CONCLUSIONS
A spectrophotometric method for the total polyunsaturated fatty acids containing the cis-methylene-interrupted diene structure is based on a most specific type of reagent, the enzyme lipoxidase. Fats, oils, hydrogenated fats, fatty acids, esters, blood plasma, microorganisms, and plant seeds can be assayed directly for their total methylene-interrupted cis-polyunsaturated fatty acid content. As a routine analvtical procedure, tlic lipoxidase method should serve as
a valuable adjunct to the available analytical techniques. It is a micromethod, allowing for the accurate determination of as little as 57 of linoleic acid. LITERATURE CITED
(1) Am. Oil Chemists’ Soc., Tentative
Method Cd 7-48, rev. April 1956, “Official and Tentative Methods of the American Oil Chemists’ Society,” Chicago. (2) Brown, J. B., Frankel, J., J . d n a . Chena. Soc. 60, 54 (1938). ( 3 ) Holman, R. T., Arch. Biochena., 15, 402 (194i). (4) Holman, R. T., ”Methods of Bio-
chemical Analysis,” Vol. 11, p. 113, D. Click, ed., Interscience, New York, 1955. (5) Holman, R. T., Bergstrom, S., “The Enzvmes,” Vol. 11, Part I, p. 574, J. B. Sumrier and RI. Myrback, eds., Academic Press, S e w York, 1951. (6) Rlitchell, J. H., Jr., Kravbill, H. R., Zscheile, F. P., 1x0. EKG, CHEx., ANAL. ED. 15, 1 (1943). ( i )Siddiqi, 4.M., Tappel, A. L., J . A m . 0~1Chernzsts’ Soc. 34, 529 (1957). RECEIVEDfor review June 19, 1958. Accepted October 10, 1958. Federation of American Societies for Experimental Biologv, Chicago, Ill., April 1957, and Fourth International Conference on Biochemical Problems of Lipids, Oxford, England, Julj 1957.
Spot Tests for Aromatic Compounds Using 2,4,7-Tri nitrofluo renone H. T. GORDON and M. J. HURAUX Department of Entomology and Parasitology, University of California, Berkeley, Calif.
Many aromatic compounds form colored complexes when 10 y are spotted on filter paper and sprayed with 0.5% 2,4,7-trinitrofluorenone in benzene. Some of the complexes also show brilliant ultraviolet fluorescence. Qualitative information about the structure of unknown compounds can be derived very easily by observing the color and fluorescence of the trinitrofluorenone complex on paper, and by determining the solubility of the complex in iso-octane and ethyl alcohol. The trinitrofluorenone reaction is also useful for the detection of aromatic compound: on paper chromatograms.
compleses are formed 2,4,7-trinitrofluorenone u-hen (TKF)is mixed with various aromatic compounds, either as solutions in ethyl alcohol or benzene (6, 7) or as solids in the Kofler mixed fusion technique (3-5). The complexes usually are in a 1 to 1 molecular ratio, are readily crystallized, have a sharp melting point, and are intensely colored. The colors commonly range from yellow to red, the darker colors usually indicating either many fused aromatic rings (as in anthracene or perylene) or the presence of polar groups (as in catechol or aniline). Empirical rules for predicting whether an aromatic compound will react n ith TNF have been derised (4). The molecule must be relatively planar and not substituted with bulky groups (such as tert-butyl). Complex formaOLECULAR
302
ANALYTICAL CHEMISTRY
Table l.
Color, Fluorescence, and Heat Stability of Trinitrofluorenone Complexes on Filter Paper
Color” Sutural Kith T S F
Heatb Fluorescencea 5 &lin,a t Xatural Kith TSF 110” C.
