Figure 3. Plate sprayed with chiorirnide reagent. Oversprayed with iodopiatinate
and a list of color reactions is included (Table I). Also described are the color reactions of a wide range of antioxidants tested involving both phenolic and amine types (Table JI). Only those antioxidants based on phenylene diamine gave a color reaction with the iodoplatinate reagent. Colors produced are distinctively different-cyclamens, purples, and greens. An overspray of sodium bicarbonate, immediately following the chlorimide spiay, and then overspraying with
nothio)benzothiazole (Santocure MOR); and mercaptohenzothiazole (MBT). Chromatograms should be observed a t intervals through a period of 48 hours. Colors tend to become more definite or fade, leaving a pale spot with a discolored center. This would appear to he characteristic of accelerators treated with the iodoplatinate reagent, and could serve as a distinguishing feature when analyzing accelerators in the presence of antioxidants. The presence of guanidines should he confirmed hy developing a third plate in acetone/ammonium hydroxide and spraying with sodium hypochlorite. This is necessary because some peptizers give a similar color reaction to guanidines with the iodoplatinate reagent, and also remain a t the point of application on the chromatogram. Finally, extracts from compounded stocks, and not pure material, must be used as reference, as the heating effects during compounding can result in chemical changes in some accelerators. All extracts and reagents are stored under refrigeration.
ACKNOWLEDGMENT The technical assistance of G. Di Giantomasso is acReceived for review September 18, 1973. Accepted De cember 26,1973.
Gas Liquid Chromatographic A,nialysis of Alkyl Alcohols, Alkyl Cyanoacetates and Alkyl 2-Cyanoacrylates Ramchandra K. Kulkarni, Eric C. Johnson,' and Clarenc e w. n. waoe U.S. Army Medical Bioengineering Research &DevelopmentLaboirat The wide spread interest in alkyl 2-cyanoacrylates as promising tissue adhesives in surgical practice made it necessaly to set down a quick and easy method of identification and analysis of these chemical compounds and their mixtures. The gas-liquid chromatograph with hydrogen flame-ionization detector has been used for the quantitative estimation of these compounds in this report. It is known that the molar response measured in recorded chromatographic peak areas for members of homologous series of hydrocarbons, carboxylic acids, alcohols, esters, ethers, etc. is linearly related to the number of carbon atoms in the molecules, when the individual samples do not differ in number and kind of functional groups (2-5), structural or stereo isomerism or variation in molecular structure, other than the increase or decrease in the numPresent address, Continental Oil Co., Ponea City, Okla. (1) H. Brudereck, W. Schneider, and I. H a i k z , Anal. Chem., 36, 46173 (1964). (2) G. Perkins, Jr., G . M. Rauayheb, L. D. Liverly. and W. C. Hamilton. Gas Chromatog. 3rd International Symposium, pp 269-285. (3) R. F. Addison and R. G. Ackman. J. Gas Chromatogr., 6 , 135-7
(1968). ( 4 ) A. Janik, J. Chromarogr., 54, (3) 21-25 (1971). ( 5 ) L. S. Ettre,J. Chromarogr., 6,525-30 (1962)
teria, the logarithms of the retention volumes in the flame ionization detector are also linearly related to the carbon number. Applying the same principle, the normal straight chain alcohols and their esters with cyanoacetic acid and 2-cyanoacrylic acid were studied for retention volumes and molar responses, and the instrument, calibrated with respect to these parameters, was used to evaluate the composition of the known synthetic mixtures, with sufficient accuracy, a s reported in the present article. The chemical analysis of alcohols and alkyl cyanoacetates is easy but time consuming, but alkyl-2-cyanoacrylates do not lend themselves to chemical analysis without the use of thiophenol, which has a strong stinking odor making analysis difficult. The gentle techniques, like liquid chromatography, have not been successful in the quantitative evaluation of these compounds within the admissible error limits. Therefore the gas-liquid chromatography, when previously investigated and worked out as regards columns, liquid phase, solid support, physical condition of the phases, temperatures, eluting gas, and other incidental details in the technique of gas chromatography, becomes the method of choice for quick evaluaANALYTiCAL CHEMISTRY, VOL. 46, NO. 6 . MAY 1974
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Figure 1. Response in area units per pM X of Flame lonization Detector of F&M 700 gas chromatograph, vs. carbon numbers of alkyl-2-cyanoacrylates, alkyl cyanoacetates, and alcohols
tion of the unknown samples containing these compounds. The extensive investigation of this technique with regard to these high boiling and easily polymerizable alkyl-2-cyanoacrylates has resulted in the data presented in this article, which can be used by workers in this field.
