I
also be utilizrtl for the t.hoit.c. of suitatilc ellis for nonayuf'oll nO:I])olal~pha5.c fo For a more accwal(~eLstiniation of thc\ optimal solvent composition, it n-nuld be advantageous t o detrrinine the Imrtition coefficients in thr oihinuni solvent tein estimated from chroniatogral)hic data and introtluce an appropriate rorrection in the conilio,.ition of the mixed phase, if necessary.
CODEINE
LITERATURE CITED
( 1 ) Jusink, FmrioN
Figure
5.
Weight distribution curve after a 24-transfer run
Mobile phase, 23 v./v. formamide
70cyclohexane
each alkaloid in thi. contents of the e l t ~ n i t ~ nSt o . 0 . T h e weight distrihution of alkaloids is presented in Figure 5. A bin the former sj-stem an almost conil)lcte scparation of the model mixture \vas obtained, which was also confii,mcd ti)- paper cliromatographic~ anal!.& of the fractions. 130th alkaloid. orcurred only in fractions S o . 9 to 11, thc \wight of which i.i only a very small 1iai.t of the original mixture. l'he ohit it ion of thf, first niaximum is i n very good agreement with that calrulated from Figure 3b, A' = 5 . For tho other alkaloid, the niaximum ocrurrctl at a lower fraction number
I,., Folia Soc. Sei., Lukili~
(Poland), in press. ( 2 ) Jusiak, L., Soc.zewii'ski, E., \l.aksmundzki. A,. ~ l c t uPolon. Pharvi. 19.
in benzene; stationary phase,
than exiiected, No. 16 instead of S o . 20 calculated from Figure 3b. This divergence must have tieen caused by nonfulfillnient of the assuni1)tions made in the theoretical treatment : nonlinearity of the partition isotherin, differentw between thc dynainir 1)artition coefficient in the chromatograi)hic ~)rorc+, and the equilibrium partition coefficients which o w u r in the cabcade processes, t,tc. Changes in the composition of the mixed nonpolar phase were in this rase much smaller as the difference of volatilities of cyclohexane and henzrne ip murh lo\vcr. The resulti of cxlwiinents indicate that paper chromatographic data may
(Polmd), in press. ( 1 0 ) \Vaksniundzki, h.,Soc.ze\\-i.'ski, J5.,
Continuous Separation by the Method of Thin Layer Chroma tog ra p hy S. TURINA, V. MARJANOVIC-KRAJOVAN, and
M. OBRADOVIC
lnstitufe for Inorganic and Analyfical Chemistry, University o f Zagreb, Yugoslavia
b A new method has been developed for the continuous separation of a mixture b y thin layer chromatography. Two different systems of solvents are fed from different sides to the thin layer spread on a triangular glass plate, while the mixture to b e separated i s introduced inear the apex of the triangular plate. The separated fractions leave the lolate at its base. 1he separation of iron and cobalt has been successfully carried out on this apparatus.
Figure 1. The principle of continuous thin layer chromatography
VOL. 36, NO. IO, SEPTEMBER 1 9 6 4
o
1905
.
,
m 5. csr
.A'
Figure 2.
Diagram of apparatus Figure 3. ADDoratur matography
Detoil A: Section of the rim of the plate; Detoil 8: Section of the strip carrying the solvent
cornponcnts, A , B , and C , is fed to the apparalus and if thc distribution coefficients of these components betu-ccn the liquids S , and 4 are different, a separation of the components will take place, :is shown in t,he diagram. Matho matically this can he represented by the equation: o(
=
fW,VL, u.)
