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 t h e 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 h u m a n 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. T h e 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, NO. 10, SEPTEMBER 1964
1907
special bottle holders described by Woods et al. (22) and centrifuged a t 2500 r.p.m. for 5 minutes. Ten-milliliter aliquots of the upper organic phase were transferred to 40-ml. centrifuge tubes containing 5 ml. of 0.1X HC1, re-extracted into acid by shaking for 15 minutes at 280 oscillations per minute, and centrifuged a t 2500 r.p.m. for 5 minutes. The lower organic phase was removed by aspiration and the 0.LY HC1 extract containing the drug was scanned by ultraviolet spectrophot,omet,ry. ;ipproximately 4-ml. aliquots of the ethylene dichloride containing 25yc isobutanol extract were transferred to 15-ml. conical centrifuge tubes and evaporated to dryness on a steam bath under a stream of nitrogen. The residue was dissolved in 25 to 100 p1. of methanol, from which suitable aliquot,s were subjected t,o thin layer and gas liquid chromatography. In order to achieve maximal recoveries of the narcotic analgesics. modifications of the above extraction procedure were required as follows : 1. For the iminoethanophenanthrenes, use 15-ml. of pure ethylene dichloride without isobutanol as the exf'racting solvent. 2. For the diarylalkoneamines, wash the organic phase with 3 ml. of 0.2.11 N a H P 0 4 , p H 4.3, and determine absorbanre in the organic solvent. 3. For the arylpiperidines, adjust the p H of the sample to 7.0, buffer with 3 ml. of 1.11 K 2 H P 0 4 ,pH 7.5 and extract with 15 ml. of pure ethylene dichloride without isobutanol. In the case of ketobemidone the regular extraction iroredure was used. 4. For the benzomorphans, use 15 ml. of pure ethylene dichloride without isobutanol as the extracting solvent. Total morphine ,{free plus conjugated) was determined by autoclaving a n aliquot of the sample with a
PROCEDURE. The free base of the nonextracted pure drugs (25-500 pg./ ml.) were dissolved in either 0 . 1 s HCI, absolute ethanol, or 25% isobutanol in ethylene dichloride (Ib-ETCI2). For l-3-methoxymorphinan, pethidine, dlalphaprodine, and piminodine the hydrochloride salts were used, but the concentrations were calculated as free base. The 0 . 2 s NaOH samples were prepared by adding 50 pl. of 1 9 s KaOH to 0 . 1 S HC1 samples in glassstoppered silica cells, and absorption spectra were determined with 0.2N NaOH in the sample beam and 0 . 1 S HC1 in the reference beam. .Ilkali ( 1 9 s NaOH) was added to the 0 . 1 S HCI samples to determine whether the bathochromic shift of the wavelengths of phenolic substances due to the formation of phenolates (?) would occur. Ultraviolet absorption spectra were then obtained in 0.l.V HC1 extracts of the drugs from human biological materials and were compared to authentic nonextracted drugs over the region 360 to 225 mp. For the diarylalkoneamines the ultraviolet spectra were determined in the organic solvent (IbETC1,) extract. Usually 15 to 200 pg. ml. of free drug in human biological material was required for ultraviolet spectrophotometric analysis. Thin Layer Chromatography. - 1 ~ PARATUS. -411 chromatography was performed on 200 X 200 m m . glass chromatoplates and developed in rectangular tanks ( D e i a g a ) , 8 V q X 4l x 8l inches. An adju+table applicator was used to apply a 250micron absorbent layer to the plate. REAGENTS. Silica gel G (E. AIerck, Germany) 30 grams dissolved in 60-ml. of glass double distilled water, and MN-cellulose powder 300-G (llacherey Nagel I% Co., Germany), 15 grams in 90-ml. of 0.1M phosphate buffer, pH 8.0. Chromatoplates were made as described by Stahl (20). Apparatus