Equipment a n d Conditions of Analytical Determinations. The F&M Model 700 Gas Chromatograph with duel hydrogen flame ionization detector, equipped with Model L S l l A Westronics recorder on which the disc chart integrator of Disc Instruments Inc. was installed to automatically record the integrator counts as a measure of area units in the peaks from the base line, was used for all the determinations. The basic parameters held constant for the chromatographic equipment were: a ) 6-ft, 44-in. 0.d. 0.178-in. i.d. stainless steel columns with chromosorb P as solid phase with 10% liquid phase on it. b) Carrier gas (helium) at pressure of 26 psi and at 3 rotameter level with 40 ml/min constant flow, at 170 "C. c) Compressed air at 18 psi, 2.6 rotameter level and hydrogen at 18 psi, and 1.6-2.1 rotameter level. d ) Injection port temperature at 210 "C, detector temperature at 220 "C, and column oven temperature at 170 "C. Column oven temperatures should be higher than 150 "C for alkyl-2-cyanoacrylates to avoid the danger of polymerization in the columns, and about 30 "C less than the highest recommended stability of the liquid phase. e) Response level range a t 102 with attenuation, 1, 5, or 10. f ) Chart speed at 1.25 in. per minute. Different liquid phases tested in the preliminary exploratory runs were: Carbowax, polyphenyl ether, silicone oil DC-200, Silicone XF-1150, and silicone gum nitrile (G.E. XE-60), all deposited on the solid support chromosorb P, at 10% level. The liquid phase XE-60 could give separation of the chemical compounds involved with maximum resolution and minimum tailing effect, so the standardization was carried out, with this liquid phase at 10% level, after the recommended equilibration a t about 200 "C for a 24-hr blank run of the chromatograph, to eliminate bleeding of the liquid phase during analysis. Sample Preparation. The alcohols, alkyl cyanoacetates, and alkyl 2-cyanoacrylates were all double distilled, from the supply or after the syntheses. The initial purity of the materials was tested by injecting a small sample dissolved in methylene chloride, and observing the peaks. It was ascertained that each compound gave rise to a single peak without any shoulders or to additional impurity peaks not amounting to more than 0.5% of the total peak area. The purity of the compounds was independently ascertained by measurement of refractive index. elemental analysis, and parachor (6). (6) F. Leonard, R. K Kulkarni, G . Brandes, J. Nelson and J . J. Cameron, J. Appi. Poiyrn. Sci., 10, 259-72 (1966)
ANALYTICAL CHEMISTRY, VOL. 46,
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The accurately weighed quantity of each chemical was dissolved in 25 ml of methylene chloride, in a volumetric flask, to make a 0.02-molar solution. The sample was then injected into the column with a microliter Hamilton syringe in amounts of 2, 4, 6, 8 and 10 pl, successively. The integrator counts were measured in each case and were plotted us. the Fmoles of the sample present. The slope of the straight line obtained was reported as the integrator counts in area units per pmole. This micromolar response is tabulated in Table I and plotted us carbon number in Figure 1. The integrator counts were all converted to those at attenuation 1, when different attenuations were used in the chromatographic work, to obtain consistent uniformity of the results. The response in area units per microgram sample was calculated and tabulated in Table I and plotted us. carbon number in Figure 2. This plot can be alternately used to evaluate the amount in micrograms of sample directly. This plot is evidently not linear. The retention volume was obtained in each case by multiplying the retention time in minutes by the flow rate of the carrier gas in milliliters per minute. The logarithm of the retention volume is plotted 1's. carbon number in Figure 3 and tabulated in Table I. In order to test the validity of the calibration data presented in Table I and the Figures 1 and 3, the different monomeric compounds (three of them each time) were accurately weighed and mixed together to form a synthetic known mixture which was dis-
Table I. Response to Alkyl Cyanoacetates, Alkyl 2-Cyanoacrylates and Alcohols of Flame Ionization Detector
Carbon no.
