zirconium and niobium is expected by the K edge of strontium, the L I edges of bismuth and lead, and the L I11 edge of thorium. General absorption becomes serious when the ratio of line intensity to internal s,tandard intensity deviates significantly from unity. Hence, it would be most pronounced for samples containing little or no molybdenum or zirconium. The effects of various elements on the determination of niolybdenum and zirconium were studied. Solutions were prepared to contain 0 or 40 mg. of molybdenum and zirconium; 100 mg. of uranium; and various amounts of lead, mercury, copper, cadmium, or sulfuric acid. Mercury, cadmium, and copper were selected as typical elements having high, medium, and low mass absorption coefficients, respectively, for the K swies radiation of zirconium and molybdenum, and various sulfuric acid concentrations were
used to study the effect of variable solvent concentration. Lead is expected to give high recoveries for the determination of zirconium because of selective absorption of the K, line for niobium. Large concentrations of elements having high mass absorption coefficients interfere with the determination of zirconium, as do large concentrations of sulfuric acid (Table VIII), and with the determination of low concentrations of molybdenum (Table IX). Each value shown is the result of duplicate determinations on one solution. No interference &-as assumed if the average of duplicate determinations was within two standard deviations of the quantity added. ACKNOWLEDGMENT
The authors gratefully acknowledge the assistance of C. F. Metz, under whose direction this work was per-
formed, and of G. C. Heasley who performed the carbon analyses. LITERATURE CITED
(1) Flikkema, D. S., Schablaske, 1%.T.> “Proceedings of the 6th Annual X-Ray Conference-Industria1 Applications of X-Ray Analysis,” W. XI. Mueller,
ed., pp. 387-98, Denver Research Institute. 1957. (2) Hakkila, E. A,, AKAL. CHEM. 33, 1012 (1961). (3) Liebhafsky, H. -4.,Pfeiffer, H. G., Winslom, E. H., Zeniany, P. I>., “X-Ray Absorption and Emission in Analytical Chemistry,” pp. 314-17, %ley, Yew York. 1960. (4) Peedd, W. F., Wright, FT. B., Jr., Rogosa, G. L., E. S. Atomic Energy Comm. Rept. ORNL-1419 (Ilec. 6, 1952). RECEIVEDfor review March 9, 1964. Accepted July 13, 1964. Work performed under the auspices of the V. S. Atomic Energy Commission. Presented at The Seventh Conference on Analytical Chemistry in Suclear Technology, Gatlinburg, Tenn., Oct. 8-10, 1963.
Gas Chromatographic Separation of Amines and Amides JERRY F. O’DONNELL and CHARLES K. M A N N Department o f Chemistry, Florida State University, Tallahassee, Fla.
b A method is descriibed for the chromatographic separatiion of mixtures of aliphatic amines, aromatic amines, and aliphatic amides on columns utilizing Dowfax 9N9 with 2’/270 N a O H as a partition liquid. Data are given to allow comparison of Dowfax 9N9 columns with similarly treated columns of Carbowax 400 and 20M for the separation of aliphatic amines. Retention data for ‘other basic compounds are included.
T
of aliphatic amines presents many problems to the chromatographer. The large number of primary, secondary, and tertiary amines, both straight and branched chain, necessitates the use of a column of considerable resolving power to achieve separation. Such columns, however, normally employ Chromosorb or other diatomaceous packing with highly active surfaces, which causes severe tailing of the amine peaks. Yumerous remedies have been offered to avoid the problem of tailing of amine peaks. Among these are: use of a support with an inert surface, such as Teflon; treatment of the surface with a basic organic compound or the use of a basic organic liquid phase; and treatment of the support or liquid phase HE SEPARATION
32306
with an alkali hydroxide to decrease the tendency of the column to adsorb amines. The use of Teflon as a support for the resolution of amines is described by Landault and Guichon ( 2 ) . These investigators found that columns of various polyglycols coated on Teflon gave fair resolution of amine peaks and that tailing of the peaks was reduced considerably. The use of tetrahydroxyethylethylenediamine (THEED) and tetraethylenepentamine (TEP) as both a liquid phase and tail reducer for other liquid phases was reported by Sze, Borke, and Ottenstein (4). They found that the use of these mixtures reduced tailing of amine peaks and permitted resolution of the methyl amines and ammonia. The use of a support treated with a n alkali hydroxide was suggested by Decora and Dineen ( I ) and was extended by Smith and Radford ( 3 ) . Smith and Radford used liquid phases of Carbowax with KOH coated on Chromosorb W for the resolution of aliphatic diamines with satisfactory results. Confronted with the necessity of separating complex mixtures of aliphatic amines, the writers compared the efficiency of various types of columns. From previous experience it was known that Dowfax 9 x 9 was, for many
