Table 1.
Determinationof Cyanoacetic Acid in Known Solutions
CAA, pg./Ml.” Taken Found 0.100 0.101 0.500 0.500 0.700 0.690 0.800 0.810 1.000 0.950 1.200 1,200 1.400 1.400 1.600 1.580 1.800 1.790 Each value represents three determinations.
Error, $6
+o. 1
... -0.12 +0.12
... ...
-0.12 -0.55
average of
conditions (room temperature). T o establish the evidence of stabilization of the color with sodium hydroxide, a number of stability curves were run at intervals of 30, 90, 150, 210, 290, 330, 390, and 480 seconds. The results showed that the color remained stable a t the end of 480 seconds (Figure 1). As a change in p H from 2 to 10 caused no instability in the color when sodium hydroxide was used, experiments were carried out at pH 10. The modified method was tested for accuracy and precision by determining the cyanoacetic acid content of solu-
tions of known concentrations (Table I). The reproducibility of the modified spectrophotometric procedure was such that the absorbance values of replicate tubes agreed within 4%. The method as modified was applied to in vitro and in vivo blood plasma samples. LITERATURE CITED
(1) Sievert, H. W., Lipton, S. W., Strong, F. M., Arch. Biochem. Biophys. 86, 311 (1960). HUBERT G. LOVELADY Institute of Agriculture University of Minneaota St. Paul 1, Minn.
Separation and Identification of Some Sympathomimetic Amines by Gas Chromatography SIR: Smith and Radford (6) described the separation of aliphatic diamines by gas chromatography using Carbowax 20M as the liquid phase on a solid support with potassium hydroxide. The present communication extends the use of a similar column to the separation of sympathomimetic amines directed toward practical applications in toxicology. As indicated in Table I, positive results were obtained with nine of the sympathomimetic amines and their salts, in every case at more than one temperature. The stationary phase was 5% Carbowax 20M on 5% potassium hydroxide-coated firebrick, 100 to 120 mesh. The apparatus consisted of a Pye argon chromatograph equipped with an ionization 8-ray (strontiumg0) detector and a 10-mv. recorder, the column being contained in a Cfoot borosilicate glass tube of 5 mm. i.d. The argon flow rate was 40 ml. per minute, the detector voltage 1750 volts, and the attenuation X 1. Responses were obtained from 0.1 to 3 pg. of the free base or salt. The free bases and salts were dissolved in ethanol or acetone to make concentrations of 10 gg. per pl,, with the exception of the catechol amines which, being difficultly soluble in mixtures
0 1 2 3 4 5 6 7 8 9 10
15
20
Minutes
Figure 1. Separation of mixture containing: ethanol (A), metamphetamine. HCI (B), methoxyphenamine.HC1 (C), ephedrineSHCI (D), phenylpropanolamine.HCI (E), and benzphetamine.HCI (F). Oven temperature, 190’ C.
of ethanol and acetone, were partially suspended. The sympathomimetic amine salts
Table 1.
