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Studies of pyrazines as their n-π charge-transfer complexes with some nitro aromatic compounds
Note
Studies 4 pyrazines as their arematic compounds
n-x
charge-transfer
complexes
with
some nitro
arid D. B. F’ARUUR’ Fwd Research Luborafmy, Mysore (India) received September 24th, 1979; re&ed muluscript received February 6th. 1980)
T. S_ VASUNDHAR4
D&exe (Fiit
Pyrazincs arc flavour compounds formed in roasted foods as a result of the interaction of carbonyis with amino compounds. DiEerent techniques such as gas chromato_graphy (GC) and mass, infrared and nuclear magnetic resonance spectroscopjl have been employed for their separation, identification and determination1-5. in GC it is difEcuIt to resoIve isomeric pyrazines using a single column. Dietrich and Mavief employed paper chromatography for the separation of pyrazine carboxylic acids. Sizer’ tried to separate some isomeric alkyipyrazines that are diEcuIt to resoIve by GC by employing thin-Iayer chromato_mphy (TLC). Even with TLC, in spite of employing diEerent solvent systems, dif?&uIties are encountered in the separation of pyrazines that have the same or very close RF values. Pyrazines are n-z donors and are known to form charge-transfer complexes with iocheb. Nitra aromatic compounds are powerful z-acceptors. Siiica gel G impregnated with z-acceptors such as2,4,6_trinitroaaisoIe (TNA), 2,4MrinitrotoIuene (TNT), 2,4,Wrinitrobenzene (TNB), picric acid (PA) and picramide (PM) has been used for the TLC separation of a variety of basic compoundse”. In this work advantage was taken of n-z complexation of pyrazines with 2,4,6trinitroresorcinoI (TNR), picry chloride (PC), 2,4,6-trinitrophenetole (TNP) and TNT for their separation on TLC plates. Pyrazines form complexes both in soIution and on thin-layer pIates impregnated with these nitro compounds. The complexation results in a distinct spectral shift in solution and a change in the RF vdues. The resolution as n--t complexes of 25 pyrazines that are generally found in roasted foods has been readiiy eEcted. In particuiar, the separation of those pyrazines which have the same or close RF values in Merent solvent systems has been achieved as 2 result of complexation. The complexes have distinct W maximaPyrazines could be detected on the TLC plates by spraying with Dragendo&-s reagent” followed by 0.5% suIphuric acid with a detection Iimit of 2.5pg, and dete.rmined after liberating them from the complexes with ethanol at levels down to 5 .ug by using a UV s_pectrophotometer_
0021-9673j80;oooooooo!sO~
$J 1980 Else*
!XcntiEc l’ubIish+ tirnpa~y
25.5
EXPERIMENTAL
Au”Jzenticpyraxines (l-25, Table I) were received from Naarden International Research Centre (Naarden, The Netherlands) and Pyrazine Speciahies (Atlanta, GA, U.S.A.). Styphnic acid, picryl chloride, trinitrotoluene and trinitrophenetole were of analytical-reagent grade. SiIica gel G (E. Merck, Darmstadt, G.F.R.) was used as the adsorbent. All solvents were dried and freshly distilled. The following eluting solvents were used with the ascending technique: I, chloroform-ethyl acetate-ethanol (60:3S:2j; 11, benzene-acetone-ethanol (90:8:2). DragendorfYs spray reagent was prepared as reported by Stahl’4 A Perkin-Elmer Model 124 ultraviolet spectrophotometer was used for sp&tral measurements and a Chromatolite W lamp (short-wave length radiation, 254 ma) for locating the compounds on the plates.
Preparation of plates, impregnation, spotting and elution A fine slurry of silica gel G (50 g) in distilled water (100 ml) was poured on to glass plates (35 x 25 cm) and spread uniformly by tilting the plates from side to side. The plates were dried at room temperature overnight and activated at 110 “C in an oven for 1.5 h before use. The average coating thickness was 6.5 mg/cm*. The plates were impregnated with a 0.01% solution of the nitro aromatic compound in carbon tetrachloride by the ascending technique. The plates were dried at room temperature and standard solutions of pyrazines (5 ,og in 10 pl of carbon tetrachloride) were spotted. The compounds were also spotted on an untreated plate, which served as a control. The control plates were elutecl with solvents I and 11, but for the elution of the impregnated plates these solvents also contained 0.01% of the corresponding z-acceptor- After elution the plates were air dried. The compounds were located under Ill light and by spraying with DragendorlPs reagent followed by 0.5 % sulphuric acid, appearing as pink spots on both unimpregnated and impregnated plates. Fig. 1 shows two typical chromatograms obtained on an impregnated plate for some pyrazines with very close RF values; on the picryl chloride-impregnated plate they were well resolved. Table I gives the RF values of 25 pyrazines, and of Lt-n complexes with four acceptors in two solvent systems.
