ОРИГИНАЛЬНЫЕ СТАТЬИ
УДК 543
REVERSED PHASE CHROMATOGRAPHIC SEPARATION AND ISOLATION
OF TAUTOMERS OF NAPHTHOQUINONEOXIMES BY HPLC. EFFECT OF pH OF MOBILE PHASE ON SEPARATION OF 3-CHLORO-2-HYDROXY-4-NAPHTHOQUINONE-1-OXIME © 2014 Yogesh Shinde, Sunita Salunke-Gawali1
Department of Chemistry, University of Pune Pune-411007, India 1E-mail: sunitas@chem.unipune.ac.in Received 19.07.2012; in final form 26.12.2013
Reversed phase HPLC separation and isolation of isomers of 3-chloro-2-hydroxy-4-naphthoquinone-1-oxime was investigated. At pH 7 with acetonitrile as a mobile phase, no separation occurs. Using various buffer solutions the pH of the mobile phase was maintained and the effect was studied between pH ~ 2.5 and ~10.5. Two distinct peaks are observed in chromatograms in the pH range studied. These peaks are assigned to para-tautomer 3,3-chloro-2-hydroxy-4-naphthoquinone-1-oxime and ortho-tautomer 4,3-chloro-4-hy-droxy-2-naphthoquinone-1-oxime. Tautomeric equilibrium was found in acidic pH of mobile phase, whereas at pH 6.8 and basic pH of mobile phase the tautomers are well separated from each other. Isolation of the tautomers was carried out by preparative HPLC technique. The solids of tautomers 3 and 4 were obtained as ammonium acetate adducts.
Keywords: naphthoquinoneoxime, keto—enol tautomerism, separation of isomers, pH effect, HPLC, 3-chlo-ro-2-hydroxy-4-naphthoquione-1-oxime.
DOI: 10.7868/S0044450214120159
Naphthoquinones are important constituents of plant families such as Plumbaginaceae, Juglandaceae, Ebenaceae and many commercially important tropical hardwood species. The first report of HPLC separation of non-isoprenoid naphthoquinones was published by Marston et al. [1]. The isoprenoid naphthoquinones are best resolved by reversed-phase HPLC, using aqueous methanol mobile phases. However, non-isoprenoid naphthoquinones require high water content for separation by reversed-phase HPLC with methanol—water systems [1]. The effect on the selectivity of different mobile phase compositions, e.g., methanol—water and acetonitrile—water binary mixtures and methanol—acetonitrile—water ternary mixture has been investigated. The retention order of the compounds with methanol—water as eluent is interpreted on the basis of intramolecular hydrogen bonding in the solute versus intermolecular hydrogen bonding between the solute and the solvent. The hydrogen bonding pattern has been studied using quantum chemical calculations [2]. The separation of thirteen naturally occurring naphthoquinones and anthraquinones as well as a pyranonaphthalene derivative achieved on a reversed phase column by Steinert et al. [3] under isocratic and
gradient conditions. The compounds are known as constituents of the heartwood of Tabebuia avellanedae (Bignoniaceae). Their chromatographic behaviour is compared to that of fUranonaphthoquinones known from the inner bark of T. avellanedae.
Y. Hsieh et al. [4] isolated plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) from root of Plumbago Zeylanica L. The crushed roots in powder form of P. Zeylanica L. were boiled with water, 50 or 95% eth-anol. The authors carried out chromatographic separation using a ZORBAX, Extend-C18 column. The authors also represented the results as liquid chroma-tography coupled with tandem mass spectrometric (LC-MS-MS) method for the determination of plumbagin form herbal medicines. Y. Wang et al. [5] investigated plumbagin's anti-#. pylori activity and developed a reversed-phase HPLC method for quantification of plumbagin from P. Zeylanica L. Reversed-phase HPLC was performed with a gradient mobile phase composed of water and methanol, and peaks were detected at 254 nm.
O
O
CH3
OH O
Juglone
OH O
Pulmbagin
Babula et al. [6] used liquid chromatography coupled with diode array detector for determination of naphthoquinones in plants. The authors also studied the effect of pH on the cultivation medium on content of plumbagin in Dionaea muscipula. The simultaneous analysis of the most commonly occurring naphthoquinones was done (1,4-naphthoquinone, lawsone, juglone and plumbagin) by HPLC coupled with diode array detector. Petra Novotna et al. [7] developed for
identification and quantitation of nine natural quino-ne dyes and applied to historical textile fibers. A Puro-spher RP18e column was used with a convex gradient of methanol in a mobile phase of 0.1 M aqueous citrate buffer (pH 2.5) and spectrophotometric diode-array detection at 270 nm. For identification of alizarin, purpurin and xanthopurpurin, occurring together in the madder plant, an isocratic method was used with a methanol—0.2 M acetate buffer (pH 4.3) as the mobile phase.
There were no reports found in literature for separation of naphthoquinoneoximes by HPLC although the chromatographic separation of naphthoquinones is known. The oxime derivative of naphthoquinones shows antioxidant properties and can be present as ste-reoisomers, tautomers, viz. syn, anti and amphi (vide infra).
