any particular peak is difficult. The identification of any one component would now seem to be achieved more certainly, so that two or more retention times may be confidently assigned to it. SUMMARY
The odor of large numbers of materials can now be assayed by direct, nondestructive sampling of the vapor above the material. Thus, for the first time, direct correlation with sensory evaluation is possible. The techniques described have potential broad application in odor research, perfumery, deodorization, the tracing of off-odor development, and quality control. ACKNOWLEDGMENT
The authors gratefully acknowledge the substantial contribution to this research by Elinor Cohen, who carried out a large part of the expeiimental work. They also appreciate the opportunity for frequent conferences with Robert Heggie of the American Chicle Co., whose advice and guidance have been most helpful. LITERATURE CITED
(1) Carroll, R. B., O’Brien, L. C., “GasLiquid Chromatography of Whiskies,” Abstracts of Papers, 135th Meeting, ACS, p. IOA, Boston, Mass., April 1959. (2) Carson, J. F., Wong, F. F., “GasLiquid Chromatography of the Volatile Components af Onions,” Advances in
I x IO-’~AMF:
Figure 12. voltages
Discrimination of cigarette smoke at low
Argon carrier gas; 8-foot column, 10% PEG 1500 on Celite; 75’ C.
Gas Chromatography, preprints of symposium presented before Division of Petroleum Chemistry and Division of Analytical Chemistry, 132nd Meeting, ACS, New York, September 1957. (3) Hainer, R. M., Axthur D. Little, Inc., private communication. (4) Harrison, J. W. E., et al., J . SOC. Cosmetic Chemists 4 ( l ) ,9-32 (1953). ( 5 ) Hewitt, E. J .,,, Mackay, D. A. M., Lewin, S. Z., Physicochemical A?; proaches to the Study of Flavor, “ F l a y Research and Food Acceptance, Reinhold, New York, 1958. (6) Lovelock, J. E., ANAL.CHEM.33, 162 (1961). (7) Lovelock, J. E., “Ionization Methods for the Measurement of Low Vapor Concentrations in Gas Chromatography,” 10th Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, March 1959. (8) Lovelock, J. E., J . Chromatog. 1, 35 (1958). (9) Lovelock, J. E., Lipsky, S. R., J . Am. Chem. SOC.82 ,,431 (1960).
(10) Mackay, D. A. hi., Hewitt, E.‘J., Bazinet, M. L., Bailey, S. D., Abstracts of Papers, 134th Meeting, ACS, p. 35A, Chicago, Ill., September 1958. (11) Mackay, D. A. M., Lang, D. A, Berdick, M., Proc. Sci. Sect. Toilef Good8 Assoc., No. 32, 7 (1959). (12) ,Sahaydak, M., American Chicle Co., Drivate communication. (13) Semmler, F. W., Arkiv Pharm. 230, 443 (1892). (14) Stahl, W. H., “Gas Chromatography and Mass Spectrometry in the Study of Flavors”, Chmislry of Natural Food Flavors, symposium sponsored by N a tional Academy of Sciences-National Research Council for Quartermaster Food and Container Institute for the Armed Forces and Pioneering Research Division, May 1957. RECEIVEDfor review October 5, 1959. Resubmitted February 17, 1961. Accepted July 5, 1961. Division of Agricultural and Food Chemistry, 135th Meeting, ACS, Boston, Mass., April 1959
Spectrophotometric Determination of Blood Barbiturate GEORGE W. STEVENSON Oepartmenf of Pharmacology and Toxicology, School o f Medicine, University o f California, 10s Angeles 24, Calif. Barbiturate is determined spectrophoiometrically by using its absorbance a t 260 mp in 1N sodium hydroxide before and after addition of ethylenediamine. 2HCI. Butyl eiher extract of blood is washed twice wiih borax buffer before sodium hydroxide extraction. The sum of amobarbiial, peniobarbital, and secobarbital obtained is clinically much more significant than toial barbiturate obiained by the faster Goldbaum procedure. Measurement of the 2 4 0 - m ~absorbances of ihe borax washes enables preliminary identification, recognition and identification of many mixtures, and determinaiion of the more polar barbiturates (including phenobarbital). Barbiturates present are partially resolved into fractions suitable for further analysis.
