Identification of Substituted Barbituric Acids by Gas Chromatography

measured. The area for saturates is subtracted from saturates plus olefins to determine olefins area. The areas are multiplied by thermal conductivity factors and normalized to 100%. ANALYTICAL RESULTS

Accuracy was determined by analyzing two synthetic blends, each containing about 50 pure grade hydrocarbons. ‘The blends were prepared to simulate gasoline as closely as possible. The analyses, as averages of three runs, and the prepared compositions n-ere : Blend 1, Prepared Found Saturates 49 7 49 9 Olefins 22 7 22 4 Aromatics 27 6 27 7

Blend Prepared 41 4 34 3 24 3

2.

5

Found 41 0 34 6 24 4

The difference betn een the prepared and found values average about 1% of the amount found. The excellent accuracy indicated by these analyses is higher than nould be evpected for routine analyses w t h this method. As a further indic:ition of accuracy, seven naphthas oi widely differing conipositions were analyzed by gas chromatography, FIA, and mass spectrometry-each in duplicate. The analyses by the thrc>e methods, given in Table 111, show relatively good agreement. Actually, the gas chromatographic method agrecs more closely with

either of the other two methods than the other tn-o agree between themselves. These analyses of naphthas of widely differing compositions demonstrate the broad applicability of the gas chromatographic method. The standard deviations average about 0.670 absolute; they are highest for olefins and lowest for aromatics. The deviations are about those expected from measurement of peak areas. CONCLUSIONS

Advantages of the new method are short analysis times and applicability to full range gasolines and other hydrocarbon mixtures containing any amount of low boiling hydrocarbons. Disadvantages are the determination of olefins by difference. the limitation to naphthas with end points not far above 400’ F., and the failure to separate saturates into paraffins and naphthenes. Several evtensions of the method are apparent. One is to divide the saturate fraction further. %-Paraffins could be separated from branched paraffins and naphthenes with 31olecular Sieves (’7). Separation of naphthenes from paraffins by selective dehydrogenation of naphthenes ( 2 1 , l4j might also be possible. The present gas chromatographic method could be simplified and improved if an absorber were available that would remove aromatics from sat-

urates plus olefins. The nitrile column then would not be needed. The method also could be improved if a n olefin absorber were available that could be made to release olefins quantitatively for direct determination. LITERATURE CITED

(1) Am. SOC.Testing Materials, “ASTM Standards, 1961,” Method D-875-60T,

Part 7,p. 361. (2) Ibid., Method D-1319-61T, Part 7, p. 702. (3) Ibid., “ASTM Standards on Petroleum

Products and Lubricants,” p. 1128, 1961. (4) Brown, R. A , A s ~ L .CHEM.23, 430 (1951). (5) Coulson, D. >I., Ihzd., 31,906 (1959). (6) Criddle, D. IT., LeTourneau, R. A., Ibid., 23, 1620 (1951). (7) Eggertsen, F. T., Groennings, S., Ibid., 33, 1147 (1961). (8) Ferrin, C. R., Chase, J. O., Hurn, R. W.,Intern. Gas Chroniatography Symp., Michigan State Cniversity, June 1961. (9) Frisone, G. J., ,Vature 193,370 (1962). (10) Frisque, A. J., Grubb, H. M., Ehrhardt, C. H., T’ander Haar, R. W., ANAL.CHEM.33, 389 (1961). (11) Keulemans, A. I. M., Voge, H. H., J . Phys. Chem. 63,476 (1959 ). (12) Martin, R. L., AXAL. CHEW 33, 347 (1961). (13) McNair, H. hl., DeVries, T., Ibid., 33, 806 (1961). (14) Rowan, R., Ibid., 33, 658 (1961). RECEIVEDfor review January 31, 1962. Accepted Bpril 20, 1062. Division of Analytical Chemistry, l i l s t Meeting, A4CS,JT7ashington, 11. C.. March 1062.

Identification of Substituted Barbituric Acids by Gas Chromato g ra phy of The ir Pyrolysis Produc ts DONALD F. NELSON‘ and PAUL L.