Conipoimds with 3 or more fused rings
3-Aleth~~lcholanthrene Pyrene (1,8)- or (4,5)-dimethylerienaphthalene Chrysene
3,4-Benzoquinoline !,6-Benzoquinoline i,8-Benxoquinoline Phenanthrene 2-hlethylphenmthrene 2-Bcety lphenanthrene 9-Bromophenanthrene 4,shfet hylenephenanthrene Retene (isopropylphenanthrene) 9-Phenanthroic acid Phenant hrenequinone Thioanthrene Aceanthrene Anthracene Anthraquinone 1,8-Dihydroxyanthraquinone Fluorant hene Fluorene Fluorenone 2-A4cetylaminofluorene 9-Methy1-2,3,i-trihydrosy-6-fluo-
rone Xanthene Xanthone Thioxanthone Acenaphthene Acenaphthenol Acenapht hylene Acenapht henequinone 9,10-Dihvdroacridine Thiodiphenylamine (phenothinzine) Rotenone
Reserpine Colchicine
++ GR o ++ ++ R +o ++ + 1-l+ Y ++ 1++ I++ 1++ 1+ YO +o ++I.1+o
++ RBI Gv -
++ YY ++ + y z!=
RO
++ 0 + y
=!=Y 10
+++ +o ++ 1-0 + Gy
++ G =tBrR
+1 YG 0
+ B
+0
tion is enhanced by electron-donating (0.p-directing) substituents such as -SH2, -OH, or -OCH3, xiid blocked by elec.tron-~~-ithdrai\ing groups such as --SOr or -C-R.
Table I. Color, Fluorescence, and Heat Stability of Trinitrofluorenone Complexes on Filter Paper (Continued) Colora Satural K i t h T S F
I1 0 The work reported here is a study of the color and fluorescence reactions obtained by spotting solutions of aromatic compounds on filter paper and spraying with a solution of TNF. This type of spot test is often sensitive to a fex micrograms of a n aromatic compound, and it may give useful qualitative information about the structure of a n unknonn. It can be used to detect many aromatic compounds on paper chromatograms. MATERIALS AND METHODS
Color, Fluorescence, and Stability to Heat. Over 300 organic compounds were spot tested. M a n y of these compounds were impure, and very difficult t o purify. Most of them were used without purification. Solutions were made u p a t a concentration of 10 t o 20 nig. per nil. in ethyl alcohol or benzene. Two 1-111. spots of a solution (10 to 20 y) were applied on a strip of 1-inch wide K h a t m a n S o . 4 filter paper (other papers are also satisfactory). One of the spots was sprayed with a 0.5% solution of trinitrofluorenone (Eastman 7135) in benzene. The spots were examined in long-wave (360 mp) ultraviolet light and fluorescence (if any) was recorded. Color in visible light was also recorded. The strip was then heated for a few minutes to about 110" C. in an oven (a hot plate may also be used, or even a Bunsen flame). .4ny change in the color of the TXF-spraa r d spot arid the control spot was recorded. The intensity and hue of the TXF compleves in Table I are of course subjective and approximate, and different observers may record somewhat diffcrent color values for a given spot. I n some instances. exposure of the spot t o ammonia vapor alters the color. Separability of Trinitrofluorenone
Complexes by Solvent Extraction. Only a small number of compounds
'
n e r e tested by this method. Onemicroliter spots were applied t o 1-inch wide strips of K h a t m a n KO. 4 paper a n d sprayed with TNF. T h e strips were hung in a glass tube, t h e lorrer end of t h e paper dipping in a n organic solvent, T, hich ascended by capillarity for 30 minutes, so that thc spot of the coniplcx was being continuously extracted by thP solvent. Fire solvents w r e tried: iso-octane (2,2,4-trimeth?;lpentane), cyclohexene, ethyl alcohol, 1-chlorobutane, and benzene. Three different patterns of extraction were obtained: (1) The T K F complex remained unaltcred a t the initial spot, (2) The T S F complex dissolved slightly, forming '1 colored streak extending from tlit. initial spot upnard for several centimeters, hut far behind the solvent front.
Fluorescence" Satlira1 Kith TSF
Heath
j 1lin. a t
lloo
c.
