24 May / June 2021

Evaluating an Underutilised Technique:

High Flow Rates to Accomplish Shallow Gradients in Less Time

by Mark A. Stone* and Timothy C. Koehn Bausch Health, Petaluma, California 94901, United States


In the separation of species which are structurally similar, such as the impurities of a drug compound, the use of shallow gradients is often beneficial. Shallow gradients can be obtained either by increasing the gradient time or the flow rate. The former is generally used as increasing the flow rate can result in excessive pressures and peak broadening. However, under the right circumstances, the alternative of using high flow rates becomes feasible, and this is quite beneficial as it allows the shallow gradient to be obtained with a shorter run time. In this work, a column packed with fused core particles of 2.7 µm diameter, run at elevated temperature, was used to generate a high-flow-rate-induced shallow gradient. The use of elevated temperatures typically results in a significant reduction of any shape selectivity that may be offered by the column. This is unfortunate as shape selectivity can also be quite valuable in the separation of related substances. In this work, we show that by use of columns containing a biphenyl stationary phase it was possible to maintain a reasonable degree of shape selectivity at elevated temperatures. Therefore, it was possible to improve the separation by reducing the steepness of the gradient without increasing the run time; and simultaneously, obtain a reasonable degree of shape selectivity. We argue that this would be less feasible with sub-2-um columns and hence the use of ultra-high pressure liquid chromatography may be overvalued in cases where shallow gradients are desired.

1. Introduction

In our laboratory we frequently develop methods for the quantitation of degradation products or process impurities of an active pharmaceutical ingredient. Such methods must be capable of separating compounds which are very similar in size, structure, and polarity. It has been our experience that methods which utilise very shallow gradients and which offer a degree of shape selectivity can be particularly beneficial in this regard. The shallower gradient can be accomplished by increasing either the gradient time or the flow rate. The latter is preferable if a shorter run time is desired. In this work, we evaluated columns packed with 2.7 µm fused-core particles, run at elevated temperatures, to allow shallow gradients to be obtained in less time. Furthermore, the use of a biphenyl stationary phase made it possible to, simultaneously, obtain a degree of shape selectivity in the separation, despite the elevated temperature. We tested this approach using one of the more challenging samples we have recently encountered in our laboratory.

2. Background

The significance of gradient steepness has been addressed in the literature [1-6] including at elevated temperatures [7]. From the standpoint of improving resolution, a specific definition of gradient steepness is relevant, which can be defined as the gradient rate (Δɸ/tG

) divided by the linear

velocity of the mobile phase. Mathematically speaking, we could express this as per relationship (1), where Δɸ is the fractional change in mobile phase strength during the gradient, tG

is the time over which the

gradient is executed, F is the mobile phase flow rate, and dc is the inner diameter of the chromatographic column.

interparticle porosity, and other terms are as defined above.

The difference is that in relationship (1) column length has been removed and the constant terms have been dropped so as to express a simple proportionality. The column length term was removed as a reduction in column length would not be beneficial for the present purpose, and could potentially compromise the separation [8-10].

It may be noted that this is very similar to gradient steepness as defined by linear solvent strength theory [6], presented as relationship (2); where L is the length of the column, S is a constant which increases with the molecular weight of the analyte, ε is the

From equation (1), it may be thought that reduction of Δɸ could be another approach to reducing the steepness of a gradient. However, this approach is generally not ideal as reductions in Δɸ may also have a negative effect on the separation. In this work, modifications to Δɸ will be made for a different purpose, which is to maintain an equivalent mobile phase elution strength when the column temperature is changed [11-12].

Considering the options of increasing the

Page 1  |  Page 2  |  Page 3  |  Page 4  |  Page 5  |  Page 6  |  Page 7  |  Page 8  |  Page 9  |  Page 10  |  Page 11  |  Page 12  |  Page 13  |  Page 14  |  Page 15  |  Page 16  |  Page 17  |  Page 18  |  Page 19  |  Page 20  |  Page 21  |  Page 22  |  Page 23  |  Page 24  |  Page 25  |  Page 26  |  Page 27  |  Page 28  |  Page 29  |  Page 30  |  Page 31  |  Page 32  |  Page 33  |  Page 34  |  Page 35  |  Page 36  |  Page 37  |  Page 38  |  Page 39  |  Page 40  |  Page 41  |  Page 42  |  Page 43  |  Page 44  |  Page 45  |  Page 46  |  Page 47  |  Page 48  |  Page 49  |  Page 50  |  Page 51  |  Page 52  |  Page 53  |  Page 54  |  Page 55  |  Page 56  |  Page 57  |  Page 58  |  Page 59  |  Page 60