separations, superior to Carbowax 2011. Dowfax 9 S 9 on Teflon \vis found to be ineffectire in separating complex amine mixtures: most peaks also exhibited moderate tailing. I t was found. however, that mixtures of 9x9 with S a O H on Chromosorb-P provide better resolution of the lower aliphatic amines and less tailing than could be obtained with Carbowax-SaOH mixtures and that the resolution was generally hcttc,r than with THEED-TI*:I’ mistureh. Moreover, 9x9 has a higher safe maximum temperature (225’ C.) than all Carbowaxes except 2011 and its ability to resolve amine mixtures is sufficient to permit seliaration of the lower aliphatics at teml)eratures fa? in excess of their boiling points. EXPERIMENTAL
Chemicals. T h e Chromo>orb-P 60-80 mesh, the Carbowax 400 and Carbonau 2011 were obtained from the F and 11 Scientific Corp -411 amines were Eaqtman 11 hite Label and n e r e uqed a \ received T h e methyl amine- and ammonia n e r e used in aqueoub solution a > rrceirrd. Apparatus. .ill a n a l r v i nere performed with a Chromalj zer-100 chromatograph u m g a hot nire dctcctor a t a filament current of 175 ma Chromatogramb were recorded on a Sargent SR 1-mr. recorder a t a VOL. 36, NO. 1 1 , OCTOBER 1964
2097
chart speed of 1 inch per minute. The sample size was 2 ~ 1 in. each case. Preparation of Columns. Three columns were made, one each of Dowfav 9N9, C a r b o w x 400, and Carbowax 2051, according to the following procedure: 5 grams of the liquid phase and 0.5 gram of S a O H were stirred in 10 ml. of melhanol t)o complete solution. This was poured onto 20 grams of Chromosorb-P, sufficient methanol was added to make a slurry, and the excess solvent was removed by evaporation. The packing was dried in a vacuum oven, with no special attjempt to avoid pickup of carbon dioxide or water vapor. The dried packing was screened t,o 60-80 mesh and was loaded into a 3ilG-inch o.d. X 9-foot copper tubing. Columns were allowed to stabilize overnight in t'he chromatograph a t 90" C. with 35 ml. per minute helium flow. A 3 i I 6 inch X 5-foot 9 S 9 / S a O H column was prepared similarly for separation of aliphatic amides.
1
I
e
RESULTS A N D DISCUSSION
The relative abilities of the three columns to separate an eight-component mixture of aliphatic amines is shown in Figure 1. The chromatograms represent the best separation obtainable without incurring extreme peak distortion. The greater resolving power of the 9 S 9 column is evident, as is the efficiency of NaOH in reducing tailing on all three columns. Separation of the earlier peaks with the Carbowax columns is greatly improved a t lower temperatures but the later peaks develop leading edges. This distortion would probably be eliminated by temperature programming. Decreasing the temperature of the 9 x 9 column to 120" C. gives improved separation, but the retention times of the later peaks become excessice. At 135" C.,ammonia and methylamine are also separated, the net retention times being 0.31 and 0.50 minutes, respectively. These com-
Table I.
Mixture 1
3
I
I
I
I
0
2
4
6
8
Figure 1 . mixtures
I
IO
Component Ammonia Trimethylamine Dimethylamine N-lleth) laniline Aniline S,S-Dimethvlaniline N,N-Dimethylformamide S,K-Dimeth> lacetamide l;-AIeth\ lforrnamide Acetamide
0
2098
ANALYTICAL CHEMISTRY
I
14
16
I
I
1
18
20
22
Lower: Dowfax 9N9 at 135' C. Center: C a r b o w a x 20M at 1 0 5 ' C. Upper: C a r b o w a x 400 a t 90' C. All columns 3/IG" X 9', 20% liquid phase, Z ' / z % NaOH on 60-80 mesh Chromororb-P. Flow rote 45 ml./min. in each case. Identification of peaks: 1. Air, 2. n-Propylamine, 3. Diethylamine, 4. Triethylamine, 5. Ira-butylamine, 6. Di-n-propycamine, 7. Impurity in n-amylamine, 8. n-Amylamine, 9. Tri-n-propylamine, 10. Impurities in tri-n-propylamine, 1 1 . Di-n-butylamine
pounds were not included in the mixture in Figure 1 because the water in IT-hich they were dissolved tails sufficiently to interfere \with the 6-minute region. The 9hT9-SaOH column has been found effective in separating a wide variety of basic organic compounds.
Retention time,a min. 0 59 1 05 1 65 1 11 13 15
86
4 8
Flos rate, ml./min.
