Anal. Chem. 1989, 6 1 , 1149-1151
1149
Effect of Column Temperature on the Direct Determination of (RS)-a-Cyano(3-phenoxypheny1)methyl (RS)-2-(4-Chlorophenyl)-3-methylbutyrate Optical Isomers by High-Performance Liquid Chromatography/Diode Array System Euphemia Papadopoulou-Mourkidou Agricultural Chemistry Laboratory, Aristotelian University, P.O. Box 19678, Thessaloniki, Greece
The effect of column temperature on the dlred analysls of the optical Isomers of fenvalerate, (RS )-a-cyano( 3-phenoxypheny1)methyl (RS )-24 4-chlorophenyl)-3methylbutyrate, was lnvestlgated by using a high-performance liquid chromaiography (HPLC)/dlode array system wlth a chiral column containing ( R )-N-( 3,5-dlnRrobenroyl)phenylglyclnellquld phase eluted wlth a moblle phase conslstlng of 0.2% 2-propanol In hexane. The retentlon times of the separated Isomers SR, RS, SS, and RR were substantially decreased as the column temperature was Increased from 2 to 35 OC. I n general the separatlon factors between enantiomers (RS/SR and R R / SS ) decreased and between diastereomers (SS/RS ) remained unchanged as the column temperature was increased. The resolution between all the pairs of adjacent peaks was best when the column was thermostated at 15 OC. Plots of In V , versus 1/T were not llnear throughout the entlre temperature range examined (2-35 "C) bearing a distlnct break at 10 OC Indicating thus that there were two phenomena governlng the chromatographlc behavlor of fenvalerate Isomers for the column temeprature regimes of 2-10 and 10-35 OC, respectlvely.
INTRODUCTION Fenvalerate is a pyrethroid insecticide having two asymmetric carbon atoms in its molecule, and the analytical as well as the technical grade materials are mixtures of four isomers. The isomer with the SS configuration is the most active insecticide. The direct separation of fenvalerate optical isomers by high-performance liquid chromatography (HPLC) has been already reported (1-4). In all cases chiral columns containing (R)-N-(3,5-dinitrobenzoyl)phenylglycineeluted with mixtures of 2-propanol in hexane ( I , 2 , 4 ) or hexane-dichloroethaneethanol ( 3 ) were used. The role of the column temperature on the retention of fenvalerate isomers analyzed by HPLC on chiral columns has never been studied before even though temperature was recognized as a factor in improving the resolution between pairs of isomers of pyrethroids (4). However, during the last decade numerous studies have been published concerning the effect of temperature on the retention and general chromatographic behavior of different groups of compounds analyzed by HPLC. Few examples are cited (5-10). Recently, the effect of temperature has been recognized also as an important parameter in improving and optimizing the resolution between enantiomeric pairs of isomers analyzed by HPLC on chiral columns (11,12). Therefore, the effect of column temperature on the chromatographic behavior of fenvalerate enantiomers was undertaken. EXPERIMENTAL SECTION Apparatus. The HPLC system consisted of a Waters Asso-
ciates Model 6000A solvent delivery system, a Rheodyne Model 0003-2700/89/0361-1149$01.50/0
2120 valve type injector equipped with a 10-pL loop, and a photodiode-array detector, Model 2140 (LKB, Sweden),equipped with a 5-pL cell. The detector was operated in the range of 19C-370 nm and was connected to an Olivetti (Italy) Model M24 personal computer operated with LKB software (Wavescan program). The data recorded by the computer were fed to a Cannon (Japan) Model PJ-1080A color printer. The Bakerbond (J.T. Baker Co.) covalent chiral cholumn (250 X 4.6 mm i.d.) containing (R)-N-(3,5-dinitrobenzoyl)phenylglycine covalently bound to aminopropyl5-pm silica gel was maintained at a certain temperature by use of a home-made