The retention behaviour and separation ability of normal and reversed phase HPLC with one non-polar and two polar mobile phases, have been studied by measuring the retention constants of a series of newly synthesized 1,2,4-triazole derivatives. The results are discussed in terms of the nature of the solute, eluent and stationary phase

Ačanski Marijana M.

Dimova Vesna

Perišić-Janjić Nada U.

Marijana Acanski

Nada Perisic-Janjic

Vesna Dimova

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Normal and reversed phase high performance liquid chromatography of some new 1, 2, 4-triazole derivatives

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83 UDC 543.544.5 : 547.79 APTEFF, 34, 1–148 (2003)  BIBLID: 1450–7188 (2003) 34, pp. 83–92 Original scientific paper NORMAL AND REVERSED PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY OF SOME NEW 1,2,4-TRIAZOLE DERIVATIVES Marijana M. Ačanski, Nada U. Perišić-Janjić and Vesna Dimova The retention behaviour and separation ability of normal and reversed phase HPLC with one non-polar and two polar mobile phases, have been studied by measuring the retention constants of a series of newly synthesized 1,2,4-triazole derivatives. The results are discussed in terms of  the nature of the solute, eluent and stationary phase. KEY WORDS:  High performance liquid chromatography (HPLC), normal phase,     silica gel column, reversed phase, C-8 bonded silica gel column,    1,2,4-triazole  derivatives INTRODUCTION 1,2,4-triazoles are a well known group of physiologically active compounds. They have found application as herbicides, fungicides (1), plasticizers, adhesives and chemi-cals in the photographic industry. Preliminary investigations have shown that newly syn-thesized 1,2,4-triazole derivatives are biologically active against Escherichia coli, Bacillus subtilus, Salmonella enteritidis and Aspergillus niger. Antimicrobial investigation were performed by the filter paper disk methods (2). The compounds had the highest activity against  Salmonella enteritidis. The primary intention in chromatography is to achieve satisfactory resolution of the sample components. The retention, and consequently resolution in liquid chromatogra-phy (LC), is determined by the interactions of the solute with both mobile and stationary phases of the system. Thus, the molecular structure of a solute, the polar and non-polar function of the stationary phase and the mobile phase composition influence retention in    Dr. Marijana M. Ačanski, Assist. Prof., Department of General and Inorganic Chemistry, Faculty of  Techno-logy, University of Novi Sad, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia and Montenegro, e-mail: marijana@tehnol.ns.ac.yu; Dr. Nada U. Perišić-Janjić, Prof., Institute of Chemistry, Faculty of Science, Univer-sity of Novi Sad, Trg Dositeja Obradovića 3, 21000 Novi Sad, Serbia and Montenegro; Vesna Dimova, M. Sc., Assist., Faculty of Technology and Metalurgy, R. Boškovića 16, 91000 Skopje, Macedonia 84 LC. It is well known that the same position of different substituentsin a molecule can have significantly pronounced effects on retention in chromatography. Because of that, in this work we studied the retention behaviour of two series of 1,2,4-triazole derivatives by normal- and reversed-phase HPLC on silica gel and C-8 modified silica gel columns, respectively, using non-aqueous and aqueous mobile phases.  The structures of the investigated compounds are presented in Table 1. Table 1. Structural formulas of the 1,2,4-triazoles studied  EXPERIMENTAL New 1-(arylaminomethyl)-1,2,4-triazoles (Table 1) were synthesized by the reaction of 1-hydroxymethyl-1H-1,2,4-triazole with the coresponding aromatic amine. They were characterized by determining melting points, IR and NMR spectra and elemental analysis. Separations were performed with a Milton Roy liquid chromatograph (Riviera Baech, FL, USA) consisting of a model Consta Metric 3000 pump and a Milton Roy Spectro Monitor 3160 variable-wavelentgh UV-Vis detector set at 254 nm. Samples were injec-ted using a Rheodyne 7125 valve (Cotati, CA, USA) fitted with a 20 µL loop. The columns 85 used were commercially available Spherisorb SI (250×4 mm i.d.; 5 µm) and LiChrosorb RP-8 (150x4 mm i.d.; 5 µm (both, E. Merck, Darmstadt, Germany).  