Syn-anti isomerization of 2,4-dinitrophenylhydrazones of
volatile carbonyl compounds in capillary gas
chromatographic-mass spectrometric analyses
Citation for published version (APA):
Uralets, V. P., Rijks, J. A., & Leclercq, P. A. (1980). Syn-anti isomerization of 2,4-dinitrophenylhydrazones of volatile carbonyl compounds in capillary gas chromatographic-mass spectrometric analyses. Journal of Chromatography, A, 194(2), 135-144. https://doi.org/10.1016/S0021-9673(00)87289-1
DOI:
10.1016/S0021-9673(00)87289-1
Document status and date: Published: 01/01/1980
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SYN-ANTI ISOMERISAZTQN OF 2,4-DINITROPHENYLHYDRAZONES QF
VOLATILE CARBBNYL COMFCWNDS IN CAPILLARY GAS CHROMATO-
GRAPHZC-MASS SPECFROMETRIC ANALYSES
v_ P. URALFfs’
ldtitute of OrgamdKemenf Cornpad, Acariemy of Sciences, ~MOSCOW (U.S.S.R.)
and
3. A. RLKS and P. A. LECLERL”Q*
L?borarc?ly of Iti- nl Akdysis. Eiidkorwn University of Techmlogy, .Eidha ven (The Nethedm~) (Reshed January 31st, 1980)
SUMMARY
Diuitropheuylhydre derivatives of volatile aldehydes and ketones were investigated by high-performance liquid chromatography (HPLC) and by gas chroma- tography-mass spectrometry (GC-MS). HPLC separations were slightly better than those obtained by existing procedures, but high-resolution GC is the method of choice. The equilibrium of the syn-rmti isomerization of the compounds is iniluenced by the nature of the solvent, the deactivation of the column and the injection aud columu temperatures. The GC profile can therefore be controlled by careful selectibn of the experimental conditions. It is shown that this phenomenon, in addition to the occurrence of characteristic vaIues of the difference in the retention indices of syn and mti isomers for a given compound, can facilitate the identification.
Chemical ionization (methane) mass spectra of the compounds were found to contaiu more information than conventional electron impact spectra. Characteristic fragment ions are tabulated.
INTRODUCX-IQN
VoJatiIe carbonyl compounds are important fiavour ingredients in many food products. For analytical purposes, these compounds are often convexted into stable 2,44uitrophenylhydrazones (DNPHs), which can be sekctively isolated from corn-. plex mixtures of flavour components_ ’
After isolation of the DNPHs, the mixture can be separated chromatograph- icaIIy either as such or after regeneration of the free carbonyl compounds. Gas chromatographic (cc) studies on the liberated compouml9” show that this method is not suitable for quantitative trace analyses. Moreover, reactions with the liberating agents may give arkfact@,
%Rese& akkss: Iastitute of Nutrition. Academy of Medical Sciences, Us&&y projezd 2/14, MascQw 109240, UsS_R_
136 V_P_URALEB.J_kRIIK$P_kLECLERCQ
Many attempts to analyse DNPHs directly by GC have been rcportedbto. In
the gas chromato_m doubling of peaks has been observed in several instamx~W~~~~~~~, even when low-rcsoiution packed columns were used. Obvious double peaks have been recorded with -high-resoIution capiIlary columna’8.‘9_ Pur&at.ion of carbony compounds before derivatization to avoid peak doubling was not successfulxg_ The doubling effects were attributed to either decompositioniO@~ZO or isomerization*7p18 of the compounds.
The occurrence
of
s)n and anti isomers of DNPHsof
aiiphatic aldehydes wasobserved in IS61 by Van Duin”, who studied these phenomena by conventional liquid chromatography_ Published as a thesis, this work was apparently overlooked in the more recent GC studies cited above.
High-performance liquid chromatography (‘HPLC) has also been applied to the auaIysis of DNPHs 16_z-x. This merhod is very attractive *use the separation can he carried out at low temperature and the compounds can he detected with relatively high sensitivity. However, HPLC has a poor separation power in comparison with capiIlary GC_
Electron impact (EI) mass spectrometry (MS) has been applied to the identi- fication of DNPHsZ4-‘6 after separation by thlu-layer or conventional column chroma- tography. EI mass spectral dam are available for many DNPHs of saturated and unsaturated aldehydes and ketones -26--29_ Chemical ionization (CI) mass spectra of DNPHs were not found in the literature.