volume of concentrated hydrochloric acid for 25 minutes a t 15 pounds of pressure (acid hydrolysis). The p H was adjusted with 1 O S S a O H and the extraction performed by the procedure described for the iminoethanophenanthrofurans. Recoveries of 25 t% 200 pg./ml. of the narcotic analgesics from urine and suitably diluted plasma and arranged according to chemical families were (range is standard error of the mean): iminoethanophenanthrofurans,88.5 k 2.6%; iminoethanophenanthrenes,87.2 f 6.2%; diarylalkoneamines, 92.5 f 7.1%; arylpiperidines, 81.3 i 5.1%; benzomorphans, 84.5 6.776. REAGESTS. Ethylene dichloride ( I , % dichloroethane) was redistilled and just prior to use was passed over a column (580 X 20 mm.) containing silica gel, 6- to 12-mesh (290 X 20 mm. from Davison Chemical Div., Baltimore, Ald.), and aluminum oxide (290 x 20 mm. from Merck & Co., Inc., R a h a a y , S . J.) suitable for chromatographic adsorption. I n automatic siphoning unit supplied the solvent to the column. Potassium phosphate buffer, p H 10.4, was iveiiared bv mixinrr a 35% aaueous solut'ion' of ar;h.drous K2HP04' with 5% ! X ~ P O ~ . Z H ~ O . Ultraviolet SDectroDhotometrv. - 4 ~ PARATCS. dllAultra 4 p.hi., and I0 P.M. daily. Individual urine samples were obtained during a 24-hour period at various time intervals antl were concentrated in a flash evaporator. 1)ul)licate 6-1111. samples were extracted for free morphine, and approximately 3 t o 6 nil. of t.he organic extracts from the duplicate samples were rvaliorated t o dryness under a stream of nitrogen and t,he residue was dissolved in 25 j~1. of methanol. d b o u t 1 pl. from each sample at each time int,ervalwas spotted on chromatoplates which were developed in E T O H : PYI)> ETOH : KH3 antl n-13UT:H.lC. Total morphine (free IIIUR conjugated) was obtained as described under t,he exl)erimental section. Six millilitt~rsof the organic phase extract from each a d hytirolyzed sami)le was eval~orafedto dryness under a strcani of nitrogen and the drug was dissolved in 100 pl. of methanol. About 1 p l . of each sanil)le o b t s i n d a t each t,iiiie interval vas sliottc~lon chroniato1)lates which were devcloped in E T O H : IT11 and E1‘OH:SHB. Figxres 2.1 and B 1)resent bhe data for free and total morphine in two of the solvent used. 11orl)hine was easily detectcd in each urinp sarnlile ohtained diiring the 24-hour collection period. Ac*tiially as little tis 1 pg. of morl)hinc may 1)t. detected on the c~hroinatol)latcusing tho iodoplatinate reagent. G a s Liquid Chromatography. T h e ret w t i o n tla t a o 11 t ai n(v I \vit h the f r e e haw of t h e narcotic anslgcsic antl the e. t eri fit i1 c o lu nin t lcr i vat i IT% oh t ai ned
Ultraviolet spectra of authentic and extracted drug (conc. as free drug in 0.2N N a O H
____ Authentic drug in 0.1 N HCI
- .-
Extracted drug in 0.2N N a O H Extracted drug iri 0.1 N HCI A . Comparison o f 1 0 0 pg./ml. of outhentic urine o f addicted patient 8 . Comparison of 100 pg./ml. of authentic drug extracted from urine C. Comparison of 500 pg./ml. o f authentic a 10% (w./v.) human liver homogenate D . Comparison o f 100 pg./ml. o f authentic pg./ml. of Levorphanol extracted from plasma
-. . .