Name of compound
4 5 6 7 8 9
Methyl cyanoacetate Ethyl cyanoacetate n-Propyl cyanoacetate n-Butyl cyanoacetate n-Amyl cyanoacetate n-Hexyl cyanoacetate n-Heptyl cyanoacetate Methyl-2-cyanoacrylate E thyl-2-cyanoacrylate
10
5 6 7 8
9 10 11
12 4 5 6 7 8 4 7 8
Area units, counts per pmole sample X 10-3
414.7 604.2 782.8 758.5 864 . O 955.7 1030.6 556.4 666.1 790.2 893 . O 919.9 920 . O 1056 . 3 1084.6 1417 . O 1530.3 1663 .4 1669.5 1706.0 1685.9 1055 , 2 1185 . 6
41.10 68.33 99.50 107.10 134.10 161 .70 188.90 61.82 83.33 110 .oo
n-Propyl-2-cyanoacrylate
n-Butyl-2-cyanoacrylate n-Amyl-2-cyanoacrylate n-Hexyl-2-cyanoacrylate
136.80 153.80 166.70 206.30 227.00 105 .OO 135 .OO 170 .OO 194.00 222 .20 125 .OO 149.00 181.80
n-Hept yl-2-cyanoacrylate n-Oct yl-2-cyanoacrylate
n-Butyl alcohol n-Pentyl alcohol n-Hexyl alcohol n-Heptyl alcohol n-Octyl aicohol sec-Butyl alcohol sec-But yl-cyanoacetate sec-Butyl-2-cyanoacrylate
Retention volume, ml
Area units, counts per p g sample
188.8 214 . O 310 . O 408.8 575.2 884 . O 1304.0 132.8 144.8 211.2 280.8 406 .O 585.2 888 . O 1200 .o 38.4 50.4 66.0 87.2 114.4 34.4 314 . O 236 .O
Table 11. Gas Chromatographic Analysis of Prepared Known Mixtures, on the Calibrated Instrument Mixture DO.
1
Components in the mixture
Methyl-2-cyanoacrylate n-Propyl-2-cyanoacrylate
2 3 4
n-Butyl-2-cyanoacrylate Methyl-cyanoacetate n-Propyl-cyanoacetate n-Butyl-cyanoacetate E thyl-cyanoacetate n-Propyl-cyanoacetate n-But yl-cyanoacetate n-Amyl-2-cyanoacrylate n-Hexyl-2-cyanoacrylate n-Hept yl-2-cyanoacrylate
Percentage mixture
Area units of peak at 10% - 1
28.40 36.40 35.20 28.15 33.90 37.95 27.90 33.80 38.30 35.41 35.78 28.81
4630 8270 8720 8400 18020 22470 12000 18170 22990 16780 17800 15800
solved in methylene chloride and made to 25 ml in a volumetric flask. This synthetic mixture was injected in the column of the chromatograph, and the integrator counts in area units were measured at attenuation 1 and range lo2 on the electrometer response level. The counts of area units and the micromolar response read off from Figure 1, us. the carbon number, gives the number of gmoles of the substance present. The percentage composition of the mixture as found from the chromatographic analysis, is given in Table 11, along with the original composition. In the four synthetic samples analyzed, the chromatographic analysis is seen t o be correct within 1-270 of the original. In this analysis, the components present must all be known. However, any individual cyanoacetate, or 2-cyanoacrylate, or alcohol in an unknown mixture could be evaluated in grams per cent by measurement of the integrator counts in area units of the peak produced in the chromatograph.
RESULTS AND DISCUSSION The procedure elaborated above, when carried out on a precalibrated gas chromatograph, gives the identification of an alcohol, cyanoacetate, or 2-cyanoacrylate, by reference to a plot of logarithm of retention volume us. carbon number, shown in Figure 3. The response in area units per micromole, determined and plotted us. the number of carbon atoms (Table I and Figure 1) for the homologous series, helps to find out the amount of any member of the series in a mixture by referring the response of the mem-
Sample found
pg
8.297 10.659 10.121 20 ..303 25.169 27 ,589 19.959 25 ,378 28.228 17.985 18.019 15.052
Percentage composition
28.5 36.7 34.8 27.8 34.5 37.8 27.1 34.5 38.4 35.2 35.3 29.5
ber to the molar response in Figure 1. Any change in the system, however, introduces change in the parameters of the system, which seriously perturbs the analytical results. The air flow, hydrogen flow, carrier gas flow, as well as the amount and physical condition of the solid and liquid phases, must remain the same to yield the same results. Also, if the same column is used for a very long time, the errors are introduced due to increase in the liquid phase concentration toward the detector end. The rubber septum in the injector port has to be renewed also after every 10-15 determinations to ensure uniform results and avoid gas leakages. The normalization of the area units in terms of integrator counts by the assumption of a convenient area value per pmole or pg for a standard reference compound, such as methyl cyanoacetate, can be made. However, no such attempt has been made in the present investigation because the direct readings in integrator counts given by the Disc Integrator, a t a given attenuation, gave satisfactory and reproducible values that could be used directly in the calculations. The use of an internal standard in these determinations was tried by using n-hexane-nitrile or n-pentane-nitrile, along with the sample. However, the method did not imporve the results obtained. The chromatographic conditions used during analysis can be reproduced easily by ANALYTICAL CHEMISTRY, VOL. 46, NO. 6, MAY 1974
