Mobilephase Elimination Sfcftir

All of the commonly used supercritical fluid mobile phases are gaseous at STP. Thus, analytes can be trapped in some way at the restrictor outlet (connected to the column outlet) as depressurization and evaporation of the mobile phase occur. This is the basis of the mobile-phase elimination approach. Griffith's research group was the first to demonstrate the potential of this methodology in 1986. They realized that this approach had a number of advantages over using a flow cell. Firstly, as most SFC applications were aimed at involatile materials, most of the analytes could be trapped on an IR transparent material at ambient temperature for FTIR analysis. Secondly, as the mobile phase could be easily removed, the complete condensed-phase IR spectrum could be measured without interference from mobile-phase absorbance bands. Thirdly, a large number of scans of the trapped analyte could also be added together to improve the signal-to-noise ratio significantly. Lastly, they recognized that the technique was potentially amenable to mobile phases (and modified mobile phases) that could not be used with flow cells.

Offline Studies

Initial work involved depositing each separated component on a moving glass plate on which a layer of powdered KCl had been laid down from a methanol slurry. A 1 mm i.d. silica packed column and 2% methanol in carbon dioxide was used to separate a mixture of quinones and diffuse reflectance spectra of the separated quinones were measured by moving the substrate into the focused beam from an FTIR spectrometer. Later work by the same group and others showed that it was not necessary to use a pow dered substrate and that superior results could be obtained using capillary SFC simply by depositing the eluted compounds on to a zinc selenide or potassium bromide window. The window could then be mounted on the sampling stage of an FTIR microscope and each spot located visually under the microscope. Once located, the transmittance IR spectrum of each spot could be measured by directing the focused FTIR beam through the sample within the microscope.

Important aspects of mobile-phase elimination SFC/FTIR are illustrated in Figure 5. With this approach, the mobile-phase flow from the end of the column is usually split to permit simultaneous detection by another means (for example, using flame ionization or UV absorbance detection) and thus permit identification of the deposition period. When an FID is used, the flow rates of each of the restrictors must be adjusted so that the FID detects the component first. The zinc selenide window can then be moved so that deposition takes place on a fresh area of the window.

Most early depositions were performed offline with the substrate stationary beneath the restrictor. These experiments allowed the shape and dimension of the deposit to be characterized and demonstrated that it was possible to deposit compounds emerging from a capillary SFC column as spots approximately 100 |im in diameter. From a spectroscopic standpoint, minimizing the spot diameter is the key to obtain the maximum FTIR sensitivity since, for a given sample quantity, the average thickness varies inversely with the cross-sectional area. Keeping the spot diameter as small as possible enables the analyte to be deposited as thickly as possible and results in greater IR absorbance according to the Beer-Lambert law.

Figure 4 Flow cell SFC/FTIR analysis of an UV-curing coating. (A) Gram-Schmidt reconstructed chromatogram. (B) Gram-Schmidt reconstructed chromatogram with added basis vector 346 to remove the baseline drift from the increased CO2 absorbance due to the mobile-phase pressure programming. (C) Selective FTIR detection of carbonyl stretching region of an aromatic ketone (1670-1690cm~1). (D) IR spectrum of photoinitiator detected in the 1670-1690cm-1 reconstructed chromatogram. Conditions; 10 m x50 |im i.d. open tubular column; poly(biphenylmethyl)siloxane stationary phase; 65°C; CO2; pressure programme from 100 to 250 atm at 2.5 atm min~1 followed by 15 min isobaric hold. (Reproduced with permission from Bartle et a/., 1989.)

Figure 4 Flow cell SFC/FTIR analysis of an UV-curing coating. (A) Gram-Schmidt reconstructed chromatogram. (B) Gram-Schmidt reconstructed chromatogram with added basis vector 346 to remove the baseline drift from the increased CO2 absorbance due to the mobile-phase pressure programming. (C) Selective FTIR detection of carbonyl stretching region of an aromatic ketone (1670-1690cm~1). (D) IR spectrum of photoinitiator detected in the 1670-1690cm-1 reconstructed chromatogram. Conditions; 10 m x50 |im i.d. open tubular column; poly(biphenylmethyl)siloxane stationary phase; 65°C; CO2; pressure programme from 100 to 250 atm at 2.5 atm min~1 followed by 15 min isobaric hold. (Reproduced with permission from Bartle et a/., 1989.)

In practice, deposition characteristics depend on the physical properties of the analyte, the temperature of the SFC restrictor (and the window) and the flow rate of the mobile phase leaving the restrictor. Most analytes that are solids can be successfully trapped on a window at room temperature, even though they may exit the restrictor in liquid form. In SFC, the restrictor must be heated to 100-250°C to prevent it becoming plugged with analytes as they come out of solution from the depressurizing mobile phase. Analytes that are liquid are often too volatile and do not solidify rapidly enough on a surface at ambient temperature; they may be blown off or spread out over the window surface by the expanding mobile

Figure 5 Mobile-phase elimination SFC/FTIR. (A) Offline approach: after deposition on a ZnSe window, the substrate is placed in the focused beam of an FTIR microscope and the spectrum of the eluite is measured with the window stationary. (B) Online approach: after deposition on the moving window, the eluite passes through the focused IR beam.