1374
ANALYTICAL CHEMISTRY
T
barbiturate determination described is one of a series of analyses of drugs and toxic substances, integrating a few simple and selective extractions with spectrophotometry. An earlier paper describes the determination of salicylate (6). Although ultraviolet spectrophotometric determination of barbiturate in the blood of patients acutely poisoned by barbiturate has proved very useful, the correlation between total barbiturate concentration and the state of anesthesia of the patient has not been satisfactory because of the wide range of potency of the barbiturates. This correlation is very much improved when the identity of the particular barbiturate present is known. Locket (6) and Sunshine and Hackett (8) have compared mean blood levels of various barbiturates HE
in poisoned patients grouped according to their anesthetic states. Correlation of the anesthetic state with barbiturate concentration is of particular importance when barbiturate poisoning is coexistent with other illness, trauma such as head injury, or poisoning due to other substances, since all or none of the depression may be due to the barbiturate. A given blood level of a more potent barbiturate can produce coma, but the same level of phenobarbital may have little effect. A series of blood samples from patients hospitalized for either acute barbiturate poisoning or coma of undetermined origin was analyzed by armodified Goldbaum procedure (2) for quantitative determination and methods developed in this laboratory for identification. I n many cases the true identity of the
barbiturate was not known prior to the analysis. An alternative to complete identification was sought because of the additional time required to carry out the identification. The liquid-liquid partition procedure described in this paper, which separates polar from nonpolar barbiturates, offers such an alternative. In the course of development of the system of identification the partition coefficients of the barbiturates available on prescription in this country were measured. In general, the higher the solvent-aqueous partition coefficient of the barbiturate, the higher its potency (or the lower its blood concentration for a given ancsthetic state). I n the procedure described the barbituratecontaining butyl ether extract of blood is extracted with two portions of borax buffer and then with 1N sodium hydroxide solution. Most of the barbiturate of lowest potency (including phenobarbital) is in the first borax extract, some is in the second borax extract, and very little is in the sodium hydroxide, while most of the barbiturate of highest potency (including amobarbital, pentobarbital, and secobarbital) is present in the sodium hydroxide. These four comprise the vast majority of barbiturates used in this country, and others are seldom encountered in analysis. Amobarbital, pentobarbital, and secobarbital must have similar potencies, if the sum of their concentrations is to be useful. Since potency comparisons for these purposes must be in acutely poisoned humans, precise comparison is difficult. Amobarbital is the least potent of the three but has a t least half the potency of the others. I t has been much less commonly found in blood samples here than the other two or its mixture with an equal part of secobarbital. The absorbances due to barbiturate i n the three extracts can be used to calculate blood concentrations of any of the tabulated barbiturates, to give a preliminary identification of the barbiturates present, and to indicate the presence of a mixture. The partition procedure is also the first part of a system of identification. APPARATUS AND REAGENTS
The apparatus has been described (6), with the exception of 40-ml. round-
