Automated Gas Chromatographic Procedure to Analyze Volatile Organics in Water and Biol ogicaI Fluids B. J. Dowty, L. E. Green,’
and J. L. Laseter*
University of New Orleans, Department of Biological Sciences,Lakefront, New Orleans, La. 70 122
The gas chromatographic analysis of low molecular weight volatile organic compounds in body fluids and tissues has in recent years been found to be an efficacious tool in the study of certain clinical problems (1-5). The analysis of such volatile components has presented numerous analytical challenges. Clinical profiling of volatile body fluid constituents borrowed heavily on existing head-space technology used in flavor analysis (6).Efforts to adapt these techniques to the analysis of biological fluids have resulted in modifications of this technology. Pauling, Robinson, and co-workers, in connection with their studies in orthomolecular medicine, did some of the initial work in this area (7). Urine samples were heated and purged with nitrogen to displace volatile components. The compounds thus extracted into the nitrogen gas stream were concentrated in a cold trap held a t liquid nitrogen temperatures. Water vapor generated by heating the urine sample interfered with the procedure by freezing and plugging the cold trap. Later the head-space analysis technique was modified by insertion of a Chromosorb-101 trap prior to the cold trap to absorb some of the interfering water (8).The cold trap was connected to the injection system of the gas chromatograph, as before, and the components concentrated in this manner were then swept onto a steel capillary column for chromatographic resolution and detection. There was still room for improvement as adsorption on the Chromosorb was not limited exclusively to water. Further modification of these techniques resulted in the procedure which is currently in use for volatile analyses uide infra (1-5). Recently this type of analysis has proved to be very successful in the monitoring of compounds present in drinking water supplies (9-12). The procedure has allowed the analysis of volatile organics in the low-parts-per-billion range from 1 liter of tap water. Because thermal extraction has been shown to be a more efficient method for the study of low molecular weight components in water requiring smaller volumes and shorter extraction times, the EPA has recently adopted this type of methodology (13,14). I t was our desire to try to simplify the sample collection, data acquisition, and reduction process as much as possible in an effort to make the procedure more adaptable for incorporation into monitoring programs a t the state and local levels. At the same time, these modifications represented the first step in an effort to simplify analysis procedures to the extent that they would be practicable on a routine basis in a clinical chemistry laboratory. Previous Methodology Prior to Automation. In volatile analyses using Tenax adsorbent, volatile organics thermally displaced from the sample are swept through a condenser by an inert helium gas stream sparging the sample and onto Tenax adsorbent contained in a glass reservoir with a fritted disc base. All of the components of this glassware system are either joined by ground glass joints or ball and socket joints to form a leak-tight system. Conventionally, the gas chromatograph operator transfers the Tenax containing the adsorbed organics from the reservoir to a glass injection port liner. This liner is then placed in the injection port, springloaded, and held in place by a hex nut cap fitted with a Teflon gasket. The injection port is maintained at 200 OC, and the Hewlett-Packard, Chemical Applications Laboratory, Route 41 and Starr Road, Avondale, Pa. 19311. 946
ANALYTICAL CHEMISTRY, VOL. 48, NO. 6, MAY 1976
adsorbed organics are thus desorbed a t this elevated temperature. Carrier gas exiting from the injection port passes through a pre-column and exits into a 4-way valve housed in the chromatographic oven. Simultaneously with placement of the liner in the injection port, the precolumn is immersed in a dry ice-methanol bath. At that time, the valve is positioned to vent the carrier gas from the precolumn into the oven. After collection in this manner for a period of about 5 min, the valve in the chromatographic oven is switched to the second position to allow the gas flow to come from the precolumn onto the chromatographic column. Simultaneously, the dry icemethanol bath is removed and the temperature programming is initiated. The above-described method of analysis gives very good results, but it requires a high degree of skill on the part of the chromatograph operator to consistently coordinate activities and obtain highly reproducible results. T o circumvent this variable, an automated system of analysis has been devised which does not require sample manipulation by the operator. T o develop such a system, selected modifications were made to the Hewlett-Packard 5830 Reporting Gas Chromatograph. In addition, a digital processor incorporated into the gas chromatograph initiates all operation parameters and timed sequences, ensuring consistency from one analysis to the next.