Ultravidet spectra of n+zz complexes in solutitm Solurio~~~ of 1 - 10B6M of each of the pyrazines and nitro aromatic compounds were prepa& in carbon tetrachloride. The solutions were mixed in various pro-
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5 _ __ __- _ _. -- ._Wm” Y___ .i_ Fig. 1_ Thin-layer chromatognms showing the separationof pyrazines having vczy close RF VaIucsa
Cbomam y without impregnation with p&y1 chloride; chroiit2tow Y. impregnatedwith piayl chkxide. A = 2,3-DiznethyIpynzine; B = 2,Sdknet&y!pyzhxe: C = 2,Wylpyrarint; G = 2-m&&D = kthyl-5-incthy~~mzkc; E = pyrazke; F = Z-ine*&oxy-34ipmpylpyrarinc; ox~-3-sec.-butyipywke; H = 2-methyIthi~kthyipymine; I = 24hoxy-3-methyipynt;ix; J= 2-iutyi-3-methylpyrarine; K = 2-propyl-3,adimtthylp>~; L = 2+~5yMmcthyl~. Adsorbent: silica .gd G (IS_Mercfr). SoIwnt system: Ch!orofonndhyl aatatKthanoI(60:38:2) for chromatogram X; for Y the system also contained 0.01% of picry chioride.
portions uld the W spadrum of each mixture was recorded together with those of the individual compounds. Table I gives the 3?_ valves of the n-z compIexes and of the individual compounds. Figs. 2-5 show the absorption patterns of some typical n-z complexes. Ifiiac-ctetermination
of pym.2ke.s
of 5 pg of each pyrazine were dorrbly spotted on two half-sides of a picryi chloride-impregnated piate and eluted with solvent I containing 0.01% of this nitro compomzd. After elutioa the first half was sprayed Gtb Dragendorff’s reagent followed by OS% sulphuric acid_ The spots corresponding to the first half of the plate were marked on the second half of the plate_ The spots on the second half (unsprayed) portion were also visible nrrder W light These were scraped with a micro-spatulainto 8 sintered-glassfunnel (1-D. OS cm). pyrazines were liberated from their complexes by treatment with ethanol, the pyrazines being found in the filtrate whereas tke nitro compound remained adsorbed on the silica gel. The SOIVU.I~ wzs careremoved fro& tlie filete under a partial vacuum, the residue was dissolved in carbon tetrachloride uld the volume made up to 10 ml_ The absorbance at the L_ values were measuii The vnounts were cakulatesl from pnzviously drzwn standard absorption graphs for each pyrazine- The recovery of the pyrazines was 97-98 ‘A and the results were reproducible. Amounts
c
Wavelength
(nm)
F&Z.2. K.i.ispectra of 2-~~~&yIthi~~3-isopmpy~pys&~~~ (x), P& (A).
5
Fig- 3. W
styphnic acid (0)
and their n-z com-
Weverecqth(nm)
spectraof 2-methoxy-3ethyipyrarine( x), picryl chIoride (0) and their n-zz cornpI= (Ah
Coftr~tnctntl
2 3 4 5 6 7
2,3~Dlmolhylpyrnzinc 2,s.Dimcthylpyrazino 2,6_Dln~ctl~ylpymzino 2~EthyLS=mcthylpyrazinc 2,3,5=Trimclhylpyrazino 2,3,5,6-Tclmmcthylpyrazinc
-_-w-w-1 2.Molhylpyrazinc
No. 01
R, vhc,s
of
chlorklc
plcryl
U/I A,,,,
,Tfy[J/lJdC NCkl
ncld corti. frrrprefnnred
sryphlk
A,,,,
ol’
rldc-frrlpreg.
k9/1tcrte
c/Jk~= rrhrltro-
Hr vallrcs art A,,,,
pfcryl
hrcrrr.lffl-
pherlclole
Irlnltro-
Rr vnhres 011 A,,,,; of rrlJhJb
HP vahuson rlclole h-
rrlrdlru~he-
266 274 275 275 290 278 279
0.30 0.36 0838 0,40 0.44 0836 0,41
0835 0837 0,39 0,39 0.52 0,55 0.37