KIRK

School o f Criminology, University o f California, Berkeley, Calif

b The pyrolysis products of 27 substituted barbituric acids, which may be used therapeutically, have been studied with the Pye argon gas chromatograph. Free acids, their sodium salts, and mixtures of free acid with anhydrous potassium carbonate were compared. Pyrolysis offers a practical method of identifying barbiturates.

T

HE retention time of a barbiturate, measured under specified gas-chromatographic conditions, has been shown (4) to be a useful addition to the list of properties which are available for its identification. By combining pyrolysis with gas chromatography, each one of a

On leave from the Dominion Laboratory, X e n Zealand.

group of substituted barbituric acids has been made to yield, under given conditions, a reproducible succession of peaks whose retention times and relative sizes form a recognizable pattern and which, when considered together, approach the requirements for individualization. Study of the pyrolysis products of organic substances by gas chromatography has been reported by a number of workers, including Davison, Slaney, and Wragg ( I ) , who suggested it as a method for the identification of polymers. Janak ( 2 , s) described the apparatus and procedure by which he pyrolyzed substances in the carrier gas stream so that the products immediately entered the column. H e recommended its use for the identification of organic substances, and described the

results of pyrolyzing certain natural products such as fats, proteins, and alkaloids and synthetic compounds such as amino acids and barbiturates. He found quantities of sample below 0.1 mg. sufficient for analysis. From the study of 14 barbiturates he reported that fragments of the molecules present are always highly specific, both qualitatively and quantitatively. I n the work here presented the pyrolysis products of 27 substituted barbituric acids were separated by gas chromatography. EXPERIMENTAL

Procedure and Apparatus. T h e barbiturates which are the subject of this study are listed in Table I, together with common synonyms, the VOL. 34, NO. 8, JULY 1962

899

A l l y l b o r b i t u r i c acid Alphenol

Arnoborbitol Aprobo r bit0 1

B

B o r b lo1 But0 borbttal

Butolbitol Butollylonol Butethol

i y c oborbitol

C

Figure 1.

Pyrolysis unit

syc opal D ~ o i l y l b o r bt d r i c acid

A.

Foil heating cup 6. Wires to variable voltage e.m.f. C. Ground joint connection to top of chromatographic column

Heptoborbitol Hexethol Hexoborbitol

substituents on the barbituric acid ring, and the melting points. Most of the barbituric acids were obtained in pure form, i.e., free of excipients, from drug manufacturers. Some were obtained from other laboratories. A few were extracted from tablets or capsules. Several individual drugs were obtained from more than one source, for comparison. The free barbituric acids were purified, in most cases, by recrystallization from aqueous ethyl alcohol until a constant melting point was obtained which agreed with published values. Three of the drugs (butallylonal, narconumal, propallylonal) were received in small quantity and were not recrystallized. After recrystallization the purified drugs were dried in a desiccator under high vacuum. Sodium salts were prepared by reaction, in ethyl alcohol solution, of equivalent weights of sodium hydroxide and the barbituric acids. The solvent was evaporated a t room temperature under the vacuum with an air current impinging on the surface of the liquid. I n most cases the salts crystallized as the ethyl alcohol evaporated, especially when aided by rubbing with a glass rod. I n some cases the salt dried as an amorphous glass which was easily crushed. The salts were desiccated under vacuum. The apparatus used was a Pye argon gas chromatograph (W. G. Pye and Co., Ltd., Granta Works, Cambridge, England) equipped with an ionization (strontium-90) detector and a 10-mv. Minneapolis - Honeywell - Brown recorder. The glass column, 4 feet long and of 5-mm. internal diameter, was packed with 5% silicone oil (No. 500) on Chromosorb W 80 t o 100 mesh. The pyrolysis unit is illustrated in Figure 1. It is attached t o the standard socket joint a t the top of the column. The central feature of the pyrolysis unit is a shallow horizontal cup about 1.5 X 3 mm. in size, formed from 0.001inch platinum foil and welded a t its ends to two 18-gage platinum wires through which an electric current may be supplied. (In the figure this portion of the apparatus has been rotated 90" on its axis, for the sake of clarity.) Loading, manipulation, and cleaning of the apparatus are facilitated by the