1,2,4-triazole derivatives were dissolved (0.02 %) in methanol and the solutions fil-tered through a 0.2 µm Chromafil filter (Macherey-Nagel, Düren, Germany).  A binary solvent system (hexane - propan-1-ol) was used as a mobile phase on the silica gel column. Binary mixtures methanol - water, and  acetonitrile - water, containing a  volume fraction (ϕ) of organic solvent between 0.4 and 0.9 were used as mobile phases on C-8 modified silica gel column. The eluents used to prepare mobile phases were of analytical grade and were filtered through a 0.45 µm filter and deggased before use. The flow rate was 1mL min-1 at room temperature. The retention factor, k, was calculated as  00tt tkr −= , where tr  is the retention time of the solute and  t0  the column void time of methanol. Each tr  value was measured in triplicate and averaged.  RESULTS AND DISCUSSION Two series of 1,2,4-triazole derivatives were studied. In series I  there were deriva-tives  which have different substituent at the  p- position of nitrogen atom (position 7), while in series II consisted of derivatives which have different substituent bound to the nitro-gen atom at position 7 (Table 1). All derivatives were examined by normal and reversed phase HPLC on the silica gel and C-8 bonded silica gel columns.  Normal phase chromatography The retention data for the 1,2,4-triazole derivatives obtained on silica gel column with hexane - propan-1-ol (8:2; v/v) are as eluent given in Table 2. Table 2.  Retention data for the 1,2,4-triazole derivatives on a Spherisorb SI column, using hexane-1-propan-ol as the eluent; designation of the compounds is as in Table 1 Compound  log k I1  -0,623 I2  -0,608 I3  -0,240 I4  -0,067 I5  -0,177 II1  0,351 II2  0,247 II3  0,038 II4  -0,646 II5  -0,666 86 The retention orders of the compounds of the particular groups were as follows: Series I:   I1 ≤ I2 < I3 < I5 < I4 Series II : II5 ≤ II4 < II3 < II2 < II1 In series I the least retention had compounds I1 and I2. These two compounds were not resolved. Compound I1 at p- position has a Cl atom, and compound I2 a Br atom. These data did not confirm our former observations that the retention of derivatives pos-sessing a halogen atom at the same position decreased from fluorine to iodine (3, 4). On the other hand, in these investigations, like in previous ones (3, 4), retention of halogen derivatives was low. Compound I3 which has a methyl group at the p-  position was stronger retained than compounds I1 and I2, but less than compound I5 which has a polar ester group at the  same position. In series I the most retained was compound I4 having a  polar nitro group at the p- positon. In series II the least retention had derivatives II4 and II5, which were not mutually resolved. The most retained was compound II1 because the molecule of pyridine at position 7 is more polar compared to the molecule of thiazole at the same position. The sequence of separation on the silica gel column of all compounds was: II5 ≤ II4 ≤  I1 ≤ I2 < I3 < I5 < I4 < II3 < II2 < II1 On the basis of the separation sequence, it is shown that the compounds having in the molecule different nonpolar groups at the same position were not resolved because the nonpolar substituents did not affect the retention. Compounds from series I were gen-erally less retained than compounds from series II. The most polar solute in series I, compound I4, was less retained than compound II3 which is in series II of middle polarity. The compounds that have a nitrogen atom as the substituent in their molecule, capable to form hydrogen bonding with silica gel surface, were the most retained (compounds II3,  II2 , II1).  