Compounds with 3 or more fused rings
Verati ine Morin Physostigmine Phenazine idibenzopvrazirie) Dibenzothiophene @-Estradiol Eigosterol Estione ('ompounds
0
++
\\
ith 2 fused rings (not heterocyclic)
+ +
Kapht lialene
1-Saphthol 2-Sapht hol 1,4-Saphthalenediol 1,3-Saphthalenediol 1,5-?;aphthalenediol 1,B-Xapht haleriediol 1,?-Saphthalenediol 2,S-Saphthalenediol 2,6-Yaphthalenediol tert-8myl-2-naphtho1 Tribromo-2-napht hol 1,2,3,4-Tetrachloro-l ,2,3,4-tetr:ihvdronaphthalene l,%Dimethylnaphthalene 1,3-Dimethylnaphthalene 1,l-Dimethylnaphthalene lJ6-Dimethylnaphthalene l,?-l)imeth> lnaphthalene 2,3-Dimeth\ lnaphthalene 2,A-Dimethj Inaphthalene
++ ++ ++ ++ ++ + 0 ++ ++ +
2,7-Dimethylnaphthalerie 2,3,6-Trimethylnaphthalenc 1,4-Dimethouynaphthalene 1,5-Dimethoxynaphthalene 2,3-Dimethox~-naphthalene 2,7-Dimetho\vnaphthalene
++ +++ +
2-Chloronaphthalene 2-Iodonaphthalene 2-Fluoronapht halene 1-Fiuoronaphthalene 1-Sitronaphthalene 1,1-Dichloronaphthalene 1,5-Dinitronaphthalene 1-Xaphthalmeacetic acid 2-Naphthoxyacetic acid 1-Saphthaldehyde
-
+ ++ ++ 0
2-Napht hylamine N-Phenyl-1-napht hylamine
0 0
S-Phenvl-2-naphthylamine
1-Nitro-2-naphthylamine 1-Benzoylnaphthalene l,l'-Dinaphthvl 2,2'-Dinaphthyl ether Di (2-napht hT-1)-p-phenylenediamine Phenvl naphthyl ketone 2-Kapht hoylacetonitrile Indene Tetralin Ijecalin Camphene Camphor 1.2-Sanhthoouiiio11e 2:~1et~gl-lJ4~naphthoquinone kj-Hydroxy-l,-l-napht hoquinone 2-H~~ro\-v-3-meth!-I-Z,i-naphtho-
quinone
+
0
+ -
* 1-
Compounds rrith 2 fused rings (one ring heterocylic) +y + Y
Quinoline Isoquinoline Lepidine 2,l-Dimethylquinoline
-
fI
-
+T
-
+ 1-
+ 1- 1+ 1+A
+++ +++
(Contitaued on page 304)
VOL. 31, NO. 2, FEBRUARY 1959
303
Table I.
Color, Fluorescence, and Heat Stability of Trinitrofluorenone Complexes on Filter Paper (Continued)
Color5 Xatural With T N F
Heatb Fluorescence” - j &lin.at Satural With T N F 110” C.
Compounds with 2 fused rings (one ring heterocyclic) 2,i-Dimethylquinoline + Y f Y 2-Isobut ylquinoline i Y 3-Isobutylquinoline 6-Isobut ylquinoline E‘ 2-Chloroquinoline fY 6-Chloroquinoline +k’ 8-Quinolinol Y A’-Met hvl-Qquinolone + Y 1-Methylquinolinium chloride 1-Ethylquinaldinium iodide 6-Dimethylaminoquinaldine G Indole RO Indole-3-acetic acid +R BrR 5-Hydroxyindole-3-acetic acid 3-Methylindole BrR Gramine Br Tryptophane i R =tY Coumarin 4-hydroxy coumarin i-hydroxy coumarin i YO f Y Dicoumarol Thiocoumarin + Y f Y Benzimidazole i Y 5-Methylbenzimidazole 2-Aminobenzimidazole Br i Y Benzothiazole 2-Aminobenzothiazole +o iY 2-Chlorobenzot hiazole Benzoxazole 2-Chlorobenzoxazole 2-(o-Hydroxyphenyl) benzoxazole Y f Y Benzoxazolone 7,8-Benzoflavone + Y BrR Isosafrole f Y Piperonal Piperonyl butoxide +O Sesoxane, 2- (3,4-methylenedioxy 10 phenoxy-3,6,9-trioxyundecane) Rutin Y Compounds with 2 or more rings (not fused) Biphenyl o-Terphenyl m-Terpheny 1 p-Terphenyl 4-Acet yl-o-terphenyl Chlorotriphenylmethane 0-.4minobiphenyl o-Xitrobiphenyl o-Phenylphenol 4-Chlorohiphenyl 4-Fluorobip henyl 2,2 ’-Dihydroxybiphenyl 4,4’-Dihydroxybiphenyl 2,2’-Dimethoxybiphenyl 3,3’-Dimethylbiphenyl 4,4’-Dimethoxybiphenyl 4,4’-Dibromobiphenyl 4,4’-Difluorobiphenyl o-Tolidine 3-Phenylsalicylic acid 5-Phenylsalicylic acid Diphenylmethane Hexachlorophene Dibenzyl trans-Stilbene I,I-Di henylethylene cis-Stifbene p-Hydroxystilbene Diethylstilbestrol Stilbene dibromide Diphenylacetylene 1,4-Diphenyl - 1,3-butadien 1,2-Diphenoxyethane Benzophenone 4Hydroxybenzophenone p-Aminobenzc phenone Furfuryl phenyl ketone (Continued on page $05)
