BIOMEDICAL CHROMATOGRAPHY, VOL. 9, 135-139 (1995)

Effect of Column Temperature and Nitrogen Flow Rate on the Separation of cis-Permethrinic Acid Isomers on a (2,3,6-Tri-O-Methyl)- P-C yclodextrin Coated Capillary Column

Karoly Payer, Esther Forgacs* and Tibor Cserhati Central Research Institute for Chemistry, Hungarian Academy of Sciences, H-1525 Budapest, P.0.B.17, Hungary

The separation efficiency of a capillary column coated with (2,3,6-tri-O-methyI)-~-cyclodextrin was determined at the temperature and flow rate ranges of 80-120 "C and 0.3-1.5 mL/min respectively using (+) and (-) cis-permethrinic acid methylesters as test compounds. The dependence of the retention differences, sum of peak widths and separation factor on the temperature and flow rate was calculated with step regression analysis. The column exhibited acceptble isomer separation capacity at each temperature and flow rate proving the excellent separation power of the cyclodextrin coating. The differences between the retention times of isomers depended on the logarithm and on the reciprocal value of temperature, the effect of flow rate was negligible. The sum of their peak widths depended reciprocally on the temperature and logarithmically on the flow rate, the impact of temperature being higher than that of flow rate. The significant impact of the reciprocal value of temperature can be explained by the inclusion complex formation between cyclodextrin and cis-permethrinic acid methylester isomers. The resolution depended on the square of column temperature and flow rate, indicating local optimums at each temperature and flow rate.

INTRODUCTION

In recent decades cyclodextrins have gained a growing acceptance and application in many fields of chroma- tography (Szejtli et al., 1987). Owing to the inclusion formation between the solute and cyclodextrin (Szejtli, 1982) the latter can modify the retention behavior of the solute, resulting sometimes in improved separation (Vigh et al., 1989a,b). Recently, cyclodextrins has been used in reversed-phase thin-layer chromatography (Cserhhti et al., 1989) to determine the relative inclu- sion complex stability of some barbituric acid deriva- tives. They have also been used in high performance liquid chromatography to find correlations between inclusion complex stability and retention time of di- substituted aromatic compounds (Arnold et al., 1989). Cyclodextrin derivatives have been successfully applied also as stationary phases in micropacked column (Smolkovii-Keulemansovii et al., 1988) and capillary gas chromatography (GC) . The per-n-pentylated Q-

cyclodextrin and P-cyclodextrin pentylated at the hy- droxy groups in positions 2 and 6 and acetylated in position 3 exhibited a marked selectivity towards enan- tiomers (Konig et al., 1988a, 1989). They can be used without degradation up to above 200 "C (Konig et al., 198%). Permethylated /3-cyclodextrin coatings have also been applied to separate isomers in capillary G C (Juvancz et al., 1987, 1988; Venema and Toisma, 1989; Novotny et al., 1989). The studies in GC dealing with the use of cyclodextrin stationary phases have been generally limited to the exploration of their separation capacity and did not investigate the underlying separ- ation and retention mechanism in detail.

* Author to whom correspondence should be addresscd

The aim of our study was to separate the (+) and (-) isomers of cis-permethrinic acid on a capillary column coated with a cyclodcxtrin stationary phase under vari- ous experimental conditions and to calculate the depen- dence of retention and separation efficiency vs. temper- ature and flow rate. The Monte-Carlo simulation has been very recently applied to predict retention time and peak half widths in GC (Schneider and Zinn, 1989), however, the method requires the knowledge of some physicochemical parameters of the compounds to be separated which are generally not available.