and the corresponding free bases had the same retention times even on a nonalkaline column, possibly because
Retention Times of Some Sympathomimetic Amines
Retention time, minutes Column temperature 210’ c. 120” C. 170’ C. 190’ C. M.1.p.” Base Salt Base Salt Base Salt Base Salt
Compound d-Desoxyephedrine . HCl (NE’). 328 d-N,a-Dimethyl henethylamine hydroc&oride Amphetamine.HzSOc (USPY 72 dl-a-Methyl henethylamine sulEte Phenethylamine 793 8-Phenethylamine Methoxyphenamine . HC1 (NND)c 669 o-Methoxy-N,a-dimethyl henethylamine h drocborlde Ephegine . H2SOn.(USP) 404 a4 I-Methvlammoethv1)benzyl dcohol sulfa&‘ 1-Ephedrine Ephedrine. HC1 403 Pseudoephedrine. HC1 869 a-(1-Methylaminoethy1)benzyl alcohol hydrochloride d-Isoephedrine Phenylpropanolaniine . HC1 805 CZ-( 1-Aminoethy1)beneyl alcohol hydrochloride Benzphetamine. HCl Methoxamine. HC1 (USP) 668 e-( +minoethyl)-2,5dimethoxybenzyl alcohol hydrochloride 1,PButanediamine (Putrescine)
11.6 11.5
2.4
12.3 12.3 14.5b 25.6
2.7b 4.2
1.6
1.6
1.7
1.6
1.8b
2.4 6.2
3.4
3.4
2.0
2.0
8.2
8.2
6.9 6 . 8 3 . 8 3 . 8 13.2b 13.1b 6.7b 6.7b 13.2 6.8 6 . 1 6 . 2 3.4 3.4 13.4b 13.3b 6.6b 6.5* 4.6
4.6
8.5* 8.6b
17.2 17.3
26.0 26.5 11.8 11.9
1.8
23. 4b
2.0 3.4b
1.4 1.6
2.w
lI5-Pentanediamine 781 1.7 2.8 1.7 (Cadaverine) 37. 6b 4.gb 2.7b Page number in the ‘‘Merck Index,” 7th ed., 1960, Merck and Co., Inc., Rahway, N. J. Major peak, by area. e NF: “The National Formulary XI,” American Pharmaceutical Assoc., Washington, D. C., 1960. USP: “Pharmacopeia of the United States of America,” 16th Rev., 1960. NND: “New and Nonofficial Drugs,” J. B. Lippincott Co., Philadelphia, 1961. 0
VOL 34, NO. 10, SEPTEMBER 1962
1345
of sufficient residual basicity of the Chromosorh W, acid-washed, solid support. No responses were observed with the alkaline chromatographic system for the catecholie and phenolic amines, presumahly because of their acidic character. Injections of 10 pg. of the salts and free bases of the following sympathomimetic m i n e s failed to elicit responses:
- -
Phenolic. Hydroxyamphetamine. HBr [p (2 aminopropyl)phenol1 ("Merck Index," p. 59); metaraminol. HnC&06 (m - hydroxynorephedrine) (660) ; 1-phenylephrine.HC1 [lm-hydroxy a (methylaminomethy1)henzyl alcohol] (802); tyramine.HC1 (4-hydroxyphenethylamine) (1078). Catecholic. l-Arterenol.H,C40,0. [l c (aminomethyl) 3,4 dihydroxybenzyl alcohol] (104); epinephrine [3,4 dihydroxy a - (methylaminomethy1)henzyl alcohol] (405) ; ethylnorepinephrine [a-(l-aminopropyl)protocatechuyl alcohol] (59) ; isoproterenol. HCI [n-(iiopropylsmmomethyl)protocatechuyl alcohol] (580).
- -
- -
- -
-
The double peaks observed, as indicated in Table I, for ephedrine, pseudoephedrine, and phenylpropanolamine were associated with decomposition on standing and in some instances resulted from treatment with base. Cadaverine (1,spentanediamine) and putrescine (1,4butanediamine), which
might he encountered in the standard toxicological extraction of tissue, gave multiple sharp pesks which did not interfere with the sympathomimetic amine determinations. Figure 1demonstrates the separation of a mixture of sympathomimetic mines. Linear responses-peak height and area-were observed for the amines under the operating conditions described No interference was encountered from akaloids, tranqnilizing drugs, or harhiturates using the operating parameters described. Barbiturates (4), b e i i acidic, did not emerge from the alkaline column. A nonalksline column utilizing a lower percentage of liquid phase (Carhowax 2QM, 1%,on Chromcsorb W, acid washed, 60 to 80 mesh) and higher temperatures (20O0 to 260' C.), which had proved very useful for screening toxicological extracts for alkaloids (1, 3), harhiturates, and tranquilizing drugs @),was tested with the sympathomimetic amines. At the higher temperatures for screening, however, these amines emerged very early and were not separated. These screcning investigations were made with the Aerograph Model A-600, Hy-Fi, gas chromatograpb. As a part of the continuing effort in this laboratory to systematize analytical toxicology, gas chromatographie operating parameters (liquid phases, inert supports, and higher tempera-
tures) are currently being investigated to determine conditions favorahle for emergence of the phenolic and catecholic sympathomimetic mines.