900

ANALYTICAL CHEMISTRY

I

Met hoi1o l o I Methorblla

,I

llareonimal

I

I I I

I

I I I

Tiiapenlal >J n:aibitol

I

I l l

1

I

I I

I

I

I

I

I

I

I

I

'Ihiornylol

I1 I

I

I l l

Proborbilol

Seccborb.fol

I

I

PteroborPitol

Propollylonol

I

I l l

I I

F2n:obcrbi'cI

MID"t2S

I

I II

Mepbaborb - c l

I l l

I

I I

I

I

I

1

0.I

I l

I I I 1

I

l

I.o

1

1

10

100

Figure 2. Results of chromatographing at 150" C. pyrolysis products of sodium salts of barbituric acids

fact that the platinum cup and wires are permanently attached to a glass stopper of standard taper. This mounting is a modification of the device reported by Strassburger et al. (6). Glass wool below the pyrolysis chamber prevents solids from falling into the column. In the pyrolysis, a current of about 6 volts and 3 amp. was used, which heated the foil cup t o a dull or medium red in about a second. The 110-volt house current was transformed by a 12-volt battery charger controlled by a Variac. The sample to be pyrolyzed was not weighed, but judged by eye on the basis of trial and error. A few tenths of a milligram of the sample were placed in the cup, which was then inserted in the pyrolysis chamber, and the ground glass joints were secured by clamps. After the argon flow had been turned on and the recorder was registering a stable base line, the sample was pyrolyzed by turning on the electric current. The circuit was wired so that the time of the pyrolysis was recorded on the chromatogram. Pyrolysis appeared t o be complete in 2 to 3 seconds, but heating was normally continued for about 10 seconds. The passage of the pyrolysis products through the strontium-90 detector a t the base of the column was recorded on a chromatograph chart moving a t the rate of 40 inches per hour.

RESULTS AND DISCUSSION

The pyrolyzates of each individual barbiturate produced a series of peaks which were reproducible in retention time and relative size so long as the column temperature and the flow rate were kept constant. The results are summarized in Figures 2 and 3, the retention times being indicated on a logarithmic scale. Figure 2 shows the retention times and relative peak heights, a t 150" C., of the pyrolysis products of the 27 barbiturates studied. The peaks produced by each sample are represented by vertical lines of three lengths, which indicate an arbitrary classification of the peak heights as large, medium, and small. Small is a rather broad category including some very small peaks which might escape notice and others which would almost qualify as medium. Since, in the interpretation of the chromatograms, retention times are more significant than peak heights, the latter values have been dealt with in this casual and subjective way because their chief value lies in establishing a visual pattern for the guidance of the investigator. The location of these vertical lines on the graph indicates the retention times, in minutes, of the pyrolysis products. Two limiting characteristics of the

Bulc'lylonol

I

i

I

5

I ,

t

1

A

I

l

I ,I

,I

I l l 1

I , /

, / 1

I , , ,

I

111 I 1 6 1

II

I 1 J l I l l

l

8

1

1

G

I

/

B;!ethol

I

I

I

I C ) cloborbital

1

A

l

l

I

0

1

1

I

I

Diollylbarbituric acid

A

I

5

I L I I I

%

8

I

I

I

I I

I

I I I I I

c i I I I

A

1

S

I

c

I I

Heptobarbital

I

I I

I I

Hexelhcl

h',',,feS

0.1

I33

10

1.0

M i n ules

0.I

1.0

10

100

0.I

10

10

100

Proborti!ol Hexoborbital

Propollylcnal Mephobarbital

I , Secotartiiol

Thiarrylal Metharbital

A

I

I

c

I I I i

A

I

0

I

, I

I

I ,

I

I /

N?r:onumot

Pm'obcrbital

B

c

1 1

I

I

1

I

I 1

I

, I

Minutes

Figure 3. products

Pienoborbitol

A. hlinu!e5

01

I

.c

6. C. 10

13;