+ ++ +++ ++ + ++ +
++ ++ ++ ++ ++ ++ ++ i-
++ 0
+
++
+
++
304
ANALYTICAL CHEMISTRY
+++ ++ +++
++ ++ ++ +++ 4 ++ +++ ++
+++ ++ ++ t 0 ++ 0 + + ++ + ++ 0 0 -
++ +++ + -
Some of the original compound (either unconiplexed excess or partly dissociated from the complex) moved with or near the solvent front, and could be detected there by spraying with TNF, (3) The T N F complex was completely dissolved and dissociated, no color being detectable anywhere on the strip. A simpler extraction method is to place the spotted paper strip in a tube or flask, cover with solvent, and note the degree of elution after 30 to 60 minutes. Results are similar to those from continuous extraction. RESULTS AND CONCLUSIONS
Color and Fluorescence of Trinitrofluorenone Complexes on Paper. T h e d a t a are summarized in Table I. T h e formation of colored trinitrofluorenone complexes on filter paper obeys many of the rules previously formulated for mixed fusions (2, 3 ) . -4t least one aromatic ring is necessary. Heterocyclic rings are less reactive than benzene rings. .4t least one of the hydrogen atoms of the aromatic ring must be replaced by a n activating group which can induce a negative charge on the ring. Dialkylamino and amino groups are most effective, giving deep orange or red trinitrofluorenone colors. A hydroxyl group gives pale yellow, and t x o or more hydroxyls give darker orange or red color. One methoxyl or methyl group gives no color, but two or more give a weak yellow color. Substituents (such as nitro, cyano, or formyl groups) that induce a positive charge on the aromatic ring never give a trinitrofluorenone color, and when added to a ring having an activating group they may weaken or abolish the trinitrofluorenone color. Substituents such as vinyl, phenyl, or other groups having double bonds conjugated to the aromatic ring also enhance trinitrofluorenone color formation, presumably because in one of the possible resonance forms the ring can acquire a negative charge. These substituents give yellow trinitrofluorenone colors, but in complex molecules like lJ4-diphenylbutadiene, anthracene, or pyrene the linkage of t x o aromatic rings by a complex resonating system makes possible even greater negativity of one of the rings, so that trinitrofluorenone colors deepen to orange, red, or gray-green. 2,4,7-Trinitrofluorenone has a pale yellow color, and its aromatic rings are positively charged. The color of trinitrofluorenone complexes represents a shift of the 2,4,7-trinitrofluorenone absorption maximum from the nearultraviolet (pale yellow color) to the visible blue (deep yellow color), the blue-green (orange color) , and the green (red color). The visible fluorescence of trinitrofluorenone complexes in ultraviolet light
.
