Anal. Chem. 1980, 52, 1003-1005
30
*0
50
63
-0
.io03
matograph, where the carrier gas may be directed on line and/or through the pyrolyzer with quick switching. Injections may be performed with either the micropyrolyzer or a normal syringe without adjustments to the system. Thus extracts (e.g., of biogenic amines, drugs) may be analyzed in rapid succession by either or both application methods, and reference compounds of suspected degradation products may be injected between pyrolyses. T h e small pyrolysis chamber (less t h a n 0.5 mL) minimizes peak broadening and the small stainless steel body provides relatively fast heat exchange (heating and cooling). Four analyses per hour of ACh and Ch (as its butyryl ester) using Pyr-GC-FID and eight analyses per hour using Pyr-GC-CI-MID can easily be performed. For the quantitative analysis of other nonvolatile molecules (e.g., drugs, small biomolecules) using appropriate internal standards (preferably homologues), this type of simple a n d inexpensive pyrolyzer should also be applicable.
I 80
diperes
Figure 3. (a) Influence of platinum wire length (+ 18 mm, A 15 mm, 0 10 mm) and current intensity on ACh response. (b) Influence of current intensity on peak height ratios ( 0 ,0 )using a 15-mm long wire
ACKNOWLEDGMENT We thank H. Dollenmeier for helpful discussions on the electronics of our system and F. Lamprecht (Memo AG, Wallisellen, Switzerland) for the use of a high temperature thermocouple.
constant (ACh/PCh) or vary only slightly (BCh/BMCh) over quite a wide current range (Figure 3). T h e temperature on t h e surface of the Pt wire, when applying optimum current for a given wire, was measured with a 0.5-mm 0.d. NiCr-Ni thermocouple (Tastotherm D 1200, BT-l202d, Gulton, Frankfurt, GFR) to be 330-370 "C. T h e thermal demethylation of choline esters begins a t about 200 "C, and temperatures of 220-250 "C have been used for direct inlet-MS (12) or slow pyrolysis on a glass probe before GC-MS (8). With the filament transformer used, the temperature rise time (TRT) was 5-7 s. A faster T R T could be attained by connecting a capacitor discharge circuit (15)to the filament, but for the determination of choline esters it is not required. T h e upper temperature limit for the Pt filaments was measured with the NiCr-Ni thermocouple to be about 1150 "C (7.5 A with a 15 mm X 0.3-mm 0.d. wire). By increasing the temperature (amperage) further, filaments broke. By optimal pyrolytic conditions, the detection limit for ACh and the butyryl derivative of Ch is 5 ng of the QA cation using a n FID detector and 10 pg using CI-MID (12) with packed glass columns. A further increase in sensitivity by the use of open tubular glass columns is in progress. T h e advantages of the syringe micropyrolyzer here described may be summarized as follows. T h e total micropyrolyzer assembly including electrical current source may be constructed a t a cost of about 10% of a commercial one. It is easy to manipulate and may be used with any gas chro-
LITERATURE CITED (1) Irwin, W. J.; Slack, J. A. Analyst (London) 1978, 103, 673-704. (2) "Analytical Pyrolysis", Jones, C. E R., Cramers, C. A., Eds.; Elsevier: New York, 1977. (3) Irwin, W. J J . Anal. Appl. Fyrol. 1979, 7 , 3-25, 89-122. (4) A.: Green. J. P.: Brown. 0. M.: Maraolis, S. J . Murochem. . , Szilaavi. P. I. 197%- 19, 2555-2566. (5) Schmidt, D. E.; Speth, R. C. Anal. Biochem. 1975, 67, 353-357. (6) Fidone, S. J.; Weintraub, S. T.; Stavinoha, W. B. J . Neurochem. 1976, 26. 1047-1049. (7) Sziiagyi, P. I:A.;Green, J. P. I n "Analytical Pyrolysis", C. E. R., Jones, Cramers, C . A., Eds.; Elsevier: New York, 1977; pp 417-418. (8) Polak, R. L.; Moienaar, P. C. J . Neurochem. 1979, 32, 407-412. (9) Maruyama, Y.; Kusaka, M.; Mori, J.; Horikawa, A,; Hasegawa, Y. J . Chromatogr. 1979, 164, 121-127. (10) Jenden. D. J.; Hanin, I.; Lamb, S. I.Anal. Chem. 1968, 40, 125-128. (1 1) Jenden. D. J.; Roch, M.; Booth, R . J . Chromatogr. Scl. 1972, 10, 15 1-1 53. (12) Mikeg, F.; Boshart, G.; Waser. P. G. I n "Recent Developments in Mass Spectrometry in Biochemistry and Medicine-VI" (Proceedings of the 6th International Symposium on MS in Biochemistry and Medicine, Venice, June 1979). Frigerio, A., Ed.; Elsevier: Amsterdam, 1980, in press. (13) Kosh, J. W.; Smith, M. B.; Sowell, J. W.; Freeman, J. J. J , Chromatogr. 1979, 163, 206-211. (14) Polak, R. L.; Molenaar, P. C. J . Neurochem. 1974, 23. 1295-1297. (15) Levy. R. L.; Fanter, D. L.; Wolf, C. J. Anal. Chem. 1972, 4 4 , 38-42.