Ahhough some attempts were made to optimize HPLC for the separation of DNPHs, l &e main purpose of this work was to study the Gc bchaviour of DNPHs on
glass capillary columns_ In particular, factors influencing isomerization phenomena,
such as the nature of the solvent, deactivation of the column wall and the injection and column temperatures were investigated_ CI mass spectra, using methane as reagxlt gas, are discussed_
EXPERIMENTAL
2,4_Dinitrophenylhydradne was used as a reagent for preparing derivatives of aldehydes and ketones. The following procedures were applied for the preparation of DNPHs: (a) the conventional method and recrystalliztion from ethanol? (b) deri-
varitation on a column filied with neutral sorbent and the rcagentzl; (c) derivatization in pyridine so1ution2’_
Liquid chromatography
lie HPLC equipment us.4 is described elsewhere?. A variable-wavelength Model PM 2D LC W detector (Carl 2Zss, Jena, G.D.R.) was applied. .Absorbances were measured at 358 nm. The stationary and mobile phase systems were similar to those described earlier for the separation of DNPHF_ Adsorption chromatography was prformcd on LiChrosorb SI-60 with ethyl aceta+&octane (1:49) or isooctanc- methylene chloride (3:2) as solvents. Reversed-phase chromatography was carried out on LiChrosorb RP-8 with acetonitriie-water (3:2) or dimethylformamidc-water
CiGW OF qyn-arr&DNPHs OF CARBoNn COhJPOUNDS 137 Gas chromatogruphy
An I~~~EGIG% IGC 120 FB gas chromatograph @ternmat Instruments, Pavil- lons sous Bois, France) equipped with a flame-ionization detector and an all-glass Tnoving needle” injectoF was used at injection block temperatures of 2oo-400 “C (mainly 225 “C). Four Duran 50 glass capillary columns (40 m x 0.4 mm I.D.), rinsed with dry methylene chloride and dried with a gentle stream of dry nitrogen, were coated by a static procedure with E-30 stationary phase using a 0.4% (w/w) solution in n-hexam?. Different treatments of the glass surface were applied before coating, in order to deactivate the column wall, with the exception of cohnnn 1, which was coated without prior treatment. Column 2 was deactivated with a 1% ~01utiou of benzyftriphenylphosphouium chloride (BTPPC) in methylene chloride, as described by Rutten and Luyten =- Column 3 was deactivated with a 5 o/0 solution of Carbowax 2OM in methylene chloride according to Blomberg35- Column 4 was deactivated by Carbowax 2OM vapouE”6~ f7. In all experiments the injector was deactivated essentially according to the same procedure as the column. The columns were operated isothermally between 200 and 260 “C, but mainly at 225 “C. The carrier gas was nitrogen at a flow-velocity of 10-20 cm/set, the detector temperature was 280 “C and the sample size was 5- IO-’ to 5- IO-r0 g per component in 1 ~1 of an appropriate solvent-
Mass spectrometty
A Fir&an Model 4000 quadrupole mass spectrometer (Finn&an, Sunnyvale, CA, U.S.A.) was used in the CI mode. The mass spectral data presented were acquired under the following conditions: ionizing electron eucr,g, 79 eV; electron current, 020 mA; scan time, 1 see per scan; and source temperature, 250 “C. Methane reactant ,g~ was introduced via the make-up gas line. The ion source pressure was maintained at O-15 Torr gauge reading. Samples were analysed by CC-MS using column No. 2 and helium as carrier gas. The column was coupled directly to the ion source via a platinum-iridium capillary (60 cm x 0.1 mm I.D.). The column was operated iso- thermally at 230 “C and the platinum-iridium interface was maintained at 250 “C. Samples were injected on to the column as described above at 258 “C.
RESULTS XND DISCUSSION
Fig. 1 shows part of a representative gas chromatogram of DNPHs derived from pentanal and heptanone-3 on capillary column No. 1. Two distinct peaks cot- respond to each compound. The baseline in between the pairs is elevated and has a slope. The composition of these complicated chromatographic zones was studied by
GGMS in the EI and CI (methane, isobutane) mode for DNPHs of different alde- hydes and ketones- The mass spectra recorded at different points of one pair of peaks and in between were identical with and similar to those obtained by direct insertion of the sample into the mass spectrometer. Therefore, the peak doubling cannot be attributed to decomposition10J6J0.
