Anal. Chem. 1989, 67, 961-965
961
Multidimensional Chromatography Using On-Line Microcolumn Liquid Chromatography and Pyrolysis Gas Chromatography for Polymer Characterization Hernan J. Cortes,* Gary L. Jewett, Curt D. Pfeiffer, Steve Martin, and Charles Smith The Dow Chemical Company, Analytical Sciences, 1897 Building, Midland, Michigan 48667
An on-line coupled microcolumn slze excluslon chromatography-pyrolysis gas chromatography system utliizing a 10-port valve and a glass interface for the analysls of nonvolatlle compounds Is described. Experimental variables, Including flow-rate stability, Interface temperature, split ratios, and reproduclbillty were studied. A quantitative example of the characterization of a styrene-acrylonltrlle copolymer is presented. Relatlve standard deviation found on the ratios of acrylonltriie and styrene generated upon pyrolysls across the molecular slze range was found to be 2.4%.
INTRODUCTION Multidimensional chromatography has been shown to be a powerful separation tool (l),especially when dealing with complex matrices that may require unattainably high theoretical plate counts for adequate resolution or when dealing with samples that require tedious cleanup steps prior to analysis. The combination of liquid chromatography (LC) and gas chromatography (GC) utilizing microcolumns for LC in an on-line mode (2-6) has been used primarily for the determination of trace components in complex matrices, where the micro-LC is used as a highly efficient cleanup step, and a section of the chromatogram containing the components of interest is transferred to a GC for further resolution and quantitation. A similar system has been used to separate components by class, followed by separation of individual components within each class (7). One limitation to the use of this technology is that it can only be applied to compounds that can be analyzed by GC; that is, they must be sufficiently volatile to be transported in the gaseous mobile phase. Nonvolatile or highly polar compounds can be analyzed by GC if they are chemically treated (derivatized) to convert them into a more suitable form (8). An example of on-line coupled LC-GC using postcolumn derivatization has recently been presented (9). Another alternative is the use of pyrolysis GC to examine the volatile pyrolysis fragments of a nonvolatile molecule. In the characterization of polymers, the combination of size exclusion chromatography (SEC) and pyrolysis gas chromatography enables the determination of average polymer composition as a function of molecular size and provides valuable information that can be used to understand polymer properties and polymerization chemistry. However, this type of information is difficult to obtain, since fractions eluting from a SEC system are usually collected manually, evaporated, redissolved in an appropriate solvent, and manually transferred to a pyrolysis probe via a syringe. This report describes an on-line coupled microcolumn (packed capillary) SEC/pyrolysis GC system utilizing a 10-port valve and a glass chamber interface. A block diagram of the system is presented in Figure 1. This initial work concentrates on the analytical instrumentation and provides a quantitative
example of its application to the characterization of a styrene-acrylonitrile copolymer.
EXPERIMENTAL SECTION Liquid Chromatography. The liquid chromatographicsystem consisted of an Isco p-LC 500 solvent delivery system operated at constant flow rate (Isco, Lincoln, NE), and a Jasco Uvidec V detector (Jasco International, Japan) equipped with a modified cell (10) whose illuminated volume was calculated as 6 nL from the capillary diameter and slit size. Detection wavelength used was 220 nm at 0.08AUFS. Injections were made with a Valco Model NI4W injection valve (Valco Instruments, Houston, TX). The injection volume was 200 nL. Columns were constructed of fused silica capillaries with an internal diameter of 250 pm (Polymicro technologies, Phoenix, AZ) and a porous ceramic bed support (11). The column length used was 50 cm. Columns were packed at 400 atm with Zorbax PSM-1000 of 7-pm particle diameter, as a sluny in acetonitrile (51). The column was evaluated and calibrated by using narrow distribution anionic polystyrene standards (Polymer Labs, England). Mobile phase was HPLC grade tetrahydrofuran (THF) (Fisher Scientific, Fairlawn, NJ) at a flow rate of 2.0 pL/min, which yielded a column head pressure of 85 atm. Interface. Transfer of the fractions of interest from the SEC system to the pyrolysis