Instrumentation for Packedcolumn SFC

The instrumentation used for packed-column SFC can be somewhat more complex than capillary SFC as shown in Figure 5. Most of the hardware, including injection valves, columns and some detectors, are the same or similar to that used for HPLC. Binary and ternary fluids are the norm, although low polarity solutes can often be eluted with pure carbon dioxide. Changing the mobile phase composition is the most effective way to change solute retention. Composition gradient elution is most common, although pressure, temperature and even flow can also be programmed. Selectivity is often most easily changed by changing the temperature. Pressure is a secondary control variable.

Figure 5 Instrumentation for packed-column SFC.

Columns Standard HPLC columns are used for packed-column SFC. The most common have inner diameters of 2 to 4.6 mm, and are 15 to 25 cm long. Both 1 mm and micropacked fused silica are also sometimes used. The most common packings are 3 to 5 |im diameter totally porous silica, although many other materials have been used. SFC is a 'normalphase' technique, making the use of more polar bonded phases commoner. The biggest change from HPLC is the growing use of longer packed columns, as much as 2.2 m in length.

Pumps The most common pumping systems in packed-column SFC use multiple high pressure reciprocating pumps operated as flow control devices. Since the main fluid is supplied in a high pressure gas cylinder, gradient switching valves cannot be used to mix modifier with the fluid in SFC. One high pressure pump is used to deliver the main fluid (SF pump) and another is used to pump modifier(s). Used in conjunction with a back-pressure regulator, properly designed pumps allow independent control of flow, composition and pressure.

The head of the SF pump is chilled to ensure the fluid is pumped as a liquid. However, even as a liquid, the fluids used still have a compressibility much higher than normal liquids. To accurately pump these fluids, the pumps need both an extended compressibility compensation range, and the ability to dynamically change compressibility compensation when the pressure and temperature (and composition) are changed. Slightly modified HPLC pumps usually have an inadequate range of compressibility compensation, and never dynamically compensate for the changes in compressibility which accompany pressure and/or composition changes. Using HPLC pumps for SFC results in highly inaccurate and non-reproducible flows and compositions.

Injection Injection is accomplished using standard HPLC injection valves operated in either fixed or partial loop modes. Care must be exercised in choosing the volume and polarity of the sample solvent. SFC is usually a normal-phase technique. Common sample solvents, like methanol, are stronger solvents than the mobile phase. If too much of such a polar solvent is injected, peaks can be distorted and broadened. Injection volumes up to 10 | L can usually, but not always, be tolerated. Smaller amounts of water can be directly injected but only under carefully chosen conditions. Decreasing the polarity of the sample solvent or injecting smaller amounts reduces or eliminates the problem.

Oven The most common temperature range for packed column SFC with binary or ternary fluids is 30

to 50°C, although both higher and lower temperatures are used. With pure carbon dioxide, the temperature is usually held at >60°C.

Since temperature adjustment often has dramatic impact on selectivity, control of the column temperature is more critical than in HPLC. For the separation of enantiomers, subambient temperatures are often desirable. Ovens similar to GC ovens with cryogenic cooling, Peltier heated/cooled ovens, and recirculating baths are all used.

Pressure control Pressure is controlled using a backpressure regulator mounted at the downstream end of the system. The BPR is used in conjunction with a pressure transducer and feedback loop. Early regulators were mechanical but today are electromechanical, allowing complex programming of the system outlet pressure. Several commercially available backpressure regulators have small internal volumes which allow peak collection after the device.

Detection Pure carbon dioxide and the FID and often the UV detector can be used to study fats, waxes, silicones, hydrocarbons and other low polarity solutes, where modified fluids are unusual.

Many other GC detectors are compatible with both pure and modified fluids and have been used with either packed or capillary columns. These include: the electron capture detector (ECD) for the study of explosives, flame retardants, pesticides, derivatives of fatty acids, etc.; the thermionic or nitrogen-phosphorus detector (TID or NPD) for the study of pesticides, caffeine in beverages, and many small drug molecules; the sulfur (petroleum, pesticides) and nitrogen chemiluminescence (similar applications to the NPD) detectors; the flame photometric detector (FPD)(petroleum), as well as others. These detectors are often used with packed columns by splitting off a small fraction of the total column effluent through a fixed restrictor.

There are many hundreds of applications of both pure and modified mobile phases using the UV/VIS detector in the speciality chemical, petroleum, pharmaceutical and agricultural chemical sectors. Standard HPLC UV detectors, including photodiode array detectors, need only a high pressure flow cell to become compatible with SFC (fluid density in the cell approaches 0.9-1 gcm"3). Carbon dioxide is completely transparent to below 190 nm. Mixed with acetonitrile or methanol, short wavelengths can be used to maximize detection limits.

Fluorescence detectors also require a high pressure flow cell, complicated by the need for light paths at 90° to each other. Several high pressure 'chiral' detectors are available for differentiating d and l isomers. Other detectors that have been successfully used with SFC include: mass spectrometry (MS), Fourier transform infrared (FTIR), and the evaporative light-scattering detectors (ELSD). Most atmospheric pressure ionization HPLC-MS interfaces work equally well, or better, as SFC-MS interfaces.

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