-. .-- --
274,335 271 276 276,334 272,335 273,335 281,337
0,26 0,38 0,40 0,42 0653 0.40 0,54
plexes In phre
0.32 0.35 0,37 0,39 0,57 0,61 0.32
275 270,310 270,310 280 275 274 282
0.40 OAI 0845 0849 0,53 0.44 0.4G
0839 0040 0,42 0,44 O,SI) 0.63 0.42
con~~lexcsrtnrcff phfc
269,315 271,309 272,314 271,314 272 271 282
0,4l 0,43 0846 0,49 0.52 0.40 OA5
0.43 260,315 0,40 271,310 0,45 212,314 0,48 270,313 OS9 273 0,so 282 OS41 204
cortr~lcxesprrgtlnred
0,34 0,39 OA3 0.44 0.48 0.32 0.37
0436 0.40 0.42 0.43 0.49 0.59 0,38
co,trpbcsprc~rtnfcrl _.. .. _._ _.___hl saltr- p/ore SdSJ’. sohrflort y;- - ---r’-” In J&. Ir,solir. plm . .__-__-..__-wJt blefu - -- - .-.---.-- ,lorr Sol. Solflon (na) - m I II w/It wt fnm, Sol- Sol. VCfll wr tnn,) f$ f$ fd 1 II I I1 wit vent I I 1; II -_..-e-..___ -.-.__-_-_--_..__..-- ._.-____I__ ..__-___- 11_1__^__ - -_._______
HJI vdrtcs
(nrrr) -.-
rzI”X,
Pure ~Jyrurhcs
Solvent systems: I =.acl~lorofor~n-ethylacclr~tc-cllmol (ffi:38:2); II := bcnzcnc-uccronc-clhanol (90:8:2), Adsorbent: silicn l~cl 0 (E, Merck). Whon tho nilro compound.imprcgnntcd phtcs wcro clulcd, the solvent nlso contained 0.01% of the rcspcclivc nitro compound, A,,,,,, values in carbon tctrnchlorido:styphaic II&~,278 nntl 333 nm; picryl chloride, 263 nnd 333nm; trinilrotolucno,262nm; trll~itrol~l~cnclolc, 20 nm, -__--..-___--------_-____ . .. _._._- --- -- ._.I___. _... .__-_._ -. - ... .. _..___- ____ _.. _ ._. .. ___._._._._ .- _ _ .. _. -__ _ _ . __.-_.__ --._-
RF VALUPS OF PYRAZINES AND ‘IIIEIR n-n COMPLEXES WITH STYPHNIC ACID, PICRYL CLILORIDE,‘~RINlTROTOLUENBAND TRINITROPI-IBNEI’OLE, AND AUSORM’ION MAXIMA IN CARIIONTETRACIILORIDE
TAllLE 1
ES’0 99.0
OIE‘ZLZ LE'O
IS'0 89'0 9LZ 8S'O OIE'ZLZ ZS’O 99'0 Z8Z E9’0 IS’0 W'OLZ 8LZ wo 08Z CL'0 88'0 08Z 88'0 06'0 EEE'ILZ 18'0 96'0 'OZZ 19'0 6AZ 9b'O 01'0 tr8Z 99'0 (9'0 IEE'trLZIS'0 69'0 6LZ 6S'O 08Z EVO EL'0 OIE'99Z E9'0 9L'o OIC'9LZ IV0 99'0 OLZ OSE 91E 88'0 09z 29'0 88'0 SIE'O9Z 96'0 P8'0'SIE'E9Z11'0 ZG'O 'GbZ 9IC IL'0 91E'bSZ 9S'O S8'0 LIE'9SZ t8'0 19'0 EEE'SLZ 99'0 18'0'ECZ zzz 011 es*0 OS'0 IEC'LLZ ES'0 L9'0 %LZ OIZ 18'0 9L'O SEE'b8Z S9'0 08'0 081
LIE 8S'O
OS'0 81'0 9LZ SE'0 06'0 081 LE.0 61'0 OK'991 1IE'OES'0 OLZ 8S'0 EG'O SLZ
IlZ SP’O GS’O ZIE'6EZ 6VO LL'O 9LZ SS'O 9L'o IrLZ 09'0 18'0
19'0 81'0 SIE'OBZ bS'0
9L’o 98’0
58'0 26'0 08Z 99’0 wo 09'0 fi8Z zvo ova 19'0 ZIE'OLZ 9E'O
GS’O 19’0 19’0 VL'O
081 081 t9z OLZ SLZ
SL'O E6'0 SEE'9LZ 18'0 06'0 Z6Z Z9'0 19'0 SEE'OLZ OS'0 85'0 99Z SE'0 OP'O t1z IS'0 wo 192 ttL'0ZG'O SEE;081 01'0 18'0 OGZ OS'0 16'0 OK'OIZ 9S'O Z8'0 16Z
99’0 OGZ 98'0 S8Z SL'O ILZ 8p'O VLZ 09'0 08Z
IS'0 6S'O OL'O wo SL'O
611 691 011 611 f6Z
99'0 9S'O 9EE'LLZ ES’0 11’0 08'0 SEE'OIIZ09'0 Ot'O 81'0 89Z PE'O LVO EVO EEC'OLZ LE'O t'8'0t's'0IEC'9LZ WO
05’0 09’0 8LZ 9S’O 18'0 08Z C9'0 OL'O OIE'OLZ 8C'O ZS'O ZIE'OLZ ob'o 11'0 bLZ 95'0 SS'O 8C'O IV0 GE'0
115'0S8'0 8LZ EL'0 $8'0 081 Eb'O 08'0 09'0 S9'0 SIE'89Z Zb'O ES'0 SIE'L9Z 119'019'0 9t'O Of"0 91E'8SZ wo LE'O IZE'E9Z 9S'O 9vo 19'0 $8'0 18Z 09'0 'GL'O 08Z S6'0 E8'0 GLZ 99'0 98'0 99z IS'0 IL'0 69'0 68'0
Qu~zuJ~d~Xt~~3w.~~xoq~~~Z
81 LI 91
:I
:r II 01 6 8
Fig. 5. UV qzctra of pyrazke (x) ~Horide (A).