thermostated compartment. The column was inserted into a dorm-size refrigerator controlled by a Crison (Spain) Model 242 electronic thermometer. Also a light bulb inserted into the refrigerator and connected to the thermometer helped to attain and maintain the desired temperature. The thermometer temperature probe was tied to the column while a small blower inserted into the refrigerator helped in the faster air mix. With this column compartment temperatures in the range of 2-35 "C (10.15 "C) can be attained. Reagents. All solvents used were HPLC grade (J. T. Baker Co.). Fenvalerate analytical standard material (99% purity) was purchased from Bromchem (West Germany). Solutions of fenvalerate standard were made in hexane. Procedure. The HPLC system was operated in an air-conditioned room with the room temperature ranging from 20 to 22 "C during the period that this experiment was conducted. The mobile phase consisted of 2-propanol-hexane (0.2/99.8 (v/v)). A stock of 20 L of mobile phase, adequate to run the whole experiment once, was made in a 50-L sealed glass jar and 1-L portions were taken daily which were filtered through 0.45-pm pore filter (Millipore) and degassed under vacuum before use. The mobile phase flow rate was monitored periodically to maintain a constant flow at 1mL/min, and additional degassing of the same patch of mobile phase was made when necessary, usually after 5-6 h of operation of the HPLC system. This operation of the mobile phase handling (filteringand degassing) was repeated day after day starting always each day with a fresh batch of 1 L of mobile phase. The column was conditioned at a certain temperature setting (2, 5, 10, 15, 20, 25, 30, and 35 i 0.15 "C) without a mobile phase flow for at least 24 h. At each temperature setting the system was equilibrated for 8 h by using a fresh batch of mobile phase. The equilibration process was considered complete when straight base lines were obtained in the chromatograms recorded at 190 nm. The following day, the system operated again with a new batch of mobile phase, three to five injections of 10 pL of standard fenvalerate solutions in hexane (0.5-0.1mg/mL) were made, and the retention times of the resolved isomers were recorded. The column dead volume was measured at each column temperature setting by injecting 5 pL of benzene.
RESULTS AND DISCUSSION The retention times of fenvalerate isomers separated under the chromatographic conditions described in the Experimental Section are given in Table I. There is a considerable decrease of retention times as the column temperature is increased from 2 to 35 "C. Also the shapes and peak widths of the eluted peaks are substantially changed as the column temperature is changed. The highly broad and tailed peaks eluted a t 0 1989 American Chemical Society
*
1150
ANALYTICAL CHEMISTRY. VOL. 61. NO. 10, MAY 15, 1989
I) n
a
1
280,
235
1w 1000
1600
1.00
1800
2000
2200
2400
SCC.
Flgure 1. Sample isograms from the analysis of 5 fig of fenvalerate analyzed under the chromatographic conditions described in t b Experimental
Section. Table 1. Retention Times of Fenvalerate Isomers temp,
SR
O C
2 5
10 15 20 25 30 35
*
1777.3 22.7 1599.5 i 10.1 1334.3 20.5 1305.0 15.2 1245.7 4.2 1208.0 12.8 1203.0 4.0 1188.0 f 3.0
** ** *
retention times: B RS ss 1957.7 12.1 1769.0 8.2 1463.7 24.1 1424.6 18.1 1353.0 i 3.5 1308.5 i 13.5 1295.7 i 5.0 1272.0 3.0
* **
*
2183.0 i 22.0 1966.5 10.9 1614.3 f 26.0 1578.6 19.2 1500.3 i 5.0 1450.5 i 15.8 1439.0 f 4.0 1417.0 i 4.0
*
*
RR 2313.0 f 16.0 2081.5 i 12.8 1698.3 f 26.0 1659.0 f 21.8 1583.0 12.2 1512.3 f 14.5 1505.0 f 6.0 1482.0 i 5.0
*
'These are mean and standard deviation values calculated from respective retention time values recorded from three to five consecutive iniertinna.