In these investigations, like in previous ones (4, 5) we showed  that silica gel did not always indicate the real polarity of the compound, but it can be said that  the retention behaviour of new 1,2,4-triazole derivatives on silica gel were in accordance with general principles in normal-phase liquid chromatography.   Reversed phase chromatography The change in compounds retention of the 1,2,4-triazole derivatives with increasing volume fraction of the modifier in aqueous mobile phases was in accordance with the well known equation, generally accepted in partition chromatography:   0 log log kk m ϕ =−  [1] where ϕ is the volume fraction of the organic component of the binary aqueous mobile phase, log k0, is the value of  log k extrapolated to ϕ  = 0, and m is a constant. The rela-tionship between the retention factor, log k, of the investigated compounds and volume 87 fraction, ϕ , of the modifier in the aqueous mixture was linear (Table 3). The compounds were more mobile in the eluent with acetonitrile as the eluent modifier than with meth-anol owing to the lower polarity of acetonitrile. It is empirically known that a change from methanol to acetonitrile generally decreases the selectivity (Figs. 1 and 2). Table 3. Constants /m/ and log k0 of Equation [1] for the relationship between retention and mobile phases composition; designation of the compounds is as in Table 1 Methanol-water Acetonitrile-water  Compound log k0  /m/ r  log  k0  /m/ r I1  1.6527 2.403 0.9663 1.5836 2.327 0.9983 I2  1.7890 2.478 0.9550 1.6910 2.430 0.9990 I3  1.3890 2.034 0.9942 1.2759 1.953 0.9936 I4  1.2340 2.255 0.9983 1.3138 2.166 0.9929 I5  1.9430 2.999 0.9995 1.5142 2.344 0.9986 II1  0.9220 2.020 0.9765 0.9120 0.899 0.9986 II2  0.5785 1.790 0.9921 0.3239 1.258 0.9835 II3  0.6197 1.620 0.9630 0.2828 0.786 0.9998 II4  4.3450 4.795 0.9991 3.5405 4.265 0.9886 II5  2.6741 3.431 0.9872 2.4070 3.308 0.9913 The numerical values of the absolute value of constants m and log k0  for each exa-mined compound and used mobile phases, containing water and methanol or acetonitrile as modifier, are given in Table 3. Correlation coefficients from linear regression analysis of experimental log k values varied from 0.9550 to 0.9998. The retention data obtained for separation on the C-8 bonded silica gel column are generally typical of reversed phase chromatographic behaviour: less polar solutes are more strongly retained. The retention order of the compounds with both mobile phases were mainly as follows: Series I:    I4 <  I5  ≤ I3 < I1 < I2 Series II :  II2 ≤ II1 < II3 < II5 < II4 Compounds II4 and II5, which were not resolved on silica gel column, were clearly resolved on C-8 bonded silica gel (Figs. 1b and 2b), because the molecule of naphthalene at the position 7  is more hydrophobic than the molecule of diphenyl at the same position. Besides, the halogen atom contributed to the increase of hydrophobicity 1,2,4-triazole derivatives. Because of that, compounds I1 and  I2 were the most retained in series I in both aqueous mobile phases; in all cases these two compounds were clearly resolved on reversed phases showing a higher retention of comound I2 (Figs. 1a and  2a). It is apperant from data in Table 3 that on C-8 bonded silica gel column and with both mobile phases the constant  log k0 and the absolute value of the constant  m increase with increasing compound retention (Figs. 3 and 4). There are, therefore, linear relation-ships between these two constants, with high correlation coefficients.   