Retention in reversed-phase HPLC

Obtained P values for the description of benzodiazepine s retention in reversed-phase HPLC are used. The equation... 

The separation of substituted benzene derivatives on a reversed-phase C-18 column has been examined [78]. The correlations between the logarithm of the capacity factor and several descriptors for the molecular size and shape and the physical properties of a solute were determined. The results indicated that hydrophobicity is the dominant factor to control the retention of substituted benzenes. Their retention in reversed-phase HPLC can be predicted with the help of the equations derived by multicombination of the parameters. 

Solute retention in reversed-phase HPLC is dependent on the different distribution coefficients established between a polar mobile and a nonpolar stationary phase by the peptidic components of a mixture. Although there are many similarities between reversed-phase HPLC separations of peptides and the classical liquid-liquid partition chromatographic methods, it is debatable whether the sorption process in reversed-phase HPLC arises due to partition or adsorption events, i.e., whether the nonpolar stationary phase functions as a bulk liquid or as an adsorptive monolayer. These aspects and the theoretical models for reversed-phase HPLC are discussed in a subsequent section. 

A simple rule for retention in reversed-phase HPLC is that the more hydrophobic the component, the more it is retained. By simply following this rule, one can conclude that any organic ionizable component will have longer retention in its neutral form than in the ionized form. Analyte ionization is a pH-dependent process, so significant effect of the mobile-phase pH on the separation of complex organic mixtures containing basic or acidic components can be expected. 

In this chapter we discussed the influence of most known secondary equilibria effects as well as the utilization of the organic eluent component absorption on the surface to describe the analyte retention in reversed-phase HPLC. 

Ionization and HPLC retention in reversed-phase HPLC... 

Solute retention in reversed-phase HPLC (RPLC) varies with the volume fraction of organic solvent in the mobile phase as (15)... 

The understanding of retention and selectivity behaviour in reversed-phase HPLC in order to control and predict chromatographic properties ai e interesting for both academic scientists and manufacturers. A number of retention and selectivity models are the subject of ongoing debate. The theoretical understanding of retention and selectivity, however, still lags behind the practical application of RP HPLC. In fact, many users of RP HPLC techniques very often select stationary phases and other experimental conditions by experience and intuition rather than by objective criteria. 

Enthalpy-entropy compensation has been investigated in reversed-phase HPLC with octylsilica stationary phase [77]. The compensation temperatures were determined for this system, and the results show that their change with the composition of the mobile phase is almost similar to that with octadecylsilica stationary phase. It can be concluded that the retention mechanisms of the separation of alkyl benzenes is the same in both systems with the mobile phase exceeding 20% water content. 

RETENTION TIMES, fR (MIN) IN REVERSED-PHASE HPLC OF NAPHTALENE SULPHONIC ACIDSa... 

In reversed-phase HPLC, column temperature is a strong determinant of retention time and also affects column selectivity. A column oven is therefore required for most automated pharmaceutical assays to improve retention time precision, typically at temperatures of 30-50°C. Temperatures >60°C are atypical due to concerns about thermal degradation of the analytes and column lifetimes. Exceptions are found in high-throughput screening where higher temperatures are used to increase flow and efficiency. Ambient or snb-ambient operation is sometimes found in chiral separations to enhance selectivity. Column ovens... 

Jinno, K., and K. Kawasaki, Correlation Between the Retention Data of Polycyclic Aromatic Hydrocarbons and Several Descriptors in Reversed-Phase HPLC. Chromato-graphia, 1983 17, 445-449. 

In reversed-phase HPLC on a Cl8 column, only six peaks are usually found, and their order of elution is 5-tocotrienol, (3- and y-to-cotrienols, a-tocotrienol, 5-tocopherol, (3- and y-tocopherol, and a-tocopherol. Figure D 1.5.4 is an example of a reversed-phase chromatograph of tocopherols and tocotrienols in rice bran oil. The tocols with unsaturated side chain have shorter retention time than those with saturated side chain. The methyl substituents on the chromanol ring also affect the retention times of tocopherols and tocotrienols. However, the effect is reversed, compared with the normal-phase HPLC method. 