w t 0 1.04
1.021
column temperatures of 2 or 5 " C are gradually transformed into sharp and symmetric peaks a t higher column temperatures. In Figure 1are presented two sample isograms recorded at column temperatures of 30 and 2 "C, respectively, showing the effect of the column temperatures on the solute retention times, peak shapes, and the resolution between adjacent peaks. The four isomers of fenvalerate are eluted in the order of SR, RS, SS, and RR (3). The effect of column temperature on the separation factor and the resolution between adjacent eluting peaks are demonstrated by the plots presented in the Figures 2 and 3. The separation factor (a) between two adjacent peaks was calculated from the formula a = k B / k A where k A and ks are the capacity factors of two adjacent peaks, respectively, calculated from the formula k = ( t - to)/to,where t is the retention time of an isomer and to is the retention time of benzene equal to 221 s. The resolution (RJ between two adjacent peaks was calculated from the formula R, = 2(tB- tA)/(wA+ WB)where (tB- tA)is the retention time difference between two adjacent peaks and W A and WB are the respective base-line peak widths. Both the separation and resolution values were calculated from the chromatograms recorded a t 200 nm from injections made a t each column temperature setting. The effect of the column temperature on the solute separation factors is demonstrated by the plots presented in the Figwe 2. The separation factors between enantiomers (RRISS and RSISR) are decreased as the column temperature is increased from 2 to 35 "C while the separation factor between the diastereomers (SSIRS) is not affected by the column temperature change except for the 10 and 35 OC temperature settings where a slight decrease and increase, respectively, are noticed. The effect of column temperature on the resolution between isomers is shown in Figure 3. The resolution between enantiomers is increased as the column temperature is increased from 2 to 15 "C and then is slowly decreased as the
'
0
10 20 30 40 COLUMN TEMPERATURE(eCl
Flgure 2. Effect of column temperature on the separation factors between adjacently eluting isomers of fenvalerate. 3.L
L
0.6 0
IO 20 30 40 COLUMN TEMPERATURE ( * C l
Flgure 3. Effectof column temperature on the resolution between adjacently eluting isomers of fenvalerate. column temperature is further increased from 15 to 35 "C. The resolution between the diastereomers RS and SS is increased with any column temperature increase in the range 2-35 oc. Plots of In VN versus 1/T where VN is the net retention volume of an isomer and Tis the absolute column temperatwe are not linear throughout the entire temperature range examined. For all four isomers, these plots are linear for the temperature range from 2 to 10 OC with correlation coefficients
ANALYTICAL CHEMISTRY, VOL. 61, NO. 10, MAY 15, 1989 10.2,,
10.3
,
I
-
T a b l e 11. T h e r m o d y n a m i c F u n c t i o n s F o u r I s o m e r s of F e n v a l e r a t e
-AH f SE," K J mol-'
10.4 -
10.5
-
-
10.6
-
>
10.7 -
z
t
,
c
10.8
a
A
109 -
11.0-
IO'K/T
Figure 4. In V , versus 1 / T plots of fenvalerate isomers analyzed under the chromatographic condltions described in the Experimental Section.
being higher than 0.99 and from 10 to 35 "C with correlation coefficients being higher than 0.97. The In VN versus 1 / T plots are presented in Figure 4. All the lines are distinctly broken at 10 OC and their respective sections are parallel to each other. Nonlinearity of Van't Hoff plots has been reported recently by Hirukawa et al. (13)for a series of alkane, alkylbenzenes, and polyaromatic hydrocarbons analyzed by a reversed-phase liquid chromatographic system and by Jansson and Johansson (14) for a series of phenoxypropylamines analyzed by ion-pair chromatography. From the In V , versus 1 / T plots the thermodynamic functions AH (enthalpy) and A S (relative entropy) of fenvalerate isomers retention can be calculated based on the equation In VN = In n, + AS/R - AH/RT, where VN is the solute net retention volume, AS is the relative entropy change, AH is the enthalpy change, R is the gas constant, T is the absolute temperature, and In n, is a negligible factor (15). The estimated thermodynamic functions AH and AS for the four isomers of fenvalerate are given in Table 11. For all four isomers both AH and A S are decreased as the column temperature is changed from 2-10 "C to the 10-35 OC regime. The differences of AH and A S between isomers and for both column temperature regimes are not significant and all lie within the fiducial limits of one standard error. However, the data presented in Table I1 may be useful in understanding the mechanism of retention of fenvalerate isomers under the chromatographic conditions examined. It appears that the retention of fenvalerate isomers is an exothermic process favored by the column temperature decrease. This is in agreement with the data presented in Figures 2 and 3 where it is clearly shown that under subambient (15 "C) column temperature optimum resolution between all adja-
isomers
2-10 " C
10-35
SR RS SS RR
4.3 f 0.6 5.1 f 0.5 4.7 f 0.5
27.4 f 0.7 27.4 f 1.3 28.1 f 1.3 28.6 f 1.5
4.9 f 0.6
O C
1151
AH and A S for t h e -AS f SE," J K-' mol-' 2-10 "C 10-35 " C
106 f 2 108 f 2 105 f 2 106 f 2
187 f 2 186 f 5 188 f 5 189 f 5
SE denotes standard error.
