Exchangeable Injection Port Cartridge for Gas Chromatographic Determination 01 Volatile Substances in Aqueous Fluids Gerhard Fteund Department of Medicine, University of Florida College of Medicine, Gainesville, Fla. 32601
VOLATILE SUBSTANCES in liquid biological samples frequently require preliminary separation procedures preceding gas chromatographic analysis, because organic material may contaminate the colurnn or interfere with detection, or because the sensitivity of the method is inadequate (1). Equilibration of liquilS with the gas phase outside the gas chromatograph (2), fractional distillation (3), solvent extraction (4), or water-retaining pre-columns (5) have been used to overcome these difficulties with variable success. It is the purpose of this communication to describe an exchangeable cartridge in the column entrance permitting direct on-column injection of biological samples. The exchangeable cartridge design facilitates continuous operation and easy cleaning. This in turn makes it practical to have small interior cartridge dimensions, which facilitate uniform heating of the injecting needle, minimal dilution of the sample by carrier gas, and rapid charring and retention of organic material.
I
I
1
II-t-D
Figure 1. Injection port
E;XPERIMENTAL Apparatus. The apparatus used was a Model 402 gas chromatograph ( F and M Scientific Co., Avondale, Pa.) equipped with hydrogen flame detectors and a MinneapolisHoneywell Model 15 single channel recorder. The Hamilton microsyringe of 10-11 capacity, Model 701N, was used to inject blood and aqueous solutions (Hamilton Corp., Whittier, Calif.). The column was 5 feet long, 0.125-inch 0.d. stainlesssteel, packed with ethylvinylbenzene (Porapak Q) 150-200 mesh (Wilkens Instrumcnt and Research, Inc., Walnut Creek, Calif.). The injection port WiiS made of an outer shell of 0.25-inch 0.d. stainless-steel tubing, 3 inches long, and a cartridge which was removable through the septum entrance for replacement and cleaning after every 30 injections (Figure 1). This cartridge was made of a thin stainless-steel tube, silver-soldered into the center of a wider tube. The outer tube consisted of 0.175-inch o.d., 2.5 inches long, stainless-steel tubing. The center tube was of identical length and had an i.d. of 0.060 inch. The top of the cartridge was drilled to a concave shape to facilitate the insertion of the injection needles. Operating Conditions. Temperatures in the injection port: 240" C ; column: 115' C ; detector: 180' C. Flow rates of nitrogen: 15 rnliminute; hydrogen: 25 rnliminute; air: 350 mliminute. Paper speed was 0.5 inchiminute. Procedure. Two-microliter blood, urine, or aqueous standards were injected with a 10-pl syringe by aspirating in the following order: 1.0 p1 of water (for rinsing), 1 pl of air, 1.4 1 1 of blood, (0.6 1 1 blood contained in the needle), and 1 pl of air (to prevent premature escape of the sample). After each injection the syringe was cleaned immediately by aspirating water or by burning out clotted blood with the flame of a match. ~
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(1) G. Machata, Mikrochirn. Acta, 4,691 (1962). (2) S. Natelson and R. L. Stellate, Microchem. J.,9, 245 (1965). (3) D. Lester, ANAL.CHEM., 36, 1810 (1964). (4) R. A. Davis, J . Forensic Sci., 11,205 (1966). (5) R. Bonnichsen and M'. Linturi, Acta Chem. Scand., 16, 1289 (1962).
A: B: C: D: E:
Swagelok tube fitting, 0.25 inch 0.d. Cartridge. Stainless-steel tubing, 0.25 inch. Swagelok reducer, 0.25-0.125 inch Column, 0.125 inch, 0.d.
The column was standardized with aqueous solutions of acetone, methanol, or other volatile compounds containing 5 ng/Ml, or more, and with 0.5 ml of 10-ppm propane in nitrogen (Matheson Corp., Rutherford, N. J,), injected wih a 1-ml gas-tight syringe (Hamilton). The peak heights were plotted against concentrations, and unknown concentrations were determined from the calibration curves. Blood samples were collected in Vacutainer tubes (Becton, Dickinson and Co., Rutherford, N. J.) containing Versene anticoagulant. Capillary blood was drawn after skin puncture directly into heparinized micro-hematocrit glass capillary tubes (Clay-Adams, Inc., N. Y . ) ,and sealed with clay after completion of the collection. The sealed part of the capillary tube was broken off immediately before the injection of the sample into the gas chromatograph. RESULTS AND DISCUSSION
The retention times of several volatile substances in minutes were as follows: propane, 2.5; methanol, 1.5; water, 3; ethanol, 6; acetone, 12; carbon disulfide, 24. Freshly prepared acetoacetic acid was quantitatively converted to acetone in the heat of the flash evaporator. Beta-hydroxybutyric acid, when injected directly into the flash heater, did not produce a measurable peak under the conditions of the experiment. Precision of the Method. A representative example of the results obtained with aqueous solutions of acetone is shown in Table I. The precision of the method using replicate determinations of aqueous acetone standards expressed as absolute standard deviation (S.D.) was 0.207-inch peak height (mean relative S.D., approximately 3 %). The recovery of acetone in aqueous solution was 99.6 f 2.6% (1 S.D., see VOL 39, NO. 4, APRIL 1967
545
Table I. Acetone in Aqueous Solution Acetone Concn., ng/d
Pl
injected
3
40
1 2
80
injected
Range: 1 attenuation
Peak height, inches
20
2
40
2
3.5 3.5 3.8 6.6 6.4 6.6 9.5 9.5 9.6 7.5 7.1 7.0 6.6 7.4 6.7 6.8 5.2 5.0 5.0 6.5 6.0 5.8 5.9 3.2 3.3 3.3 6.6 6.5 6.7 4.6 4.7 4.8 6.2 6.3 6.5
ng
60
2
40
2
80
4
3
120
8
4
160
8
1
2 3 4
80
8
160 240 3 20
8 16 16
-
Recovered Mean, ng
z
21
105.0
38
95.0
59
98.3
41
102.5
79
98.8
118
98.3
158
98.7
82
102.5
158
98.7
238
99.2
318
99.3
X
Standard deviation: 0.207 inch
Table I). The precision and accuracy of replicate determinations of methanol and ethanol in aqueous solution and blood were in the same range. The accuracy of the method was determined by the addition of aqueous solutions of acetone to serum. The peak height of acetone normally present in whols blood and serum blank was subtracted from the peak height obtained in the analysis of acetone added to blood. In blood frctm nonfasting healthy subjects this blank was usually below a peak height of 2 inches at attenuation 2, corresponding to