Reversed-phase HPLC can separate polyphenolics of extracts on the basis of polarity. HPLC easily produces better resolution among chemically similar compounds in extracts than conventional chromatographic methods. The operating temperature of the column during reversed-phase HPLC analysis should be controlled for data reproducibility. A change in temperature produces only a minor effect, however, on band spacing in reversed-phase HPLC and produces essentially no effect in normal-phase HPLC (Lee and Widmer, 1996). A range of ambient temperatures is widely used, and elevated temperatures are often applied. The retention times of the peaks are dependent upon the type of column and the combination of various solvents used in the method. 

Most HPLC applications used for phenolic analysis simply allow the room temperature to determine the operating temperature of the column, but elevated temperatures of between 30°C and 40°C are often applied for phenolics and derivatives in apples (14), carrots (15), apple juice (6,13), bilberry juice (16), and for cis-trans isomers of caffeic and p-coumaric acids in wines (17). Generally, a change in temperature has only a minor effect on band spacing in reversed-phase HPLC and has essentially no effect in normal-phase separations. Thermostatic control of the column temperature is generally recommended to provide reproducible retention. 

Lin, J. T., and McKeon, T. A. 2003. Relative retention times of the molecular species of acylglycerols, phosphatidylcholines and phosphatidylethanolamines containing ricinoleate in reversed-phase HPLC. /. Liquid Chromatogr. Rel. Technol., 26, 1051-1058. 

Spicer, V. et al. Sequence-specific retention calculator a family of peptide retention time prediction algorithms in reversed-phase HPLC applicability to various chromatographic conditions and columns. Anal. Chem. 2007, 79, 8762-8768. 

The log Ofj term reflects the differences in capacity ratios of the two peptide solutes Sj and Sj which differ by a functional group and is analogous to the term used to predict selectivity differences for the classical liquid-liquid partition chromatography of peptides. The influence of functional group behavior on the retention of polar solutes in reversed-phase HPLC has been the subject of several recent articles and similar trends are apparent with peptide derivatives (29-31). 

In a binary eluent system (acetonitrile-water), an adsorbed organic phase with finite thickness and composition different from the bulk mobile phase is preferentially accumulated near the surface of the bonded phase. The organic layer accumulated near the bonded ligands could behave as a liquid stationary phase in reversed-phase HPLC, and it contributes to the overall analyte retention process. 

Retention of lonizible Analytes in Reversed-Phase HPLC... 

In reversed-phase HPLC with water/organic eluents, ionic interactions always play an important role in regard to analyte retention, solvation, ionic equilibria, and other processes. To some extent, chromatographic effects and practical use of ionic interactions have been discussed in the previous sections of this chapter. In this section the influence of the ionic additives in the mobile phase on the retention of ionic or ionizable analytes will be discussed. 

S. D. West, The prediction of reversed-phase HPLC retention indices and resolution as a function of solvent strength and selectivity,/. Chromatogr. Set. 25 (1987), 122-129 and S. D. West, Correlation of retention indices with resolution and selectivity in reversed-phase HPLC and GC, J. Chromatogr. Set. 27 (1989), 2-12. 

R. LoBrutto and Y. V. Kazakevich, Retention of ionizible components in reversed-phase HPLC, in S. Kromidas (ed.). Practical Problem Solving in HPLC, Wiley-VCH (2000), New York, pp. 122-158. 

S. M. C. Buckenmaier, D. V. McCalley, and M. R. Euerby, Rationalization of unusal changes in efficiency and retention with temperature shown for bases in reversed phase HPLC at intermediate pH, J. Chromatogr. A 1060 (2004), 117-126. 

The above-given Martin equation form the basis for the Kovats retention index system in gas chromatography as well as for several HPLC retention prediction schemes. It must be noted here that the relationships between retention parameters and carbon numbers are usually linear at some limited range of the aliphatic chain length up to 6-8 carbon atoms in reversed-phase HPLC [491. 

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