Design Studies for a Biomedical Gas Chromatograph E. C. Homing, C. D. Pfaffenberger, and A. C. Moffatl Institute for Lipid Research, Baylor College of Medicine, Houston, Texas 77025 An instrument was constructed for the purpose of studying the parameters involved in the design of a new gas chromatographic separation system. Three stages are involved in the gas phase process: separation of solvent(s), reagent(s), and all compounds with MU value up to a point predetermined by the separation conditions; backflush transfer of the sample to be analyzed to an analytical column; and analytical separation of sample components by isothermal or temperature programming techniques. The separation of function of the usual GC column into two serial functions permits reconsideration of most of the elements of design which limit the performance and usefulness of present day gas chromatographs in biologic and medical studies.
THEBASIC DESIGN of currently available laboratory gas chromatographs is virtually the same as that used in the early experimental work of Martin and James. Technological improvements have been made in many ways. Electrically heated air baths have replaced the jackets heated by boiling liquids; sensitive and stable detectors are now routinely used; syringe injection into the closed system has replaced the dropping of a micropipet into the open chromatographic tube; and a variety of options, including the use of capillary columns and temperature and pressure programming, have been developed for modifying the chromatographic column and its mode of use. The basic design of the system has remained unchanged, however; the sample (liquid or support-carried) is introduced into a heated chamber o r zone of the chromatographic column, and the entire sample, usually consisting of solvent(s), reagent(s), and material(s) to be separated, is sent through the column and into the detector. This mode of operation is adequate for many purposes, but it is not particularly satisfactory for repetitive analyses of complex samples of biologic orgin. There are many reasons, familiar t o scientists whose experience lies in biologic and medical areas of work, for this circumstance. The substances to be separated are almost always in derivative form, and this means that a variety of reagents and solvents are usually present. Some of these may be beneficial with respect to column preservation, but others are often harmful. The drenching of a thin-layer (1-2z liquid phase) packing with solvent is not beneficial, and the filling of the system with solvent and reagent vapors which Present address, Home Office Central Research Establishment, Aldermaston, Reading, Berks., England. 2
ANALYTICAL CHEMISTRY, VOL. 44, NO. 1, JANUARY 1972
are removed by exponential dilution rather than by the chromatographic process is also undesirable. The analytical sample may amount to only a few micrograms o r less, but current sample handling procedures necessitate the use of a narrow range of liquid volumes (usually 1-5 pl). Metalor glass-supported sample introduction techniques have been advocated fo1 some purposes; in our experience these methods work perfectly with reference samples, but often poorly or not at all with samples of biologic origin. These difficulties have led to two divergent opinions about G C methods. In one view, the column provides little more than a way of introducing a sample into the detector. Great emphasis is placed o n prepurification of the sample by other chromatographic methods before the G C process is carried out. Analytical procedures of this kind usually have as their objective the estimation of a single compound, often through the use of a n electron capture detector. Another view is that the column should function as a separating device with as high a resolution as possible, and that prepurification should be limited to as few low resolution separation operations as possible. The objective is generally to carry out multicomponent “profile” analyses. F o r both groups, a necessity for repetitive analyses during a biologic study leads to difficulties: in one instance the prepurification steps with attendant losses becomes the least satisfactory part of the procedure, and in the other instance, column deterioration and unsatisfactory performance due to solvent-reagent flooding is a frequent occurrence. In order to determine if a design change would result in a superior instument for use in biologic studies, a n experimental GC unit was constructed for the purpose of studying gas phase separation problems associated with multicomponent analyses. A two-column system, with backflush transfer of the analytical sample, has been tested for functional soundness. Problems relating to automatic sample introduction and use of samples of biologic orgin are under study. EXPERIMENTAL
Instrument. Figure 1 shows a flow diagram for a threecolumn GC separation system, arranged for use with either hydrogen flame ionization detection or electron capture detection for analytical samples. A thermal conductivity detector was used to monitor the removal of solvent(s) and reagent(s) and separations occurring on Column I. In the initial studies reported here, the exit line from the analytical
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ANALYTICAL CHEMISTRY, VOL. 44, NO. 1, JANUARY 1972
