30 August / September 2016

Practical Effects of Sample Diluent on Peak Shape Using sub 2um Columns in Achiral SFC

by Victoria Coulthard*, Alex Brien

Reach Separations Ltd., BioCity Nottingham, Pennyfoot Street, Nottingham. NG1 1GF *Corresponding author:

It is well known that for optimal peak shape, maximum detection sensitivity, and highest observed chromatographic efficiency, analytes should be dissolved in sample diluent matching the initial mobile phase composition [1]. This laboratory best practice serves both reverse phase achiral and chiral liquid chromatography. The impact of sample diluent is particularly important in preparative chromatography where reproducible retention times are critical to enable stacking of injections. For chiral separations performed via supercritical fluid chromatography (SFC), the sample diluent effect is less pronounced than in reversed-phase HPLC, see Figure 1. It is possible, even on coated chiral stationary phases, to inject mixtures of dichloromethane (DCM) and methanol with little detrimental effect to the chromatography, or observing the expected deterioration in performance of the stationary phase.

of methanol was far more pronounced on the 50 mm length column than on the 100 mm length column, see Figures 2a and 2b. These results suggest the peak deformation is either inversely proportional to the amount of time spent on the column which is a function of the bed length or the percentage of modifier required to elute the peak, both of which are less for the 50mm column.

Figure 1: Racemate dissolved in 1:1 mixture of MeOH and DCM and 350 µL injected onto the SFC - into a modifier stream of methanol. An injection is made every 165 seconds, no distortion of peak shape is ob- served and the increase in co-solvent at the time of injection has no impact on the retention time of material already on the column [2].

When studying the effect of sample diluent on achiral ultra-performance SFC columns it is evident that it does have a major effect on chromatographic performance as previously reported [3]. This is something we have reproduced in our laboratory using the Acquity UPC2

with the Torus 2-PIC column.

Due to the physical nature of carbon dioxide at atmospheric conditions, it is impossible to use the mobile phase as an injection diluent in SFC. As with other studies, we overcame this challenge by mimicking the physical properties of super/subcritical CO2

by using

something of similar polarity, in this case, heptane.

We have carried out an experiment, incrementally increasing the injection volume of the same compound, 7-Diethylamino-4-

Column 1: 2.1 mm x 50 mm, 1.7 µm. Column 2: 2.1 mm x 100 mm, 1.7 µm.

In each case, a gradient method of 3% to 35% methanol in CO2

run over 1.25 minutes

(50 mm length column) and 2.5 minutes (100 mm length column) was performed.

The results from both columns arrived at the same conclusion, the methanol diluent provided the worst peak shape and heptane/ IPA provided the best. The detrimental effect

methyl-coumarin (1 mg/mL) in three different diluents; methanol, isopropyl alcohol (IPA) and a one-to-one mixture of heptane and IPA. In each case, multiple injections were carried out on two different dimensions of the Waters Torus 2-PIC:

As expected this data set confirms that peak shape and plate count can be optimised by using a non-polar diluent, see Figure 3. In this case the theoretical plate count, calculated as in Figure 4, was taken to be a measure of efficiency and therefore a judge of the quality of the chromatography. The most significant contributing factor to the theoretical number of plates in this case was the width at half peak height, peak distortion on the shorter column lead to half height peak widths of up to 8 seconds. A high number of theoretical plates and therefore efficiency is desired as the narrower the peaks the less selectivity required to give adequate resolution.

N = 5.545 (tR wh ) 2

N = number of theoretical plates tR

= retention time

Wh = peak width at half height Figure 4: Equation to calculate the theoretical number of plates [4].

To produce the most efficient chromatography, a diluent closely representing the mobile phase should be chosen. However, the often limited solubility of pharmaceutical compounds in heptane or even heptane/IPA mixtures

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