RESULTS
TNT
AND
znd n-z compkzes
(Cl) and trinitropknetole
of
pyrazk withsQ@nic acid
(.),
piqyl
(O)_
DIXXJSSION
As is evident from Table I, pyrazines form charge-transfercomplexes with titro aromatic compounds both in solution and on TIC plates, which is reflectedin tie spectraIshifts and in the RF values. For most of the compounds a large spectral shift is acconpzr?ied by 2 sigdkant change in theiz R, V&XS. Fyrazines aren donors_ The nitrogen atoms in the ring deactivateall of the tig carbon atoms towards electropbilicsubstituentsand activate them towards nucleophilicsubstituents Hence nucleophihcsubstituentssuch as all& alkoxy and thioalkyl groups on the pyrazine ring further modify the donor capacity of the molecule. Of the four nitro aromaticcompounds tried as acceptors, viz., styphnicacid, PC, TNT and TNl, PC gave the best separation of the complexes. The chlorine atom attachedto the nitro-aromaticring furtherenhancedthe acceptorproperties_ Theoretically,pyrazines could form 1 :I, 1:2 and 2: 1 complexes with nitro aromatic compounds. From a Job’s plot in solution it was observedthat all of the pyraies formed only 1 :l complexes.In some instancesthe complexesshowed more than one absorptionband in solution. This could be due to multiplecharge transfer occurringbetweenthe closely relatedWed orbitalsof the donor moleculeand closely relatedunfilledorbitals of the acceptor molecule. Silicagel G was suitableas an adsorbent.Of the two solventsystemsexamined, solvent I gave betterresolutionsof the complexes_The compounds could be detected with Dragendorff’sreagent‘bothon unimpregnatedplates and on platesimpregnated with nitro aromatic compounds. Ibis propertyof n-z complexatioc on TLC plates was found to be of great utility in the resolutionof those pyrazineswhich had either identicalor very close RF valuesFor example, the following groups of pyrazineshad very close RF values in both solvent systems (Table I)_ Group I -2,3-dimethylpyrazin~ 2&iimethylpyrazine; pym.zine, 2&dimethylpyrazine; Group II -2ethyl-S-xethylpyrazine, Group III -2-metboxy-3-isopropylpyrazine, 2-methoxy-Zkec.-butylpyrazine ylpyrazine 2methylthio-3-&y1pyrazine; Group IV -2*thoxy-3-methylpyrazin~ 2-acetyi-3-meth-
ylpyraziq 2-propyW&dimetby~pyrazine,2effiyl-6-metby~pyrazinepyra6ne. However, as can be seen from Fig_ L, as the IHE complexesthey gave very distinctseparations. The proceduredescribedis very u&X for the separation of pyrazines that are difikralt to resolve. it is possible to detect and determinethem with limits of 2-5 and 5 p& respectively.
The authorsare gratefulto Dr. I?. K. Vijayaraghavan,Director, Defence Food Research Laboratory, Mysore, for his keen interest and encouragement. The generous help rendered by Dr. H. 3. Takken, Naarden International Research Laboratory, The Netherlands and Dr. M. A. Gianturco, Coca Cola Company, U.S.A., in supplyingauthentic pyrazinesas gifts is gratefuliyacknowledged. REFERENCES 1 R. C. Buttery, R. M. Seifert, D. CL Guadqni and L. Co Ling _?m Agr. Food Chem., 19 (1971) 969. 2 P. E. Kothkr, M. E. Mason and I. A. Newell, 1. A&. F&d Ckem., 17 (1969) 393. 3 J. P_ Wahdf A. 0. Pitet, T. E. Kinlin, R. Murali Dhara and A. Sazderson I. Agr. Food Chem.. 19 (1971) 972. 4 H. A. Badarmkh. P_ Frkdel. V. Krampl, J. A. Remer, F. W. Skphzd and M. A. Gianturco, J. Agr. Food Ckem., 15 <1%7) 1093. 5 Mmvan Razg, M. S_ Stein and M_ S_ Tibb&.s_J. &r_ Fmd Cherrz., 16 (196s) 1005. 6 P. Dietrich and D. hhvier, f_ Chrm~~~ogr_,1 (1958) 67. 7 C- E. !3izer.impubliskd resuk 8 V. G. Krishna and Mihie Chowdhy, 1. Phys_ Gem_, 67 (1963) lofj7. 9 A. K. Dwiveciy, D. B. Pzrihar, S. P. sharma and K. K. Vega, J. Ckromarogr.. 29 (1967) lZO_ 10 D. B. Pi&bar, S_ PmShyma and K. KmVesma, J. Ckromatogr., 31(1967) 120. 11 D. B. P&har, S. P. shanna and K. K. Vexma, J. Forensic SE SK., 10 (1970) 77. 12 E. Stahl, lIih dyer Chromatognzphy, Springer Verlag, New York, 1969.