morphine with 53.1 pg./ml. of morphine extracted from dihydrohydroxycodeinone with 4 0 pg./ml. o f the same pethidine with 160 pg./ml. of pethidine extracted from Levorphanol (I-3-hydroxy-N-methylmorphinon) with 3 2
Conditions. The alkali curves were obtained b y adding 50 PI. of 19N N a O H to 0.1 N HCI samples in the silica cells. Either 0.1 N HCI or 0.2N NaOH was in the sample beam and only 0.1 N HCI in the reference beam
of human liver. The bathochromic shift, was not observed due t o the absence of a phenolic: hydroxyl in this compound. The rather peculiar triple absorbance ]leak \vas also observed with: noriicthidine; piminodine; dl-alphalirodine; d-l)ropoxyphene; and 1acetylmethadol, The identical comparison of authentic l-3-hydroxy--Ymethylmorphinan (Levorphanol) in acid and alkali with the same drug extracted from plasma (diluted 1:5) appears in Curve D. T h i n Layer Chromatography. I n Table I11 t h e R j values obtained on silica gel G for t h e various narcotic analgesics in seven ,different solvent systems are presented. Separation a n d partial purification of mixtures of narcotic analgesics in extracts of biological materials may e a d y be accomIilished bl- selecting t,he tippro-
Successful tn-o dimensional c’hroniatograms of narcotic analgehics wcre obtained using NeOH: 13ESZ and either
A
8 /
P
O
N
\
J S O L V E N T
\ T
. . - - -*- _ .
0
I
.
.
e
.
.
.
b
*
0 8
I O I 3 5 6 IO 12 3-6 P IM T AM
T
AM
8 10 I 3 5 6 10 I 2 3-6 7 T r A M PM AM
in sel)arating t h e commonly ahused narcotics-i.e., niorl)hine, codeinex, dihydrohydroxycodeinone, and pethidine. VOL. 36, NO. 10, SEPTEMBER 1 9 6 4
191 1
hydroxy - S - methylmorphinan, 13 - hydroxy - iV - allylmorphinan, ketobemidone, l-acetylmethadol, piminodine, and the benzomorphans partially reacted wit,h acetic and propionic anhydride. All other narcotic analgesics reacted completely to form t8he esterifipd column derivatives. The ret,ention times of the esterified derivatives were always greater than the corresponding free base. In the case of morphine, the retention time of the first acetylated peak was similar ' b u t not identical to 6-monoacetylmorphinc: however, the retention time of t,he second peak was identical to that, of diacetylmorphine (heroin). The first peak may represent either 3-monoacet,ylniorphine or a mixture of 3- and 6-monoac~etylmorphine. ,in attempt t'o determine t,he nature of this peak was unsuccessful, since the
following t h e injection of the anhydrides are given in Table IV. T h e relat,ive retention times (RRT) for the entire iniinoethanophenanthrofuran family were greater than codeine and norcodeine. I n all other chemical familics the RRT were less t,han codeine, except' for piminodine (3.66) and phenazocine (2.1I ) . The degree of reactivity of the drugs with the anhydrides varied within and among chemical families. Where est,erification with the anhydrides was theoretically not possible, as with heroin, dihydroodeineone, 1-3 - met'hoxyAY- rnctli~liiiori~hinan.dl - methadone, d-proI)osyphene, pethidine, and &alphaprodine, no change in the retention times was obswved following thc injcction of acetic and propionic anhydride. Compounds such as codeine, et,hylmorlhine, dihydrocodeinone, 1-3-
Ill.
'osle
preparat,ion of the free base of 3-monoacetylmorphine vielded morjihine. Further evidence 6; the effectiveness of t,he peak shift technique ( I ) may be seen in comparing the identical retent,ion times of acetylated 6-monoacetylmorphine with diacetylmorphine. Acetic and propionic anhydride were primarily used to form the esterified column derivatives of the narcotic analgesics; however, morphine was subjected to a homologous series of anhydrides. -4s the homologous series was ascended, the retention times for t'he first and second peaks increased as follows: acetic, 8.06, 11.34; propionic, 10.50, 18.37; n-butyric. 12.75, 29.06; valeric, 16.87, 48.19: and hexanoic, 22.31, 79.50. .ittempts to 'form esterified derivat'ives of morlihine with nheptanoic, glutaric, succinic, and trifluoroacetic anhydrides were unsuccessful.