Figure 5 Mobile-phase elimination SFC/FTIR. (A) Offline approach: after deposition on a ZnSe window, the substrate is placed in the focused beam of an FTIR microscope and the spectrum of the eluite is measured with the window stationary. (B) Online approach: after deposition on the moving window, the eluite passes through the focused IR beam.

phase. This was demonstrated by one group who analysed 21 common polymer additives varying in molecular mass from 225 to 1178. Only Topanol OC was unretained on the window, due to its high vapour pressure. The other additives were either deposited as liquids or solids, with spot sizes that ranged from 200 to 300 |im in diameter. By cooling the window, it is possible to capture volatile compounds, but this significantly complicates an otherwise simple interface. High quality IR spectra were typically measured from a 100 ng deposit by adding 1000 scans using a quality IR microscope with an MCT detector. However, it is important to take a suitable background IR spectrum from an area of window where a blank deposit, made during a time when no analytes have eluted, has been collected. This is because the mobile phase can contain hydrocarbon or other contaminants from the pumping system or column that may be deposited along with the analytes. The IR spectrum of the pure analyte is obtained by subtracting the background IR spectrum from that of the crude analyte.

To date, the interface has been demonstrated by a number of groups, with samples containing phenolic carboxylic acids, steroids, polycyclic aromatic hydrocarbons, indoles, quinones and barbiturates. Most measurements have employed capillary columns for the separation. This is because the commonly used 50-100 |im i.d. open tubular columns operate at very low linear velocities and the gaseous flow rates produced by the expanding mobile phase are only in the region of 2-5 mL min"1. Because of this, application of the methodology to packed column separations using carbon dioxide and modified carbon dioxide has been questioned. It is indeed difficult to deposit analytes using conventional 4.6 mm i.d. packed columns, because the gaseous flow rates are simply too high (&500-1000 mL min"1). However, reducing the diameter of the packed column to 1 mm i.d. reduces the volume flow rate by a factor of (4.6)2, or 20 times, and allows successful analyte deposition. This was demonstrated for the analysis of sulfanilamides using a 1 mm i.d. Deltabond-CN packed column with 0.1% water in carbon dioxide as the mobile phase. It was possible to obtain high quality IR spectra from &120 ng sulfisoxazole.

Online Studies

The logical development of the offline work discussed above has been the commercialization of online mobile-phase elimination interfaces, in which deposition occurs on a moving window that then passes through the infrared beam. The Bio-Rad Tracer interface (originally designed for GC/FTIR) was adapted for use with SFC: a 50 |im i.d. fused silica transfer line was used with an integral restrictor fabricated at the end of the tube. The end of the restrictor was placed 75 |im from the ZnSe window (60x30 mm) and heated to 100°C by a cartridge heater. The ZnSe window was cooled to — 10°C with a methanol-dry ice mixture to trap compounds efficiently without condensing the mobile-phase. The speed of translating the deposition window was selected to maintain an appropriate compromise between chromatographic resolution and spectroscopic sensitivity. The best compromise was obtained when the distance moved by the window during the time equal to the full width at half height of the narrowest peak in the chromatogram was equal to the diameter of the spot deposited on the stationary window. For most of the SFC work, the window was moved at a rate of 100 immin"1. In order to remove the expanded mobile phase, 0.1 mTorr pressure was maintained in the interface using a 170 L s"1 turbo-molecular pump. This pumping speed was fast enough that virtually no absorption due to CO2 was observed at gaseous mobile-phase flow rates less than 5 mL min"1 at STP.

The lowest MIL reported to date for caffeine, using online direct deposition SFC/FTIR, is 600 pg. This is significantly lower than can be achieved using a flow

Figure 6 IR spectra measured from (A) 3ng, (B) 600pgand(C) 300 pg caffeine during online mobile-phase elimination SFC/FTIR. (Reproduced with permission from Norton and Griffiths, 1995.)
Figure 7 Chromatograms of six cholinesterase inhibitor pesticides. (A) SFC/FTIR Gram-Schmidt reconstructed chromatogram; (B) FID traces for the six pesticides injected individually. (Reproduced with permission from Norton and Griffiths, 1995.)

cell interface. The IR spectra measured from 3 ng, 600 pg and 300 pg caffeine are shown in Figure 6. The interface has been further demonstrated for online measurement of Gram-Schmidt and functional group reconstructed chromatograms of cholines-terase inhibitor pesticides (Figure 7) and polycyclic aromatic hydrocarbons using carbon dioxide and for triazine herbicides using carbon dioxide modified with 2% methanol. Clearly, the possibility of using modified mobile phases is one of the great advantages of the mobile-phase elimination approach.

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