-
RECEIVED for review November 1, 1979. Accepted January 21, 1980.
Stocastic Variability of Noise with the Hall Electrolytic Conductivity Detector for Gas Chromatography Reginald K. S. Goo,' H. Kanai, V. Inouye, and H. Wakatsuki Laboratories Branch, Hawaii State Department of Health, Honolulu, Hawaii 968 13
T h e use of an electrolytic conductivity system in conjunction with a combustion furnace for the detection of chlorine, nitrogen, and sulfur compounds was first described by Coulson ( I ) . H e has shown that the controlled oxidative or reductive pyrolysis of gas chromatographic eluants produced simple inorganic gases which may be subsequently dissociated in water a n d detected conductometrically. Coulson's detector, besides being unwieldy, had one serious drawback for pesticide residue analysis, its sensitivity was low. Since then, there has been a steady stream of improvements 0003-2700/80/0352-1003$01 .OO/O
(2-5). Then Hall (6) developed a sensitive and selective microelectrolytic conductivity detector. Since then, this electrolytic conductivity detector has been used in pesticide residue analysis because of its selectivity for chlorine, nitrogen, and sulfur. Also its superior discrimination against polar co-extractives makes it a desirable detector for complex matrices. Many laboratories such as ours, doing daily routine pesticide residue analysis, require consistent signal-to-noise ratios from day to day. Some of the factors which affect noise and sen-
e
1980 American Chemical Society
1004
ANALYTICAL CHEMISTRY, VOL. 52, NO. 6, MAY 1980 c
Table I. Change in Isopropyl Alcohol Concentration with Time
1 A +
--&L_=id+
e
a
--TIIT
(HECD). EXPERIMENTAL Apparatus. A Tracor Model 220 gas chromatograph equipped with a Model 700 HECD was used. The gas-liquid chromatographic column consisted of a 6-ft by 4-mm i.d. glass column packed with 5% OV-101 on 80/100 mesh Chromosorb WHP. Oven temperature was maintained isothermally a t 200 "C with helium carrier gas flow at 60 mL/min. Inlet and transfer line temperatures were 225 "C. A '/,-inch by 71/2-inchquartz. combustion tube operated at 830 "C with hydrogen flow at 20 mL/min was used. The ion-exchange resin was AG 501-X8 (BioRad Laboratories). Operating parameters used were similar to those described in the FDA Pesticide Analytical Manual ( 7 ) . Changes in isopropyl concentration was determined using a Tracor Model 220 gas chromatograph equipped with a flame ionization detector (FID). Separation was attained using a 3-ft by 4-mm i.d. glass column packed with 80/100 Porapak Q. Inlet and detector temperatures were 250 "C. Hydrogen, air, oxygen, and helium carrier gas were 150, 60, 20, and 60 mL/min, respectively. Under these conditions, isopropyl alcohol gave a retention time of approximately 5'/, min. A Haake Model E52 Bath controller-circulator was used for temperature controlled experiments. The reservoir was a cylindrical glass vessel 10 inches deep and 8 inches in diameter. A Precision Scientific Co. stop watch, measurable to 0.1 s, was used for flow rate studies. The water jacket is shown in Figure 1. The two glass tubes were each 21/2-ft by 7/16-inch0.d. These glass tubes were held in place by clamps affixed to two ring stands. The jacketed portion of the Teflon tubing ('/16-inch 0.d.) was 41/2 feet in length. All connections were made wit,h 7 / l s inch 0.d. Tygon tubing. Procedure. Change in Concentration of Conductivity Solvent. Isopropyl alcohol-water solution, 25% (v/v), was prepared a t 20 O C . The HECD solvent reservoir was filled with this solvent and the gas chromatograph set to standard conditions as previously specified. The cell exit line connected to the reservoir cap was positioned such that no bubbling of solvent occurred. After an hour of operation, 2 mL of solvent was removed and put into a 10-mL volumetric flask. Then 1.00 mL was transferred to a 200-mL volumetric flask and diluted with 20 O C distilled-deionized water. The isopropyl alcohol concentration was then determined by GLC-FID. The recorder response of 2-chloroallyl diethyldithiocarbamate (CDEC) and its corresponding short term noise levels were monitored on the GLC-HECD. Similar procedures were done every 24 h for a period of 5 days. Table I summarizes the results of this experiment. Comparison of Peak Height, Noise, and Conductiuity Solvent Flow Rate over a n 8-Hour Period. The three different systems compared are (1)25% isopropyl alcohol without a temperature controlled water jacket, (2) 100% isopropyl alcohol without a temperature controlled water jacket, and (3) 100% isopropyl alcohol with the temperature controlled water jacket ( 2 5 "C) as illustrated in Figure 1. In each of the systems described above, every 2 h, 4 injections of 12.8 ng CDEC and 3 solvent flow rate determinations were made. Noise levels were determined at the beginning and ending of the experiment. Table I1 and Figure 2 summarize the results of this experiment. Dependence o f Conductivity Soluent Flou Rate xith Concentration. The conductivity solvent flow rate studies were done
