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elution the gradient delay volume will effectively change the co- solvent composition on the column from that programmed into to the SFC system and for different SFC systems this could result in different elution times for analytes. The nature of the relationship between retention time and co-solvent composition means that this will be more impactful in SFC compared to HPLC.
Temperature
The next most significant parameter is temperature, where the van’t Hoff relationship still applies, assuming that all other parameters are equal, however it should be stated that within SFC it has been noted by a range of authors that this relationship is only linear over a small temperature range [8, 9].
Viscosity Where;
K - retention factor, ΔS - entropy
T = absolute temperature,
ΔH = enthalpy (note negative term), R = Gas constant ø= phase ratio
Assuming that the phase ratio does not change with temperature, the result is a linear relationship between Ln(k) and 1/T. The gradient is indicative of either an exothermic or endothermic adsorption isotherm, meaning that the retention time can increase or decrease with increasing temperature. Since different compounds can have different enthalpies and entropies, it is feasible that at a certain temperature co-elution of the compounds can exist, and that increasing or decreasing the temperature will result in a separation occurring. This concept is particular useful to know when separating chiral compounds, where temperature is often used to enhance a separation [10, 11]. It should also be noted a large pressure drop across the column can result in a high temperature drop across the column, although this is limited for SFC to highly compressible areas in the temperature pressure phase space [1], which from a practical perspective when using co-solvents is not applicable.
Pressure
Pressure has been shown to affect the retention time of certain compounds in LC, however the examples within the academic literature are not common [12,13]. In SFC it is well know that the pressure can have a substantial impact on the retention time [14, 15], and can be used by the separation scientist to reduce analysis times. This effect is more pronounced at lower co-solvent compositions, and as an example Berger [16] showed that at 5% MeOH, retention is nearly halved when the BPR pressure is increased from 100 to 300 bar. However, at higher MeOH concentrations, pressure becomes progressively less important. The use of smaller particles and/or higher flow rates can results in higher pressure drops across the column suggesting that the assay may become more sensitive to smaller modifications that can occur in method transfer.
The viscosity of a fluid is affected by temperature, which in turn will affect the diffusion rates of analytes within the mobile phase. In liquid chromatography, this does affect the optimal flow rate but it is not impactful, however in SFC this has a greater implication in the retention time of individual analytes. Table 1 gives a summary of the typical properties of the three states off nature that are commonly employed within a chromatographic system. It can be seen that the difference in the viscosity between a liquid and a supercritical fluid is quoted as a factor of ten. It should be noted that this is very dependent on where in the phase space the parameters are chosen, and that the properties of a supercritical fluid should be considered as a continuum rather than being discrete values. This highlights one of the issues associated with the use of SFC in that the range of viscosities and diffusions that are present within a system can be very large and so consequently the system impact can be very pronounced, if care is not taken in considering the effects that the pressure drop associated with tubing etc. It should also be stated that in many cases though this is not an issue the analytical scientist needs to be aware of the potential consequences so that it can become an effective part of the separation scientist’s toolbox for troubleshooting.
Diffusion
One of the advantages of SFC is the relatively high diffusion rates than are observed in the physical state when compared to liquids. For small changes that may be observed when scaling or transferring a method the diffusion term is not impactful, however it should be noted that by varying the temperature or pressure and moving around the phase diagram that very different diffusion rates can be observed which will affect the fluid dynamics, with the dispersion being very dependent on the molecular diffusion rates.
Density
One of the most controversial parameters often highlighted as an important variable, particularly in earlier texts, is density, where early authors believed that altering the density could change the properties of the mobile phase from very non-polar to a polarity akin to isopropyl alcohol. One approach suggested that could be used to modify the density was the addition of small amounts of alcohol to the mobile phase. This, it was speculated, would modify the density but not the elutropic strength of the mobile phase, thus the retention time would alter due to the pressure differences. It was concluded that, actually, the elutropic strength of the solvent did indeed alter significantly with the addition of small amounts of additives, although it should also be stated that the retention time is affected by the density of the mobile phase, however not to the extent that early researchers believed.
Density, as a control parameter has been discussed at length in a previous edition of Chromatography Today. The conclusion is copied here as a summary of a very nice article by Berger on the topic [16].
“The relationship between density and viscosity of MeOH/ CO2
mixtures used in SFC is complex. In fact, at higher modifier concentrations, or higher pressures, the relationship is confused or essentially opposite to most users’ perceptions. This makes density less than useless, and, in fact, incorrect in determining retention or
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