bottomed and glass-stoppered centrifuge tubes, which are available from the Corning Glass Works. n-Butyl Ether. The solvent must be purified (6). Spectro grade butyl ethyl with ultraviolet cutoff of 235 mp suitable for either salicylate or barbiturate determination without further purification is available from the Matheson Co. Borax B d e r , 0.05M. Dissolve 19.05 grams of reagent grade sodium
borate decahydrate in water to make 1 liter. Store in elass-stoDDered bottles. The pH is approximately 9.2 at; 25' C. Sodium Hydroxide, 1N. Make up an amroximatelv 8N solution bv rapid& weighing- and dissolving t h i pellets in distilled water. Titrate a dilution of this solution to determine its exact normality. Dilute the solution to lh'. Store both 8 X and 1N sclutions in paraffin-lined glass bottles. (I! stored in a n unlined glass container, the IN solution is usable for only a few days.) Ethylenediamine.2HC1, 2.62M. Add sufficient water to 34.8 grams of Matheson 7857 to make 100 ml. of solution. Centrifuge if not perfectly clear. Barbituric Acid Solutions. The acids were obtained as the pure solids from their manufacturers and used without further purification. Solutions of lO+M barbituric acids were prepared by adding the solid acid to a small volume of sodium hydroxide solution containing a slight excess of base. After shaking a t room temperature for a few minutes, a solution of the monoanion was obtained. After diluting nearly to volume with distilled water, hydrochloric acid was added to give a slightly acid solution which was then diluted to volume. I n a few instances a small volume of methanol was added to the solid, most of it evaporated off,and the residual concentrated solution rapidly diluted with water. Solutions stored in polyethylene bottles a t 3' C. for two years were within 2% of their original concentrations. PROCEDURE
Pipet 5 ml. of whole blood and 25 ml. of butyl ether into a glass-stoppered 40-ml. centrifuge tube. Rapidly invert the tube approximately 200 times. Centrifuge it until the ether layer is free of aqueous droplets. Determination of Sum of Amobarbital, Pentobarbital, and Secobarbital. Pipet 20 ml. of the ether extract and 20 ml. of fresh ether for the blank into separate centrifuge tubes and add to each 5 ml. of 0.05M borax buffer. Equilibrate layers, using 100 rapid inversions. Centrifuge the tubes. Remove all the buffer, using a Propipette and 5-ml. pipets. Expel air while passing the pipet through the ether layer. Withdraw the last portion of the buffer slowly with the tip of the pipet a t the very bottom of the tube. After wiping the ether from the outside of the pipet with tissue, expel the buffer into a vial and sto per the vial (Borax 1). A)dd another 5-ml. portion of borax buffer to each tube and repeat the above procedure, using the same pipets to remove the buffer layers and transfer them to vials (Borax 2). Pipet 5 ml. of 1N sodium hydroxide into each tube and equilibrate the layers, using 100 rapid inversions. After centrifugation, pipet 1 ml. of the sodium hydroxide from both sample and blank
tubes into 1-ml. absorption cells, using 1000-pl. micropipets and a mouth tube. Occlude the top of the pipet while inserting it through the ether layer, expel air to remove ether from the tip, and wipe ether from the outside of the pipet with tissue after filling it. Reserve pipets for mixing the respective solutions as below (NaOH 1). Measure the absorbances of the alkali a t 260 (Azm), 255, 250, and 240 mp. It is also desirable to determine absorbances a t longer wave lengths. Add 250 pl. of 2.62M ethylenediamine. 2HC1 to both blank and solution. Using the corresponding wet 1000-pl. pipets above, mix the contents of the cell by filling the pipet and expelling solution into the cell several times, and by washing down.the insides of the cell. Measure the absorbances of the solutions a t 260 ( A z a b ) , 245, 240, 235, and 230 mp. If fullsized cells are used, use 3 ml. of the 1X sodium hydroxide extract instead of 1 ml., and add 750 p1. of the 2.62M ethylenediamine. 