Basics of Supercritical Fluid Chromatography

SFC Basics Course

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1 - What are Supercritical Fluids and the History of SFC

2 - SFC Instrumentation and Advantages of SFC

3 - SFC Analytical Operating Conditions Part I Stationary Phases

4 - SFC Analytical Operating Conditions Part 2 Mobile Phases and Other Parameters

5 - Preparative SFC

 

Welcome Back!

Welcome back to this SFC basics introduction course. We hope you have found it informative and useful to begin your journey into SFC. In the previous sessions, we have looked at the analytical advantages of SFC, the modules required to run an SFC, as well as the history of SFC and what makes a supercritical fluid. In this final part of the course, we will look at using SFC in a preparative system.

Preparative SFC

GC and HPLC are commonly used to pre-separate samples, but SFC can be used in the same way as well. Preparative separation by SFC offers the following advantages.

  1. (1) Posttreatment after Preparative Separation

Supercritical carbon dioxide evaporates at ambient temperature and pressure conditions, which eliminates the need for posttreatment.

  1. (2) Solvent Cost for Large Preparative Separation Quantities

Using inexpensive and environmentally-friendly CO2 can reduce the cost of purchasing and disposing of solvents.

  1. (3) Preparative Separation Recovery Rates

After preparative separation, HPLC requires posttreatment steps, such as solvent evaporation or concentration. In contrast, SFC solvents evaporate easily, which minimises the need for posttreatment and prevents component fragmentation or decomposition during posttreatment.

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  • When SFC is used for preparative separation, an organic solvent capable of dissolving the target components is sometimes added after column separation to prevent their precipitation within flow channels. Gas-liquid separators can then be employed to remove these solvents, and a  variety of such devices are currently being developed by various manufacturers.
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  • Similar to preparative HPLC, preparative SFC begins with analysis-scale optimisation before scaling up. This involves switching to a preparative column, adjusting the mobile phase flow rate, and modifying the sample injection volume. Equivalent separation can be achieved by increasing flow rate and injection volume in proportion to the column’s cross-sectional area. Fig. 1 shows an example of changing from an analysis scale to a preparative scale. By using a column with the same stationary phase, separation equivalent to the analysis scale can be achieved.

Analytical to Prep

Fig. 1 Migrating from analysis scale to preparative scale with SFC

Preparative LC is a technique used to purify samples for specific target components. It is used in a wide range of fields, including chemicals, pharmaceuticals, and food testing. Preparative LC serves as a powerful tool for achieving higher purity and recovery rate levels of target components, but it requires drying and powderising steps.

SFC can improve preparative workflow efficiency by significantly reducing the amount of work involved in the powderisation process after preparative purification. The Nexera UC product line includes three systems—a stacked fraction system intended for large-volume fractionation, a multi-fraction system for separating multiple peaks, and an analytical fraction system intended for analysis-scale fractionation.

These products also feature Shimadzu’s unique gas-liquid separator (patented) that inhibits sample dispersion and carryover to obtain high recovery rates. Therefore, excellent recovery rates can be achieved even for highly volatile compounds, such as the fragrance linalool, regardless of the flow rate or modifier concentration. The following sections describe the three product lines and the gas-liquid separator.

 

  • Stacked Fraction System

    This system is optimised for large volume fractionation that involves repeatedly injecting compounds with up to several components (Fig. 2). The FRS-40 injection and collection unit can function as both an injector and fraction collector for repeatedly injecting the same sample to collect gram-level preparative fractions. It can inject volumes up to 20 mL* and collect ten fractions. It supports flow rates from 10 to 150 mL/min and connecting columns with an internal diameter from 10 to 30 mm.

  • Stacked fraction system

    Fig. 2 Nexera UC Prep stacked fraction system

 

  • Multi-Fraction System

    This system is suitable for applications involving fractionation of multiple components in samples with many peaks detected, such as for impurities in pharmaceuticals (Fig. 3). Volumes up to 2 mL* can be injected using an autosampler that holds up to 162 samples (when using 1.5 mL vials). The FRC-40 SF fraction collector can collect up to 540 fractions (using 10 mL vials). It supports flow rates from 10 to 150 mL/min and connecting columns with an internal diameter from 10 to 30 mm.