.-
.. ._
LITERATURE CITED
-.
--.. A. palea, n. IVI., nignec, P. F., Vandenheuvel, W. J. A., Wildman, W. C., J . Am. C h .Soe. 82,3791
(I) uoya, n. . I - .
1
(2 Parker, K. D., Fontan, C. R., Kirk,
CHEM. 34,757 (1962).
*N g:A .rL.
(3) Parker, K. D., Fontan, C. R. Kirk, P. L., "The Separation and Id~nti6cstion of Some Alkaloids and Relate; Corn ounds by Caa Chromatngrs. hy
Ed-
unpu\linhed data, 1961, School of inology, University of California Berkeley 4, Calif. (Preeented at the fall meeting of the California Association of Criminahta, San Frmeisco, Calif., nA-k-"CWVIi'
4n.2,
'JVA.,
,
K. D., Kirk, P. L., ANAL. CBEM.33, 1378 (1961). : 5 ) Smith, E. D., Rdford, R. D., Ibid., 1160 (1961). KENNETE D. PnaKea CMRLESR. FONTAN PAUL L. KIRK School of Criminology University of California Berkeley 4, Calif. I:4) Parker,
Work supported by grante from National lnntitutra of Health, U. S. Public Health Sewice. EF 21[C31 iformerlv RC-4372 and R&&OZ). 'and from th; Research Committee, University of California. Reported a t the fall meeting of the califcrnia Asao:i!tion_ of- ~ i n a l i s t a , S m NrBnCisM, Calif., VctOber 1961.
Thin-Layer Chromatography on Microslides SIR: Thin-layer chromatography can be used as a sort of ~croprocessfor identification of indieduel substances or mixtures of a limited number of components The method use8 microscope slidcs as support plnks, small cylindrical jars as chromatographic chambers, and frequently can be douc in a few minutes. The necwsrv rquipment is thus reducal to a rninimurn,some available in evcrylahoratury, the rest can hc made Iiy n n v while . . -. . . ... worltshop. Tlus process IS illustraterl by chromatograms of essential oils, morphine, rutin, usnic acid, and aminupyriue. The method is especially useful for the examination of rnedicina and toxicological substances.
22 mm. i.d., and 25 mm. in height, the lower base being recessed on one side by 0.2 mm. At a right angle to this recess, and parallel with the base, a straight piece of the same material as the
EXPERIMENTAL
The basic glass plates are flat microscopic slides, 75 X 25 mm. in length and width, and 1 or 1.5 mm. in thickness. For the extension of the thin layer, they are placed, narrow sides together, on a baseplate of wood or plastic, against a raised edge to keep them aligned (Figure 1). The applicator is a small cylinder of bronze or stainless steel or even plastic of 33 mm. ad.,
1346
ANALYnCAl CHEMISTRY
Figure
1.
Close-up of
applicator
cylinder is soldered or glued, so that its lower edge extends beyond the base by about 2 mm. The dimensions of this piece are ahout 90 X 6 X 2 mm., and it serves solely as a guide rail during the movement of the applicator over the glass slides (Figure 2). For the preparation of the thin layer, about 3 grams of silica-gel G from Merck or similar products are mixed with water in the conventional way for thin-layer chromatography. The liquid is placed in the applicator, already on the first slide, and after a moment, the applicator is moved slowly and continuously over the slides which.are subsequently dried in the conventional manner. Normally, up to three star& points are marked a t a distance of 10 mm. from the lower edge of the slide, and the frontline is marked a t 50 mm. from the startpoints. About 0.5 to 1 pl. of the solution to be analyzed is plaeed on each startpoint, taking care that the spot formed does not exceed 2 mm. in diameter. Normally 5 to 10 pg. of each substance may be applied. The chromahgrnphic chamber is a cylindrical jar of ahout 35 mm. i.d. and 80 mm. in height, with a cork or glass stopper or aground g l w plate as lid. On the bottom, a glass rod of the same length as the inner diameter