2HC1 instead of 250 pl. Calculate concentration of barbiturate using Equation 1. Preliminary Identification of Barbiturates. Measure the absorbances of Borax 1 and Borax 2 a t 245, 240 (A*& and A2@d), and 235 mp, using the borax washes of the fresh ether a8 blanks. Calculate Ratios 1 and 2 using Equations 2 and 3. The values of these ratios for the pure barbiturates are shown in Table I. Determination of More Polar Barbiturates. Equation 4 is used for the most polar barbiturates (including phenobarbital) and Equation 5 can be used for the barbiturates of intermediate polarity. Determination of Total Barbiturate. This is like the determination of amobarbital, pentobarbital, and secobarbital, except t h a t the butyl ether s extracted only with 1N sodium hydroxide. A fivefold volume of butyl ether should be used for extraction of the blood but volumes other than the one-fourth volume of I N sodium hydroxide may be used to extract the butyl ether, the same volume relations being used with the blank ether. Calculate barbiturate concentration using Equation 6. CALCULATIONS
Barbituric acid, mg. per cent =
kp
X
AA2wa-b)
(1)
where AA2a(s--b) = Azm. - 1.25 X AZab and k, is a constant for the given barbiturate from Table I. The average k, for amobarbital, secobarbital, and pentobarbital is 7.43. Ratio 1
= A~/AAzaoc~-b)
Ratio 2
=
A240e/A240d
(2) (3)
Barbituric acid, mg. per cent kd(A2lOo
- A24Cd)
(4)
ka(Aztoc
+
(5)
A240d)
where kd and k, are constants for a particular barbiturate from Table I. VOL. 33, NO. 10, SEPTEMBER 1961
1375
c la
Barbituric acid, mg. per cent =
VNnOIi #*Tlb.e. (&o. - 1.25 Aisof)
(6)
where kr is a constant for a given barbiturate from Tnble I. When the identity of the barbiturate is not known, it is recommended that the kt value for pcntobarbital be used. TINROA and Vb are the volumes of NaOH and butyl ether used in the 1N sodium hydroside extraction of the butyl ether. A ~ Mand , A2af are analogous with AzWn and Azsob.
...
k, = 100 k t / ( % in NaOH 1) kd
kr = lo4 dil. M . W . / A Erecov. ~~ = lo6 dil. M.W./staorecov.
(per cent in Borax 1 per cent in Borax 2)
-
(7)
(8)
(9)
k, = 106 dil. M.W./sZIOrecov.
(per cent in Borax 1
+ per cent in Borax
2)
(10)
where dil. is dilution factor = 1.25; recov. is per cent estracted from blood into butyl ether; and M.W. is molecular weight of the acid. RESULTS
-. SI
.-
.-GI
B
-1
d
01
ir
3
1376
ANALYTICAL CHEMISTRY
8
Ultraviolet Absorption Properties. The first three columns of molar absorptivities shown in Table I were determined by dilution of equal volumes of the barbiturate solutions and 2 N sodium hydroxide, followed by further dilution to give solutions with absorbances a t 260 mp of 1.5, 1.0, 0.5, and 0.15. Absorbances were measured before and after addition of a one-fourth volume of ethylenediamine dihydrochloride solution. The Ae values were constant. A similar study was carried out to give the €240 values of the boras buffer solutions of the barbiturates, and these were nearly constant a t the various concentrations. For greatest accuracy these extinction coefficients or the constants in the equations must be determined for a particular spectrophotomctcr. Blanks. Blood samples collected in B-D oxalate tubes from 25 random hospitalized patients were analyzed. Mean values are shown in Table I1 and exclude two samples in which barbiturate was found. Nineteen post-mortem blood samples mere analyzed using the total barbiturate procedure. Excluding one of these containing salicylate and two containing barbiturate, the mean mas 0.05 f 0.12 mg. yoof apparent barbiturate. Recoveries. Barbiturate solutions diluted in saline were mixed with whole blood to give solutions of 2.0 and 0.5 mg. yoin each of two different samples of blood bank blood. The percentages of each of the barbiturates recovered using the total barbiturate procedure are given in Table I.