Thin-layer Chromatographic Data for the Various Narcotic Analgesics Arranged According to Chemical Families
Cornpound (free base)
ETOH, P?il)
ETOH, HAC
R / values ( X 100) in viLrious solvent systerns ETOH, XleOH, t-hXI YI,, SHY UESZ n-BE
n-BI'T, H.4C
n-BYT, HC1
54
34 62
IhlI?jOETHANOPHEN.~~Tl~ROF~R.4~~
29 8
30 12 37 71 16 11
33 46 15
17 46
3s
27 48 29 50 35 55 24 21 25 29 21 25
24 40
11 4
21
86.1
7
89 13
25 0
76 35 2-5 17 46
35 67 15
4s 86 59 90 88 76 65 84 63 67 76
34 10 41
87 64
13
27 24 10 19 29 29
STRb 8 6 15 25
25 01 56 95
66 53
92 85
45 41
96
STR STR
30
45 43 42 32 37
49 32 41 26 25 33 28 29 23 34 37
51
60
63
61
%59
e53
IMINOETHAZOPHENANTHRESES 11
47
5
68
13
43
19 01
I
65
38 9s
-
6.i
70
80
10 10
7
ST11 41
PTR 44
72 55 66 64
17
17 38 56
55 52 53
62 62 61
20
44 63
s
8-
80
59 81
73
I)IARYI,AI,KoNE.4MISES
34
50
64 -.>
60
09 09
40
i3
6s
97
,54
42 12 31 3!) 8S
41 65
ARYI.PIPERII)ISES 07 51 39 47 40 $13 73 99
36 10 24 34 85
12 20 76
46 58 42 42 69
11
40
40 58
BENZOMORPHASS
1912
ANALYTICAL CHEMISTRY
88
87
97
82
70
76
"I
12
36
-lniorphinan
4 12 4 12 3 19 3 19 5 72
dl->lethadone 1-Acetylmethadol d-Propoxyphene
3 32 3 65 3 56
0.55 0.61 0.59
Pethidine Norpet hidine Ketobenridone dl-Alphaprodine Piminodine
1 22
0 0 0 0
I-3-H ydroxy morphinan
l-3-hlethoxy-.V-met hylm orphinan l-3-hlethoxyniorphinan
1 2 1 21
22 53 22 94
3 32 3 65 3 56
ARYLPIPERIDINES 1 3 2 1 3 66 36
20 20 42 20
26 34 18 94
14 67
I~~ISOETHA~OPHENANTHRENES 0.69 4.12 4 59 4 12 0.69 12.97 0 53 3 19 0 53 9 28 0 95 5 72 6 56 5 72
l-3-Hydroxy-.Y-me thglmorphinan
12 56
3 65
20 44 3 19
11 06
8 25
3.32 3.56
22 01
53 22
2 53
38
21 94
5 81
4 50
1 22 3 71 1 22
3 25 40 GB
BE\ZOMORPHA\S
dl-2’-Hydroxy-j,S-dinie t hyl-212 66 phene thyl-6,7-benzomorphan 1-2 ’-Hydroxy-2,5,0-t rime t hy1-6,71 87 benzornorphan 2’-Hy~~rosy-5,9-tlilriethyI-2-( 3,s4 27 dinlet hyla11y1)-6,i-henzornorphan 2’-Hydroxy-5,9-dime thy I-Z-cyclo3 78 propylme t hyl-6,7-benzoinorphan Retention time of the free unreacted drug. * RRT, retention time relative to free codeine.