probe was made with a Valco 10-port valve Model NIlOWT equipped with a 1.0-pL sample loop and a 5.0-pL flush loop made of 50 pm i.d. fused silica tubing (Figure 2A). The valve was operated in the load position so that the micro-LC effluent flowed through the 1.0-pL loop and to waste until a section of the SEC chromatogram containing the components of interest was present in the loop. The 5.0-pL flush loop was filled manually with THF. (The flush loop volume was experimentallydetermined to be the minimum volume required to prevent carryover in subsequent injections.) At the appropriate time, determined by observing the detector response as represented in the recording device, the valve was switched to the inject position, so that carrier gas entered the valve and displaced the contents of the flush loop into the sample loop and through the transfer line (10 cm X 50-pm i.d. fused silica) to the surface of the pyrolysis ribbon. (Hewlett-Packard Model 18580A pyroprobe operated at 700 "C for a 1-s interval at a ramp rate of 20 OC/ms.) The pyrolysis probe was placed inside a glass chamber constructed from a 65 mm X 9 mm 0.d. X 7 mm i.d. tube and two 30 mm X 6 mm 0.d. x 0.4 mm i.d. tubes welded perpendicularly to each other (Figure 2B). Swagelok reducing unions, fittings, and graphite-Vespel ferrules (1/4 in. to 1/16 in. for inlet and outlet lines, and 3 / 4 in. to connect the glass chamber to the pyrolysis probe) were used to connect the fused silica transfer lines from the 10-port valve and to the outlet of the glass interface chamber. Secondary flow of carrier gas was introduced into the glass chamber via a two-hole ferrule placed at the inlet of the glass chamber parallel to the transfer line from the 10-port valve. The chamber was wrapped with heating tape and heated after transfer of the components of interest from the 10-port valve to 180 OC (outside surface temperature). The outlet from the glass chamber was connected to a four-port switching valve (Valco model N4WT) via a 10 cm X 250 pm fused silica capillary. In the vent mode, the carrier flowing from the chamber following introduction of the sample from the micro-LC was directed to atmosphere in order to allow rapid venting of the solvent while carrier gas was introduced from the valve to the rest of the system. In the inject
0003-2700/89/0361-0961$01.50/00 1989 American Chemical Society
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9 GC Oven 10 4.Port Switchirg Salve 11 sp1mer 12 Micro Melemg Valve
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Flgure 1. Block diagram of on-line microcolumn liquid chromatography-pyrolysis gas chromatography system. 6
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Flgure 3. Calibration curve for microcolumn SEC system: column, 50 cm X 250 pm i.d. fused silica packed with Zorbax PSM-1000 ( d p = 7 pm); eluent, THF; flow rate, 2.0 pL/min: detector, Jasco Uvidec V at 220 nm; injection size, 200 nL: standard concentration, 10 mg/mL.
supply carrier to the capillary GC column. The outlet of the four-port valve was connected to a low dead volume three-way tee used as a splitter (SGE, Melbourne, Australia) via a 10 cm X 250 pm fused silica section. The analytical GC column was connected to the outlet of the tee, and a piece of 250-pm fused silica tubing used as the split vent was connected to a micro metering valve to control the split ratios. Gas Chromatography. A Varian Model 3700 gas chromatograph (Varian Instruments, Walnut Creek, CA) with a flame ionization detector was used. The analytical column used was a 50 m X 0.20 mm i.d. 5% phenyl methyl silicone of 0.33 pm film thickness (Hewlett-Packard Instruments, Avondale, PA). Temperature program used was 50 to 240 "C at 10 OC/min. The temperature program was initiated at the time of pyrolysis, which took place after any residual solvent had eluted from the system. Once the transfer of the components of interest was made via the 10-port valve as described in the interface section, the solvent was vented through the outlet of the four-port valve. At the time that the 10-port valve was switched to allow introduction, the heating element was turned on. When the majority of the solvent had been removed, the four-port valve was switched to direct the flow from the glass chamber to the split tee and the analytical column. After elution of any remaining solvent, the residue was pyrolyzed and the GC temperature program was started. The carrier and auxiliary gas used was helium, and make-up gas to the detector was nitrogen at 20 mL/min.
