Analytical SFC

Figure 7.5 Separation of a cis/trans isomer mixture by SFC using 6% methanol, isocratic elution, (a) Analytical SFC separation. Conditions column 250 X 4.6 (i.d.) mm Berger NH2 flow rate 2.5 mbmin oven temperature 35°C nozzle temperature 40°C outlet pressure 120 bar sample concentration 5 mg/ml in methanol injection volume 5 pi UV 220 nm. (b) Preparative SFC separation. Conditions column 150 X 21.2 (i.d.) mm Berger NH2 flow rate 50 mPmin oven temperature 35°C nozzle temperature 60°C outlet pressure 100 bar sample concentration 50 mg/ml in methanol injection volume 1 ml UV 220 nm.

The separation was developed on an analytical SFC system (Series SF3 Gilson System) with a 4.6 x 250mm analytical column packed with the CSP Chiralcel OD. The impact of the organic modifier, operating temperature, and pressure was studied on analytical equipment. The retention time and selectivity change with the eluent composition (percentage of IPA), and these variations are presented in Figure 12.19. The back pressure of the column was set at 80 bar and the temperature at 20 °C. 

Analytical SFC units are perfectly suited for optimizing the chromatographic conditions to maximize the process throughput. The separation of TSO racemate developed on an analytical system was therefore successfully extrapolated to a pilot unit equipped with a 50-mm id DAC column (System Supersep 50, Novasep) and integrating a CO2 recycling loop. 

Very recently, the separation of polar analytes has also been performed by using pure water under subcritical conditions. Subcritical water has several unique characteristics. For example, the dielectric constant, surface tension, and viscosity of water are dramatically decreased by raising the water temperature while a moderate pressure is applied to keep water in the liquid state. At 200 -250°C, the values of these physical properties are similar to those of pure methanol or acetonitrile at ambient conditions. Therefore, subcritical water may be a potential mobile phase for polar analytes. SFC mobile phases other than CO2 are reviewed separately in this encyclopedia. 

A schematic of an analytical SFC is shown in Fig. 4. There are two high pressure pumps one to deliver carbon dioxide and the other to deliver normal liquids as modifier. The modifier pump delivers normal liquids and is, for all practical purposes, an HPLC pump. 

Many well-known chiral chromatographers (39 6) have published articles or reviews about the significant saving in time and effort possible using analytical SFC in place of HPLC, both in method development and in routine work. Most major pharmaceutical companies have appreciable numbers of SFCs for this type of analysis. 

The combination of high-capacity autosamplers and an analytical SFC pumping system allows the collection of purity data on up to eight 96-well plates per day with peak capacity as high as 40. A series of diasteriomers was successfully separated using slightly less aggressive conditions, as shown in Fig. 12. 

An enclosed collection cassette has four individual compartments, into which a robot places the various tubes/collection vials, as shown in Fig. 8. After a fraction is collected, the robot returns the fraction to the original position of the tube/vial. This design limits the user to no more than four fractions per injection. However, this hardware is intended to be used with analytical SFC-MS, which identifies the correct peak and its retention time. A spreadsheet is created which relates analytical retention time windows to the desired peak. The chromatography is directly scaled to the semiprep level and the retention time windows are passed to the semiprep hardware. In most cases, a single fraction is collected. The analytical SFC-MS is intended as a form of triage to eliminate as many marginal samples as possible to minimize the number of prep runs. With MS tracking, the intent is to collect one peak per injection. 

On the subject of sample preparation, analytical SFC can save the analyst considerable time, as illustrated by the SFC profile of the composition in a nutraceutical capsule containing sawtooth palmetto berry extract (Fig. 12). In this case, the extract was dissolved and diluted with a minimal amount of hexane and directly injected into the chromatograph. By density programming the CO2 mobile phase, a high-resolution chromatogram can be facilitated. The complexity of the sawtooth palmetto berry extract is apparent and... 