2 11
12 66
14 62
0 31
1 87
2 06
1 87
2 66
0 71
4 27
4 97
4 27
6 34
0 63
3 78
4 50
3 78
6 SI
19 12
VOL. 36, NO. IO, SEPTEMBER 1 9 6 4
1913
The
gas liquid chromatographic of extracts of biological fluids containing analgesic drugs is illustrated in Figure 3. 'The retcnt,ion data for free niorphine (unhydrolyzed) and total morl)hine (acid hydrolyzed) were obtainrd from the 3 P.M. and 8 .4.51. urine samples of the 1 niorl)hine as de Little, if any, interference from urinary or plasnia constituents were present in the unhgdrolyzed extracted samples. Ho~vever,acid h j d i d y s i s of the urine as 1)ert'oriiied tmoobtain total morphine does /)resent some difficulties in gas chromatography due to the extraction of urinary constituents which tend to break down following hydrolysis and appear. as extraneous peaks. Several cst rancxus peaks appeared during the formation of acetates and propionates of mori)hine on the column. This kind of intcrirwnce does not prevent the use of the gas chromatographic technique on hydrolyzed extracts of biological materials, but does require adequat,e cont,rols caoiisihting of extract:: of known normal biological fluids and tissues to be run concurrently with the unknon-n samples:. Since some variation in the control material might be expected, it is advihnble that co-gas rhromatography be 1)erforinecl. by simply adding the suspected drug to t>hebiological sample and rc,chroiiiato~rai,hiIi~. Gas chro-
matographic analysis of free anti esterified derivatives of several analgesic drugs from h>iman tihsue extracts did not present difficulties. ACKNOWLEDGMENT
Grateful acknowledgment is made to Leo ;1. I'irk, Hoffmann-La Roche, Inc., for the iminoethanophenanthrene conipounds, to 11. J. Levenstein, Endo Laboratories, Inc., for tlihydrohydroxyniorphinonc~and dih?.droc.odeinone; to Karl Pfister, llerck, Sharp & I h h m e Research Laboratories, for et,hylmorphine; and to the Clinical Chemistry Section a t St. Joseph for samples cf Hospital, Lexington, Ky., human plasma and tissues. LITERATURE CITED
(1) hiders, 31. W., Xlannering, G. J., A N A L .CHEM.34, 730 (1962). (2) Bradford, L. W., Brackett, J. W., Jfzcrochitn. A4ctw 3, 332 (1958). (3) Brochrnann-Hanssen, E., Svendsen, A . B., J . Pharm. Sci. 51, 1095 (1962). (4) Clarke, E. G. C., H u l l . .\-arc. 11, 27 (1959). ( 5 ) Clarke, I;. C:. C., Williains, XI,, / b i d . , 7, 33 (1955). ( 6 ) Cochin, J., Daly, PV. J., Experientia 18, 294 (1962). ( 7 ) Coggeshall, S . O., (ilassner, A. S., Jr., J . A m . ( ' h e m . Soc. 71, 3151 (1040). ( 8 ) Erhlich-Rogoziiisky, S., Cheronis, X. D., J/icrochem. J . 7, 336 (1963). (9) Farmilo, C. G . ) Levi, I)., Oestreicher,
10) Farmilo, C. G., Oestreicher, P. Ill. L., Levi, L., /bid.,6, 18 (1954). 11) 'Genest,,dK., Farmilo, C. G., / b i d . , 11, 20 (1959). 12) Goldbaurn, L. R., Kazyak, L., J . Pharinacol. Ezvl. Therav. 106. 388 (1952). 13) Kazyak, L., Knoblock, E. C., Asar,. CHEZI.35, 1448 (1963). 14) L1oyd;IS. A,, Fales, H. XI., Highet, P. F., Vanden Heuvel, W.J. A,, M'ildman, \V, C., J . .Am. Cheni. Soc. 8 2 , 3791 (1960). (1.3) lIannering, G. J., Jlixon, -4.C., Carroll, S . V., Cope, 0 . B., J . Lab. Clin. Jfed. 44, 292 (1954). (16) Sakamura, G. R., R d l . S a r c . 12, 17 (1960). (17) Oestreicher, P. M. L., Farmilo, C. G., Levi, L., [bid.,6, 42 (1954). (18) Parker, K. I)., Fontan, C. R., Kirk, P. L., ASAL.CHEM.35, 346 (1963). (19) Sobolewski, G.) Sadeau, G,, ("/in. Chetii. 