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prevents the use of these solvents as diluent and as such; a more practical approach is required.


If we ignore the difference in N between the two columns (the difference in retention factor makes this direct comparison unfair) we can compare the similarities by analysing the results in Figure 5:


• Deterioration in theoretical plate count is observed as the injection volume increases, this is true for all three diluents analysed on both columns.


Figure 2a


• On both columns, the effect is most pronounced when injecting in methanol and least in heptane/IPA.


• The effect of using IPA as a diluent is only marginally worse at larger injection volumes than using a mixture of heptane/IPA.


Figure 2b


Figure 2: Comparison of increasing injection volume (0.2 (Black), 0.4 (Blue), 0.8 (Green), 1.6 (Aqua), and 3.2µL (Pink)) on a 50mm (2a) and 100mm (2b) x 2.1 mm, 1.7µm column, using methanol as the diluent.


In conclusion, injection diluent does play a major role in peak deformation when using sub 2 µm ultra-performance SFC columns. This effect can be minimised by using a solvent of similar polarity to the gradient starting conditions, although this is not always possible or practical due to poor solubility.


Compromises must be made regarding:


• Column length versus run time, ultra- performance SFC is usually chosen due to the possibility of using very short gradient methods for screening and scouting, so a 50 mm column may be essential.


• The concentration of the sample versus the volume to be injected. Often dependant on the solubility in the diluent.


One solution we have found to enable a ‘one size fits most’ approach is dissolve the sample at ~1 mg/mL in IPA and inject 0.5 -1.0 µL on a 100 mm column, running a 2.5 minute gradient. Although not the scientifically best approach it is the most practical for our laboratory.


Figure 3: A series of 0.2 (Aqua), 0.4 (Pink), 0.8 (Green), 1.6 (Blue) and 3.2µL (Black) injections of Diethylamino-4-methyl-coumarin (1 mg/mL in heptane/IPA) on 2.1 mm x 50 mm, 1.7 µm.


For information: Reach Separations is an outsource chromatographic purification laboratory specialising in both achiral and chiral separations of small molecules. From their well-equipped labs in Nottingham (UK) they are keen advocates of the benefits of SFC particularly with its growing reputation for achiral separations.


References:


[1] J.Ruta, S.Rudaz J. Chromatogr. A, 1217 (2010) 8230-8240


[2] C.White, J. Chromatogr. A, 1074 (2005) 163-173


[3] J.N.Fairchild et al, LCGC North America, Vol. 3, Iss. 4, 326-333


Figure 5: Number of theoretical plates (N) plotted against injection volume for the three diluents trialled.


[4] Agilent Technologies https://www.agilent.com/ cs/library/Support/Documents/f39250232446.pdf


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