Instrument Modifications and Methodology for Automated Volatile Analyses. The Hewlett-Packard 5830 Reporting Gas Chromatograph was easily modified to allow automated collection of volatile organics and their subsequent resolution by capillary chromatographic methods. This instrument is equipped with a digital processor and offers the advantage of being both a gas chromatograph and a data system. Commands establishing parameters of operation of the gas chromatograph and methods of data treatment are given via a keyboard. The printer-plotter records all keyboard commands and lists the analysis conditions as well as plotting the chromatogram. Events can be time-programmed and the printer-plotter makes a note of such events on the chromatogram. Basic Instrument Modifications. Figure 1 illustrates some of the GC system modifications which have been developed in this study. The glassware system (stripper) containing the sample to be analyzed, is coupled directly to the gas chromatograph via two automated valves and the Tenax adsorbent column. After the sample flask has been installed, all remaining operations are completely automatic, including sample stripping, trapping of volatiles, sample injection, analysis, and printing of the report. A three-way solenoid valve (V3) directs carrier gas from the “A” flow controller of the GC through the stripper and adsorber via Valves 1 and 2, which are maintained a t 200 “C in a heated compartment. The adsorber is a %inch 0.d. stainless steel tube packed with Tenax and plugged a t both ends with silylated glass wool. During the analysis cycle, the adsorber must be controlled a t three different temperatures and the valves must be positioned in three different configurations. These changes are made automatically by time programmed commands from the GC signal processor. Methodology. Figure 1 also illustrates the positions of
1
Step
S T R I P P I l l G A N 0 C O L L E CT 1 O N
Carrier G a s (A)
+
Stripper 1
I
Step
I
Carrier
2
I N J E C T I O N TO
G.C.
Carrier Gas
3
Step
Cor4 D 1 T I O 11 I N G A O S O R B E R
5834 GC
I
v1
Stripper
1
J
i
Carrier Ga S
I
1
Figure 1. Instrument configurations
Valves 1 , 2 , and 3 and the resultant gas flow patterns during the three stages of the analysis. During the stripping and collection cycle (Step 1) carrier gas from the "A" flow is directed by Valve 3 through the stripper and through the adsorbent column to vent. Carrier gas from the "B" flow controller flows via Valve 1 directly into the capillary column in the GC oven. During this time, the adsorber block is controlled at 35 "C, the printer-plotter chart drive is off, and the GC oven is controlled a t 40 "C. The stripping operation continues for 50 min, and then the oven temperature automatically is cooled to -20 "C by the addition of liquid nitrogen. At 60 min, the stripping operation is complete and the GC oven has equilibrated a t -20 "C. At this point Valves 1 and 2 are positioned as shown in Step 2 and the cooling air sup-
plied to the adsorber block is switched off. This causes the "B" carrier gas to backflush the trapped volatiles into the GC column as the adsorber block is heated rapidly to 200 "C. At the same time, the printer-plotter chart drive is started and the integrator is turned on by a positive Area Reject Command. Carrier gas "A" flow also is diverted from the stripper and the sample flask may be removed. At 65.0 min, Valve 1 and 2 are positioned as shown in Step 3. This causes carrier gas "A" to flow through the adsorber column to vent as the temperature is raised to 350 "C. In this manner, any residual heavy organics are stripped from the adsorber before the next analysis. The GC oven is held a t -20 "C for 10 min more; then it is programmed rapidly to 25 "C. A t 82.5 min, the oven is again programmed upward a t 2 "C/min until a temperature of 170 ANALYTICAL CHEMISTRY, VOL. 48,
NO. 6,
MAY 1976
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I LITER TAP WATER
w
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z
0 Q
cn w
a a W
0
a
0
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. .