Studies 4 pyrazines as their arematic compounds
n-x
charge-transfer
complexes
with
some nitro
arid D. B. F’ARUUR’ Fwd Research Luborafmy, Mysore (India) received September 24th, 1979; re&ed muluscript received February 6th. 1980)
T. S_ VASUNDHAR4
D&exe (Fiit
Pyrazincs arc flavour compounds formed in roasted foods as a result of the interaction of carbonyis with amino compounds. DiEerent techniques such as gas chromato_graphy (GC) and mass, infrared and nuclear magnetic resonance spectroscopjl have been employed for their separation, identification and determination1-5. in GC it is difEcuIt to resoIve isomeric pyrazines using a single column. Dietrich and Mavief employed paper chromatography for the separation of pyrazine carboxylic acids. Sizer’ tried to separate some isomeric alkyipyrazines that are diEcuIt to resoIve by GC by employing thin-Iayer chromato_mphy (TLC). Even with TLC, in spite of employing diEerent solvent systems, dif?&uIties are encountered in the separation of pyrazines that have the same or very close RF values. Pyrazines are n-z donors and are known to form charge-transfer complexes with iocheb. Nitra aromatic compounds are powerful z-acceptors. Siiica gel G impregnated with z-acceptors such as2,4,6_trinitroaaisoIe (TNA), 2,4MrinitrotoIuene (TNT), 2,4,Wrinitrobenzene (TNB), picric acid (PA) and picramide (PM) has been used for the TLC separation of a variety of basic compoundse”. In this work advantage was taken of n-z complexation of pyrazines with 2,4,6trinitroresorcinoI (TNR), picry chloride (PC), 2,4,6-trinitrophenetole (TNP) and TNT for their separation on TLC plates. Pyrazines form complexes both in soIution and on thin-layer pIates impregnated with these nitro compounds. The complexation results in a distinct spectral shift in solution and a change in the RF vdues. The resolution as n--t complexes of 25 pyrazines that are generally found in roasted foods has been readiiy eEcted. In particuiar, the separation of those pyrazines which have the same or close RF values in Merent solvent systems has been achieved as 2 result of complexation. The complexes have distinct W maximaPyrazines could be detected on the TLC plates by spraying with Dragendo&-s reagent” followed by 0.5% suIphuric acid with a detection Iimit of 2.5pg, and dete.rmined after liberating them from the complexes with ethanol at levels down to 5 .ug by using a UV s_pectrophotometer_
0021-9673j80;oooooooo!sO~
$J 1980 Else*
!XcntiEc l’ubIish+ tirnpa~y
25.5
EXPERIMENTAL
Au”Jzenticpyraxines (l-25, Table I) were received from Naarden International Research Centre (Naarden, The Netherlands) and Pyrazine Speciahies (Atlanta, GA, U.S.A.). Styphnic acid, picryl chloride, trinitrotoluene and trinitrophenetole were of analytical-reagent grade. SiIica gel G (E. Merck, Darmstadt, G.F.R.) was used as the adsorbent. All solvents were dried and freshly distilled. The following eluting solvents were used with the ascending technique: I, chloroform-ethyl acetate-ethanol (60:3S:2j; 11, benzene-acetone-ethanol (90:8:2). DragendorfYs spray reagent was prepared as reported by Stahl’4 A Perkin-Elmer Model 124 ultraviolet spectrophotometer was used for sp&tral measurements and a Chromatolite W lamp (short-wave length radiation, 254 ma) for locating the compounds on the plates.
Preparation of plates, impregnation, spotting and elution A fine slurry of silica gel G (50 g) in distilled water (100 ml) was poured on to glass plates (35 x 25 cm) and spread uniformly by tilting the plates from side to side. The plates were dried at room temperature overnight and activated at 110 “C in an oven for 1.5 h before use. The average coating thickness was 6.5 mg/cm*. The plates were impregnated with a 0.01% solution of the nitro aromatic compound in carbon tetrachloride by the ascending technique. The plates were dried at room temperature and standard solutions of pyrazines (5 ,og in 10 pl of carbon tetrachloride) were spotted. The compounds were also spotted on an untreated plate, which served as a control. The control plates were elutecl with solvents I and 11, but for the elution of the impregnated plates these solvents also contained 0.01% of the corresponding z-acceptor- After elution the plates were air dried. The compounds were located under Ill light and by spraying with DragendorlPs reagent followed by 0.5 % sulphuric acid, appearing as pink spots on both unimpregnated and impregnated plates. Fig. 1 shows two typical chromatograms obtained on an impregnated plate for some pyrazines with very close RF values; on the picryl chloride-impregnated plate they were well resolved. Table I gives the RF values of 25 pyrazines, and of Lt-n complexes with four acceptors in two solvent systems.
Ultravidet spectra of n+zz complexes in solutitm Solurio~~~ of 1 - 10B6M of each of the pyrazines and nitro aromatic compounds were prepa& in carbon tetrachloride. The solutions were mixed in various pro-
_ ._ -_ _~
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:
_.
__
:
‘-7’
--. >
L
.
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__
*_.-,
-
_
;
.
-
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6-C
j
i_
g
&
&
i
-2 -i
i_d_A‘_8
.;_(.-
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__ _
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-_:_ -_. =;
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=_.b
.:
g
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it-
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.