Barbiturate in Butyl Ether Extracts. Butyl ether extracts of diluted solutions of the pure barbituric acids were extracted with two one-fourth volumes of borax buffer and one of 1N sodium hydroxide. Concentrations of barbiturate in the three extracts were calculated in each case from their absorbances and t h e previously determined e values. Mean calculated percentages are given in Table I, with the barbiturates listed in order of polarity] the least polar a t the top. The ratios were obtained from the above-measured absorbance values. Blood samples to which the various barbiturates had been added were analyzed in the same way. Subtracting absorbances due to the blood blank, the percentages of the barbiturates were within 2% of the values in Table I. Mixtures of Barbiturates. If the presence of a mixture of both polar and nonpolar barbiturates is indicated by the values from Equations 1 and 4 and Ratios 1 and 2, the ratios will be somewhat, in error, since some of each barbiturate will be present in Borax 2 whose AZm is used to calculate each ratio. Methods of calculation which can be used to improve the values are illustrated, using data from two cases of barbiturate poisoning. EXAMPLE1. AAzoo(s-b) = 0.296. A24oE = 0.250. Az(od = 0.090 Ratio 1 = 0.090/0.296 = 0.30 Ratio 2 = 0.250/0.090 = 2.8
Mg. yo as secobarbital (from Equation
1) = 2.2 Mg: % ' a s phenobarbital (from Equation 4) = 0.9
Presence of a t least two barbiturates is indicated. Ratio 1 indicates the presence of a nonpolar barbiturate and Ratio 2 a polar barbiturate. Since the value of Ratio 1 will be increased by the presencc of polar barbiturate in Boras 2, the nonpolar barbiturate is most likely secobarbital. Likewise, the above Ratio 2 volue must be lower than that of the polar barbiturate present. It is Dial or a more polar compound. Assuming sccobarbital, the value of Ratio 1 of secobarbital, 0.15, is inserted into Equation 2 and the Auod due to secobarbital is calculated. A240d = 0.15 x 0.296 = 0.044 0.250 - 0.044 = 0.206 0.206 = Az,o, (due to polar barbiturate) 0.044 = 0.046 0.090 0.046 = (due to polar barbiturate) Ratio 2 recalculated = 0.206/0.046 = 4.5.
-
The value of Ratio 2 indicates phenobarbital. Other tests confirmed secobarbital.
EXAMPLE 2. AAzao(s-b) = 0.172. A2(oo = AUod = 0.386 Ratio 1 = 0.386/0.172 = 2.24 Ratio2 = 1.478/0.380 = 3.8
1.478.
Mg. % ' as pentobarbital from Equation 1 = 1.3
Table II. Blood Barbiturate Blanks. Mean Absorbance f Std. Dev. 0.024 f 0.017
A?4Cd0.014 f 0.009
A Y O [ ~I , )O.Ud6 f 0.016
Apparent Barbiturate, Mg. 7' f Std. Dev. Inter mediatec Nonpolard Polarb (as phenobarbital) (as phenobarbital) (as pentobarbital) 0.06 f 0.04 23 samples. * From Eq. 4. 0 From Eq. 5. From Eq. 1.
0 . 1 4 f 0.09
0.05 f 0.12
0
*
Mg. yo as phenobarbital from Equation 4 = 6.2
Presence of a t least two barbiturates is indicated] one a t least as polar as phenobarbital and the other a barbiturate considerably less polar than Nostal, since the polar barbiturate in Borax 2 increases Ratio 1 considerably. Using the value of Ratio 1 for phenobarbital (a possible and the most common polar barbiturate) and Equation 2, the Azaod due to phenobarbital = 1.478/4.4 = 0.336. The AA260(a-t,b) due to phenobarbital = 0.336/6.5 = 0.052. The and AAzko+b) values due to the nonpolar barbiturate are: 0.172 0.052 0.120 Their Ratio 1 = 0.050/0.120 = 0.42.
Table 111.