  • Multi fraction system

    Fig. 3 Nexera UC Prep multi-fraction system

 

  • Analytical Fraction System

    This system enables analysis-scale fractionation for applications that only require fraction quantities up to several milligrams, such as for checking synthesis (Fig. 4). By connecting an FRC-40 SF fraction collector to the Nexera UC system, the same system can be used for applications ranging from determining analytical conditions by method scouting to preparative separation of about a few milligrams. With a maximum flow rate of 5 mL/min, it supports using analytical size columns.

  • Analytical fraction system

    Fig. 4 Nexera UC Analytical fraction system

 

  • LotusStreamTM Gas-Liquid Separator

    When carbon dioxide transitions from the supercritical fluid state to the gas state during preparative SFC, its volume immediately expands by about 500 times, which can cause eluate to splatter from the column, a factor leading to lower recovery rates. The newly developed LotusStreamTM (patented) gas-liquid separator (GLS) uses multiple flow channels to limit the flow rate without increasing the tubing diameter. As a result, the carbon dioxide is discharged externally and the liquid travels along the column and then drips directly below without the eluate splattering (Fig. 5). This improves the recovery rate without compromising on sample dispersion and carryover. There is also a LotusStreamTM GLS available for the analytical scale, which allows for fractionation of small volumes, even into vessels such as 1.5 mL vials. The volatile optical isomers of linalool in Fig. 6 illustrates high purity fractionation using the LotusStreamTM separator.

  • LotusStream Separator

    Fig. 5 Gas-liquid separation with and without the LotusStreamTM Separator

 

LotusStream Linalool

Fig. 6 Fractions of the optical isomers of linalool reanalysed after using the LotusStreamTM

 

Did you know?

Prep Workflow

The workflow for preparative analysis is similar between both HPLC and SFC modalities (Fig. 7). The sample is first chromatographed using a scouting gradient. This is then optimised to meet the separation criteria, which means adjusting the %B/min change, the temperature, the backpressure regulator settings and other operating parameters. Upon completion of the method on the analytical scale, a loading study should be performed to ensure there isn’t any issue with the chromatography on a larger scale based on sample concentration. Finally, the method can then be scaled up geometrically for the new column dimensions by adjusting the injection volume and flow rate so the compound of interest can be purified.

Prep-Workflow

Fig. 7 Simple flow diagram for the preparative process

Prep Considerations

  1. (1) Volume of CO2.

The amount of supercritical CO2 needs to be carefully controlled. It is important to have a CO2 monitor installed to detect gas leaks. This is relevant for both prep and analytical scale.

  1. (2) Stationary phase chemistry.

Ensure that the analytical scale stationary phase is available in the preparative scale format.

  1. (3) Loading studies.

It is important to perform loading studies from an analytical scale up to prep scale. It is important to know about the solubility of the sample in question. The injection volume is increased incrementally until acceptable resolution has been lost or loss of pure fractions.

  1. (4) Scaling.

The injection volume and flow rate are geometrically scaled to maintain peak shape.

 

So that is the final session in this introductory SFC course. We thank you again for your participation and wish you much success in the final test. Click on the link below to take you through to the quiz to gain your Certificate of Attendance.

If you have enjoyed this course, then feel free to inform your colleagues, employees, friends, and acquaintances who work with or have an interest in SFC who want to gain a basic understanding. The next start will be announced through our newsletter or on our homepage. We will also be looking at generating a preparative course. To be the first to hear about it, click here.

Your Shimadzu Chromatographic Team

 

References

1Cross-Pharma collaboration on the development and evaluation of a new mid-scale preparative supercritical fluid chromatography instrument, M. Biba, M. Wong, A. Akin, E.T. Manning, L. Schaffter, L. Miller, Y. Zhang, W. Farrell, J.O. DaSilva, L. Nogle, B. Hritzko, F. Riley, R.P. DePianta, K. Barry, D.A. Gao, E. Seest, M. Goel, L. Chung, J. Paulson, H. Lee, D.B. Moore, S. Dong, W. Leister, N. Fukushima, A. Sasaki, T. Lee, T. Iriki, M. Nishimura, M. Tomita, M. Owa, K. Tanaka, T. Shagawa, T.J. Moran, T. Bamba, C.J. Welch, Org. Proc. Res. Dev., 2020, 24, 1271-1280

 

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