(under the conditions of this inveatigation) requires a strong absorption band near 360 mp, and the fluoresced light is alwaj-s in the range of 600 to 700 mp. All fluorescent trinitrofluorenone complexes are yellow to orange-Le., have a n absorption band in the 400- to 450nip range. The structural requirements for fluorescence, lion ever. are much more restrictive than those for color. Substituents such a5 amino, hydroxyl. or methoxyl prevent fluorescence, while alkyl or aryl groups usually enhance fluorescence. This suggests that the absence of a strong fixed dipole, and the presence of many new relatively equivalent resonance states (in addition to the benzenoid ring resonance), as in durene
e other forms o r biphenyl f
-=a *
=== f
*
_ .
other forms
make fluorescence possible. The strong fluorescence of the relatively synimetrical isomers (2,3-, 2,6-, and 2,7-) of dimethylnaphthalene, the Iyeaker fluorescence of the less symmetrical isomers (1,2-, 1,3-, 1,6-), and the lack of fluorescence of the 1,7- isomer all tend to support this vie!?. Hon-ever, the weak fluorescence of the 1,4- isomer and the intense fluorescence of 2,3,6trimethylnaphthalene suggest that i t is substitution on carbons 1,4,5, and 8 t h a t depresses fluorescence, while substitution on 2,3,6, and 7 enhances it. The importance of such position effects would probably become clear if a much larger number of compounds were tested, but many compounds of interest are difficult to obtain. There is a correlation between melting point and trinitrofluorenone complex fluorescence, because all the dimethylnaphthalenes having a t least one methyl group on a n a-carbon are liquid a t room temperature, while all those having no methyl group on a n a-carbon are solids. There is a similar correlation nith the distribution coefficient between ta o immiscible organic solvents (such as iso-octane and b-methoxypropionitrile), since the a-methyl substituted isomers partition more in favor of the less polar phase (iso-octane). This suggests that molecular polarizability is the factor that makes possible a stronger and more flexible interaction of the molecules of the P-isomers with one another (higher melting point), with a semipolar solvent
Table I.
Color, Fluorescence, and Heat Stability of Trinitrofluorenone Complexes on Filter Paper (Continued)
Colors _Natural With TXF Compounds trans-l,2- Dibenzoylethylene Benzoin Remil Benzyl ether Benzylaniline Benzalaniline Salicylaldazine Diphenylamine Hydrazobenzene Azobenzene Azoxybenzene p-Aminoazobenzene Diazoamino benzene s-Diphenylurea 1,3-Diphenylguanidine Phenyl salicylate
Heatb Fluorescence" 5 >lin, at Natural With T N F 110" C.
with 2 or more rings (not fused)
-
-
fT -
-
-
+Y
-
+Y =t
-
T
-
-
-
+ Br +Y ++Y +GGy Br +Y ZkY +YBr YBr
+
+ -
++Br
f Y
f Y ++T -
-
-
-
-
-
-
f Y -
-
-
-
++ ++ + 0 f
0