Partition Coefficienis" of Various Substances Butyl Ether CHC1, 3.7 Salicylic acid 50 4.4 Benzoic acid 14 4 Phenobarbital 4 Theophylline 0 01 0.24 6* 200b Dicumarol Nikethamide 0.09 43 17 Caffeine 0 04 0 02 1 0 Sulfadiazine Solvent-water of un-ionized form. Partition coefficient of total Dicumarol (solvent-pH 6.86 buffer). a
b
0.386 0.336 0.050
This is probably one of the common nonpolar barbiturates, but the large escess of polar barbiturate makes the above calculation much less precise than usual. Other tests confirmed pentobarbital and phenobarbital. Pentobarbital (corrected) mg. % = 7.49 X 0.120 = 0.9 mg. %. DISCUSSION
Presence of barbiturate is proved by the absorbance peaks both a t approsimately 240 mp in pH 9 or 10 solutions and a t approsimately 255 mp in I N sodium hydroside solution. Peaks are demonstrated by additional measurements 5 mp above and below these wave lengths. Barbiturate may be present as calculated using Equations 1 or 4 even in the absence of peaks but in their absence the identification cunnot be considered conclusive. Normal sodium hydroside is used in place of 0.45N in the Goldbaum procedure because higher pH of the solution gives a higher proportion of the masimal barbiturate dianion absorption spectrum and because it is also desirable in the system for identification. The more concentrated alkali requires a more Concentrated acid buffer than the boric acid or ammonium chloride solutions currently in use in other laboratories. The much greater solubility of ethylenediamine dihydrochloride and its pK. of 10 make it
ideal for measurement of the barbiturate monoanion absorption a t pH 10. The 0.05M borax buffer used is fivefold concentrated NBS boras standard buffer and differs only slightly from it in pH. It is a n ideal buffer and the pH of properly prepared and stored buffer need not be measured. This p H stability helps to ensure constancy of the partition coefficients. Chloroform-water partition coefficients of the available barbiturates were measured and were used initially in this laboratory for identification] but butyl ether was later found to be preferable. Helldorff indicated that chloroform-water partitions might be useful for identification of barbiturates (3). Dybing published values of the partition cocfficients of several barbiturates between chloroform and water (f), and Leyda, Lamb, and Harris, the partition coefficients of various barbiturates between water and 60% chloroform40Q/o iso-octane (4). The partition coefficients of the barbiturates between various solvents and water have been tabulated (7). The disadvantages of chloroform include: high volatility, its precipitation of blood proteins, its position beneath the blood in the extraction tube, emulsification of its blood extracts with alkali, and its decomposition in sodium hydroxide to carbon monoxide whose bubbles interfere with absorbance readings. Butyl ether is a more specific exVOL 33, NO, 10, SEPTEMBER 1961
* 1377
tractant of barbituric acids than chloroform, as illustrated in Table 111. The solvent-aqueous partition coefficients of most compounds are considerably higher with chloroform than with butyl ether, but the butyl ether-aqueous and CHC18-aqueous partition coefficients for a given barbituric acid are nearly the same. Acids stronger than the barbituric acids, such as salicylic acid, are exceptions, but usually only a low proportion of such a one present in blood is extracted into butyl ether, and all of this is removed from the ether by Borax 1. Many partition methods utilizing the partition coefficients of the barbiturates for their identification were tested. Some of these were superior t o the method described for differentiation of certain barbiturates but were not as generally applicable. The use of the simple 240-mp absorbances for Borax 1 and 2 is not as accurate as the use of the Goldbaum absorbance difference-type procedure, since substances other than barbiturates absorb a t 240 mp. Addition of 8N sodium hydroxide to these and use of their 260-mp absorbance differences have been tried, but are usuaily not required. The use of Equation 4 for determination of phenobarbital is nearly as accurate as the total barbiturate procedure for clinically significant concentrations of 2 mg. % or more and has the great advantage that it can be used in the presence of mixtures of the other common three, since the contributions of the latter t o the absorbance of Boras 1 and 2 are nearly the same. Equation 5 is less satisfactory but it need not be used, since Equation 4 can be used for compounds more polar than Nostal and Equation 1 for com-