Preparative separation of the isomers of acetaldehyde DNPH was achieved by HPLC on LiChrosorb SI-60, using isooctane<thyl acetate (49:l) as solvent_ The identiQ of the isomers was established by pro&xx NMR. The isomer eluting first in both CC and HFLC appeared to be the syn isomer:
The occurrence of a single -peak far DNPHs of formaldehyde, acetone, diethyl ketone and other symmetrical cartionyl compounds will be easily understood.
The raised baseline between two DNPH isomers is evidently due to isometi- tion in the column during chromatography_ This results in intermediate retention for compcnents passing one pxt of the c&unn as a SKI! and the other as an mrfi isomer,
Attempts have been made to suppress isomerization in the chromatographic coIumn. Fig_ 1 shows si_gnificant on-cslumn isomeriz-ition (elevated baseline behwxn
DNPH isomers) on column No. 1, w&h was not treated with deactivating agents. Lcs-s isomcrization was observed with column No. 2, which was h-catcd with BTPPC as described above. Fig_ 2 represents the separation of a DNPM mixture on this column_ The cohnnn eEciency for heptanone-2 DNPH (capacity ratio, k’ = 6.5) was MO theoretical plates per metre (2300 for n-CUHso, k’ = 5.4). In contEaS& carbowax treatment (columns No. 3 and 4) decrea_ ted the column performance with respect to
Gc-MsoF5yzwma~~OF-~cQMPouN5s a39
the separation of DNPHs. Rapid isomerization was observed, resulting in enormous peak broadening, although separations of other compounds (hydrocarbons, pesticides,
steroids) were suffessfitl. Column No. 2 was used in further expe+nent.s.
Fig 2. t3rornatogram of a mixture of 2&dinitropbenylbydrazoenylhydrarones from a gfass capillary column coated with SE-30 (coiunm No. 2). Column and injector temperature, 225 “C; linear carrier gas (nitrogen) &o&y, XOmm/sec; Peaks: 1 = methartal; 2 = etbimal (syn); 3 = e&anal (mxfi); 4 =
propanal (SPY: 5 = acetone; 6 = pmpanal (mxti); 7 = butamI (syn); 8 = butanone (syn); 9 = bu- tanone (onii); 10 = butanal (car@ t 2-methyIbutanal (syn); 11 = = 3-uxethylbutanal (cgm); 12 = pemmoae-2 (qm); 13 = 2-methylburax.d (cud); 14 = 3aethyIbutanal (mri); 15 = pentanal (qn); 16 = pentaaonc-2 (am-); 17 = 4-methyIpentanone-2 (syn); 18 = pentanal (mui); 19 = B-methyI- pentanone-2 (antii; 20 = kxanone-2 (syn); 21 = bexanoixe-2 (anti); 22 = heptauone-2 (qn); 23 = beptanonc-2 (anti) -f- beptanal (syn); 24 = JxxenaI-2; 25 = beptanal (anti); 26 = octanone-2 (xv-n); 27 = octanon+2 (a&; 28 = kptenal-2; 29 = mfanal (anti) f nonmone-2 (gm); 30 = nouauone+2 (a?ui-).
Within the column temperature range of 20&2&I “C, increasing temperature promotes the isomerization rate-The slope between the pairs of isomer pe-aks increases signikantly more than can be accounted for by decreas ing retention times at higher temperatures. Temperatures of 220435 “C are preferable, providing reasonable
thation times and acceptable isomerization.
The injector temperature si_@kantly infhxences the syn/arz?i ratio, as shown in Fig. 3 for derivatives of propanal, pentawl and heptanal. It should be noted that the injected sample contained mainly mti isomers, corresponding approximately to the ratios as in chromatogram A at a low injector texq~rature. Rapid isomerization was observed at 400 “C (Fig- 3B), which resulted in increased amounts of the syn isomers.
The initial ratio of isomers depzuds substautially on the derivatization proce- dure?‘. Conwzsious using a neutral adso&entll yield high fractions of syn DNPHs (JOoh. Derivatives obtained from a pyridkte sdution*’ were mainly anti isomers (96 “A. The pemtagcs given zefm to butanal DNPH, as determined with GC column No. 2 at 225 “C and the injeaor also at 225 “C.