Results of chromatographing at 1 10' C. pyrolysis

from free barbituric acids from sodium saltsof barbituric acids from mixture of free barbituric acid carbonate

and anhydrous potassium

VOL 34, NO. 8, JULY 1962

901

logarithmic scale used heIe may be noted: It does not permit the location of zero (the time a t which pyrolysis was initiated), and it suggests, in its lower reaches, a n unrealistic degree of precision. For example, there is no significant difference in this study between 0.35 and 0.40 minute. The location of the large initial peak (which represents an unresolved mixture) indicates the column-volume replacement time. The relative retention times of the pyrolyzates of any one barbiturate were found to be independent, generally, of large changes of flow rate, minor changes of column temperature, and changes of column dimensions, provided that the same stationary phase was used. The retention times recorded in Figure 2 suggest that a t 150" C. some of the pyrolysis products are comparatively nonvolatile. This n-as indicated, also, by the appearance on the walls of the pyrolysis chamber (which was a t room temperature) of deposits which, however, may have been unpyrolyzed barbiturates. This might be obviated by heating the pyrolysis chamber, or by

Table 1.

Official Name Allylbarbituric acid (NNR) Alphenal Amobarbital (USP) Aprobarbital Barbital (CSP) Butabarbital (XNR) BII t d hi t,al ... . Butallylonal (XU'NR) Butethal (NNR) ~

Cyclobarbital (NNR)

Minutes

Diallylbarbituric acid (NSR) Heptabarbital

pyrolyzing the samples within the oven. Either modification, however, mould increase the complexity of the apparatus and might result in volatilization before pyrolysis. The apparatus used in this study gave enough peaks for identification. Figure 3 compares the results of chromatographing, a t 110" C., the products obtained by pyrolyzing each of the barbituric acids as -4,the free acid; B, the sodium salt; and C, a

Barbituric Acids Studied

11.p.5

" c.

Common Synonyms Sandoptal, Itobarbital

Substituents 5-allyl-5-isobutyl

Lubergal, Prophenal Amylobarbitone (BPC), Aniytal Alurate Barbitone (BP), Verona1 Butisol Lotusate, Talbutal Pernoston Butobarbitone (BPC), Neonal, Soneryl Cyclobarbitone (BPC), Hexemal, Phanodorn

5-allyl-5-phenyl 5-ethyl-5-isoamyl

158 159

5-allyl-5-isopropyl 5,j-diethyl

141 190 168

Allobarbitone (BPC) Dial hledomin

Hexethal (XNR) Hexobarbital (NXR)

5-ethyl-j-sec-but yl

5-allyl-5-sec-butyl

5-(2-bromallyl)-5-sec-butyl

5-butyl-5-ethyl

ANALYTICAL CHEMISTRY

140

110 6

124

5-( l-cyclohexenyl)-5-ethyl 173 5-a11yl-5-(2-cyclopenten-

141

1-vl\

5,d i h y l

175

5-( l-cyclohepten-l-y1)-5ethyl 5-ethyl-5-n-hexyl 5-( l-cyclohexen-l-yl)-l,5dimethyl

175

Ortal, Hebaral Hexobarbitone (BP), Cyclonal, Evipal Mephobarbital ( N S R ) Phemitone (BP), bkbaral, l-methyl-5-ethyl-5-phenyl Prominal llethallatal (USP) hlosidal 5-ethyl-5-(2-methylally1)2-thio Genionil l-methyl-5,5-diethyl Metharbital (XNR) Narconumal ... I-me thyl-5-allyl-5isopropyl Pentobarbitone (BP), 5-ethyl-5-( 1-methylbutyl) Pentobarbital (LSP) Nembutal Phenobarbitone (BP), Phenobarbital (USP) 5-ethyl-&phenyl Gardenal, Luminal Ipral Probarbital (XNR) 5-ethyl-5-isopropyl Xostal, Noctal 5-( 2-bromoallyl)-5-isopropyl Propallylonal (NNR) Quinalbarbitone (BP), Secobarbital (NKR) 5-allyl-5-(1-methylbutyl) Seconal Surital 5-ally1-5-( 1-methylbuty1)Thiamylal (XNR) 2-thio 5-ethyl-5-( 1-methylbuty1)Thiopentone (BP), Thiopental (USP) Pentothal 2-thio Vinbarbital (KNR) 5-ethyl-5-( 1-methyl-IDelvinal butenyl) a Fractional degrees have been omitted. Sample insufficient for recrystallization.