Compounds with only one (homocyclic) ring and no amino or hydroxyl groups directly on the ring 1,2,4,5-Tetrarnethylben~ene~ *Y +E' 0 Pentamethylbenzene" f O 0 +Y Hexamethylbenzene' Z k O 0 +O Hexaethylbenzene" 3,4-Dimethoxybenxyl alcohol +O Benzylamine Br 0 3,4,5-Trimethoxybenzoic acid +Y 0 2,4-Dichlorophenoxyaceticacid fT Thiosalicylic acid 0 ++Y 2,3-Dimethoxybenzaldehyde i Y 3,4-Dimethoxybenzaldehyde m-Thiocresol +Y p-Tolyl methyl sulfide +OR Compounds with only one (homocyclic) ring and a t least one amino group Ani1i ne RY 4-Nitroaniline ++Y +YO 4-Chloroaniline +RBr 4-Bromoaniline ++RBr 2,4,6-Tribromoaniline +O o-Toluidine +RBr p-Toluidine RI4-Methyl-3-aminoaniline 0 ++Br 4-Methyl-2-aminoaniline f Br Gy o-hminophenol Zk Br Br 0 m-ilminophenol 0 Br Anthranilic acid +O ++B j=B p-Aminobenzoic acid +RO 3-Hydroxy-4-aminobenzoic acid +O 4-Aminosalicylic acid f Br Br SY *I' 0 m-ilminoacetophenone f O YG f YG 0 4-Acet ylaminoaniline =!=R N,N-Dimethylaniline +R p-Dimethylaminobenzaldehyde Br & B Compounds with only one (homocyclic) ring, no amino group, and a t least one hydroxy group fT Phenol 2,5-Dimethylphenol =kY 3,4-Dimet hylpheno 1 +YO $. 2,4-Dichlorophenol =kY fY 2,3,5-Trimethylphenol ++I* 2-Is0propy1-5-methylphenol +Y 2-Allylphenol fY 2-Amylphenol i Y 4-Ally1-2-methoxyphenol $ 0 4-Propenyl-2-methoxyphenol Br 2->1ethosv-4-acetylphenol 1Salicylic acid fY 5-Chlorosalicylic acid +I' +B 5-Bromosalicylic acid +Y ++B Salicylaldehyde Y Y o-Hydroxyacetophenone fY *B Pyrocatechol =kY 3-Methyl-2- hydroxyphenol +YO 2,3-Dihydroxybenzoic acid f YO i. B ZkB 3,4-Di hydroxybenzaldehyde f Y Resorcinol f OR (Continued on page 306)
+
+
+"
++
+
++ ++ ++ ++ ++ ++ ++ + ++
+ ++
+
+
+
+
+
++
++
++ ++ ++ ++ ++ ++ ++
+
++
VOL. 31, NO. 2, FEBRUARY 1959
++ ++
305
Table 1.
Color, Fluorescence, and Heat Stability of Trinitrofluorenone Complexes on Filter Paper (Continued)
Heat* Fluorescencea j \lin, at Satural With TFI? 110" C.
Colora Satural With TSF
Compounds v,-ith only one (homocyclic) ring, no amino group, and a t least one hydroxy group 3,4-Dihydroxybenzoic acid fk' 2-Nitroresorcinol i.Y i.0 0 4Hexylresorcinol +O Hydroquinone i R 2,5-Dihydroxyacetophenone 4 1Pyrogallol 0 T Y IT 2,3,4-Trihydroxyacetophenone 2,3,4-Trihydroxybenzoic acid fk' Phloroglucinol
+
+ +
Compounds 2-hydroxy pyridine 3-Hydroxypyridine 2-Methylpyridine 4-Methylpyridine 2,4,6-Trimethylpyridine 2-Amino-3-methylpyridine 2-Amino-4methylpyridine 2-Amino-6-methylpyridine 2,6-Diamino-Pmethylpyridine 4-Pyridinepropanol 2-Methylpyrazine 2-Aminothiazole 2,4-Di hydroxythiazole a
with only one (heterocyclic) ring -
-
-
-
-
-
-
-
-
-
++OR
-
-
-
++o ++o ++Br -
-
-
-
-
-
-
+ T
t
Color and fluorescence symbols.
B.
Blue
-
Orange Pink Red Violet Y. Yellow
0. P. R. V.
Bk. Black
Br. Brown G. Green Gy. Gray
i
++ +
S o color or fluorescence Faint color or fluoresenre Distinct color or fluoresence Intense color or fluorescence
indicating effect of heating on color and fluoresence: +Symbols + Color completely disappears + Color becomes fainter
0 Color changes only slightly or not a t all 50 to 100 y necessary. No color reaction with 10 y. Bpparent inconsivtencies in data may be partly ascribed to nonuniforniity of the heating operation, and partly t o presence of volatile or nonvolatile impurities in sample. c
~~
Table 11.