EXPERIMENTAL

A Perkin Elmer (Bodenseewerk, Germany) gas chromato- graph (Model F22) equipped with a flame ionization detector was interfaced to a Perkin Elmer Sigma 10 integrator. The column was a 13.7 m X 0.2s mm i.d. glass capillary column coated with (2,3,6-tri-O-methyl-/3-cyclodextrin. Nitrogen was used both as carrier and make-up gas (15 mlirnin). Both injector and detector temperatures were 200 "C, the split ratio was 1 : 100. The (+) and (-) cis-permethrinic acids(3 - (2,2 - dichloroethenyl) - 2,2 - dirncthylcyclo-propanecarboxy- acid) were synthesizcd at the 'Chinoin' Chemical and Pharmaceutical Works, Budapest, Hungary. This compound served as a basic material for the synthesis of some new pesticides. The (+) and (-) cis-permethrinic acids were scparately methylated with diazoinethane as previously de- scribed (Kiinig et al . , 1986). The methylated derivatives were separately dissolved in diethylether at a concentration of 3mg/mL. A mixed solution was also prepared containing 3 : 3 mg/mL of each methylester in diethylether. The amount injected was 0.1 UL in each case. The carrier flow rate was varied between 0.3 and 1.5 niL/min in steps of 0.3 mL/min;

CCC 0269-3879/95/030135-05 0 1995 by John Wiley & Sons, Ltd.

Ruceiiied 20 December 1994 Accepted I 1 January 19%

136 K. PAYER E T A L

A B

, . min

C D

23.06 24.24

67.6 ,

i- min

Figure 1. Separation of cis-perrnethrinic acid methylester isomers on cyclodextrin stationary stationary phase. A, 120 "C, flow rate 1.5 mllmin; B, 120 "C, flow rate 0.3 mL/min; C, 80 "C, flow rate 1.5 mL/min; D, 80 "C, flow rate 0.3 mL/min.

the column temperature was changed between 80 and 120 "C in steps of 10 "C. The solutions of the individual cis- permethrinic acid inethylesters and also the mixtures were injected three times at each temperature and llow rate pair. The retention time ( t ) was accurately measured by the inte-

grator, the peak half width (W) was graphically determined from the chromatogram. Since separation efficiency depends on the differences in retention times (tl-* = t , - t2) and on the sum of the peak half widths (ZW= W,+ Wz) these two parameters were also calculated. The resolution (Rb) was

t

Figure 2. Effect of column temperature and nitrogen flow rate on the retention difference between cis-permethrinic acid methylester isomers. Temperature. 80-120 "C; flow rate, 0.3-1.5 mLlmin.

SEPARATION OF CIS-PERMETHRINIC ACID ISOMERS 137

Table 1. Dependence of the difference of the retention between cis-permethrinic acid methylester isomers ( y = t,-Z) on the column temperature (x, ) and flow rate (x2). Results of stepwise regression analysis.

No. of independent variables Parameter 1 2 3

b 2809 53.3 - 1.89 Sb 822 19.6 0.34 Path coefficient (%) 52.72 42.05 5.23 y = a + b,. l/x, + b2.10g xi +&.log x, n = 24 a = - 134.3 F,,, = 56.30 ? = 0.8941.

calculated from the parameters listed above for each column temperature-flow rate pair.

As it has never been proved that the temperature depen- dence of retention is the same on cyclodextrin as on tradi- tional stationary phases (Kaiser and Rackstraw. 1983), the temperature dependence of the t,, value was calculated with the help of a stepwise regression analysis (Mager, 1982): t,_> values were taken as dependent variables, the linear, logar- ithmic, quadratic and reciprocal values of flow rate and column temperature (altogether 8 variables). The acceptance level of the single independent variable was set to 95% and the number of accepted variables was not limited. In the common multivariate regression analysis the presence of independent variables exerting no significant influence on the dependent variable lessens the significance level of the inde- pendent variables, significantly influencing the dependent variable. To overcome this difficulty the stepwise regression analysis automatically eliminates from the selected equation the insignificant independent variables, thus increasing the information potential of the calculation. The dependence of the sum of peak width and that of the resolution on the column temperature and flow rate were calculated in the same manner.

t

"C

RESULTS AND DISCUSSION

The reproducibility of the determinations of the reten- tion time and peak half width was good, the coefficient of variation never exceeded 0.5% and 17% for reten- tion time and peak half width respectively. The coef- ficient of variation did not depend on the column temperature or on the flow rate.