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column (Column 11) was connected directly t o a hydrogen flame ionization detector (FID). Column I11 (another analytical column), four-port valve V3 and selector valve Sz were by-passed. Valves Vq, SI, and S3were not used. Mass gas flow monitors (Matheson Gas Products) were used to measure carrier gas flow (nitrogen) from sources N2 (A), Nz (B), and Nz (C) into Columns I and 11. Source N2 (D), for the reference side of the thermal conductivity detector, was connected through a flow restrictor to the line from N 2 (A). The gas pressures varied with the type of columns that were employed, and with the flow rates established for the separations, but were within 25-60 psi. Constant flow regulators (Brooks) were also installed in lines from N2 (A) and N2 (B) to aid in regulating gas flows during independent evaluation of Columns I and 11. The injection devices were glass tubes resembling the upper part of Barber-Colman 5000 GC columns with cylindrical septum closures for sample injection and gas flow introduction. The exit end was a Kovar seal, followed by a Swagelok connection to l/16-in. stainless steel connecting tubing to a four-port valve VI. Column I was a 6 f t X 4 mm (3.4 mm i.d.) glass U-tube with Kovar seal ends; the packings used were 1% G E GC SE-30 o n 80-100 mesh Gas Chrom P (Applied Science Laboratories) o r 4 Dexsil300 on 80-100 mesh Gas Chrom P. The packings were prepared according to our usual method, involving size-grading, acid-washing, silanization and solution coating ( I ) . The exit line from Column I was directed to a thermal conductivity detector (Carle Instruments) by way of valve VZ. Column I1 was a n analytical column. Two types were used: a 12 f t X 4 mm glass W-tube with Kovar ends, packed with a 1% SE-30 o n 100-120 mesh Gas Chrom P packing, and a 6 f t U-tube with Kovar ends, with a n initial 3 f t of 1-mm i.d. and a terminal 3 f t of 2-mm i.d. glass tubing, packed with 4% Dexsil 300 o n 80-100 mesh G a s Chrom P. BarberColman (now Nuclear-Chicago) Model 5000 heating ovens, with Barber-Colman heat control equipment, were used for Column I and Column 11. The temperatures of the valve oven and detector ovens were controlled separately. Injection temperatures were 260 "C; the F I D temperature was 300 "C; the TCD temperature was 280 "C; the valve oven temperature was 260-280 "C. All tubing connections were l/lG-in. stainless steel. The valves, which represent a significant technological improvement in construction and function, were by Valco, Inc. Figure 2 shows details of the valve oven and valves VI, V2, and SI. The valves were actuated by air pistons; a solenoid-controlled air valve with a single throw three-position switch was used to operate VI and VZ simultaneously. Mode 1-Stage 1 Operation. With valves VI and Vz in the position shown, a sample was injected. Column I was operated isothermally. The TCD response provided a record of solvent(s) and reagent(s) removal. The temperature and flow rate were adjusted so that alkane C19 was eluted in 5-6 min and Czain 9-10 min. A relatively fast flow rate was maintained (90-100 ml/min). The temperature was usually 135 o r 150 "C. Figure 3 shows separations obtained with a Dexsil 300 column when the sample was injected in three 2 0 4 portions over 40 sec and in one 60-pl portion over 40 sec. Sample volumes from 2 to 200 p1 were used in other experiments. Column I1 was tested separately under Mode 1 operation. Figure 4 shows a n analytical separation of alkanes in singlecolumn operation, with the usual solvent front effect. Carrier gas source N z (B) was used with flow rates of 40-60 ml/min (measured with the column at 200 "C). Mode 2-Stage 2 Operation. With valves VI and Vz in the broken-line position (Figure l ) , carrier gas from source N Z (C) was sent in reverse direction (backflush) through Column (1) E. C. Homing, W. J. A. VandenHeuvel, and B. G. Creech,
in "Methods of Biochemical Analysis," Vol. 11, D. Glick, Ed., Interscience, New York, N.Y., 1963. 4
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ANALYTICAL CHEMISTRY, VOL. 44, NO. 1, JANUARY 1972
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I and into Column 11. The temperature of Column I was raised to 280 "C as rapidly as possible, while Column I1 was a t 30-40 "C (oven cooling, room air circulation). The backflush flow was usually 60-80 ml/min. At the completion of the transfer, sources N Z(B) and N2 (C) were closed (solenoid valves), valves VI and VZwere restored to Mode 1 operation, and Column I1 was heated to 180 "C as rapidly as possible. Mode 1-Stage 3 Operation. With the valves in Mode 1 position, and with source N2 (B) open, a temperature programmed separation was carried out. Column I1 may also be operated isothermally if desired. Figure 5 contains the record of a separation showing the serial stages of operation. Samples. Solutions of n-alkanes Cll, c13, C16, Clp, C2,,, CZZ,C Z ~Cz8, , c32, and C36in toluene a t a concentration of 1 pg/pl, and of CISand CXin toluene at a concentration of 10 pg/pl, were employed. RESULTS AND DISCUSSION Aims and Principles of Operation. The immediate objective of this work was to determine the feasibility of operation of a two-column GC instrument, and to study the relationship between operating parameters and design. A more distant aim is the development of a n analytical system which will accept a sample of biologic origin, in any solvent, and provide multicomponent qualitative and quantitative chemical data at the termination of the analysis, without removal of the sample from the system and without human intervention during the analysis. The separation operation would be carried out in a sub-system. The present instrument is a prototype unit concerned only with design problems which must be studied in order to construct a useful separation device. Column I is a relatively low efficiency, high capacity column whose function is that of converting the initial sample, which would normally contain solvent(s), reagent@), and unwanted substances, along with the compounds under study, into a n analytical sample to be transferred t o Column I1 in the gas phase. The analytical samples may be defined in chromatographic terms as a cut of the original sample. The initial studies have been with hydrocarbon reference mixtures, and the cut has been defined as all substances with methylene unit (MU) values (2) between 20 and 36 for the phase used ( 2 ) E. C. Homing, M. G. Homing, N. Ikekawa, E. M. Chambaz, P. I. Jaakonmaki, and C. J. W. Brooks, J. Gas Cliromatogr., 5, 283 (1967).
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