_---.
_
_.
i_
L:
-_; __*
--mm -_ _ _.____L-
5 _ __ __- _ _. -- ._Wm” Y___ .i_ Fig. 1_ Thin-layer chromatognms showing the separationof pyrazines having vczy close RF VaIucsa
Cbomam y without impregnation with p&y1 chloride; chroiit2tow Y. impregnatedwith piayl chkxide. A = 2,3-DiznethyIpynzine; B = 2,Sdknet&y!pyzhxe: C = 2,Wylpyrarint; G = 2-m&&D = kthyl-5-incthy~~mzkc; E = pyrazke; F = Z-ine*&oxy-34ipmpylpyrarinc; ox~-3-sec.-butyipywke; H = 2-methyIthi~kthyipymine; I = 24hoxy-3-methyipynt;ix; J= 2-iutyi-3-methylpyrarine; K = 2-propyl-3,adimtthylp>~; L = 2+~5yMmcthyl~. Adsorbent: silica .gd G (IS_Mercfr). SoIwnt system: Ch!orofonndhyl aatatKthanoI(60:38:2) for chromatogram X; for Y the system also contained 0.01% of picry chioride.
portions uld the W spadrum of each mixture was recorded together with those of the individual compounds. Table I gives the 3?_ valves of the n-z compIexes and of the individual compounds. Figs. 2-5 show the absorption patterns of some typical n-z complexes. Ifiiac-ctetermination
of pym.2ke.s
of 5 pg of each pyrazine were dorrbly spotted on two half-sides of a picryi chloride-impregnated piate and eluted with solvent I containing 0.01% of this nitro compomzd. After elutioa the first half was sprayed Gtb Dragendorff’s reagent followed by OS% sulphuric acid_ The spots corresponding to the first half of the plate were marked on the second half of the plate_ The spots on the second half (unsprayed) portion were also visible nrrder W light These were scraped with a micro-spatulainto 8 sintered-glassfunnel (1-D. OS cm). pyrazines were liberated from their complexes by treatment with ethanol, the pyrazines being found in the filtrate whereas tke nitro compound remained adsorbed on the silica gel. The SOIVU.I~ wzs careremoved fro& tlie filete under a partial vacuum, the residue was dissolved in carbon tetrachloride uld the volume made up to 10 ml_ The absorbance at the L_ values were measuii The vnounts were cakulatesl from pnzviously drzwn standard absorption graphs for each pyrazine- The recovery of the pyrazines was 97-98 ‘A and the results were reproducible. Amounts
c
Wavelength
(nm)
F&Z.2. K.i.ispectra of 2-~~~&yIthi~~3-isopmpy~pys&~~~ (x), P& (A).
5
Fig- 3. W
styphnic acid (0)
and their n-z com-
Weverecqth(nm)
spectraof 2-methoxy-3ethyipyrarine( x), picryl chIoride (0) and their n-zz cornpI= (Ah
Coftr~tnctntl
2 3 4 5 6 7
2,3~Dlmolhylpyrnzinc 2,s.Dimcthylpyrazino 2,6_Dln~ctl~ylpymzino 2~EthyLS=mcthylpyrazinc 2,3,5=Trimclhylpyrazino 2,3,5,6-Tclmmcthylpyrazinc
-_-w-w-1 2.Molhylpyrazinc
No. 01
R, vhc,s
of
chlorklc
plcryl
U/I A,,,,
,Tfy[J/lJdC NCkl
ncld corti. frrrprefnnred
sryphlk
A,,,,
ol’
rldc-frrlpreg.
