecules of known polarity,... 

Biodegradation calculated rate constant k = (9.49 0.41) x 10 min" from retention times in reverse phase chromatography (Umshigawa Yonezawa 1979 quoted, O Grady et al. 1985) ... 

Biodegradation rate constant k = (6.86 0.23) x 10- min- was estimated from the retention time in reverse phase chromatography (Urushigawa Yonezawa 1979) ... 

Guo, D, Mant, C. T., Taneja, A. K, Parker, J. M. R., and Hodges, R. S., Prediction of peptide retention times in reversed-phase high-performance liquid chromatography. I. Determination of retention coefficients of amino acid residues of model synthetic peptides, /. Chromatogr., 359, 499, 1986. 

Urushigawa Y, Yonezawa Y. 1979. Chemico-biological interactions in biological purification systems VI. Relation between biodegradation rate constants of di-n-alkyl phthalate esters and their retention times in reverse phase partition chromatography. Chemosphere 5 317-320. 

Various methods have been employed for the prediction of retention times in reversed-phase liquid chromatography. 

A quantitative analysis of the structure-retention relationship can be derived by using the relative solubility of solutes in water. One parameter is the partition coefficient, log P, of the analyte measured as the octanol-water partition distribution. In early work, reversed-phase liquid chromatography was used to measure log P values for drug design. Log P values were later used to predict the retention times in reversed-phase liquid chromatography.The calculation of the molecular properties can be performed with the aid of computational chemical calculations. In this chapter, examples of these quantitative structure-retention relationships are described. 

Funasaki, N., Hada, S., Neya, S. (1986) Prediction of retention times in reversed-phase high-performance Uquid chromatography from the chemical structure. J. Chromatogr. 361, 33 5. 

Urushigawa, Y., and Y. Yonezawa (1979), Chemicobiological Interactions in Biological Purification System VI. Relation between Biodegradation Rate Constants of Di-u alkyl Phthalate Esters and Their Retention Times in Reverse Phase Partition Chromatography, Chemosphere 5, 317-320. 

The above results demonstrated the possibility of the quantitative analysis of retention times in reversed-phase liquid chromatography. In addition, a cyano-group-bonded silicone phase was constructed, and MI energy values were calculated. The sum of the MIFS and cnMIES (cyano phase) energies... 

Normal-phase liquid chromatography is thus a steric-selective separation method. The molecular properties of steric isomers are not easily obtained and the molecular properties of optical isomers estimated by computational chemical calculation are the same. Therefore, the development of prediction methods for retention times in normal-phase liquid chromatography is difficult compared with reversed-phase liquid chromatography, where the hydrophobicity of the molecule is the predominant determinant of retention differences. When the molecular structure is known, the separation conditions in normal-phase LC can be estimated from Table 1.1, and from the solvent selectivity. A small-scale thin-layer liquid chromatographic separation is often a good tool to find a suitable eluent. When a silica gel column is used, the formation of a monolayer of water on the surface of the silica gel is an important technique. A water-saturated very non-polar solvent should be used as the base solvent, such as water-saturated w-hexane or isooctane. 

The elution order of phthalic esters is related to the carbon chain length. The longer the chain length, the shorter the retention time in normal-phase liquid chromatography, and the elution order is reversed in reversed-phase liquid... 

The retention times of peptides with fewer than 20 residues in reversed-phase chromatography can be predicted with a high degree of accuracy based on their amino acid composition and the characteristics of their N-terminal and C-terminal amino acids. A number of researchers (66 -75) have studied the role of amino acids in peptide retention and have established retention coefficients for the different amino acids. The retention coefficient value of each amino acid is normally calculated by regression analysis of the retention times for peptides of known composition. 

Mobile phases used in reversed-phase chromatography are frequently composed of mixtures of methanol and water or acetonitrile and water. Increasing the proportion of water causes an increased retention of the more hydrophobic solutes relative to the more polar solutes. The surface tension of the mobile phase plays a major role in governing solute retention, so an increase in temperature, by reducing viscosity, increases column efficiency and shortens retention times. 

Fatty acids have so far been analysed by CEC either as the free acids or as phenacyl- or methyl esters. Aqueous acetonitrile (50 mM) at pH 6 (9 1 v/v) was shown to be the optimal mobile phase [191]. It is generally known that in reversed-phase chromatography free fatty acids and fatty acid methyl esters separate according to the partition number, which is defined as the carbon number minus twice the number of double bonds. A double bond reduces the retention time by the equivalent... 

In spite of widespread applications, the exact mechanism of retention in reversed-phase chromatography is still controversial. Various theoretical models of retention for RPC were suggested, such as the model using the Hildebrand solubility parameter theory [32,51-53], or the model supported by the concept of molecular connectivity [54], models based on the solvophobic theory [55,56) or on the molecular statistical theory [57j. Unfortunately, sophisticated models introduce a number of physicochemical constants, which are often not known or are difficult and time-consuming to determine, so that such models are not very suitable for rapid prediction of retention data. 

The retention factor, Eq. (7.2), for each species / is calculated knowing the dead time, t(), and the retention time of species i at infinite dilution, /r,./- There are known methods in the literature for calculating the dead time or retention time for a non-retained peak in normal-phase, reversed-phase and ion-exchange chromatography [67]. For example, in normal-phase chromatography, pentane in 95 5 hexane-acetone is unretained. In reversed-phase chromatography, a common measure of void volume is from the refractive index response obtained when the sample solvent composition is different from the mobile-phase composition. 

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