N
,OH
0
1
R = Cl, 3-ClLwox
N
,O
OH
2
O4
H
R
O
Anti
N
„O-H
O
Syn
Various isomers of 3-chloro-2-hydroxy-1,4-naphthoquinoneoxime.
Ov
N......H
i
O
R
O
Amphi
Recently Zaware et al. [8] reported the single crystal X-ray structures of 2-hydroxy-3R-4-naphtho-quinone-1-oxime. All the crystal structures show predominant amphi isomer in solid state, however it has been predicted based on NMR spectroscopy that they are present as mixture of structural isomers [9]. Thus there is need to investigate further this area of research due to following reasons,
1. The isomers % compositions of syn: amphi stereoisomers reported in the literature are deduced from 1H NMR in DMSO; however, the single crystal X-ray structures of C3 derivatives of 2-hydroxy-1,4-naph-thoquinone-1-oxime shows predominant amphi isomer in solid state.
2. The tautomers 1 and 2 were discarded on the synthetic ground.
3. There were no reports for keto—enol tautomers like 3 and 4.
4. There were no reports of chromatographic separation of tautomers/stereoisomers of naphthoquino-neoximes by HPLC and preparative HPLC techniques.
The present investigation reports the separation of tautomers of 3-chloro-2-hydroxy-4-naphthoquino-ne-1-oxime (3-ClLwox). The separation was achieved varying the pH of the mobile phase. The tautomers were further isolated by preparative HPLC.
EXPERIMENTAL
Chromatographic system. Shimadzu, LC-2010CHT liquid chromatograph was used for separation of tau-tomers. The detector used was SPD-M20A diode array and auto sampler injection system, connected with LC-Solution with a multi-channel module. Shimadzu LC-8A preparative liquid chromatograph was used for isola-
tion of tautomers. The detector used was SPD-M20A diode array detector and a Rheodyne valve manual injection system (5000 ^L loop), connected with LC-Solution with a multi-channel module.
A YMC ODS-A C18 and X-bridge C18 column (length 150 mm, internal diameter 4.6 mm, particle size 5 ^m, pore size 12 nm) was used for separation of tautomers (Table 1). A YMC-ODS C18 Column 500 mm x 30 mm, particle size 10 ^m) was used for isolation of tautomers. The mobile phase consisted of water and acetonitrile in various proportions. Water and acetonitrile was acidified with trifluoroacetic acid (TFA), mixture of ammonium formate and formic acid, basified by using ammonium bicarbonate, mixture of ammonium formate and ammonia. Ammonium acetate was used for preparation of neutral mobile phase. Detector was set at 254 nm.
Chemicals and materials. HPLC grade CH3CN and CH3OH were obtained from Merck Chemicals. TFA, hydroxylamine hydrochloride and NaOH were obtained from Qualigen Chemicals. 2,3-dichloro-1,4-naphthoquinone was obtained from Sigma-Aldrich. Milli Q water was used wherever necessary. The starting materials 2-hydroxy-3-chloro-1,4-naphthoquino-ne and 3-chloro-2-hydroxy-4-naphthoquinone-1-oxime were prepared according to the published procedures^, 11].
HPLC analysis. Wavelength 254 nm was used for HPLC analysis as well as isolation of isomers. Gradient programme for analytical HPLC: 5 to 95% of mobile phase B up to tR 8 min, holds for 1.5 min at tR 8 min from tR 9.51 to 12 min 95 to 5% of mobile phase B. Gradient Programme for isolation of isomers: initially mobile phase B 10 to 35% at 22 min, holds for 4 min; tR 32 to 40 min mobile phase B 42 to 50%, then 100% mobile phase B at 60 min. For separation and isolation of the tautomers, 100 and 5000 ppm solution respectively of 3-ClLwox was used (prepared in HPLC grade methanol).
RESULTS AND DISCUSSION
Dynamic interconversion of chiral oxime compounds [12] research by gas chromatography (GC) was reported recently by Marriott et al. The authors also reported multidimensional GC method of comprehensive two dimensional gas chromatography (GC x GC) for the study of interconversion processes of oximes [13, 14].
Separation of 3-ClLwox isomers in various pH.
Separation of 3-ClLwox was achieved in various pH of the mobile phase, using appropriate buffer solutions.
pH ~ 7 (mobile phase without buffer and modifier). Chromatographic condition I (Table 1) was used for this analysis (pH ~ 7). The chromatogram obtained is shown in Fig. 1a. There is no interaction of the 3-ClLwox with column and mobile phase was observed at this pH. The data are represented in Table 2.
pH ~ 2.5 (mobile phase with acidic buffer). Chromatographic condition II was used with pH of mobile phase ~2.5. The chromatogram obtained is as shown in Fig. 1b. Two peaks of isomers were observed in the 3-ClLwox, peak shape was found to be broad for both the peaks; however the separation between the peaks was not base to base. We named the two peaks as isomer X and isomer Y (the peak with less retention time is named as isomer X and the peak with more retention time than X is named as Y isomer). The percentage of isomer X was found to be ~29% (tR = 4.965) and the percentage of isomer Y was found to be ~71% (tR = = 5.410, Table 3). The percentage of isomer
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