k9/1tcrte
c/Jk~= rrhrltro-
Hr vallrcs art A,,,,
pfcryl
hrcrrr.lffl-
pherlclole
Irlnltro-
Rr vnhres 011 A,,,,; of rrlJhJb
HP vahuson rlclole h-
rrlrdlru~he-
266 274 275 275 290 278 279
0.30 0.36 0838 0,40 0.44 0836 0,41
0835 0837 0,39 0,39 0.52 0,55 0.37
-. .-- --
274,335 271 276 276,334 272,335 273,335 281,337
0,26 0,38 0,40 0,42 0653 0.40 0,54
plexes In phre
0.32 0.35 0,37 0,39 0,57 0,61 0.32
275 270,310 270,310 280 275 274 282
0.40 OAI 0845 0849 0,53 0.44 0.4G
0839 0040 0,42 0,44 O,SI) 0.63 0.42
con~~lexcsrtnrcff phfc
269,315 271,309 272,314 271,314 272 271 282
0,4l 0,43 0846 0,49 0.52 0.40 OA5
0.43 260,315 0,40 271,310 0,45 212,314 0,48 270,313 OS9 273 0,so 282 OS41 204
cortr~lcxesprrgtlnred
0,34 0,39 OA3 0.44 0.48 0.32 0.37
0436 0.40 0.42 0.43 0.49 0.59 0,38
co,trpbcsprc~rtnfcrl _.. .. _._ _.___hl saltr- p/ore SdSJ’. sohrflort y;- - ---r’-” In J&. Ir,solir. plm . .__-__-..__-wJt blefu - -- - .-.---.-- ,lorr Sol. Solflon (na) - m I II w/It wt fnm, Sol- Sol. VCfll wr tnn,) f$ f$ fd 1 II I I1 wit vent I I 1; II -_..-e-..___ -.-.__-_-_--_..__..-- ._.-____I__ ..__-___- 11_1__^__ - -_._______
HJI vdrtcs
(nrrr) -.-
rzI”X,
Pure ~Jyrurhcs
Solvent systems: I =.acl~lorofor~n-ethylacclr~tc-cllmol (ffi:38:2); II := bcnzcnc-uccronc-clhanol (90:8:2), Adsorbent: silicn l~cl 0 (E, Merck). Whon tho nilro compound.imprcgnntcd phtcs wcro clulcd, the solvent nlso contained 0.01% of the rcspcclivc nitro compound, A,,,,,, values in carbon tctrnchlorido:styphaic II&~,278 nntl 333 nm; picryl chloride, 263 nnd 333nm; trinilrotolucno,262nm; trll~itrol~l~cnclolc, 20 nm, -__--..-___--------_-____ . .. _._._- --- -- ._.I___. _... .__-_._ -. - ... .. _..___- ____ _.. _ ._. .. ___._._._._ .- _ _ .. _. -__ _ _ . __.-_.__ --._-
RF VALUPS OF PYRAZINES AND ‘IIIEIR n-n COMPLEXES WITH STYPHNIC ACID, PICRYL CLILORIDE,‘~RINlTROTOLUENBAND TRINITROPI-IBNEI’OLE, AND AUSORM’ION MAXIMA IN CARIIONTETRACIILORIDE
TAllLE 1
ES’0 99.0
OIE‘ZLZ LE'O
IS'0 89'0 9LZ 8S'O OIE'ZLZ ZS’O 99'0 Z8Z E9’0 IS’0 W'OLZ 8LZ wo 08Z CL'0 88'0 08Z 88'0 06'0 EEE'ILZ 18'0 96'0 'OZZ 19'0 6AZ 9b'O 01'0 tr8Z 99'0 (9'0 IEE'trLZIS'0 69'0 6LZ 6S'O 08Z EVO EL'0 OIE'99Z E9'0 9L'o OIC'9LZ IV0 99'0 OLZ OSE 91E 88'0 09z 29'0 88'0 SIE'O9Z 96'0 P8'0'SIE'E9Z11'0 ZG'O 'GbZ 9IC IL'0 91E'bSZ 9S'O S8'0 LIE'9SZ t8'0 19'0 EEE'SLZ 99'0 18'0'ECZ zzz 011 es*0 OS'0 IEC'LLZ ES'0 L9'0 %LZ OIZ 18'0 9L'O SEE'b8Z S9'0 08'0 081
LIE 8S'O
OS'0 81'0 9LZ SE'0 06'0 081 LE.0 61'0 OK'991 1IE'OES'0 OLZ 8S'0 EG'O SLZ
IlZ SP’O GS’O ZIE'6EZ 6VO LL'O 9LZ SS'O 9L'o IrLZ 09'0 18'0
19'0 81'0 SIE'OBZ bS'0
9L’o 98’0
58'0 26'0 08Z 99’0 wo 09'0 fi8Z zvo ova 19'0 ZIE'OLZ 9E'O
GS’O 19’0 19’0 VL'O
081 081 t9z OLZ SLZ
SL'O E6'0 SEE'9LZ 18'0 06'0 Z6Z Z9'0 19'0 SEE'OLZ OS'0 85'0 99Z SE'0 OP'O t1z IS'0 wo 192 ttL'0ZG'O SEE;081 01'0 18'0 OGZ OS'0 16'0 OK'OIZ 9S'O Z8'0 16Z
99’0 OGZ 98'0 S8Z SL'O ILZ 8p'O VLZ 09'0 08Z
IS'0 6S'O OL'O wo SL'O
611 691 011 611 f6Z
99'0 9S'O 9EE'LLZ ES’0 11’0 08'0 SEE'OIIZ09'0 Ot'O 81'0 89Z PE'O LVO EVO EEC'OLZ LE'O t'8'0t's'0IEC'9LZ WO
05’0 09’0 8LZ 9S’O 18'0 08Z C9'0 OL'O OIE'OLZ 8C'O ZS'O ZIE'OLZ ob'o 11'0 bLZ 95'0 SS'O 8C'O IV0 GE'0
115'0S8'0 8LZ EL'0 $8'0 081 Eb'O 08'0 09'0 S9'0 SIE'89Z Zb'O ES'0 SIE'L9Z 119'019'0 9t'O Of"0 91E'8SZ wo LE'O IZE'E9Z 9S'O 9vo 19'0 $8'0 18Z 09'0 'GL'O 08Z S6'0 E8'0 GLZ 99'0 98'0 99z IS'0 IL'0 69'0 68'0
Qu~zuJ~d~Xt~~3w.~~xoq~~~Z
81 LI 91
:I
:r II 01 6 8
Fig. 5. UV qzctra of pyrazke (x) ~Horide (A).