RESULTS AND DISCUSSION
Flgure 2. Diagram of microcolumn liquid chromatography-pyrolysis gas chromatography interface: (A) 10-port switching valve and loop configuration; (B) glass chamber pyrolysis interface: (1) 10-port switching valve, (2) transfer capillary, (3) glass chamber, (4) pyrolysis ribbon, (5) heating tape, (6) transfer capillaries, (7) four-port switching valve, (8) split tee, (9) capillary GC column, (10) micrometering valve. (11) auxllary carrier gas. All components following the glass chamber were positioned within the GC oven. For further details see text.
mode, the carrier from the glass chamber was directed via the four-port valve through the rest of the system while the carrier flowing to the valve was directed to atmosphere. In this manner, rapid venting of the solvent was achieved while continuing to
The aim of this work was to develop an on-line LC/GC system that could analyze fractions from a liquid chromatograph that normally contain components which are nonvolatile, such as polymers. A micro-SEC system was chosen because the components of interest are diluted in much less volume when compared to a conventional SEC column, and this feature allows the investigation of a polymer in relatively few analyses while allowing reproducible deposition of the components of interest on the pyrolysis surface. Alternatively, microbore or conventional columns could be used; however, since the volume of effluent is much larger using these columns, only very small sections of the SEC effluent can be transferred to the pyrolysis interface. The reproducibility of the experiment when using conventional size columns is under investigation.
ANALYTICAL CHEMISTRY, VOL. 61, NO. 9,MAY 1, 1989
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Figure 4. Effect of mass injected on observed peak elution volume
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for styrene-acrylonitrlle copolymer. Other conditions are as in Figure 3.
The micro-SEC column was calibrated by use of anionic polystyrene standards, and the resulting graph is presented in Figure 3. It should be noted that the standard concentrations used (10 mg/mL) are higher than those typically used in SEC experiments and will have an effect on the resolution obtained, particularly in the high molecular weight region. However, the results obtained indicate that said concentrations do not destroy the separation. Column performance was estimated by measuring the asymmetry factor, (12) for a totally permeated molecule, in this case, toluene, and was found to be 1.04. Column efficiency was found to be 38000 plates/m obtained by measuring peak width at half height. The resolution factor (Da) obtained for this column was 0.034, considered to be as good or better than that attainable in a well-packed conventional column. The elution volume in SEC is known to increase with sample concentration due to the change with concentration in hydrodynamic volume of a polymer in solution. In addition, high sample concentrations may cause band broadening due to viscous streaming of the solute band. Figure 4 represents a plot of observed peak elution volume as a function of mass injected. For the operating conditions used, the amount of polymer injected on the micro-SEC column should not exceed 2 pg in order to obtain molecular weight distributions based on the calibration curve obtained for narrow distribution polystyrene standards. Extrapolation of the log molecular weight vs concentration curve to infinite dilution indicated a relative error of 5% at the peak molecular weight using the concentrations described. Although the error may be greater at the higher molecular weight end, for the sample analyzed in this work, the high molecular weight components represent a small portion of the total polymer distribution. It is known that instrumental flow rate fluctuations can cause large errors in the calculated sample molecular weight. Flow rate reproducibility when using microcolumns operated between 1 and 10 pL/min becomes a very important aspect of the experiment; however, little information exists on instrumental reproducibility at such low flow rates. Therefore, the flow rate reproducibility of the system was investigated by making repetitive injections of toluene over a 24-h period. At the operating flow rate used (2.0 pL/min), a relative standard deviation of 2.1% was obtained. In order to compensate for the flow rate fluctuation experienced, a small amount of toluene was added to the polymer solutions as an internal standard, which allowed corrections for the flow rate variability. Ideally, the total fraction pyrolyzed should be transferred to the GC column; however, it was found that the
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Figure 5. Effect of split ratios on peak shape and sensltivity: (A) analysis without split, (B) 1O:l split, (C) 20:l sptt, (D) 30:l split; column, 50 m X 0.2 mm i.d. 5% phenyl methyl silicone ( d , = 0.33 pm); oven