MO et al,” reporkd aa infknce of ffie solvent on the size of the secondary
(syn) peak, although later this effect was not observed18. We examined several solvents
140 V. P. URALFZL I; A. REIKS. P. A. FLECLERCQ anti
~~.3.~ilencrof~~~~~tureon~nIati~-camollnh ofsyn-andmrri_DNPHisomcrsof proptnal (l), ptanal(2) and hcptamd (3). G&s capillzy column coated wi-h SE-30 (co1uim.1 No. 2) operated at 225 ‘C. Injector tcrnperaturcr A, 235 ‘C; B, 400 “C.
over a period of 2 weeks, the s&znr~ ratio of two samples of butanal DNPH, con- taining 70 and 4% of the ~G.I isomer, respecGvely. In acetic acid, however, rapid isomerization was observed, resulting in an equilibrium corresponding to 25 ok of the s~u and 75 % of the mrri isomer for both samples. Similar results were initially obtained with chloroform. After removal of hydrochloric acid, however, this solvent no longer promoted isomerization. Hence the isomerization in solutions is connected with the soIvent acidity, as also follows from Van Duin’s datazl. It can be concluded that acidetalysed interconversion leads to an equilibrium in the sy~+znri composition. Thus, DNPH derivatives obtained in acidic medium show a constant syr@uzti ratio in solutions’s.
Consequently, it is also apparent that glass capillary column and injector deactivation procedures have to eliminate the surface acidity (Duran 50 is slightly acidic) to suppress isosnerization during chromato_graphy.
The results obtained ahow one to control the appearance of the chromato- grams. To simplify the chromatograms, derivatization procedures should be applied that yield mainly anti isomers. Neutral solvents (isooctane or carbon disulphide) must be used for further treatment and Iow injector temperatures (approximately equal to the coIumn temperature) have to be chosen to sninimIze the formation of .syu isomers. On the other hand, the identification can be aided by the presence of vn-DhTH isomers in defined quantitative ratios and with appropriate retention data. This can easily be achieved by dissolving the DNPH mixture in acetic acid or by increzsing the injector temperature. Thus, for anaIysis of an unknown mixture of DNPHs two chronatographic runs are advisable: one with suppressed SJR isomer formation and another with both isomers present.
Retention indices of J~TI- and rmri-DNPHs of 40 aldehydes and ketones are presented in Tabk I. an&DKPH isomers of n-alkanals were used as reference com- pounds for the c&uJ.ation of retention indices. Derivatives of the symmetrical carbonyl compounds, having only one isomer, and of methyl isopropyl ketone, methyl iert.-butyl ketone and a-unsaturated aldehydes producing negligible amounts of the SJX isomer, are characterized by only one retention index value.
tori Isomers, having the (larger) a&y1 soup in the mrri position with respect to the N-H group, are more exposed to stationary phase moIec&s than the corre-
GC-MS OFS)72dnfi_DNPEFS OF CARBQENYL COMPOUNDS 141 TABLE I
RETENXONiNDiCES (Z) OF 2,4-DmIOPHENYUWDRAZgNES OF CABBONYL COMPOUNDS ON SE-30 AT 225 “C
The a.&-DNPH isamers of zr-&mds serve as reference campourds. DNPNs of
-HZ=
Z AZ' DNFHs of Z AZ’
ketones
syn mrti syrr pnii
Nod 2-Metbylprapanal ZMethy~butmal 3-Metbylbutmd ZMethyIpentad 3-MethyIpentzd 2-EthyIbutzmd P=penal Butcd-2 He-2 Hepteinal-2 2&Jzkxac?ienal 188 272 366 463 559 657 755 854 399 418 476 523 469 100 200 300 400 500 600 700 800 QOO 346 449 451 533 561 526 298 468 676 787 749 12 28 34 37 41 43 45 46 48 50 33 57 38 57 Acetone Butsnane Pentauane-2 Hexanaue-2 Heptmout+2 Octanane-2 Nammane-2 fMethylb&anone-2 Per&Moue-3 4Methylpentanone+2 3,3-Disnethyibutanone-2 3-Methylpentmone-2 ZMethyIpentanane-3 2&Dimetbylpentanane-3 2-Methylhexanane-3 Keptaixans? EMethylhexanone-3 Heptiinone-3 5-Methylhexmone-2 6-Methylheptmane-3 2,6Dimethylheptanane1 Nanmane- Nonanone-4 Nonanone-3 292 370 386 442 468 SW 562 616 658 7aQ 755 805 851 436 451 451 iii 493 517 488 503 SW 547 574 591 5.52 565 60 620 586 624 649 666 657 737 758 773 776 806 16 26 33 42 46 46 28 24 15 27 13 20 38 I7 15 30 - AZ = I-, - k=-
spending syn isomers. Therefore isomers with a higher retention index have the anti structure.