88 0,50 0,55 0,60 0,65 0,70 0,75 0,80-0,6-0,4-0,20,00,20,40,6  I1 I2 I3 I4 I5ϕΜεΟΗa)log k 0,50 0,55 0,60 0,65 0,70 0,75 0,80 0,85 0,90-0,8-0,6-0,4-0,20,00,20,40,60,81,0b)  II1 II2 II3 II4 II5log kϕΜεΟΗ  Fig. 1. Relationship between the retention constant (log k) and the methanol concetracion (ϕMeOH) in the mobile phase methanol-water; designation of the compounds is as in Table 1 89 0,40 0,45 0,50 0,55 0,60 0,65 0,70 0,75-0,20,00,20,40,60,8 a) I1 I2 I3 I4 I5log kϕΑΧΝ  0,40 0,45 0,50 0,55 0,60 0,65 0,70 0,75 0,80-0,6-0,4-0,20,00,20,40,60,81,01,2 b)log k II1 II2 II3 II4 II 5ϕΑΧΝ  Fig. 2. Relationship between the retention constant (log k) and the acetonitrile concetracion (ϕACN) in the mobile phase acetonitrile-water; designation of  the compounds is as in Table 1 90 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0012345I3I4I5I2 I1II1II2II3II5II4Intercept  -1.2998Slope        1.1673r                 0.9866log k0/m/  Fig. 3. Plot of log k0 against /m/ for the mobile phase methanol-water; designation of the compounds is as in Table 1 1234501234I5 I4 I3I2 I1II2II1II3II4II5Intercept   -0.4147Slope         0.8738 r               0.9681log k0/m/  Fig. 4. Plot of log k0 against /m/ for the mobile phase acetonitrile-water; designation of the compounds is as in Table 1 91 CONCLUSION  –  On silica gel, the retention sequence of 1,2,4-triazole derivatives obtained with non-polar eluent is that predicted on the basis of polarity of the compounds. However, some retention data show that the retention on silica gel does not always indicate the real polarity of the compound. –  On C-8 bonded silica gel, the retention sequence of the compounds obtained by applying aqueous-organic mobile phases, is basically a consequence of the compound hydrophobicity. –  Compounds that are not resolved in normal phases are resolved in reversed phases and vice versa. –  For resolving 1,2,4-triazole derivatives it is necessary to have one column of silica gel and one column of C-8 silica gel. This combination guarantees resolution of the  1,2,4-triazole derivatives. REFERENCES 1.  Hansh C. and T. Fujita: Classical and Three-Dimensional QSAR in Agrochemistry, American Chemical Society, Washington, DC (1995) p. 107. 2.  Perišić-Janjić N. U., M. D. Lazarević and J. S. Balaž: A Study of Retention Beha-viour of Some Benzimidazole and Benztriazole Derivatives on Cellulose and Starch Layers.  J. Planar Chromatogr. 10 (1997) 267-272. 3.  Petrović S. M., M. Ačanski., V. Pejanović and  J. Petrović: Retention Behaviour of Estradiol and Estrone Derivatives in Normal and Reversed Phase Thin Layer Chromatography. J. Planar Chromatogr. 6 (1993)    29-33. 4.  Ačanski M. M., S. Jovanović-Šanta and  L. R. Jevrić: Normal and Reversed Phase Thin-Layer Chromatrography of New 16,17-Secoestrone Derivatives. J. Serb. Chem. Soc. 68 (2003) 57-63. 5.  Ačanski M. M.: Retention Behaviour of Some Estradiol Derivatives on Alumina in Normal Phase Chromatography. J. Serb. Chem. Soc. 68 (2003) 163-170. 92 ТЕЧНА  ХРОМАТОГРАФИЈА  ПОД  ВИСОКИМ  ПРИТИСКОМ  НА  НОРМАЛНОЈ  И ОБРНУТИМ  ФАЗАМА  НЕКИХ НОВИХ  1,2,4-ДЕРИВАТА  ТРИАЗОЛА Маријана М. Ачански,  Нада У. Перишић-Јањић  и Весна Димова У раду је испитано ретенционо понашање и раздвајање новосинтетизованих деривата 1,2,4-триазола, течном хроматографијом под високим притиском на нор-малној и обрнутим фазама. Уз извеснe ограде, резултати показују да је раздвајање на силика гелу (нормална фаза) у складу са поларношћу једињења, док је раздва-јање на октил силика гелу (обрнута фазa) у складу са хидрофобношћу једињења. Деривати 1,2,4-триазола  који  нису  раздвојени  на  силика  гелу  раздвојени  су  на октил силика гелу и обрнуто. У хроматографији на обрнутим фазама испитана је и промена ретенције деривата 1,2,4-триазола са променом запреминског удела ор-ганске компоненте у двокомпонентној воденој покретној фази.  Received 14 August 2003 Accepted 29 September 2003 

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Normal and reversed phase high performance liquid chromatography of some new 1, 2, 4-triazole derivatives

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