Chromatography factors affecting retention

P. R. Haddad, F. Hao and B. K. Glod, Factors affecting retention of basic solutes in ion-exclusion chromatography using an anion-exchange column, J. Chromatogr. A., 671,3,1994. 

Twenty previously unreported polycyclic aromatic hydrocarbons (PAHs) were synthesized by condensing mixtures of 1-phenalenone-type ketones. These PAHs were isolated and identified by using field-ionization mass spectrometry, spectrofluorometry, and UV-visible absorbance spectrometry. The spectrometry and chromatography of these and other PAHs were compared. Correlations between the steric strain and the spectral valley depths between maximums, or chromatographic retention, were found. Steric effects were not the only factors affecting retention resonance stability also appeared to increase retention. 

The pH of the mobile phase will affect retention in liquid chromatography if the structure of the solute molecules in solution is affected by the pH. This will clearly be the case if the solute species may occur in a protonated or a non-protonated form, dependent on the pH. The pH may also affect the capacity factors of ions and neutral molecules for which this is not the case, but in this case the effects are usually small. 

Capacity parameters are not often used as primary optimization parameters in chromatography. Therefore, they are only included in table 3.10 in those cases in which they are used with some frequency. It should be noted, however, that changing one of the capacity factors usually involves the use of a completely different column and is therefore unattractive. Although changing the capacity parameters affects retention in an essentially predictable way, changing the column (packing material, film thickness, etc.) may give rise to unexpected second order phenomena. This is a second reason for which capacity parameters should not be recommended as primary optimization parameters. 

K. D. Bartle, M. L. Lee and S. A. Wise, Factors affecting the retention of polycyclic aromatic hydrocarbons in gas chromatography, Chromato-graphia, 1981, 14, 69-72. 

The data listed in Tables 5.3-5.6 are simply observations concerning the effect of eluent concentration and resin exchange capacity on the retention factors of metal cations. A more fundamental approach is to examine the effect of physical and chemical variations in both the mobile and stationary phases on chromatographic behavior of ions. The factors affecting selectivity of ion chromatography have been reviewed in a recent publication [11]. 

Column temperature is an important factor in separation of analytes in liquid chromatography. Column temperature can affect retention, selectivity, peak shape, and column pressure. UHPLC separations are frequently carried out at elevated column temperature, as this helps reduce the back pressure that results from operating columns packed with smaller particles at high linear velocities. 

In practice, experimental peaks can be affected by extracolumn retention and dispersion factors associated with the injector, connections, and any detector. For hnear chromatography conditions, the apparent response parameters are related to their corresponding true column value by... 

Liquid chromatography can be operated under mild conditions in terms of pH, ionic strength, polarity of liquid, and temperature. The apparatus used is simple in construction and easily scaled up. Moreover, many types of interaction between the adsorbent (the stationary phase) and solutes to be separated can be utilized, as shown in Table 11.1. Liquid chromatography can be operated isocratically, stepwise, and with gradient changes in the mobile phase composition. Since the performance of chromatography columns was discussed, with use of several models and on the basis of retention time and the width of elution curves, in Chapter 11, we will at this point discuss some of the factors that affect the performance of chromatography columns. 

The injector and the detector have dead volumes that affect the total retention volume. In gas phase chromatography, because the mobile phase is compressible, the flow rate measured at the end of the column has to be corrected by the compressibility factor J, which accounts for increased pressure at the head of the column (c.f. 2.2). 

The pressure can also affect the thermodjmamic and kinetic parameters of the phase equilibritun. Retention factors and presumably the other coefficients of the isotherms change with increasing pressure [25-30], As a first approximation, in a pressiue range wider than the one within which preparative chromatography is carried out (z.e., 0-200 atm), the retention factors of most compounds increase linearly with increasing pressure. The slope of the variation is proportional to the difference between the partial molar volumes in the mobile and stationary phases [26,29,30], The effect of the pressure on the separation factor in homologous series has been measured [26,29,30]. It is quite negligible in practice. 

Different compounds have variable ionization intensities, which are further affected by factors such as chromatographic retention times and suppression by compounds present in the analyte fluid and sample matrix, so it is essential to use isotope-labeled internal standards, which are physicochemically identical to the molecule of interest rather than structural analogs, to normalize for effects that can lead to erroneous quantification. Isotope-labeled standards also account for any differences arising out of sample preparation and absorptive loss due to selective binding on surfaces during chromatography (32). 

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