Some of our results are shown in Fig. 1. Within the whole range of chromatographic conditions the (+) form eluted first, the inversion of retention order was never observed. Acceptable separation was achieved in each case proving the excellent separation power of the cyclodextrin stationary phase. The retention difference decreases with increasing temperature and flow rate (Fig. 2), however, the dependence differs slightly from the linear.

The parameters of the equation describing the dependence of the retention time difference on the column temperature and flow rate are compiled in Table 1. The equation fits well to the experimental data, the significance level was over 99.9% (see F,,,, value). The change in the selected independent vari- ables explains about 90% of the change in retention time difference (dependent variable: see r z value). Against expectation the retention time difference depends not only logarithmically on the column tem- perature, the reciprocal component has approximately the same impact on the difference (see path coefficient values). This finding indicates that in case of cyclodex- trin stationary phases the temperature dependence of retention may deviate from that of traditional columns. This phenomenon can be tentatively explained by the assumption that the temperature dependence of the

Figure 3. Effect of column temperature and nitrogen flow rate on the sum of peak widths of cis-permethrinic acid methylester isomers. Temperature, 80-120 "C; flow rate, 0.3-1.5 mL/ min.

138 K. PAYER E T A L

~~ ~

Table2. Dependence of the sum of peak half widths ( y = X W ) of cispermethrinic acid methylester isomers on the column temperature (x,) and flow rate (q). Results of stepwise regression analysis

Parameter

b

Path coefficient (YO) sb

No. of independent variable 1 2

8781 -39.22 769 5.23

60.35 39.65 y = a + b , llx,+b2*log x, n = 24 a = -73.8 F,,,, = 88.05 ? = 0.8935

Table3. Dependence of the separation factor ( y = R , ) of cis- permethrinic acid methylester isomers on the column temperature (x,) and flow rate (xz). Results of step- wise regression analysis.

No of independent variable Parameter 1 2

b -7.53 - 10-5 -9.28. sb 5.40. 10 2.03. 10 Path coefficient (%) 75.29 24.7 1 y = a + b,(xl )' + b , ~ ( x ~ ) ~ n = 24 a = 1.55 F,,,, = 101.97 ? = 0.9066

cyclodextrin inclusion complex stability may contribute with an additive (according to our calculation : recipro- cal) factor to the logarithmic temperature dependence of the retention. The reciprocal dependence may be due to the weaker complex formation at higher temper- atures. The impact of flow rate to the retention differ- ence is relatively low.

The three dimensional plot of the sum of peak widths (ZW) vs. temperature and flow rate (Fig. 3 ) is rather similar to Fig. 2: the peak widths increase as expected with the lower column temperature and lower flow rate. The parameters of the equation describing the dependence of the sum of peak half widths on column temperature and flow rate are compiled in Table 2. The equation fits well with the experimental data, the sig- nificance level was over 99.9% (see FCalc value). The change in the selected independent variables explains about 90% of the change in the sum of peak widths (dependent variable: see r 2 value). In this case the column temperature also has a greater effect on the sum of peak widths than has the flow rate (see path

"C

coefficient values). The three dimensional plot of the separation factor ( R , ) vs temperature and flow rate is presented in Fig. 4. It is clear from the plot that the flow rate has a markedly lower influence on resolution than the column temperature.

