Peak capacity in gradient reversed-phase liquid chromatography of biopolymers Theoretical and practical implications for the separation of oligonucleotides

Abstract Reversed-phase ultra-performance liquid chromatography was used for biopolymer separations in isocratic and gradient mode. The gradient elution mode was employed to estimate the optimal mobile phase flow rate to obtain the best column efficiency and the peak capacity for three classes of analytes: peptides, oligonucleotides and proteins. The results indicate that the flow rate of the Van Deemter optimum for 2.1 mm I.D. columns packed with a porous 1.7 m C 18 sorbent is below 0.2 mL/min for our analytes. However, the maximum peak capacity is achieved at flow rates between 0.15 and 1.0 mL/min, depending on the molecular weight of the analyte. The isocratic separation mode was utilized to measure the dependence of the retention factor on the mobile phase composition. Constants derived from isocratic experiments were utilized in a mathematical model based on gradient theory. Column peak capacity was predicted as a function of flow rate, gradient slope and column length. Predicted peak capacity trends were compared to experimental results

Estimation of the extent of the water-rich layer associated with the silica surface in hydrophilic interaction chromatography

The possible presence of a mobile phase layer rich in water on the surface of silica columns used under conditions typical in hydrophilic interaction chromatography was investigated by the injection of a small hydrophobic solute (benzene) using acetonitrile-water mobile phases of high organic content. Benzene does not partition into this layer and is thus partially excluded from the pores of the phase up to a water content of about 30%, after which hydrophobic retention of the solute on siloxane bonds is observed. In 100% acetonitrile, the retention volume of benzene was smaller than that estimated either by pycnometry or by calculation from the basic physical parameters of the column. This result might be attributable to the larger size of the benzene molecule: the elution volume of a molecule is the pore volume minus a surface layer half the diameter of the analyte molecule. However, some influence of strongly adsorbed water that remains on the surface of the phase even after extensive purging with dry acetonitrile cannot be entirely discounted. The results suggest that about 4-13% of the pore volume of a silica phase is occupied by a water-rich layer when using acetonitrile-water containing 95-70% (v/v) acetonitrile. © 2008 Elsevier B.V. All rights reserved

Investigation of the effect of pressure on retention of small molecules using reversed-phase ultra-high-pressure liquid chromatography

The effect of inlet pressure on the retention of a series of low molecular weight acids, bases and neutrals, was investigated at constant temperature in reversed-phase liquid chromatography using a commercial ultra-high-pressure system (Waters UPLC instrument). For neutral compounds, relatively small increases in retention factor of up to ∼12% for a pressure increase of 500 bar were noted; the largest values were obtained for polar solutes, or solutes of higher molecular weight. Ionisable acids and bases gave much larger increases in retention with pressure, in some cases as high as 50% for a pressure increase of 500 bar. Thus, such compounds could show increases in retention factor approaching 100% over the pressure range available in the commercial UPLC instrument. Due to these differential increases, significant selectivity effects can be obtained for mixtures of different types of solute merely by changing the pressure. © 2008 Elsevier B.V. All rights reserved

Further investigations of the effect of pressure on retention in ultra-high-pressure liquid chromatography

In this study, we investigated further the large increases in retention with pressure that we observed previously in RP-LC especially for ionised solutes. These findings were initially confirmed on a conventional silica C18 column, which gave extremely similar results to the hybrid C18 phase originally used. Large increases in retention factor of ∼50% for a pressure increase of 500 bar were also shown for high MW polar but neutral solutes. However, experiments with the same bases in ionised and non-ionised forms suggest that somewhat greater pressure-induced retention increases are found for ionised solutes. Retention increases with pressure were found to be considerably smaller for a C1 column compared with a C18 column; decreases in retention with increasing pressure were noted for ionised bases when using a bare silica column in the hydrophilic interaction chromatography (HILIC) mode. These observations are consistent with the partial loss of the solvation layer in RP-LC as the solute is forced into the hydrophobic environment of the stationary phase, and consequent reduction in the solute molar volume, while the water layer on the surface of a HILIC packing increases the hydration of a basic analyte. Finally, retention changes with pressure in RP-LC can also be observed at a mobile phase pH close to the solute pKa, due to changes in pKa with pressure. However, this effect has no influence on the results of most of our studies. © 2009 Elsevier B.V. All rights reserved

Gebler, Implications of column peak capacity on the separation of complex peptide mixtures in single- and two-dimensional high-performance liquid chromatography

Abstract Column peak capacity was utilized as a measure of column efficiency for gradient elution conditions. Peak capacity was evaluated experimentally for reversed-phase (RP) and cation-exchange high-performance liquid chromatography (HPLC) columns, and compared to the values predicted from RP-HPLC gradient theory. The model was found to be useful for the prediction of peak capacity and productivity in single-and two-dimensional (2D) chromatography. Both theoretical prediction and experimental data suggest that the number of peaks separated in HPLC reaches an upper limit, despite using highly efficient columns or very shallow gradients. The practical peak capacity value is about several hundred for state-of-the-art RP-HPLC columns. Doubling the column length (efficiency) improves the peak capacity by only 40%, and proportionally increases both the separation time and the backpressure. Similarly, extremely shallow gradients have a positive effect on the peak capacity, but analysis becomes unacceptably long. The model predicts that a 2D-HPLC peak capacity of 15,000 can be achieved in 8 h using multiple fraction collection in the first dimension followed by fast RP-HPLC gradients employing short, but efficient columns in the second dimension