Original

CHROMATOGRAPHY, Vol.34 No.2 (2013)

Original

Effect of Temperature on Retention of Diazines inReversed−Phase Liquid Chromatography
Chiharu Takemura1, Kentaro Yogo1,2, Ikuo Ueta3, Kiyokatsu Jinno1, Yoshihiro Saito1
1

Department of Environmental and Life Sciences, Toyohashi University of Technology
1−1 Hibarigaoka, Tempakucho, Toyohashi 441−8580, Japan

2

Morinaga & Co., Ltd., 2−2−1 Shimosueyoshi, Tsurumi−ku, Yokohama 230−8504, Japan3

Department of Chemistry, University of Yamanashi, 4−3−11, Takeda, Kofu 400−8511, Japan

Abstract
Retention behavior of diazines, consisted of pyrazines, pyridazines and pyrimidines, was studied at various column temperatures in reversed−
phase liquid chromatography (RPLC). An increased retention of pyrazines was observed on several types of stationary phases including octadecylsilica (ODS) phases when column temperature was elevated, suggesting an abnormal temperature effect. Similar abnormal temperatureeffect on the retention was also observed for pyridazines and pyrimidines with an acetonitrile/water mobile phase at elevated column temperatures. On the other hand, no similar trend was found for these analytes with a methanol/water mobile phase. In addition, the retention for pyrazole, imidazole and their methyl derivatives was decreased with increasing the column temperature regardless the type of organic modifier inthe mobile phase. The results suggest that the abnormal temperature effect on the retention of diazines could be mainly induced by an unusual
thermodynamic behavior between the stationary phase and the mobile phase containing acetonitrile.
Keywords : diazines, retention behavior, temperature effect, reversed−phase, liquid chromatography

in typical RPLC conditions. Thus, it is quite important to under-

1. Introduction
Reversed−phase liquid chromatography (RPLC) has been re-stand the retention behavior in RPLC, because a good understand-garded as one of the most powerful separation techniques, and has

ing of the retention mechanism that dominated the retention of sol-been widely used for the separation and isolation of various com-utes still remains as one of the major objectives in separation sci-pounds. The separation in RPLC is based on the partition of soluteence [3,4].

molecule between mobile phase (typically a mixture of organic sol-

Pyrazine and its derivatives comprise a group of heterocyclic ni-

vent and water) and stationary phase (generally octadecyl func-

trogen−containing and very volatile compounds that are useful andtional groups bound to silica surface). The explanation of the reten-

important in many industries [5,6]. They have been widely used astion mechanism and the corresponding experiments for understand-drugs and food flavors, and one of the most important flavor com-

ing it have been carried out by many researchers for a long time,pounds responsible for the cocoa taste. Typical example ofsince RPLC was developed as a conventional separation techniquepyrazines is: pyrazine, 2,5−dimethylpyrazine and tetramethylpyra-[1,2]. However, the mechanism was not completely known due tozine in cocoa products as important flavor compounds [7,8]. In

the existence of many parameters controlling the separation process

pharmaceutical industry, they are found in natural medicines andCorresponding author: Yoshihiro Saito
Tel: +81−532−44−6803
Fax: +81−532−48−5833
E−mail address: saito@ens.tut.ac.jp―９７―CHROMATOGRAPHY, Vol.34 No.2 (2013)

have anti−inflammatory and anti−thrombotic effects [9−11]. Thus,
pyrazine and its derivatives are very important compounds in ourdaily life.
Based on the results in our previous investigations on the retention of pyrazines and its derivatives [12], a theoretical interpretation and the subsequent retention prediction were also studied,
where the retention prediction models for pyrazines were developed using multiple linear regression and artificial neural networks[13]. In these previous investigations, the retention of pyrazines
was systematically determined in various conditions, including avariety of mobile phase conditions and column temperatures. Theresults suggested the existence of abnormal temperature effect on
the retention of several alkylpyrazines on a typical octadecylsilica(ODS) stationary phase with a mobile phase containing acetonitrile(ACN) as the organic component in reversed−phase (RP) conditions [12,13].
In this work, the temperature effect on the retention of diazines,
consisted of pyrazines, pyridazines, pyrimidines and their derivatives, in RPLC was further studied at various column temperaturesin the range from 0 to 50°
C. For comparison the retention for pyrazole, imidazole and their methyl derivatives was also studied.
2. Experimental
2.1. Chemicals and Reagents
All regents, solvents and sample solutes were of analytical grade
and were used without further purification process. ACN, methanol (MeOH), uracil, 2−methylpyrazine, 2,3−dimethylpyrazine,
Figure 1. Chemical structures of analytes used in this study. (1)pyrazine; (2) 2−methylpyrazine; (3) 2,3−dimethylpyrazine; (4)2,3,5−trimethylpyrazine; (5) tetramethylpyrazine; (6) pyridazine;(7) 3−methylpyridazine; (8) pyrimidine; (9) 4−methylpyrimidine;(10) 4,6−dimethylpyrimidine; (11) pyrazole; (12) 3−methylpyrazole; (13) imidazole; and (14) 4−methylimidazole.