The parameters of equation describing the depen- dence of the separation factor on column temperature and flow rate are compiled in Table 3 . The equation fits well to the experimental data, the significance level was over 99.9% (see FcdIc value). The change of the selected independent variables explains about 90% of the change of the separation factor (dependent variable: see r 2 value). In this case also the column temperature has a greater effect than the flow rate (see path coef- ficient values). However, the character of the equation is different from those in Tables 1 and 2. The quadratic dependence of the separation factor on the column temperature and flow rate indicates that an optimum separation temperature and flow rate can be found at each temperature and at each flow rate. These local opt imum can be easily calculated from the equation,

1 m[ /min

Figure4. Effect of column tempemture and nitrogen flow rate on the separation of cis- permethrinic acid methylester isomers. Temperature 80-120 "C; flow rate 0.3-1.5 mllmin.

SEPARATION OF CIS-PERMETHRINIC ACJD ISOMERS 139

since the equation has a maximum value when its first derivative is cqual to zero.

It can be concluded that the capillary glass column coated with permethylated beta cyclodextrin exhibits a high separation efficiency at wide temperature and flow rate ranges. The equations describing the dependence of the retention differences between isomers, the sum of peak widths and the separation factor on the temper- ature and flow rate fit well to the experimental data.

The equations may facilitate the search for optimum separation conditions in a wide range of chromato- graphic conditions.

Acknowledgement

This work was performcd with the financial support of OTKA 2670 grant of the Hungarian Academy of Sciences.

REFERENCES

Arnold, E. N., Lillie, T. S. and Beesley, T. E. (1989). J. Liq. Chromatogr. 12, 337.

Cserhati, T., Szejtli, J. and Bojarski, J. (1989). Chrornatographia 28, 455.

Juvancz, 2.. Alexander, G. and Szejtli, J. (1987). J. High. Res. Chromatogr. Chrornatogr. Commun. 10, 105.

Juvancz, Z., Alexander, G. and Szejtli, J. (1988). J. High. Res. Chromatogr. Chromatogr. Commun. 11, 110.

Kaiser, R. E. and Rackstraw, A. J. (1983). Computer Chromatography, Vol. 1, p. 62. Dr. Alfred Hijthig Verlag, Heidelberg, Basel, New York.

Konig, W. A,, Gyllenhaal, 0. and Vessman, J. (1986). J. Chromatogr. 356, 354.

Konig, W. A,, Lutz, S. and Wenz, G . (1988a). Angew. Chem. lnt. Ed. Engl. 27, 979.

Konig, W. A,, Lutz, S., Mischnik-Lubecke, P., van der Bey, E. and Wenz, G. (1988b). StarchlSfarke 40, 472.

Konig, W. A., Lutz, S., Hagen, M., Krebber, R., Wenz, G.. Baldenius, K., Ehlers, J. and Dieck, H. (1989). J. High. Res. Chromatogr. Chromatogr. Commun. 12, 35.

Mager. H. (1982). Moderne Regressionsanalyse, p. 135. Salle, Sauerlander, Frankfurt am Main, Germany.

Novotny, H. P., Schrnalzing. D., Wistuba, D. and Schurig, V. (1989). J. High Res. Chromatogr. Chromatogr. Cornmun. 12, 383.

Schneider, M. W. and Zinn, P. (1989). Chromatographia 28,241. Srnolkova-Keulemansova, E., Pokorna, S., Feltl, L. and Tesarik,

K. (1988). J. High Res. Chrornarogr. Chromatogr. Comrnun. 11, 670.

Szejtli, J. (1 982). Cyclodextrins and Their lnclusion Complexes. Akademiai Kiado, Budapest, Hungary.

Szejtli, J., Zsadon, B. and Cserhati, T. (1987). Ordered Media in Chemical Separation (Hinze, W. L. and Arrnstrong, D. W., eds.). Am. Chern. SOC. Symp. Ser. 342. Washington DC.

Venema, A. and Toisrna, P. J. A. (1989). J. High Res. Chrornatogr. Chromatogr. Commun. 12, 32.

Vigh, G., Quintero, G . and Farkas, G. (1989a). J. Chromatogr. 484, 237.

Vigh, G., Quintero, G. and Farkas, G. (1989b). J. Chrornatogr. 484, 251.