must instead be dissolved in pure organic co-solvent or a similar liquid is an important problem having strong impact on the eluted band profiles. We have found that there are principally three different reasons for peak distortion upon injection of a sample dissolved in the co-solvent. Only one of these is generally known, called ‘the plug effect’ and is due to the solvent strength – mobile phase mismatch [7]. We will give some examples of the ‘plug effects’ as well as the other putative distortion effects due to the injection in SFC.


The column employed was a Kromasil Diol 60 Angstrom, 5 micron (150 x 4.6 mm) and mobile phase was comprised of carbon dioxide with several different compositions of methanol (MeOH). The SFC system was a Waters UPC2

Figure 1. Schematic illustration of the principle for the (a) mixed-stream injection mode respective (b) the modifier-stream injection modes. The figure was adapted from Figure 1a and b in [10].

system (Waters Corporation,

Milford, MA, USA) equipped with a diode array detector (DAD) and a Waters single quadrupole mass spectrometer (SQD), using either APCI or ESI in positive mode. The injected solute peaks were measured with both the DAD and the SQD and was manually tuned for single ion recording to detect Deuterated MeOH molecules. The solutes injected were of analytical grade, the column temperature was 40°C, and the back pressure regulator (BPR) was set to 140 bar if not otherwise noted.

Results and Discussion

In SFC the sample cannot readily be dissolved in the CO2

based mobile phase,

so it is often dissolved in a strong organic solvent, or in the co-solvent itself. This presents a series of complications; the most obvious one being that the injection will result in solvent strength and mobile phase strength mismatches. The mismatches often result in more or less serious distortions of the eluted peak profiles. These combined effects are often described as ‘plug effects’ and are illustrated below. It is important before describing ‘plug effects’ to describe the two main injection modes in SFC.

Injection modes

In SFC there are two main injection techniques the so called mixed-stream injection mode and the modifier stream injection mode [7,8], respectively. Figure 1a shows the principle of the mixed-stream injection mode which is actually the same principle as in ordinary HPLC; the injection is performed just prior to the column, after that

the CO2 stream is mixed with the modifier

(i.e. the co-solvent). Figure 1b shows the principle of the modifier-stream injection mode; here the injection is made in the modifier-stream, which is then mixed with the CO2

sample solution had a major impact of the shapes of the eluted peaks.

stream (Figure 1b). The advantages and disadvantages of the respective technique were previously evaluated in preparative SFC by Miller and Sebastian [9] and by Yun and Rajendran [8]. However, the mixed- stream injection mode (Figure 1a) is the most commonly used injection mode in commercial SFC instruments.

Peak distortion due to the ‘plug effect’

Recently we investigated the origin of peak deformation due to injection solvent effects in SFC, using both systematic experiments and numerical modelling [10]. The experiments proved that the injection volume and the elution strength of the

Our experiments showed that the peak distortion without doubt results from the so called ‘plug effects’. Normally an SFC mobile phase contains 5-10% co-solvent in the main component, i.e. carbon dioxide. When the solute is dissolved in 100% co-solvent, the local mobile phase composition in the sample plug at the column inlet will have considerably larger elution power than the bulk mobile phase, causing strong deformations. This is especially the case for the larger injection volumes we systematically studied.

Figure 2a shows a series of injections made from a 0.25 g / L solution of antipyrine analysed on identical SFC systems but using the two different injection modes. Injection volumes were varied from 5 µL to 75 µL; the purple lines showed the resulting band profiles using the modifier stream injection mode and the black lines the resulting band

Figure 2. Comparisons between mixed-stream injections (black solid lines) and modifier-stream injection ns (purple solid lines). Injections of 5 µL, 30 µL and 75 µL sample containing antipyrine dissolved in MeOH (Left; 0.25 g/L; Right 100 g/L). It was compensated for the difference in dead volume between the two injection two techniques. The figure was adapted from Figure 2a and b in [10].

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