208
48
220
40
2
1 92 2 96
4 5 7 8
Column temp., O C. 90
01 21
53
90 n-Butyramide 97 Measured from air peak hlixtures 1 and 2 Beparated on 9' X 3/16", 2 0 5 Donfax 9 S 9 2 ' / 2 5 S a O H on 60-80 mesh Chromosorb-P AIixture 3 separated on same packing, 3/1G" X 5' column X,K-1)ipropylpropionamide
I
12
RETENTION TIME -MINUTES Resolution of 8 aliphatic amines by three liquid phase-NaOH
Retention Times of Various Basic Compounds Resolved by Dowfax 9N9 Columns
Methylamme 2
I
I
The retention times and column conditions for some of these compounds are listed in Table I. The separation of ammonia and the three methylamines is not complete at 90" C. but is usable; better separation is obtained a t 70" C. but the ammonia peak tails badly a t this temperature. Resolution of animonia and the methylamines at 90" C. is about like that given for the THEEDT E P mixture in Figxire 3 of reference (4). I n separating mixtures of amides, the peaks are symmetric with no visible tailing. The mechanism by which the NaOH reduces tailing is not, clear to the writers. A reaction of S a O H with either 9 S 9 or with the Carbowaxes would not be expected; however it appears doubtful that neutralization of acidic sites on the support surface can account for the improvemcnt in i m k shape. We find, in agreement with Smith and Radford, that treatment of Chromosorb-P with alcoholic KiOH followed by rinsing with methanol to remove excess base, is not effective in reducing tailing of aliphatic amines, although it does give satisfactory results with the more weakly basic aromatic amines. Dowfax 9 S 9 on
Teflon. a sul~posedly inert surface: yields tailing peaks for the aliphatic amines. I t would appear, therefore, that the base must be altering the behavior of the liquid phase. I t is immaterial n-hether the base is dissolved in the 9 x 9 and the mixture added to the support or whether the base and the s ~ -are 9 on the cjupl,ort separately, so long as the requisite amount of base is present.
ACKNOWLEDGMENT
The writers thank the Dow Chemical for the ’Owfax 9F9.
cO*
LITERATURE CITED
(1) Decora, A. W.) Dineen, G . U., “Gas
Chromatography,” H. J. Soebels, R. F. Wall, S . Brenner, eds., p. 33, Academic Press, Sew York, 1961.
( 2 ) Landault, C., Guichon, G . J J . Chromatog. 9, 133 (1961J. (3) Smith, E. D.>liadford, It. I>., * \ S A L CHEM. 33, 1161 (1961).
(4) Sze, Y. L., Borke, 11. L., Ottenstein, D. Ibid., 35, 240 (1963).
RECEIVEDfor review April 9, 1964. Accepted August 5, 1964. This investigation was supported by research grant GlI-10064 from the Public Health Service of the U. S. Department of Health, Education, and Welfare.
Determination of Diazepam (Valium) in Blood by Gas Liquid Chromatography J. ARTHUR F. de SILVA, MORTON A. SCHWARTZ, V. STEFANOVIC, J. KAPLAN, and L. D’ARCONTE Department o f Pharmacology, Hoffmann-la Roche Inc., Nutley, N. J .
b A method for the! determination of diazepam (Valium) in whole blood of humans involves selei:tive extraction of diazepam into ether, acid hydrolysis, and analysis b y gas liquid chromatography. The methocl has high sensitivity (0.02 to 0.03 pg. per ml. of blood), i s specific for diazepam after it i s hydrolyzed to 2-methylamino-5chlorobenzophenone, and has a very acceptable recovery and reproducibility of the order of 94% + 5.0. The method has been successfully applied to the determination of diazepam in samples obtained from a variety of clinical studies including a blood level fall-off study and the placental transfer of diazepam in expectant mothers. Blood levels of diazepam and i t s N-demethylated metabolite can be determined in a single assay and the method i s also applicable to the determination of chlordiazepoxide-HCI (Librium) in blood.
D
i-chIoro-1,3-dihydro-lmethyl - 5 - phenyl - 2H - 1,4benzodiazepin-2-one (Valium) is a new psychot~herapeutic drug of the 1,4benzodiazepine class: of compounds (11). Its pharmacological and clinical properties have been reported by Randall et al. ( 8 ) . Although diazepam is chemically related to chlordiazepoxide (Librium) it’ differs from the latter in that’ it contains a meth!.lated-S at, position 1, and both the basic side chain a t ~jo>ition2 and the -Y-oxide function a t pmition 4 (Figure I ) . (Valium and Lihrium are registered trademarks of Hoffniann-La Roche Inc. for the phariiiawutical forinu1atio:ns of diazepam and chlordiaze;,ositie-HCl, respectively.) Because of these differences, diazepam IAZEPAM,
cannot be determined by the analytical procedures developed for chlorodiazepoxide. hydrolyhis to an aromatic primary amine which is measured by a Bratton-Marshall procedure ( 7 ) or coniersion to a lactam which is determined spectrofluoronietrically ( 5 ) . Studies by Schnartz, Koechlin, and I
