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 mines 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).
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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.
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.. ._
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
Parker, K. D., Fontan, C. R., Kirk, CHEM. 34,757 (1962). (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 unpu\linhed data, 1961, School of Edinology, University of California Berkeley 4, Calif. (Preeented at the fall meeting of the California Association of Criminahta, San Frmeisco, Calif.,
(2
*N g:A .rL.
nA-k-"CWVIi'
4n.2,
'JVA.,
,
Parker, 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)
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 at 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, while the rest can hc made Iiy n n v . . -. . . ... 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
of the jar serves as separator, if two slides are placed in the chamber at the same time. Alternately, a test tube of about 30 mm. i.d. and 90 to 100 mm. in length can be used. For a larger number of slides, rectangular glass jars, with guide rails such as are used for the preparation of slides in botany, may be employed. The drying and developing of the spots follow conventional procedures. Several experiments demonstrated the practicability of the method. All were executed on silica-gel G (Merck) layers of 2Wmicron thickness. Essential oils of peppermint and sassafras diluted with alcohol to a concentration of 10% were run with benzene. The front was reached in about 5 min. The spots were developed with a saturated solution of antimony trichloride in chloroform, with subsequent heating to about 120' C. Morphine and rutine dissolved in alcohol in a concentration of 1% were run with butanol containing 10% of acetic acid by volume and saturated
I
A
2
4
B Figure 2. Schematic side ( A ) and bottom ( E ) view of applicator 1. Cylinder. 2. Recess In base. 3. Guide rail. 4. Curved extremity of guide rail to even o d small irregularities in slide width
with water. The front was reached in about 30 min. The spots were developed with Wasicky's reagent (p-dimethyl-
aminobenealdehyde in sulfuric acid) with subsequent heating to about 70" C. Vsnic acid dissolved in chloroform in a concentration of 1% waa run with benzene. The spot was examined by ultraviolet light. Aminopyrine dissolved in alcohol in a concentration of 1% was run with methanol. The front was reached in about 5 minutes. The spot was deseloped with an aqueous solution of ferric chloride at about 1%. In all cases the spots were well defined. The essential oils separated readily into their components, making possible an easy verification of adulterants. The R , values of the principal substances tested, as described, a t 22' C., were: menthol, 0.43; safrol, 0.94; morphine, 0.26; rutin, 0.6; usnic acid, 0.43; aminopyrine, 0.63.
ROBERTOWASICKY Departmenta de Farmacognoaia da Faculdade de Farmlcia e Odontologis da Univeraidade de SBo Paulo Rua Trea Rim, 363 SHo Paulo, Brad
Microdetermination of Fluoride Using Null-Point Potentiometry SIR:In the null-point poteiitiotuetric determination of fluoride previously described [O'Donnell, T. A.. Stewart, D. F., ANAL. CHEM.33, 337 (196111, fluoride solution of unknown concentration was added to one of two identical half-cells containing a cerium (1V)-cerium(II1) solution. Fluoride complexed the cerium(1V) strongly and the redox potential of that half-cell was decreased. Standard fluoride solution was then titrated into the other half-cell until there was no potential difference hetween the two half-cells. The lower limits of applicability of this method were 5 X 10-3M fluoride with an accuracy of 0.5% or 10F3M fluoride with an accuracy of 1%. In the present work the basic method has been developed as a micromethod, an accuracy of 0.5% being obtained in the determination of 5 X 10-4M (10 p.p.m.) fluoride. In addition, the experimental procedure previously reported has been simplified significantly. The improvement in accuracy a t low concentrations has been obtained by several modifications of the original method. First, improved electrical connection between the two half-cells has resulted from the replacement of the agar-KC1 salt bridge by an asbestos fiber. One of the difficulties in the original method arose from the necessity for adding water from a second buret during titration with sodium fluoride solution in order to keep the COII-
centrations of cerium(1V)-ceriuni(Il1) solutions comparable in each half-cell. This has been eliminated by adding concentrated fluoride solution from a microburet, so that the increase in volume during a titration is negligible. A third modification is one of general application for this type of potentiometric titration. By using a volume
of cerium(IV)-cerium(II1) solution in the titrant half-cell which is lsrger by a certain factor, say ten, than that in the half-cell containing the fluoride for analysis, it is possible to titrate a small quantity of unknown fluoride with ten times the amount of standard fluoride, with a consequent increase in accuracy. EXPERIMENTAL
A Figure 1. A. 6. C. D.
c
Apparatus used
Asbestos fiber connection sealed in glass Platinum foil electrodes Stirrer Microburet
Apparatus. The apparatus used is shown in Figure 1. The null-point detection was by means of a Pye portable potentiometer (Catalog No. 7569 P) with a n external Pye Scalamp galvanometer (Catalog No. 7902/S). Platinum foil electrodes were used. I n a typical analysis the internal half-cell consisted of a glass tube (6 x 6 / * inch) with an asbestos fiber sealed into the bottom. Several different tubes employing asbestos fiber, cracked glass, and sintered glass electrical connections were tested before selecting the asbestos fiber connection as being the most satisfactory-Le., having a low resistance and negligible transfer of liquid through the connection. The external half-cell was a 250ml. beaker. The microburet used had a delivery volume of 0.2 ml. and could be read to an accuracy of =tO.OOOl ml. All reagents used were of analytical grade. Procedure. For analysis of a solution 5 x lO-4M in fluoride, dissolve 0.64 gram of ceric ammonium sulfate and 0.28 gram of cerous sulfate in 200 ml. of water and 14 ml. of 18M sulfuric acid, and dilute to 1 liter. VOL 34, NO. 10, SEPTEMBER 1962
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