TIME (MINUTES) Figure 2. Gas chromatographic resolution of volatile components isolated from 1 conditions described in the text.
"C is obtained. The oven remains a t this temperature until the end of the run. At 90.0 min, the adsorber conditioning is complete, and the adsorber block heater is turned off and cooling air is introduced to return the adsorber to 35 "C for the next analysis. Finally a t 160.0 min, the run is terminated automatically and the report is printed out. Table I. Reproducibility of Chromatographic Retention
Times
I. of tap water, 10 m l of blood, and 120 m l of urine. Chromatographic
Applications. To illustrate the usefulness of this technique, volatile organic compounds eluted from a 1-1. tap water sample, 10 ml of whole blood, and 120 ml of urine were analyzed. The results are depicted in Figure 2. Those samples were chosen for analysis because they represent the most active areas of research in volatile analyses a t this time. Most recently though, studies on low molecular weight components of tissue samples have been shown to be an area of great potential ( 2 ) , and they also could be analyzed using this automated procedure.
Retention time, min Trial 1 13.09 17.53 20.40 24.27
Trial 2 12.20 12.98 17.52 20.46 24.30
29.82
29.84
12.21
940
e
Range
Range X 100/mean
0.01
0.08
0.11 0.01 0.06 0.03 0.02
0.42 0.06 0.29 0.12 0.07
ANALYTICAL CHEMISTRY, VOL. 48, NO. 6, MAY 1976
RESULTS AND DISCUSSION Analysis of volatile components is a well-established field of endeavor having applications in the areas of flavor analyses (6),water analyses (9-14), monitoring of hemodialysis patients ( I ) , transplacental passage studies ( 3 ) ,diabetic metabolic aberration studies ( 4 , 5 ) , and tissue studies (2). The method of sample introduction into the gas chromatograph is more complicated than the conventional syringe injection,
vide ut supra. Manual insertion of the glass injection port liners eventually results in chipping and cracking. This annoyance is compounded by the difficulty in finding glass tubing of the exact dimensions for insertion into the injection block, as glass tubing often varies in diameter several hundredths of a mm even along the same length of tubing. Elimination of operator manipulation of the sample collection and injection steps and replacement with complete control of all timed events by a digital processor greatly enhances the reproducibility in analyses. The unique data treatment capabilities of this instrument, modified as shown, are notable: 1)retention times on all peaks are printed directly above the peak on the chromatogram; 2) an event marker signaling the beginning and end of integration of each peak is made on the chromatogram; 3) integration of peak areas is performed and the data can be expressed in terms of area percentage evaluations, or internal/external standard calculations can be made. Figure 2 illustrates several types of samples which are analyzed for volatile components to which the automated process has been applied. As can be seen, approximately 170 lowmolecular-weight constituents were resolved in the urine sample. This resolution is significantly better than has been previously reported in the literature. Rather than attribute this improved resolution to the chromatographic column employed in the study, it is more likely that the expanded range of temperature programming (-20 to 170 "C) produced the resultant improvement. Table I shows the repeatability of retention times of key peaks obtained from replicate analyses. In summary, automation of a volatile analysis procedure by modification of a commercially available gas chromatograph has resulted in enhanced reproducibility, elimination
of the inconvenience and loss of time due to manipulation and breakage of injection port liners, and improved chromatographic resolution as a result of expanded temperature programming capabilities. The procedure is now standardized to such a degree that it can be performed routinely in any laboratory.
ACKNOWLEDGMENT The authors thank K. Patchel for instrument modifications and N. Foster and K. Geer for technical assistance.