pounds less polar than aprobarbital. If Equation 4 indicates no more than 0.2 mg. % and there is no peak at 240 mp, this may be due to blank or interfering substance. If a single barbiturate is present, the total barbiturate procedure can be used t o check the results of application of Equations 1, 4, or 5 . If this is done, the value of the expression 100 (mg. % barbiturate from Equation l)/(mg. % ' barbiturate from Equation 6) will equal the per cent in 1N NaOH in Table I and' will be more accurate for identification of the nonpolar barbiturates than the use of Ratio 1; however, this requires an additional extraction. Mixtures of barbiturates appearing close to each other in Table I are not readily identified as mixtures. It is for this reason, since mixtures are often found, that it is felt that this partition procedure alone cannot give positive identification. Likervise, it does not appear that additional partition procedures to sharpen differences between the barbiturates are worthwhile, since most values might be simulated by a mixture. Accordingly, the final goal of the procedure is the partial resolution of mixtures of barbiturates into fractions whose constituent barbiturates can be positively identified by other procedures to be described in a system for identification in a later. paper. The most significant difference between this and earlier methods for quantitative determination of barbiturate is the separate determination of classes of barbiturates of different potencies. This has proved in cases hospitalized for barbiturate poiscning to be a satisfactory substitute for identification following determination of total
barbiturate, though totai barbiturate alone is satisfactory when the identity of the barbiturate is known with certainty. Nevertheless, in some cases positive identification is desirable. It is anticipated that in forensic practice also this partition procedure will usually be sufficient without need of positive identification. ACKNOWLEDGMENT
The author thanks Frank McKee, Director of the Clinical Laboratories, UCLA Medical Center, for supplying blood samples, and Raymond Abernethy, Head Toxicologist, Los Angeles County Coroner's Office, for his kind cooperation. LITERATURE CITED
(1) Dybing, F., Scand. J . Clin. & Lab. InUeSt. 7. SUDD1. 20. 114 (1955). (2) Go1dbaum;'L. R., A&L. CHEM.24, 1604 (1952). (3) Helldorff, I., Scand. J . Clin. h Lab. Invest. 7, Suppl. 20, 127 (1955). (4) Leyda, J. P., Lamb, D. J., Harris, L. E.. J . Am. Pharni. Assoc.. Sn'. Ed. 49, 581 (1960). (5) Locket, S., Proc. Roy. SOC. Med. 49, 585 (1956). (6) Stevenson, G. R., ANAL.CHEM.32, 1522 (1960). (7) Stevenson, G. W., University of California, Los Angeles, Calif., unpublished d a h ..
(8) Sunshine, I., Hackett, E., Clilz. Chem. 3, 125 (1957).
RECEIVEDfor review May 8, 1958. Resubpitted March 2, 1961. Accepted April 26, 1961. Investigation supported by research grant B-1106 from National Institute of Xeurological Diseases and Blindness, National Institutes of Health, U. S. Public Health Service. California Association of Crirrinalists, Los Angeles, Calif., April 1958. An-erican Academy of Forensic Sciences, Chicago, Ill., February 1959.
Separation and Identification of Barbiturates by Gas Chromatography KENNETH D. PARKER and PAUL L. KIRK School of Criminology, University o f California, Berkeley, Calif.
b Separation and identification of 23 barbituric acid derivatives, in the form of free acids, are described. Microgram samples, in organic solvent, are subjected to separation in the argon gas chromatograph with an SE-30 stationary phase. Preparation of the sample, as well as class identification, involves a double basic-acidic extraction from blood, coordinated with examination by ultraviolet spectrophotometry. All of the barbiturates tested produced single peaks whose 1378
*
ANALYTICAL CHEMISTRY
retention times could be used for identification. With proper calibration, the areas may be used for quantitative estimation.
N
of distinguishing among the barbiturates have been employed in toxicology. However, the low specificity of all chemical methods and the small structural differences that exist among many of the barbiturates have made such identiUMEROUS METHODS
fications difficult unless each compound is purified carefully. Ultraviolet absorption (4, 5 ) distinguishes the common barbiturates and the thiobarbiturates as groups: the absorptions being influenced by the pH. Differential hydrolysis (9) and differential partition between solvents (1, 10) have been successful with single compounds, but are not satisfactory for mixtures. Melting points, x-ray diffraction, and infrared absorption are suitable with pure solid material, in milligram quanti-