The SFC systems are grouped into two categories analytical SFC for chemical analysis and preparative SFC for scale-up chemical synthesis and purification. On a fundamental level, the instrumentation for SFC consists of the following (1) a fluid delivery system with high-pressure pumps to transport the sample in a mobile phase and to control the pressure (2) the column in a thermostat-controlled oven where the separation process occurs (3) a restrictor to maintain the high pressure in the column (4) a detection system and (5) a computer to control the system as well as to record the results (see Figure 9.5 as an example). In SFC the mobile phase... 

Your Answer to Chiral Separation and Lhiripcation, 2001 MultiGram Analytical SFC Why SFC. 

The purification of combinatorial libraries on a Berger system is deseribed by Farrell et al. at Pfizer for their parallel solution-phase syntheses. The overall process employs as well analytical SFC in combination with mass spectrometry and nitrogen chemiluminescence deteetion off-line of the preparative-scale SFC systems. Pre-purification analytical SFC/ MS/CLND allows the triage of samples for purification, and an in-house software package analyzes data for predicted quality based on an evaluation of UV and MS data for the potential of co-eluting peaks during purification. This same software package selects a collection time window for purification, which is necessary to limit the number of fractions per sample. This system accommodates the purification of samples up to 50 mg... 

Analytical screens are performed with both reverse-phase RP-HPLC and SFC isolation techniques. Analytical SFC should be screened first unless instrumentation availability or project background specifics dictate otherwise. Screening achiral column bonded phases varying in polarity and functionality against different mobile-phase solvent choices is effective for identifying analytical methods for the purpose of impurity isolation. There are currently many unique achiral SFC bonded phase column choices commercially available (2-ethyl pyridine, diethyl amino, dinitrophenyl, pyridine urea, diol, cyano, etc.). SFC column choice provides the most impact in manipulation of relative selectivity for individual... 

Although analytical SFC was demonstrated in the early 1960s, it has only been in recent years that the availability of adequate high resolution packed and capillary SFC columns and instrumentation has led to renewed interest in the technique. Plasma emission is a natural development because of its use in GC and HPLC. A surfatron MIP sustained in helium has been employed for SFC detection, giving sulfur-specific detection at 921.3 nm with a 25 pg s limit for thiophene [28]. An argon high efficiency MIP has been interfaced with packed column SFC and the separation and detection of ferrocene and derivatives achieved with iron specific detection. Methanol modifier concentrations to 5% were tolerated in the carbon dioxide mobile phase [29]. 

In spite of the significance of the publications cited the technique of preparative SFC has received relatively little attention when compared with analytical SFC. The method has the potential capability of replacing normal phase preparative HPLC because when coupled with supercritical fluid extraction (SFE) it carries out the extraction, preconcentration and chromatographic fractionation in a single nm according to Saito et al. [13]. 

In HPLC it is a simple matter to ensure that the solubility of the sample in the chosen mobile phase is adequate. In analytical SFC the injection of small amounts of foreign solvents can often be tolerated provided that the solvent chosen is of a similar polarity to the mobile phase. Furthermore, in analytical SFC high solubility of the analyte in the injection solvent and mobile phase is not so important because much lower concentrations are employed, whereas in preparative SFC this loading can represent a primary limitation on the production rate of the purified compound. 

Sensitivity for the eluted solute peak is often much larger than with analytical SFC. In fact, as often happens in preparative HPLC, the variable wavelength UV detector must be de-tuned away from the wavelength of maximum absorption of the eluting species to prevent overload of absorption. For the vast majority of cases when using modified carbon dioxide mobile phases there is no need to look further than the simple UV detector. 

The flame ionisation detector (FID) has been used in analytical SFC and could be used in preparative SFC by by-passing a very small fraction of the mobile phase flow through to the detector. Obviously the FID could not operate in a largely carbon dioxide atmosphere and would respond to many modifiers. The photoionisation detector (PID) has been used in analytical SFC but is better suited to highly sensitive detection of specific types of compound. 

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