902

10

I

IO

100

Figure 4. Results of chromatographing pyrolysis products of sodium pentobarbital a t 50",75",1 1 0",and 150" C.

...

Cyclopal

0

125 147 181

161 155 b

130

178 2f3 84

138

158 165

mixture of the free acid with anhydrous potassium carbonate. When the free acids were pyrolyzed there ~ a aq tendency for the initial peaks to be larger than thoseobserved from the corresponding sodium salts, and for the later peaks to be smaller, or even to disappear In toxicological analysis, hon eTer, barbiturates usually are isolated as free acids. With many of the barbiturates, mixtures of free acid and potassium carbonate were found to give substantially the same pyrolyzates as did the corresponding sodium salts Hone\ er, this \{as not true of all of them; some of the allyl compounds, in particular, n ere exceptions to this rule. Production of clearly defined peaks of reasonable size, with a minimum of tailing and overlapping, and yet without unduly prolonging the retention times of the least volatile pyrolyzates, involved some compromise of sample size, flow rate, and column temperature. For chromatography a t 150" C. (Figure 2 ) a suitable standard n a s the largest peak produced by the pyrolysis of sodium alphenal. K h e n its retention time was standardized a t 3 5 minutes, the flow rate was 90 ml. per minute. At 110" C (Figure 3) the final large peak from sodium pentobarbital was chosen as the standard, the retention time being 3 minutes, and the flon rate 95 nil. per minute. KO one set of conditions \\a$ ideal. For example, comparison of Figures 2 and 3 shons that more of the later peaks mere detected a t 150" C. -it the loner temperature these mere slow in emerging, and were so spread out as to cause no perceptible elevation of the recorder chart base line. On the other hand, as the column temperature mas raised, the earlier peaks were crom-ded together, so that a t 150" C. the peaks nhich emerged nithin 0 5 minute were probably unresolved mixtures, and even up to 1 minute the resolution was only fair. The loss of peaks is illustrated in Figure 4, n hich presents the results of chromatographing the pyrolysis products of sodium pentobarbital a t 50", 'is", 110", and 150" C. In this graph 13 peaks at 50" are successively reduced

to nine a t 75", four a t 110", and three a t 150" C. At 150' C., however, a later and previously overlooked peak is detected. When very large samples were pyrolyzed, additional peaks of very small size were detected. These have been disregarded, however, in the preparation of the graphs, as being more likely to confuse than to elucidate the problem. The observation of Janak ( 3 ) that the critical factor in reproducibility is the linear velocity of the carrier gas along the pyrolysis unit is of interest here, but was not examined in this study. By plotting the retention times on a horizontal logarithmic scale, besides gaining compactness, one can establish a visual pattern for the pyrolyzates of each barbiturate which would be recognizable by another investigator using the same stationary phase under somewhat different chromatographic conditions. Obviously, it is essential for any one desiring to use this method for the identification of barbiturates t o

establish his own standards. The representation offered in the accompanying graphs may, however, serve tentatively t o identify a barbiturate and may aid in the selection of the appropriate standard barbiturate to be pyrolyzed to confirm the identification. The close correspondence between the pyrolysis chromatograms of secobarbital and thiamylal points up the fact that their structures differ only in that the latter has a sulfur atom substituted for the usual oxygen atom a t the 2 position. A similar relationship existing between pentobarbital and thiopental is reflected in the chromatograms also, though somewhat less markedly. These observations indicate the possible application of pyrolytic gas chromatography not only to identification but also to structure studies. Its value for quantitative determination should receive attention. The possible effects of metabolites and other physiological materials also remain to be studied.