RESULTS
TNT
AND
znd n-z compkzes
(Cl) and trinitropknetole
of
pyrazk withsQ@nic acid
(.),
piqyl
(O)_
DIXXJSSION
As is evident from Table I, pyrazines form charge-transfercomplexes with titro aromatic compounds both in solution and on TIC plates, which is reflectedin tie spectraIshifts and in the RF values. For most of the compounds a large spectral shift is acconpzr?ied by 2 sigdkant change in theiz R, V&XS. Fyrazines aren donors_ The nitrogen atoms in the ring deactivateall of the tig carbon atoms towards electropbilicsubstituentsand activate them towards nucleophilicsubstituents Hence nucleophihcsubstituentssuch as all& alkoxy and thioalkyl groups on the pyrazine ring further modify the donor capacity of the molecule. Of the four nitro aromaticcompounds tried as acceptors, viz., styphnicacid, PC, TNT and TNl, PC gave the best separation of the complexes. The chlorine atom attachedto the nitro-aromaticring furtherenhancedthe acceptorproperties_ Theoretically,pyrazines could form 1 :I, 1:2 and 2: 1 complexes with nitro aromatic compounds. From a Job’s plot in solution it was observedthat all of the pyraies formed only 1 :l complexes.In some instancesthe complexesshowed more than one absorptionband in solution. This could be due to multiplecharge transfer occurringbetweenthe closely relatedWed orbitalsof the donor moleculeand closely relatedunfilledorbitals of the acceptor molecule. Silicagel G was suitableas an adsorbent.Of the two solventsystemsexamined, solvent I gave betterresolutionsof the complexes_The compounds could be detected with Dragendorff’sreagent‘bothon unimpregnatedplates and on platesimpregnated with nitro aromatic compounds. Ibis propertyof n-z complexatioc on TLC plates was found to be of great utility in the resolutionof those pyrazineswhich had either identicalor very close RF valuesFor example, the following groups of pyrazineshad very close RF values in both solvent systems (Table I)_ Group I -2,3-dimethylpyrazin~ 2&iimethylpyrazine; pym.zine, 2&dimethylpyrazine; Group II -2ethyl-S-xethylpyrazine, Group III -2-metboxy-3-isopropylpyrazine, 2-methoxy-Zkec.-butylpyrazine ylpyrazine 2methylthio-3-&y1pyrazine; Group IV -2*thoxy-3-methylpyrazin~ 2-acetyi-3-meth-
ylpyraziq 2-propyW&dimetby~pyrazine,2effiyl-6-metby~pyrazinepyra6ne. However, as can be seen from Fig_ L, as the IHE complexesthey gave very distinctseparations. The proceduredescribedis very u&X for the separation of pyrazines that are difikralt to resolve. it is possible to detect and determinethem with limits of 2-5 and 5 p& respectively.
The authorsare gratefulto Dr. I?. K. Vijayaraghavan,Director, Defence Food Research Laboratory, Mysore, for his keen interest and encouragement. The generous help rendered by Dr. H. 3. Takken, Naarden International Research Laboratory, The Netherlands and Dr. M. A. Gianturco, Coca Cola Company, U.S.A., in supplyingauthentic pyrazinesas gifts is gratefuliyacknowledged. REFERENCES 1 R. C. Buttery, R. M. Seifert, D. CL Guadqni and L. Co Ling _?m Agr. Food Chem., 19 (1971) 969. 2 P. E. Kothkr, M. E. Mason and I. A. Newell, 1. A&. F&d Ckem., 17 (1969) 393. 3 J. P_ Wahdf A. 0. Pitet, T. E. Kinlin, R. Murali Dhara and A. Sazderson I. Agr. Food Chem.. 19 (1971) 972. 4 H. A. Badarmkh. P_ Frkdel. V. Krampl, J. A. Remer, F. W. Skphzd and M. A. Gianturco, J. Agr. Food Ckem., 15 <1%7) 1093. 5 Mmvan Razg, M. S_ Stein and M_ S_ Tibb&.s_J. &r_ Fmd Cherrz., 16 (196s) 1005. 6 P. Dietrich and D. hhvier, f_ Chrm~~~ogr_,1 (1958) 67. 7 C- E. !3izer.impubliskd resuk 8 V. G. Krishna and Mihie Chowdhy, 1. Phys_ Gem_, 67 (1963) lofj7. 9 A. K. Dwiveciy, D. B. Pzrihar, S. P. sharma and K. K. Vega, J. Ckromarogr.. 29 (1967) lZO_ 10 D. B. Pi&bar, S_ PmShyma and K. KmVesma, J. Ckromatogr., 31(1967) 120. 11 D. B. P&har, S. P. shanna and K. K. Vexma, J. Forensic SE SK., 10 (1970) 77. 12 E. Stahl, lIih dyer Chromatognzphy, Springer Verlag, New York, 1969.