temperature, 50 to 240 O C at 10 OC/rnin: carrier, helium at 60 cm/s: detector, FID at 320 O C ; makeup, nitrogen at 20 mL/min: P, point at which pyrolysis was made: (1) styrene. interface must be swept at a high carrier gas flow rate in order to obtain relatively sharp peaks. Because of the high pressure generated by the GC column used, sufficiently high flow rates could not be obtained; therefore a split system was designed and investigated. Alternatively, the use of megabore columns, solid adsorbents, or cryogenic trapping may allow rapid transfer of the pyrolysis products to the capillary GC system without the need for splitting, maximizing sensitivity. Figure 5 represents chromatograms obtained on a polystyrene homopolymer analyzed at various split ratios. The sample was prepared by dissolving 70 mg of polymer in 10 mL of THF. A 1.0-pL aliquot of this solution was deposited on the pyrolysis ribbon by manually filling the sample loop using a syringe. Analysis without a split (Figure 5A) yielded a higher background noise level, large, tailing solvent peak, and the peak of interest (in this case styrene) was broad and split at the top. A split ratio of 1 0 1 resulted in much improved chromatography (Figure 5B). Increasing the split ratio to 201 and 30:l did not affect the chromatography, except that sensitivity was decreased (Figure 5C,D). For the rest of the study, split ratios were set at 1O:l. Interface Temperature. In classical pyrolysis GC, the interface is heated to minimize condensation of the fragments of interest and poor chromatography. The effect of the in-
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 9, MAY 1, 1989
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Figure 7. Microcoiumn size exclusion chromatogram of styreneacrylonitrile copolymer. Fractions transferred to the pyrolysis system are indicated. Conditions are as in Figure 3.
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Figure 8. Typical pyrolysis chromatogram of fraction from micro SEC system. Conditions are as in Figure 5: (1) acrylonitrile; (2) styrene.
I/ 5OoC 50°C IOOClmin 190°C Flgure 6. Effect of interface temperature: (A) 25 OC; Conditions are as in Figure 5.
504:
(B)180 OC.
terface temperature was investigated by chromatographing the pyrolysis products of the styrene homopolymer. The sample was prepared and deposited on the pyrolysis ribbon as described above. Figure 6A represents the chromatogram obtained at an interface temperature of 25 "C while Figure 6B represents the results obtained a t an interface temperature of 180 "C. The peak shapes improved considerably a t the higher interface temperature. However, when the transfer was made while the interface was heated to this temperature, the sample tended to splatter and deposit some of the polymer on the interface walls rather than on the pyrolysis ribbon, yielding reduced sensitivity and nonreproducible results. For the rest of the study, transfer of the fractions of interest was made a t room temperature, and once the transfer was completed, the interface was heated to 180 "C. The application of on-line SEC/pyrolysis GC to the characterization of a styrene-acrylonitrile copolymer is presented in Figures 7 and 8. Figure 7 represents the micro-SEC chromatogram obtained on the polymer prepared by dissolving 10 mg/mL in THF. The various fractions transferred to the interface are indicated on the chromatogram. Figure 8 represents the capillary GC chromatogram obtained after pyrolysis of fraction number 1 from Figure 7. The molecular
weight range of this fraction was from 1800 OOO to 450 OOO and this separation is typical of the capillary GC chromatograms obtained after pyrolysis of the sections across the molecular size distribution. The relative composition of copolymer eluted in each fraction was determined by measuring the area ratios of the acrylonitrile and styrene peaks generated. In our initial experiments, the relative standard deviation found on the area ratios was 6%, which was considered unacceptable. The variability of the preliminary data was attributed to the use of a platinum ribbon as the pyrolysis surface. When pyrolysis takes place, the ribbon flexes and seldom returns to its original position. When the fraction of interest is transferred from the switching valve to the ribbon, the fraction is not deposited a t exactly the same site as the previous fraction due to the orientation change of the ribbon. Since the ribbon does not heat evenly throughout its length, the transferred fraction experiences a different pyrolysis temperature, yielding variable results. In order to confine the transferred fraction to a reproducible area, the ribbon geometry was modified by creating a well in which the transferred fraction could be deposited. In addition, by adjusting the auxiliary carrier gas flow rate, the solvent evaporation rate was increased, minimizing the opportunity for the solvent to spread on the ribbon. With these modifications to the pyrolysis interface, the relative standard deviation found on the ratios obtained across the molecular size range was calculated as 2.4%, which is
Anal. Chem. 1989. 6 1 , 965-972
Table I
fraction of molecular wt range ( ~ 1 0 3 108-45 45-18.6 18.6-8.0 8.0-2.8 2.8-1.1