The elution sequence of DNPHs is similar to that observed for free carbonyl compounds on non-polar stationary phase?. However, hydrazones give twice as much retention data from one cohmm as do free carbonyis, thus considerably increasing the possibilities of identification. The di values in TabIe I are a measure of the distinction between the hydrocarbon groups attached to the (converted) carbony group. For compounds with the same number of carbon atoms, the dl value decreases in the order n-alkanals, methyl n-a&y1 ketones, ethyl z~Ikyl ketones and so on (cf-, 2-methylbutanai, pentanal, 3-meth~lbutanal). Within homologous series the dl value increases with increasing number of carbon atoms, and approaches a constant value. These regularities may facilitate the identification of unknown components.
Separations of DNPH mixtures using E5Pk.C were similar to those repcrted by Sehm2z. A higher selectivity was obtained in the separation of DNPHs of alkanals and 2-aIkanones with the same number of carbon atoms. This was accomplished by
142 v. P. URALEFS, J- R WKS. p. A- ==w STN-GNPH CF h3?S!lo?Z-4 KCSKCH41 - N 3223 293 I ----a 277 ‘, I t d! 1; . . . . ICO 153 200 250 300 350 WI--,
Fig_ 4. chcmka! ionization mass spectrum of the 2+dinicfophexlythyaIaxxle of nonaLlone4.
kng a LiChrosorb RP-8 coiumn with dimethylformamide-water (3:l) as soIvent system. However, the relativeIy low separation power of this method, in comparison with capillary GC, restricts the use of HPLC for the analysis of DNPHs in complex ZnixtLxes.
CI (methane) mass spectra of many DNPHs were recorded. Fig. 4 shows, as an example, the CI spectrum of the nonanone-4 derivative. The spectra of the syn and lmti isomers of the investigated compounds were found to be identical- The masses and relative abundances of characteristic ions from the CI spectra of DNPH deriva- tives are given in Tables II acd IH.
Protonated molecular ions, +MH+, appear to be the base peaks in most
TABLE II
SELECl-ED iONS FROM THE CI (MEJXANE) MASS SPEchRA OF 2+DINITROPHENYLHYDRA- ZONES OF ALDEHYDES
10s with a miss below m/z 60 were not _measured.
iUH+ [MH - X0,?+ [MH - NOJ+ mjz It?4 m!z 154 R,HC=GHz R,Cz 1;H
MlZUWld (2ll)loo (181) 5 (165)12 23 8
-E&?Ild (22S)loO (195) 6 (17% 2 24 9
Propanal (239)loo @?9)10 (193) 5 21 10
Butanal (253)loo (223)13 (zo7) 5 26 19 cm29 (70) 64
2-Meffiyipropad (253)loo (223115 (2wI 3 45 2s (72~1 (70) 95
Hexanal (25l)lW (251) 5 (WI 2 2.5 4 (lcmll (98) 14 2-Metisylpultanal (2sl)mo (251) 3 (23% I 27 1 UW 6 (98) 8 2-Ethylbutanal (2sl)lao (251) 4 (235) 1 20 2 (100) 8 (98) 9 HCpWl (W5)lOO (265) 5 (249) 2 20 5 (114)lS (112) 14 (309) 51 (27!?)19 (263) 5 28 65 (12w6 (l26wm hpcnrl (237)mQ (2G7) 7 (191) 3 13 4 w-l13 BUtC&&2 (251)loo (221) 3 (205) 1 5 1 mm (68) 9 He&-2 (279:KMl (249) 3 (233) I 4 (98) 8 (96) 6 Hepteml-2 (293~100 (263) 3 (247) 1 4 : (112) 8 (110) 6 HCXd.kd (277) 48 <247)21 (231) 1 23 46 m92 (94)laQ
I44 V_P.URALEB,J~kRIJK!S,P-kJAK!L=CQ
ACKNOWLEDGEMENTS
This work was supported by the Scientific Exchange Agreement (S.E.A.). Thanks are due to Dr. I_ H. Dhont, Dr. S. van Straten, Dr. H. Morse (Central Institute for Nutrition and Food ResezrcE TNO, Zeist, The NetherIan&) and Dr. H. T. &dings and Dr- H. vat Duin (Netherlands Institute for Dairy Resemch NIZO, Ede, The NetberIands) for discussions on the analysis of carbonyl compounds. We also thank Dr. J_ W. de Haan and Mr. L. J. M. van de Ven (Eindhoven University of Technolo_gy) for the NMR analyses.
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