Analyte HPLC retention

Analyte HPLC retention is a competitive process, and in an ideal form assuming only analyte-eluent competition for the stationary phase surface and in the absence of any secondary equilibria, one can write... 

When using microbial products for mammalian metabolite identification, it is suggested to compare all the analytical data available. For example, slight differences in MS2 or MS3 spectra may indicate that the microbial products are not the same as the mammalian metabolite. Owing to matrix effects, HPLC retention time often varies from run to run, so it is good practice to spike a comparable amount of purified microbial product into the in vitro, in vivo or purified samples that contain the mammalian metabolite of interest. If the microbial metabolite and the mammalian metabolite are the same compound, then they should co-elute under different HPLC conditions, including different solvent pH, and the MS and/or UV peak area would increase accordingly. 

The decolourization of the azo dye amaranth was also investigated using atomic hydrogen permeating through a Pt-modified palladized Pd sheet electrode. The decolouration products were separated by RP-HPLC in an ODS column. The isocratic mobile phase was 0.1 M aqueous orthophosphoric acid. The flow rate was 1.2 X 10 2 cm3/s and decomposition products were detected at 236 nm. The RP-HPLC system separated two analytes with retention times of 3.4 and 4.5 min, as demonstrated in Fig. 3.47. The peaks were... 

HPLC retention times Partition coefficients can also be derived from retention times in high-pressure liquid chromatography (HPLC) analyses This approach provides some experimental advantages that simplify the analytical procedures and allow the handling of mixtures... 

First, we will explore the three fundamental factors in HPLC retention, selectivity, and efficiency. These three factors ultimately control the separation (resolution) of the analyte(s). We will then discuss the van Deemter equation and demonstrate how the particle diameter of the packing material and flow rate affect column efficiencies. 

If the HPLC mobile phase is operated close to the pA of any solute or if an acidic or basic buffer is used in the mobile phase, the effects of temperature on retention can be dramatic and unpredicted. This can often be exploited to achieve dramatic changes in the separation factor for specific solutes. Likewise, the most predictable behavior with temperature occurs when one operates with mobile phase pH values far from the pA s of the analytes [10], Retention of bases sometimes increase as temperature is increased, presumable due to a shift from the protonated to the unprotonated form as the temperature increases. As noted by Tran et al. [26], temperature had the greatest effect on the separation of acidic compounds in low-pH mobile phases and on basic compounds in high-pH mobile phases. McCalley [27] noted anomalous changes in retention for bases due to variations in their pA s with temperature and also noted that lower flow rates were needed for optimal efficiency. 

In a study presented by Jinno et al. [124], packed column capillary electrochromatography, open-tubular CEC, and microcolumn liquid chromatography using a cholesteryl silica bonded phase have been studied to compare the retention behavior for benzodiazepines. The results indicated that CEC was a promising method, as it yielded better resolution and faster analysis than microcolumn LC for benzodiazepines. Similar selectivity to HPLC was noted, except for a few solutes that were charged under the separation conditions. Columns packed with the ODS and cholesteryl phases were compared and showed totally different migration orders of the analytes. The retention on the cholesteryl silica sta-... 

An alternative (or just different) description of HPLC retention is based on consideration of the adsorption process instead of partitioning. Adsorption is a process of the analyte concentrational variation (positive or negative) at the interface as a result of the influence of the surface forces. Physical interface between contacting phases (solid adsorbent and liquid mobile phase) is not the same as its mathematical interpretation. The physical interface has certain thickness because the variation of the chemical potential can have very sharp change, but it could not have a break in its derivative at the transition point through the interface. The interface could be considered to have a thickness of one or two monomolecular layers, and in RPLC with chemically modified adsorbents the bonded layer is a monomolecular layer that is more correctly... 

Another approach to the expression of the analyte adsorbed on the surface is based on the consideration of the surface specific quantity which has been accumulated on the surface in excess to the equilibrium concentration of the same analyte in bulk solution. This allows avoiding an introduction of any model of adsorbed layer as shown later, it is a fruitful approach for the description of HPLC retention. 

R. LoBrutto, A. Jones, and Y. V. Kazakevich, Effect of Counteranion Concentration on HPLC Retention of Protonated Basic Analytes, J. Chromatogr. A 913 (2001), 191-198. 

Adsorbent surface area, pore volume, and pore diameter are the properties of significant importance. HPLC retention is generally proportional to the surface area accessible for a given analyte (Chapter 2). Surface area accessibility is dependent on the analyte molecular size, adsorbent pore diameter, and pore size distribution. 

Surface area of HPLC adsorbents is probably the most important parameter, although it is almost never used or accounted for in everyday practical chromatographic work. As shown in the theory chapter (see Chapter 2), HPLC retention is proportional to the adsorbent surface area. The higher the surface area, the greater the analyte retention, although as we discuss later, depending on the surface geometry, analytes of a different molecular size could effectively see different surface areas on the same adsorbent. 

HPLC retention expressed either in absolute values (T ) or in relative terms (retention factor) is a complex function of a multitude of parameters. Type of bonded phase is only one of them. Surface area of base material, pore size, bonding density, end-capping, and even the column history all can significantly affect analyte retention. 

HPLC retention is sometimes explained as the result of competitive interactions of the analyte and eluent molecules with the stationary phase. From this point of view the stronger the eluent interactions with the adsorbent surface, the lower the analyte retention, which leads to the term eluent strength. ... 

Many attempts to correlate the analyte structure with its HPLC behavior have been made in the past [4-6], The Quantitative structure-retention relationships (QSRR) theory was introduced as a theoretical approach for the prediction of HPLC retention in combination with the Abraham and co-workers adaptation of the linear solvation energy relationship (LSER) theory to chromatographic retention [7,8],... 

The basis of all these theories is the assumption of the energetic additivity of interactions of analyte structural fragments with the mobile phase and the stationary phase, and the assumption of a single-process partitioning-type HPLC retention mechanism. These assumptions allow mathematical representation of the logarithm of retention factor as a linear function of most continuous parameters (see Chapter 2). Unfortunately, these coefficients are mainly empirical, and usually proper description of the analyte retention behavior is acceptable only if the coefficients are obtained for structurally similar components on the same column and employing the same mobile phase. 

A stainless column packed with Lichrospher lOORP-18 was used to analyze Acanthoside-D from the solvent extraction. The composition of mobile phase in analytical HPLC was experimentally determined and it was water/acetonitrile/methanoi=80/14/6 vol.%. From the chromatogram, retention time of Acanthoside-D was found to be 12 min. Figure 1 shows the analysis of Acanthoside-D from the extraction of the trunk of Acanthopanax senticosus. The flow rate of mobile phase and injection volume were 1 nt(/min and 20pl, respectively. 

HPLC retention data for QSRR analysis are usually obtained by measuring log at several eluent compositions (isocratic conditions) and then extrapolating the dependence of log on a binary eluent composition to a fixed mobile phase composition, common for all the analytes studied, based on the Soczewinski-Snyder model ... 

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