62 August / September 2019
Chromatography Today Help Desk
The help desk has covered the issues of transferring and scaling HPLC methods in previous issues of Chromatography Today, however the issues associated with method transfer in other forms of chromatography has not been discussed. In this issue of Chromatography Today we will look at some of the issues associated with transferring assays when using Supercritical Fluid Chromatography (SFC).
SFC Method Transfer and Scaling Considerations Co-Solvent
To better understand the issues associated with method transfer in SFC it is first important to go through some of the basic properties of a supercritical fluid. The definition of a supercritical fluid is “any substance at a temperature or pressure above its critical point, where distinct gas and liquid phases do not exist, resulting in there being no transition points, i.e. boiling points”. Supercritical fluids have often been described as having higher diffusion than liquids and also greater solvation than gases, which would make them an ideal solvent for chromatographic separations, see Table 1. This statement, whilst being correct, perhaps oversimplifies the reality, as actually there is a continuum between the liquid and gas states and an analyst varying the temperature or pressure would move between the physical characteristics of a gas or a liquid without incurring the challenge of going through a phase transition.
Within SFC there are a variety of parameters that the separation scientist controls either directly through the instrumentation or indirectly as a consequence of varying another parameter. These variables are;
• Co-solvent fraction, typically % methanol • Temperature • Pressure • Viscosity • Diffusion • Density
Gas and liquid chromatographers will look at this list and relate to this, however in LC and GC, these parameters are more controlled, and their impact can be readily defined, and indeed there are a variety of popular software packages which aid the separation scientist to develop robust methods [2, 3].
Each of these parameters will be discussed in relation to the stability of the assay, as this will be indicative of how easy the assay will be to transfer or indeed to scale-up.
The most significant parameter within most chromatographic systems is the stationary phase, and this is also the case with SFC. It is, however, assumed that the stationary phase would not be a parameter readily altered during method transfer. Of the parameters that are affected during method transfer the most significant is the co-solvent fraction, for simplicity methanol will be considered to be the co-solvent throughout this article. A common relationship that is used [4-6], which relates the retention time to the volumetric methanol fraction, is given by;
Where;
tr t0
– retention time of solute peak – retention time of unretained peak
CM
– volumetric methanol fraction S, d – constants k0
– retention factor with no modifier In LC this relationship is often linear and given by;
Where; S – constant
It is evident from the effect that the co-solvent has on the elution time in SFC, that extra column volumes which cause a delay to the gradient reaching the column will potentially have a greater impact than in LC, where extra column dwell volumes have already been shown to cause issues with method transfer [7]. Thus, when transferring a method or scaling a method from analytical to preparative scale, it is important to be aware of the impact that the co-solvent can have on the elution time on individual components. In isocratic systems this will not be affected by the delay volume, however with gradient
Table 1. Properties of carbon dioxide in different physical states, liquid, gas and supercritical fluid [1]. Gas
Density (g/cm3 Viscosity (Pa-s)
) Diffusion coefficient (cm2 /s)
(0.6~2.0)×10-3 (1~3)×10-5 0.1~0.4
Liquid 0.6~1.6
(0.2~3.0)×10-3 (0.2~2.0)×10-5
Supercritical Fluid 0.2~0.9
(1~9)×10-5 (0.2~2.0)×10-3
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