2,3,5−trimethylpyrazine, tetramethylpyrazine and 4,6−dimethylpyrimidine were purchased from Wako Pure Chemical Industries,
Ltd., Osaka, Japan, and pyrazines, pyridazine, 3−methylpyridazine,pyrimidine, 4−methypyrimidine, pyrazole, 3−methylpyrazole, imidazole, 4−methylimidazole were purchased from Tokyo ChemicalIndustry Co., Ltd., Tokyo, Japan. The chemical structures of compounds studied are shown in Figure 1. Water was purified by atadecylsilica stationary phases, ULTRON VX−ODS (150 mm×4.6Milli−Q Water Purification System (Millipore, Tokyo, Japan).

mm i.d., Shinwa Chemical Industries Ltd., Kyoto, Japan) was employed, where octylsilica and triacontylsilica phases (Develosil C−

2.2. RPLC Measurements
The RPLC system was consisted of a PU−880 pump, a UV/Vis−

8 and Develosil C−30, respectively; 250 mm×4.6 mm i.d.,

990 detector (Jasco, Tokyo, Japan) and a Model 7125 injectorNomura Chemical Co., Ltd., Seto, Japan) were also used for com-

(Rheodyne, Cotati, CA, USA) with 20 µL injection loop. Column

parison. As the mobile phase, mixtures of ACN/water and MeOH/temperature was controlled by a Model BF−61 Thermo Mate Bathwater were used with compositions from 30/70 (v/v) to 50/50 (v/v).

(Yamato Scientific Co., Ltd., Tokyo, Japan), while a home−madeThe mobile phase flowrate was set at 1.0 mL/min, and the column

water bath with a temperature−controlling system was also em-temperature was changed in the range between 0 to 50°
C.

ployed for the chromatographic measurements, if needed. For theStock solutions (1000 µg/L) of the standard analytes were pre-data collection and processing, Borwin Chromatography Data

pared with either water or MeOH as the sample solvent. By dilut-Processing Software (Jasco) running on a personal computer wasing these stock solutions with the mobile phase solvents, all theused.

working solutions of 20 µg/L concentration were prepared. As thedead−volume maker uracil was used, and at least five injections

Two types of commercially available columns packed with oc-―９８―

CHROMATOGRAPHY, Vol.34 No.2 (2013)

Chiharu Takemura, Kentaro Yogo, Ikuo Ueta, Kiyokatsu Jinno, Yoshihiro Saito

were carried out for the all the chromatographic measurements.

column temperature in over all the temperature range studied. ThisThe detection wavelength for pyrazoles and imidazoles having ais the same trend as normally found for most of the analytes, suchfive−membered ring was set at 254 nm, while for pyrazines, pyri-as polycyclic aromatic hydrocarbons (PAHs), on typical ODS

dazines and pyrimidines consisted of six−membered ring, it was set

phase in RPLC conditions [14−16]. However, as typically found inat 270 nm on the basis of preliminary experiments.

Figure 2 B, where ACN/water＝70/30 was used as the mobilephase, an increased retention factors for pyrazines was observed
when the column temperature was elevated. A similar trend was

3. Results and Discussion
Typical chromatograms for the separation of pyrazines on analso observed for all the ACN−based mobile phases studied. TheODS phase are shown in Figure 2. With a mobile phase consisted

results have a good agreement with previous reports [12,13], sug-of MeOH and water (having the ratio from 30/70 to 50/50), all thegesting an abnormal temperature effect on the retentions ofretention factors of pyrazines were decreased with increasing thepyrazines with ACN/water as the mobile phase.
Figure 3 shows the van’t Hoff plots of pyrazines in the above
cases with MeOH/water and ACN/water as the mobile phase,where logarithmic retention factor was plotted against the inverse
absolute column temperature. As can be found in Figure 3, logarithmic retention factors for pyrazines are increasing linearly withthe decrease of the column temperature using MeOH as the mobilephase component, showing a normal temperature effect found in
most of the column temperature studies in RPLC [14−16]. On theother hand, with ACN/water as the mobile phase (Figure 3 B), theretention factors are logarithmically increasing with the increase ofFigure 2. Typical chromatograms for the separation of pyrazines atdifferent column temperatures. Mobile phase: (A) MeOH/water＝30/70; (B) ACN/water＝30/70. As dead volume marker, uracil (U)was used. Other conditions: column, ULTRON VX−ODS (5 µm,
150 mm×4.6 mm i.d.); flowrate, 1.0 mL/min; detection, UV at270 nm.

Figure 3. van’t Hoff plots for pyrazines. Mobile phase: (A) MeOH/water＝30/70; (B) ACN/water＝30/70. Other conditions are thesame in Figure 2; and the assignments are the same as in Figure 1.