noise level,b %
4
25.0 21.6 19.2 16.4 13.5
4.0
5
10.9
6.0
0 1
2
Figure 1. Temperature controlled water jacket. (A) From circulator, (B) to reservoir, (C) from ion-exchange resin, (D) to conductivity cell, (E) Tygon tubing, (F) glass tube, (G) Epoxy glue, (H) Teflon tubing
sitivity, if understood, can be controlled t o yield reproducible noise and sensitivity levels from day to day. It is t h e purpose of this paper t o describe some of t h e factors which cause variabilitv with t h e Hall electrolvtic conductivitv detector
isopropyl alcohol concn %a
time, day
3
0.25 0.50 1.0
S/N
ratioC 25 13 5.8
4.1 1.7 1.2
1.5
Concentration of alcohol obtained from solvent reservoir with HECD operated a t standard conditions. TemShort term perature, ambient; needle valve, 4 turns. noise and measured as average peak t o valley height as % of full scale deflection. Conductivity, 1 0 ; attenuation, 1. 1 2 . 8 ng CDEC injected. a
-Table 11. Comparison of Peak Height and Noise over an 8-Hour Period
peak heightC av. value, RSD, mm %
conductivity solvent 25% isopropyl alcohola i O O % isopropyl alcohola 100% isopropyl alcoh ol
noise,d m m initial final
151
13.0
3.8
4.9
153
5.2
3.5
3.5
155
2.4
3.7
3.7
a Without a temperature controlled water jacket. Temperature controlled water jacket, 25 'C. Conductivity, 1 0 ; attenuation, 1. Conductivity, 1; attenuation, 2. G 35C
3 300
1
0
2
-1-1_--e 6
TI*
8
1"s
Figure 2. Comparison of flow rates over an 8-h period. (a) 100% isopropyl alcohol with temperature controlled water jacket. (m)100% isopropyl alcohol without water jacket. (A)25 Yo isopropyl alcohol without water jacket
at 25 "C. Various concentrations of ethyl alcohol, isopropyl alcohol, and methyl alcohol were studied with the GLC-HECD operated a t standard conditions. Conductivity solvent measured was approximately 4 mL and time was measured to the second with a stop watch. Results are in Table 111.
RESULTS A N D DISCUSSION We have found t h e variations in t h e sensitivity that occur daily are due to t h e decrease in t h e concentration and temperature variation of the conductivity solvent. Tabie I shows not only isopropyl alcohol concentration and signal-to-noise
Anal. Chem. 1980. 52, 1005-1006
and -0.966, respectively. These relationships indicate they are inversely proportional. Further analysis of Table I11 shows conductivity solvent flow rate decreases with increasing alcohol concentration u p to 50%. Higher concentrations of alcohol results in a n increase in solvent flow as shown with ethyl alcohol at 100% and isopropyl alcohol at 75%. Explanation of this phenomenon can probably be shown by the Poiseuille equation (9)
Table 111. Change in Flow Rate of Various Conductivity Solvents with Alcohol Concentration flow ratea
alcohol,
ethyl alcohol
%
0.0 10.0 25.0 50.0
0.732 0.497 0.387
0.187
i i i
0.004 0.003 0.002 0.007
0.239
i
0.010
t
75.0 100
isopropyl alcohol 0.732 i 0.004 0.498 = 0.002 0.339 * 0.002 0 . 1 5 8 i 0.002 0.269 i- 0.004
methyl alcohol 0.732 0.615 0.479 0.408
+ 0.004 + 0.003 i
i
1005
0.002 0.002
V = -7rPr4 817
where V is the volume rate of flow along a cylindrical tube, I is the length of the tube, r its radius, P the difference of pressure at the ends, and q the coefficient of viscosity. Similar phenomenon would occur by substituting viscosity of ethyl alcohol-water mixtures obtained from the Table in the Handbook of Chemistry and Physics ( I O ) , in the above mentioned equation. At 25 "C, viscosity increases from 12.36% (v/v) to 55.93% (v/v), and from 65.56% (v/v) to 100% (v/v) viscosity reverses. T h e increase in the flow rate due to temperature can also be explained by the Poiseuille equation. Viscosity of isopropyl alcohol at 15 "C is 2.86 and at 30 "C is 1.77 (11). Substituting these values in the equation would yield faster flow rates a t higher temperatures. Generally, the conductivity solvent in the reservoir might be approximately 25 "C i n the morning and as high as 35 "C after 4 h of operation.