migration angle of an individual componcnt to the left (-) or to the right (+) of the axis of symmetry I ) = mu~flicient of distribution of the inilividnal cotn1,onent between the two phases u, = vrrt,ical wmilmrmt of migration of lihasn 1 with respect to the axis of syinnrrtry u2 = v d i i x l (:omponent of migration of ~ h s 2c with rcspect to the axis of syinmetry 01
=
for
continuous
thin
loyer
chro-
EXPERIMENTAL
T o investigate the possibility of continuous separation, the apparatrin shown in Figures 2 and 3 has been constritctcd. The base of the apparatus is a glass plate having the form of n triangle which is covered with a 1-tnm. layer of Kieselgel G according to Sbahl (E. Merck A.G.) (2). The liqiiid phases are introduced by sbrips of film Irapcr wrapped in polyethylene film (Vigrirc 2. details A and B ) . To avoid ovaporation, the thin layer is covered with a glass plate having the same dimensions as the base plate. The liquid phases are contained in reservoir lwttlrs and their rate of flow can hc controlled by raising or lowering the bottles. The glass plate is inclined under an angle of 45'. The eluents with the separated components are collected in 17 bottles connected to the apparatus by strips of filter paper. The mixtures to be separated are brought to the point P also by a strip of filter paper. For this purpose a hole is made in the upper glass plate.
1
E
B. A.
Flow of the iron ond cobalt ions o ~ r o i the i plote
1906
-
Groph showing the concentrotion of methyl alcohol. acetone, hydrochloric acid, and water in the receiving bottles
Figure 4. ANALYTICAL CHEMISTRY
Diagram and graphs of separations
C.
Groph showing the concentrotion of cobalt and iron in the receiving bottler
RESULTS
DISCUSSION AND CONCLUSION
but i x t h c ~ i ~to show the pract’ical feasibility of a sq)ai.ation on the basis of tlic h!-~)oth(.sis and to ascwtain the difficulticts in thc way of tht, I)ractical proc:cdur~s. Th(> rniit~uw of iron(1I I ) antl vobalt (1 I ) chloiitlw was rhosen becaiis.c’ the coloixtion allows the flow of t 11cs liquids to bc followd visually and t h i,ate of flow of theeluents and the inixtulc to I)t. dirwtly controlled. LVith colorless ions thc wot,k lvould doubt I c d y I)(, murh more difficwlt and tiiiic, (wisuiiiing, us t,he ions would have t o I)() tlrtcc.tod by al)i)rol)riatereagents, and the, control of rate of flow would not Ix st i,aiglitfor\vaid. The results of t,he experimrnts point to the conclusion that tmhetriangular h h : i l ) ( > of i l w glahs I)latcs i h adal)tcd to t’he contiiiilous srl)aratiori, but it is I’ossible that h~t t w rcsults might be arhieved by changing the hhal’c of thc plate. l‘he roin1)osition of the adsorbent niixt,urc. used in thin layer chromatog-
raphy can bc changcd within wide limits to adal)t it t o (#hanging circumstancch. ‘I’his is a big a t l v a n t ~ a g ~ ~ when comi )aiwl to I )aI ) r r vh IY )inatogral’hy. IIorcwver, bj- a1)I)lying a thicker layer the rat(>of flow anti, consequently, the rate of sel)aratioii cwi be increased, whivh is an advantagc in preparative chemistry or when the substance to be dctemined is prcseiit in extremely small concentrationh. LITERATURE CITED
(1) Merck, E., Ilarnistadt, “Chromatugraphie,” Bull. 108-111 (I963!;,
( 2 ) hlerck, E., Ilarrnstadt, 1 r i p r a t e fiir Dunnschicht - Chromatographie, ”
(Folder) 1963. (3) Pleuger, “Collec:tochrorri,” (Folder) 1063. ( 4 ) Pucar, Z., J . C’hron~atoy.4,261, (1960). ( 5 ) Turina, S., Krajovan, \.., ICosti)maj, T., 2. Anal. Chem. 189, 100 (1962). RECEIVED for review February 24, 1964. Accepted May 4, 1964.