Resistance of Trinitrofluorenone Complexes to Solvent Extraction
Iso-octane hIethylcholanthrene Pyrene Chrysene Salicylaldazine Benzidine 2,4-Toluenediamine o-Phenylenediamine Fluoranthene hnthracene Acenaphthylene Phenanthrene Indole N-Phenyl-2-naphthylamine
Aceanthrene 1,4-Saphthalenediol 2,4,i-Trinitrofluorenone
2-Naphthol Fluorene 9,lO-Dihydroacridine 3,4-Bmzoquinoline Phenothiazine
2-Amino-3-methylpyridine
Acenaphthene trans-Stilbene Diphenyl amine Benzylaniline
2,3,G-Trimethylnaphthalene
++ + 0
306
++ ++ ++ ++ ++ ++
++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + +++ ++ ++ ++ 0
Alcohol
++ ++ ++ + 0
1-ChloroCyclohexene butane Benzene
0 0
++ ++ 0 0 + 0 0 0
0
0 0 0 0 0 0 0
0 0 0
0 0
Complex very resistant to solvent extraction. Complex slowly extractable. Complex easily extractable.
ANALYTICAL CHEMISTRY
++ ++ ++ +++ ++ + 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
+ ++ +++ 0 0 0 0 0 0 0 0 0 0 0 0 0 0
+
0 0 0 0 0 0 0 0 0 0 0 0 0
0
0
0 0 0
0
0
0
(such as 8-methosypropionitrile), and with trinitrofluorenone. The one apparent exception to the above rules for trinitrofluorenone fluorescence is the o-formyl phenol structure (as in salicylaldehyde), in n-hich a peculiar hydrogen-bond ring nith neIv resonance forms exist. (1, 8).
The fact that o-acetyl phenols do not form fluorescent trinitrofluorenone complexes suggests that the equivalence of the hydrogen atoms in the formyl and hydroxyl groups is important. The colored trinitrofluorenone complexes formed on filter paper are not necessarilv crystalline solids nith a 1 to 1 molecular ratio. It has been shown that compounds such as p-terphenyl jield colored noncrystalline mixing zones in the Kofler mixed-fusion apparatus ( 4 ) , and that lJ4-diphenylbutadiene can form two trinitrofluorenone roniplexes (a 1 to 2 and a 3 to 1 hydrocarbon-TXF ratio) ( 6 ) . Stability of Trinitrofluorenone Complexes to Heat. The data are included in Table I. Disappearance of color on heating indicates t h a t the aromatic compound is relatively volatile, and this may be useful in characterizing a n unknown, or a mixture of volatile and nonvolatile rompounds. Action of Various Organic Solvents on Trinitrofluorenone Complexes. The data are summarized in Table 11. It is clear t h a t trinitrofluorenone complexes tend to fall into t1r.o main groups: those soluble in iso-octane, and those not soluble in iso-octane. Because trinitrofluorenone itself is relatively insoluble in iso-octane, the dissociation of the complex requires that the aromatic compound be isooctane-soluble and the binding forces between the compound and trinitrofluorenone be relatively weak. The iso-octane-soluble complexes \vi11 therefore be formed by compounds that have no strongly polar groups, and probably no more than two aromatic rings. The iso-octane-insoluble compleves ill hare strongly polar groups, or three or more rings, or both. The iso-octane-insoluble complexes can be further subdivided into tivo main groups: those soluble in ethyl alcohol, and those insoluble in ethyl alcohol. The aromatic compounds m ith strongly polar groups will be soluble. those without strong polar groups, but with many aromatic rings will be insoluble. Other solvents may be used, but usually will not give more useful information. I n this laboratory the trinitrofluorenonr reagent has been especially useful
for the detection of aromatic compounds on paper chromatograms. Because chromatography causes spots to enlarge by diffusion, quantities of 50 to 100 Y may be required for good detection on a chromatogram. LITERATURE CITED
(1) Branch, G. E. IC., Calvin, M., “Theory
of Organic Chemistry,” pp. 70 E., 118-19, Prentice-Hall, S e w York, 1941.
( 5 ) I b i d . , 30, 542 (1958).