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careful selection of column materials, preparation of the column, use of the gases of uniform quality, and setting the electrometer response and the recorder amplification a t the same value each time the determinations are made. The question of ester interchange within the column while the mixtures of esters are injected was not as serious as expected, but the ester interchange can become serious if mixtures having large retention volumes are analyzed. In resetting the chromatographic conditions with new columns, one of the reference compounds, like methyl cyanoacetate, can be used as an external standard to adjust the response to the same original value found in the first calibration. The use of a suitable internal standard to obviate the effect of temporary fluctuations in any of the parameters involved is being investigated further. The secondary butyl derivatives of the above series did not conform to the linearity of the logarithm of the retention volume or response in area units per micromole to the carbon number as expected. This is shown clearly from the values of the same reported in Table I, which do not fit in the plots of Figures 1and 3. Accuracy of Determinations. The response in peak area of the hydrogen flame ionization detector ( I ) depends linearly on the mass flow rate of the sample (at low values) described by: (1) where I = quantity of ion current in coulombs, g = quantity of carbon in grams flowing, t = time in seconds, and K = proportionality constant (coulombs/g carbon). In the present investigation, the only polarizing potential available on the instrument was 112.5 volts a t a constant value, and the relative value of the ion current was given by the peak area in the integrator counts. The proportionality of the above relationship was maintained by keeping the mass flow rate of each sample within certain limits, by adhering to the technique described in the ex-
perimental section above. In short, the samples were all 0.02 molar in solution in methylene chloride, giving approximately 1 to 2.8 pg total carbon per sample in solution per microliter, and the peak area was covered by each sample in an average time of about 20 seconds depending on the retention volume. By this method, the mass flow rate of carbon amounts to nearly 0.5 to 1.4 x lo-? gram per second per microliter injection. If the maximum volume injected each time is 10 microliters, the response as measured was within the 1-270 accuracy in the determination of the sample size from the area. However, if the sample size injected was larger than 30 pg in any compound, the linearity became uncertain and the determination was no longer reliable.
CONCLUSIONS A gas chromatograph equipped with a hydrogen flame ionization detector can be calibrated us. pure samples of normal monohydric alcohols, alkyl cyanoacetates, and alkyl 2-cyanoacrylates. Logarithms of retention volumes and of micromolar responses are linearly related to the number of carbons of the individual compound in each homologous series. The identity and the amount of any unknown compound in the same series can be evaluated by analysis of the compound on the precalibrated gas chromatograph. ACKNOWLEDGMENT The authors gratefully acknowledge the invaluable help in the analytical work by Ronald Harrison, the physicochemical assistant. Received for review July 16, 1973. Accepted December 28, 1973. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.
Gas Chromatographic Determination of Thiopental in Plasma Using an Alkali Flame Ionization Detector Lawrence T. Sennello and Fred E. Kohn Division of Experimentai Therapy, Abbott Laborafories. North Chicago. //I. 60064
Thiopental, an ultra short-acting barbiturate, is often used as a general anesthetic in conjunction with inhalation or with another intravenously administered anesthetic. Thus, it is occasionally desirable to monitor the level of the barbiturate in the blood of patients during and/or after anesthesia. Although the technical literature contains numerous procedures for the determination of thiopental and other barbiturates in biological fluids, most of the methods are relatively insensitive or time-consuming. Grochowska ( 2 ) and Oroszlan and Maengwyn-Davies (2) reported spectrophotometric procedures with sensitivities of about 20-300 pg per ml. Scoppa ( 3 ) reported a spectrofluorometric de( 1 ) 2 . Grochowska, Mikrochirn Acta. 5, 1905 (1968). (2) S. I . Oroszlan and G . D Maengwyn-Davies, J. Arner. f h a r r n A s s 49, 507 (1960). (3) P . Scoppa, Biochirn. A p p l , 13, 274 (1966).
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termination with a sensitivity of about 0.5 pg per ml of fluid. Braddock and Marec ( 4 ) published a gas chromatographic procedure for the determination of thiopental and pentobarbital in biological fluids with lower detection limits of about 1pg per ml. Because of the inherent specificity and sensitivity of the technique, it was decided to pursue a gas chromatographic approach. Here too, though, published methods tended to be somewhat insensitive or tedious. Either sensitivity was low because of sample loss during lengthy “clean up” steps, or great caution had t o be taken to prepare especially inert columns which caused little or no tailing of barbiturates. To overcome the problems of adsorption or decomposition of barbiturates resulting from interaction with the column, one must either chemically modify the drugs to (4)
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Marec J Gas Chrornatogr
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