Dete rmincr ti o n of Narcotic Ana Igesics in Human Biological Materials Application of Ultraviolet Spectrophotometry, Thin Layer and Gas Liquid Chromatography 5. J. MULE National lnstitufe of Mlental Health, Addiction Research Center, lexington, Ky.
b The rapid quantitative extraction o f narcotic analgesics from human biological material cmd the analysis of each extract b y ultraviolet spectrophotometry, thin layer chromatography, and gas liquid chromatography i s described. Maximumi and minimum absorbance values as well as molar absorptivity data were determined in 0.1N HCI, 0.2N NaOH, absolute ethanol, and 25% isobutanol in ethylene dichloride. The bathochromic shift in alkali was not observed when the free phenolic hydroxyl of the analgesic drug was altered. ‘Thin layer chromatographic (silica gel1 G and buffered, pH 8.0, cellulose powder adsorbent) R , values of the drugs were determined in seven different solvent systems. Gas liquid chromatographic retention data were obtained for the free drug base as we11 as the acetylated and propionateid column derivatives on a 2% SE-30 !;iloxane polymer column at 215” C. Unique differences were observed between 3 1 compounds in five different chemical families. Usually 3 to 5 hours were required to extract and completely analyze a biological sample for the presence of microgram quantities of narcotic analgesics.
F
years t,hc. det,ection and determination of narcotir analwerc’ largc,ly based on micro1)recil)itation incthods (5, 9, 2 f ) , quditat.ivc c d o r tests (4, 8, 19) and ultraviolet s1)ectrol)hotonietry (2, 10, 12, 1 7 ) . l‘hv surcess or failure of a n identification, especially from biological material, was largely dependent ul)on the isolation and I)urificat,ion Iirocess which preceded the identification. Paper chromatography (11, 15, 2 6) provided a siinl)lc%method whcfcby the isolation and Iiartial Iwification of some narcotic analgwicns was easily acc.oml)lishd, hut the time required for develolmient was usually 12 to 24 hours. Cochin and Daly ( 6 ) , using the Stahl t8hin layer rhroniatographic- technique (20),were able t’o seliarate and identity analgesic drugs in 4 to 5 hours. The application of gas liquid chromatograllhy ( 1 , 9, I S , 14, 18) t’o the detection Or sr1)aration of analgwic drugs has further incrcmctt the rapiditmy and .sensitivity of idcntification. It is ohvioits, howvc,r, that t,he 1)riiiiary use of thcw ana1yt;c.d nwthods was for the detertion andlor isolation of the pure drugs^ Generally data were not available concerning the quantitative recovery of these coinOR M A N Y
pounds from extracts of biologicaal materials. The extcnsivr IIHO of t8hcL narcotic analgesics in nicdicinc, t hri I toxicity, and in ~)artic.ulartlitlir wltliction liability make their railid dctwtion in microgram quantities highlj. important. I t is, th(wforo, thr, 1)url)ose of this 1)al)cr t.o 1)r(w’nt r:iI)i(l quantitative methods for t h r cstracation of the narcot ic analgc,sic*sfrom h i i n i n n biological fluids and tihsues, as \v(s11 as of eavh organic c.xtrac*l.11 a1 techniques ol‘ iiltixvioh spectrophotometry, thin layer cliromatography, and gas liquid c.hromat ography. EXPERIMENTAL
Extraction of the Iminoethanophenanthrofurans. Ihlilicate 6-ml. s:iinples of human urinc, plasma, or tissilc homogenat’e (loyo in 0.1.Y IICI) i i i 40-ml. glass-stol)l)ercd centrifugt, t,ubes were adjusted t o pH 10.0 nitli 2.5.11 NaOH and buffercvl wit11 3 ml. of potassium phosphate h f f e r , 1” 10.4. The solutions w(’re .s.nturatcd with 2 grams of Sac1 ant1 miscxtl with 15 ml. of eth!.lcne di(-lilorid(~ containing 25% isobutanol I v . Y.) . The samples were shaken for. 30 i i i i i i i i l ( t b at 280 os(-illations 11er niiiiutc, in i i i i International shaker nxic~liinc ii+inc VOL. 36, N O . 10, SEPTEMBER 1964
1907