LITERATURE CITED (1) B. Dowty, R. Gonzalez, and J. L. Laseter, Biomed. Mass Spectrom., 2, 142 (1975). (2) I. R. Politzer, S. Githens. B. Dowty, and J. L. Laseter, J. Chromatogr. Sci., 13, 378 (1975). (3) B. Dowty, J. Storer. and J. L. Laseter, J. Ped. Res., in press. (4) A . Zlatkis. H. A. Lichtenstein, and A. Tishbee, Chromatographia, 6 (2). 67 (1973). (5) A . Zlatkis, W. Bertsch, H. A. Lichtenstein, A. Tishbee, F. Shunbo, H. M. Liebich, A. M. Coscia, and N. Fieischer. Anal. Chem., 45, 763 (1973). (6) R. A. Flath, R. R. Forrey. and R. Teranishi, J. Food Sci., 34, 382 (1969). (7) R. Teranishi, T. R. Mon, A. B. Robinson, P. Cary, and L. Pauling, Anal. Cbem., 44, 18 (1972). (8) K. E. Matsumoto, D. H. Partridge, A. B. Robinson, L. Pauling, R. A. Flath, T. R. Mon. and R. Teranishi, J. Chromatogr., 85, 31 (1973). (9) B. Dowty, D. Carlisle, J. L. Laseter, and J. Storer, Science, 187, 75 (1975). (10) B. Dowtyand J. L. Laseter. Anal. Lett., 8 ( l ) ,25 (1975). (11) B. Dowty, D. Carlisle, and J. L. Laseter, Environ. Sci. Techno/., 9, 8 (1975). (12) B. Dowty and J. L. Laseter, J. Am. Water Works Assoc., submitted (1975). (13) T. A. Beliar and J. J. Lichtenberg, J. Am. Water Works Assoc., 66, 739-744 (1974). (14) T. A. Bellar J. J. Lichtenberg, and R. C. Kroner, J. Am. Water Works Assoc., 66, 703-706 (1974).
RECEIVEDfor review November 25,1975. Accepted February 12, 1976.
Remote Terminal Compatible Magnetic Tape Cassette Data Recording Device Arden W. Forrey" and Walter R. Baker' Clinical Research Center, Harborview Medical Center, School of Medicine, University of Washington, 325 Ninth A venue, Seattle, Wash. 98 104
The process of collection of data from instruments is one important step in the subsequent activities of reduction and analysis of data, and the development of quality control procedures within either the research laboratory or the small clinical-analytical laboratory. The use of computers provides one method for these laboratories to manage these data. One of the most highly advertised of the computer approaches is the laboratory minicomputer, though programmable desktop calculators may also be employed'. Minicomputers, however, are best used in situations where a high volume of data, fast sampling times, or control loops are required. Many procedures in the laboratory cannot justify the expenditures for dedicated minicomputers because of the wide variety and infrequency of the procedures which they use. A number of devices have been on the market for the logging and/or storage of data, several of which are minicomputer based. Before the latest of these devices came on the market, there was no device selling for less than $3000 which was easily configured or easy to use in the laboratory for collecting data directly from laboratory instruments. Present address, M e d i t e k Labs, Inc., 2852 Fairview Avenue
East, Seattle, Wash. 98104.
The sole availability of suitable digital components is not a satisfactory answer to this problem for the many laboratories which have no facility for assembling instrumentation. The use of remote terminals and telecommunications input, however, allows the laboratory access to that power and speed of computation which is proportionate to the task for a large segment of these laboratories; another very useful and complementary method is to process laboratory data by programmable desktop calculators. One of the obstacles to wide use of a flexible approach to data calculation using these resources is the unavailability of a device which collects data easily, accurately, and rapidly, and yet is completely plug compatible with both standard remote terminals and common desktop calculators. This account reports the development of such a device which collects data from a wide variety of laboratory devices, formats it flexibly, and records it onto standard magnetic tape cassettes. When the cassette device is plugged into a standard interface on standard remote terminals, it is then able to transmit the stored data under the control of the laboratory-located remote terminal into a data file in a telecommunications-equipped computer system for later analysis. The data on the cassette can, alternatively, be read into a programmable calculator for local data reducANALYTICAL CHEMISTRY, VOL. 48, NO. 6, MAY 1976
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