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CHROMATOGRAPHY, Vol.34 No.2 (2013)

the column temperature. During the above temperature study, noalong with the corresponding plots for pyrimidines in Figure 5.

hysteretic effect was observed for the comparison of retention fac-From these plots, an abnormal temperature effect of these analytes

tors measured when the column temperature was sequentially in-

could be observed with ACN as the organic component in the mo-creased and decreased to reach the target temperature.

bile phase. The results are quite consistent with that obtained for

In order to further investigate this abnormal temperature effectpyrazines. In contrast to above trend, the abnormal temperature ef-of pyrazines with ACN/water as the mobile phase, the separation offect was not observed for pyrazoles and imidazoles having a five−the same pyrazines mixture was carried out on other type of sta-membered ring in their molecular structure, as shown Figure 6. For

tionary phases, including octyl− and triacontyl−silica phases (De-pyrazoles and imidazoles, the retention was logarithmically de-velosil C−8 and Develosil C−30, respectively), at various column

creased with increasing the column temperature regardless the or-temperatures from 0 to 50°
C. The results clearly demonstrated that

ganic component in the mobile phase. The behaviors of this groupa similar trend was also observed for these stationary phases inof solutes have normal temperature dependence that was identicalterms of the abnormal temperature effect on the retentions ofwith the trend in the case using MeOH as the mobile phase compo-pyrazines with a mixture of ACN/water as the mobile phase. Thatnent.

means, for all the measurements with ACN/water as the mobile

By calculating the enthalpy of solute transfer from mobile phase

phase, the retention of pyrazines increased with elevated the col-to stationary phase (Table 1), it was confirmed the abnormal tem-umn temperature, suggesting an abnormal temperature effect on theperature dependence that usually associated with enthalpy−entropy

retention of these analytes with ACN as the organic solvent in thecompensation effects as reported previously [17−20]. As can bemobile phase.

seen in Table 1, all the analytes, except for pyridazines andThe same temperature studies were also carried out for pyri-pyrimidines, showed negative enthalpy of transfer from mobiledazines, pyrimidines, pyrazoles and imidazoles. van’t Hoff plots

phase to stationary phase with MeOH/water (30/70) as the mobilefor pyridazine and 3−methylpyridazine are illustrated in Figure 4,phase, while the values of enthalpy obtained with ACN/water (30/Figure 4. van’t Hoff plots for pyridazine and 3−methylpyridazine.
Mobile phase: (A) MeOH/water＝30/70; (B) ACN/water＝30/70.
Other conditions are the same in Figure 3.

Figure 5. van’t Hoff plots for pyrimidine, 4−methylpyrimidine and4,6−dimethylpyrimidine. Mobile phase: (A) MeOH/water＝30/70;(B) ACN/water＝30/70. Other conditions are the same in Figure 3.

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CHROMATOGRAPHY, Vol.34 No.2 (2013)

Chiharu Takemura, Kentaro Yogo, Ikuo Ueta, Kiyokatsu Jinno, Yoshihiro Saito

Table 1. Standard enthalpy (kJ mol−1) of transfer from the mobile
phase to the bonded phase.
Mobile phase
Analytes

pyrazine
2−methylpyrazine
2,3−dimethylpyrazine
2,3,5−trimethylpyrazine
tetramethylpyrazine
pyridazine
3−methylpyridazine
pyrimidine
4−methylpyrimidine
4,6−dimethylpyrimidine
pyrazole
3−methylpyrazole
imidazole
4−methylimidazole

MeOH/water

ACN/water

30/70

30/70

−5.33
−5.07
−5.99
−6.41
−7.40
−2.69
−4.24
−6.48
−5.93
−6.95
−9.42
−10.93
N/A
N/A

1.92
3.30
4.29
6.13
7.28
5.25
0.50
2.63
3.48
4.53
−4.49
−2.69
−15.99−11.06

N/A:not measured.

cal studies should be made to interpret the abnormal dependence of
these solutes retention on the column temperature with ACN/water
Figure 6. van’t Hoff plots for pyrazoles and imidazoles. Mobile
phase: ACN/water＝30/70. Analyte: (A) pyrazoles and (B) imidazoles. Other conditions are the same in Figure 3.

as the mobile phase solvent. However, the results suggest that70) are positive. At this stage, it can be said that the increased re-Acknowledgements

more efficient separation of diazines could be developed by tuning
up both the mobile phase and column temperature conditions.

tention of diazines (pyrazines, pyridazines and pyrimidines) at ele-

One of the authors, KY, acknowledges Mr. N. Ookawara, Mr. K.

vated column temperature might be induced by the abnormal ther-Sugiyama and all the members in the laboratory of food analysis

modynamic behavior during the partition between these stationary(Morinaga & Co., Ltd.) for their valuable help and suggestions.

phase and the mobile phase containing ACN at a certain content.
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