(6) Orchin, )I.,
Reggel, L., Woolfolk, E. O., J . -4m.Chern. SOC.69, 1225 (1947). (7) Orchin, bl., Woolfolk! E. O., Zbid.,
(2) Gillam, *4., Stern, E. S., “Introduction to Electronic Absorption Spectrescopy in Organic Chemistry,” pp. 127-8, Edward Arnold, London, 1954. (3) Laskowski, D. Grabar, D. G., >fCcTOne, w.c.,h A L . CHEM. 2 5 , 1400 (1953). (4) Laskowski, D. E., McCrone, W. C., Ibid., 26, 1497 (1954).
68, 1727 (1946).
RECEIVED for review January 18, 1955. Accepted September 2, lyj8, a o r k supported by Research Grant E-l081(c), from the Sational Institutes of Health, U.S. Public Health Service.
Analysis of Long-Chain Fatty Acids by Gas-Liquid Chromatography Micromethod for Preparation of Methyl Esters WILHELM STOFFEL, FLORENCE CHU, and EDWARD H. AHRENS, Jr. Rockefeller Institute, 66th Street and York Avenue, New York 2 1 , N. Y.
An essential prerequisite for the analysis of lipide mixtures of biological origin by gas-liquid chromatography (GLC) is the quantitative formation and isolation of the constituent methyl esters. A micromethod is described involving interesterification with methanol and hydrochloric acid. By sublimation the methyl esters are isolated from the reaction products in a pure form ready for gas-liquid chromatography. The methylation and sublimation can be done with ease on a large number of samples. This method eliminates the use of alkali and diazomethane, which may lead to isomerization or pyrazoline formation.
A
method for forination of methyl esters of long-chain fatty acids necessarily precedes analysis by gas-liquid chromatography. The requirements of an optimal micromethod are: quantitative yield, absence of change in double-bond structure of highly unsaturated acids, and technical convenience. Saponification of fatty acids esterified as glycerides, phosphatides, and sterol esters can be followed by methylation with diazomethane. However, this procedure suffers some disadvantages-yields may be poor because of the formation of addition products of diazomethane a t ethylene bonds (pyrazolines) (4); structural changes in double bonds may occur during saponification ( 1 ) ; and the isolation of soaps from nonsaponifiable contaminants is rarely completely satisfactory. Formation of fatty acid methyl esters by interesterification, on the RELIABLE
other hand, has proved in this laboratory t o be technically simpler, milder, and more quantitative than diazomethanolysis. Yields of methyl esters from glycerides, phosphatides, and cholesterol esters are nearly quantitative, alterations in double bond structures are avoided, and the technical aspects are relatively simple. Completeness of interesterification d e w n d s an acid medium and the ahsence of water. I n an acid medium,
the electron-donating Lapacity of the is greater oxygen in water, H-Q-H, than that of the oxygen in methanol, CH8-g--H, and, therefore, methylation demands strictly anhydrous conditions. In the absence of water,
H
R, Hon-eJ-er, if water is present,
R
H
As Reaction 2 has preference over Reaction 1, methyl ester formation by interesterification is hindered in the presence of water. I n the method described below, a n y nonsaponifiable contaminants which may be present are eliminated by sublimation of the methyl esters. This feature assumes considerable importance when methyl esters are formed from cholesterol esters; the mixture of methyl esters applied to the gas-liquid column can be evaluated quantitatively only if free from cholesterol. Similarly, the triglycerides isolated by silicic acid chromatography from mixtures of naturally occurring lipides (2) are frequently contaminated by free cholesterol; the methyl esters formed by interesterification are conveniently freed of cholesterol by sublimation. The procedure has proved satisfactory for interesterification of safflower oil glycerides, soybean phosphatides, and cholesterol palmitate, as well as for glycerides, sterol esters, and phosphatides isolated from human serum by silicic acid chromatography ( 2 ) . Absence of changes in polyenoic acids was verified b y ultraviolet and infrared spectrophotometry and by degradation studies ( 5 ) .
IO’ METHOD
Reagents. D r y hydrochloric acid (5%) in superdry methanol ( 6 ) . VOL. 31, NO. 2, FEBRUARY 1959
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