cently eluting isomers can be obtained. It seems that there are two phenomena determining the chromatographic behavior of fenvalerate isomers under the examined conditions. At subambient temperatures (2-10 "C) an "adsorption" physical in nature seems to predominate, while at higher temperatures (10-35 "C) a less exothermic process seems more probable. A mixed retention mechanism was also suggested by Jansson and Johansson (14) for phenoxypropylamines chromatographed on LiChrosorb Si 6 0 eluted with phosphate buffer (pH = 2.1) containing 0.001 M N,N,N-trimethyloctylammonium bromide and 0.001 M octyl sulfate.
ACKNOWLEDGMENT The valuable comments and suggestions by Dr. N. A. Katsanos, Professor of Physical Chemistry, University of Patras, Greece, are greatly appreciated. Registry No. Fenvalerate, 51630-58-1; (SR)-fenvalerate, 67614-32-8; (RQfenvalerate, 66267-77-4; (SS)-fenvalerate, 66230-04-4; (RR)-fenvalerate, 67614-33-9. LITERATURE CITED (1) Chapman, R. A. J. Chromatogr. 1983, 258, 175-182. (2) Papadopoulou-Mourkidou, E. Chromatographia 1985, 20,376-378. (3) Lee, P. W.; Powell, W. R.; Stearns, S. M.; McConnell, 0. J. J . Agric. Food Chem. 1987, 3 5 , 384-387. (4) Cayley, G. R.; Simpson, 8. W. J . Chromatogr. 1986, 356, 123-129. (5) Diaslo, R. 6.; Wilburn, M. E. J . Chmmatogr. Sci. 1979, 17. 565-567. (6) Giipin, R. K.; Sisco, W. R. J . Chromatogr. 1980, 194, 285-295. (7) Issaq,H. J.; Fox, S.D.; Lindsey, K.; McConnell, J. H.; Welss, D. E. J . Liq. Chromatogr. 1987, 10, 49-70. (8) Jinno, K.; Nagoshi, T.; Tanaka, N.; Okamoto, M.; Fetzer, J. C.; Biggs, W. R. J . Chromatogr. 1988, 436, 1-10, (9) Martin, J.; Mendez, R.: Negro, A. J . L i 9 . Chromatogr. 1988, 1 1 ,
1707-1716. (10) Tchapla, A.; Heron, S.;Colin, H.; Guiochon, G. Anal. Chem. 1988, 6 0 , 1443-1448. (11) Mazzo, D. J.; Lindemann, C. J.; Brenner, G. S.Anal. Chem. 1988, 58, 636-638. (12) Akanya, J. N.; Taylor, D. R. Chromatographia 1988, 25, 639-642. (13) Hirukawa, M.; Arai, Y.; Hanal, T. J. Chromatogr. 1987, 395, 481-48?. (14) Jansson, S O . ; Johansson, M.-L. J . Chromatogr. 1987, 3 9 5 , 495-501. (15) Katsanos, N. A.; Lycourghiotis, A,; Tsiatsios, A. J.Chem. SOC.,Faraday Trans. 1 1978, 7 4 , 575-578.
RECEIVED for review August 5, 1987. Resubmitted January 17, 1989. Accepted January 27, 1989. This work has been partially supported by a grant from the Ministry of Agriculture, Division of Animal Production, Greece.