6, 153 (1960). (20) Stahl, E,, Schroter, G., Kraft, G,, Renz, R., Pharviazie 11, 633 (1956). (21) Stewart, C. P., Stolnian, A , , eds. "Tosicology," Vol. 2, Chap. 7 , Academic. Press. Sew York. 1961. (22) Woods, L. A,, Cochin, J., Fornefeld, E. J.,-McZlahun, F . G.,Reevers, M. H., J . Pharmacal. Espl. Therap. 101, 188 (1951). RECEITED for review March 23, 1964. .kccepted June 29, 1964. Presented at 26th Annual XIeeting of Comniittee on 1)rug Addiction and Xarcotics, Sational Academy of Sciences, Xational Research Council, Washington, 1) C., February 1964
Separation and Analysis of Nonylphenoxy Polyethylene Glycol Ether Adducts by Programmed Temperature Gas Chromatography HERBERT G. NADEAU, DUDLEY M. OAKS, Jr.,l W. ALAN NICHOLS, and LAWRENCE P. CARR Central Analytical Research Services, Olin Mathieson Chemical Corp., New Haven 4, Conn.
b A gas chromatographic method has been developed for the separation and determination of component compounds (adducts) in nonylphenoxy polyethylene glycol ethers. Products containing up to 10 moles of ethylene oxide have been studied. The procedure allows for the separation of eight adducts and has been made quantitative for the first five in the nonyl phenol ethylene oxide series. The method utilizes dibutyl fumarate as an internal standard. Analysis of several products possessing different ratios of ethylene oxide to nonyl phenol shows relatively good agreement of adduct distribution with theory based on a Poisson function.
T~;:YY)~
of ethylene ha. been eshaustively ,studid by Flory ( I ) , and it ha$ been shown that the reaction products t:'I"oxYL.krrLoS
1914
ANALYTICAL CHEMISTRY
should vary in molecular size in proportions approaching a Poisson function. Work done by Mayhew and Hyatt (3) has demonstrated that the ethoxylation of nonyl phenol results in a mole ratio distribution of products (adducts) simulating a Poisson curve in a manner analogous to polyosyethylene glycols. The work of Miller, Rann, and Thrower (5) on the reaction between phenol and ethylene oside further substantiates a Poisson distribution of products. Later work by von Tischbirck ( 6 ) on alkoxylated phenols demonstrated that the distribution of adducts was to a great extent dependent ulmn the catalyst system em1)loyed. Fractionation of alkylphenoxy polyethylene glycol ether (SPEO,) compounds is tisually accom1)lished by both molecular and conventional distillation; however. the fractions obtained are not usually [lure, and the woik is tinie-con-
suming. By such methods, fractions having an average molecular weight of about 500 (KPEO,) ran be obtained. 13eyond this point, thermal degradation takes place. Fractionation of the molecular adducts of octyl phenol polyoxyethylene glycols consisting of 9.7 ethylene oside units has been accomplished, employing solution chromatography on silicic acid columns ( 2 ) . This work involved the separation of adducts by solvent elution, giving rise to fractions which were quite pure. The procedure is, however, tedious and to some extent depends upon weighted fractions for quantita: tion. In studies involving catalyst evaluation and kinetics, where it is desirable to follow the ethosylation of a phenol Present address, Wilkens Instrument B Itese;Lrc.h, In? , Walnut Creek, Calif