mL/min. Means and standard deviations are based on Temperature of conductivity solvent, 25 '(3; needle valve, 4 turns. a
3 analyses.
ratio decrease with time, but short term noise increases using a mixed solvent. Using undiluted isopropyl alcohol as the conductivity solvent in conjunction with a temperature controlled water jacket, gives the most reproducible detector response from day to day. Table I1 makes a comparison of peak height and noise over an 8-h period. T h e first line in the table shows the effects on precision and noise due to concentration and temperature changes in the conductivity solvent. T h e second line reveals the effects of temperature increases throughout the day. The noise remained the same and the precision is acceptable for noncritical work. I t should be noted that the new Tracor Model 700A HECD uses 100% n-propanol whereas the older Model 700 uses 50% (8). Therefore, laboratories using the Tracor Model 700A HECD should not have any problems with alcohol concentration changes. The third line shows the effects of constant alcohol concentration and temperature. The effect from changes in alcohol concentration is more significant than temperature because of the added noise effects from water. Explanation for the decreasing detector response over time is caused by the increasing flow rate as shown in Figure 2. The increasing solvent flow rate is due to concentration and temperature. T h e dependence of the conductivity solvent flow rate with alcohol concentration is shown in Table 111. Calculation of the bivariate correlation coefficient for isopropyl alcohol for concentrations from 0 to 50% is -0.965. Similar calculations for methyl alcohol and ethyl alcohol yield -0.952
LITERATURE CITED D. M. Coulson, J . Gas Chromatogr.. 3, 134 (1965). J. W. Dolan and R. C. Hall, Anal. Chem., 45, 2198 (1973). J. F. Lawrence and A. H. Moore, Anal. Chem., 48. 755 (1974). J. F. Lawrence and N. P. Sen, Anal. Chem., 47, 367 (1975). G.Winnett and W. L. Illingsworth, J . Chromatogr. ScL, 14, 255 (1976). R. C. Hall, J . Chromatogr. Sci., 12, 152 (1974). (7) Pesticide Analytical Manual. U.S.Department of Health, Education, and Welfare, Food and Drug Administration, Vol. I, Sec 315. ( 8 ) R. C. Hall, private communication, 1979. (9) "Handbook of Chemistry and Physics", 44th ed., The Chemical Rubber Publishing Co., Cleveland, Ohio, p 225 1, (10) "Handbook of chemistry and Physics", 44th ed., The Chemical Rubber Publishing Co., Cleveland, Ohio, p 2269. (11) "Handbook of Chemistry and Physics", 44th ed., The Chemical Rubber Publishing Co., Cleveland, Ohio, p 2261. (1) (2) (3) (4) (5) (6)
RECEIVED for review September 20, 1979. Accepted January 29, 1980.
Photoluminescence of Silica Gel GF as a Potential Problem in Liquid Scintillation Counting Andrew W. Stocklinski Department of Medicinal Chemistry, School of Pharmacy, University of Georgia, Athens, Georgia 30602
During some recent in-vitro metabolism studies in this laboratory ( I ) , significant photoluminescent effects were observed after the 254-nm ultraviolet (UV) zonal analyses and liquid scintillation counting of "C metabolites isolated from commercially prepared silica gel GF thin-layer plates. I wish to call attention to this effect because it can be a potential source of errors in t h e thin-layer radiochromatographic analyses of drugs and their metabolites obtained from biological experiments. T h e present paper describes this photoluminescent effect and some preliminary results which indicate that the duration of this effect varies significantly between two commercial forms of silica gel G F thin-layers. A method for circumventing the photoluminescent effect is also described. 0003-2700/80/0352-1005$01.00/0
EXPERIMENTAL Materials. Pre-coated silica gel G and silica gel GF thin-layer plates of 0.25-mm thickness were purchased from Analtech, Inc. (Newark, Del.) and used without modification. Powdered Merck silica gel GF, type 60, was purchased from MCB Reagents (Cincinnati, Ohio) and applied onto glass plates as an aqueous slurry with a Desaga spreader. The thickness of the dried plates was 0.3 mm. Scintiverse, a pre-mixed liquid scintillation cocktail, was purchased from Fisher Scientific Co. (Fairlawn, N.J.). 14Cdiphenylmethane (sp. act., 14.9 pCi mmol-'; 14C-methylene)was prepared by literature methods (2,3).All other reagents employed in this study were of reagent grade and used without further purification. Apparatus. Liquid scintillation counting was performed with a Beckman LS 100-C liquid scintillation countw. Operating
C
1980 American Chemical Society