ACKNOWLEDGMENT

The authors gratefully acknowledge the technical assistance of H. R. Harvey; and further, thank cooperating pharmaceutical supply houses for furnishing the pure compounds used in this study. LITERATURE CITED

(1j Davison, W. H. T., Slaney, S., Wragg, A. L., Chem. & Ind. (London) 1954,

1356.

(2) Janak, J., Natuie 185, 684 (1960). (3) Janak, J., Coll. Czechoslov. Chem. Commun. 2 5 , 1780 (1960). (4) Parker, K. U., Kirk, P. L., ANAL. CHEM.33, 1378 (1961). (5) Strassburger, J., Brauer, G. M., Tryon, M., Forziati, A. F., Ihid., 32, 454 (1960j. RECEIVED for review December 18, 1961. Accepted April 17, 1962. California AE-

sociation of Criminalists, Fall Meeting 1960, Berkeley, Calif. Work supported by grants from the Sational Institutes of Health, U. S. Public Health Service (RG-4372 and RG-5802), and from the Research Committee, University of California.

Qua Iita tive Gas Chroma t o g ra phic A n a lysis by Means of Retention Volume Constants CHARLES MERRITT, Jr., and J. T. WALSH Pioneering Research Division, Quartermaster Research and Engineering Center,

,A method for the functional group classification and subsequent qualitative identification o f chromatographic peaks from retention volume data alone i s described. The method employs two columns having different liquid phases. When appropriate liquid phases are employed, the ratio o f isothermal retention volumes, or the difference between programmed temperature retention volumes, o f compounds within an homologous series is constant. Different constants are found for the various homologous series. From criteria established from this behavior, the functionality of a given compound can b e determined. The method can b e applied to chromatograms obtained either isothermally or with programmed temperature and with either packed or capillary columns. In this study, criteria are established for the identification of members of homologous series of normal alkanes, alkyl benzenes, alcohols, aldehydes, ketones, ethers, esters, thiaalkanes and thiols.

T

HE INABILITY of gas chromatography by itself t o provide adequate qualitative data has impaired its usefulness. -4uliiliary chemical (21) or in-

U. S. Army,

strumental (1, %, 6 , 7 , 9, 10, 16) methods have been required to identify peaks obtained from unknown mixtures particularly when such mixtures consist of several components having different functionality. This situation has been due largely to the fact that retention volume data on a single column lack sufficient specificity to permit unambiguous identification. The difficulty of collecting eluted chromatographic peaks, particularly from complex mixtures or of small amounts of a component, has limited the application of subsequent instrumental analysis. The determination of compound type by means of functional group analysis (21) is limited by sample size. Furthermore, functional group analysis cannot be readily adapted to the detection of inert types of compounds such as aliphatic hydrocarbons or ethers. It is highly desirable, therefore, to have available the means for characterization of functionality of eluted components from retention volume data alone. The possibility of using data from two or more columns having different liquid phases has been reported previously (3, 6, 11-15, 19, %3), but the use of such data has been generally neglected. The purpose of this investigation is to

Natick, Mass.

shorn that retention volume data from appropriately chosen columns can be used in a systematic way to provide a method for the functional group classification and subsequent qualitative identification of chromatographic peaks. I n this study criteria have been established for the identification of members of homologous series of normal alkanes, alkyl benzenes, alcohols, aldehydes, ketones, ethers, esters, thiaalkanes, and thiols. The establishment of criteria for isomeric compounds is currently being studied and nil1 be the subject of a subsequent paper. EXPERIMENTAL

Apparatus and Procedure. Conventional gas chromatography apparatus \vas used throughout this study. The packed columns were either the stainlws steel coil type or the glass U-type. Twenty per cent by weight of the liquid phases used were coated on 60-80 mesh firebrick. The thermal conductivity detectors used were four-element bridges (tungsten filaments) operated a t 200 ma. from a 12-volt source. The ionization detectors used with packed columns employed either radium-226 or strontium-90 as the radioactive source. Capillary columns were 100-foot stainless steel capillaries coated from 5% VOL. 34, NO. 8, J U L Y 1962

903