Experimental Studies on a New Polar Bonded Liquid Chromatography Phase Eli Grushka and Edward J. Kikta, Jr. Department of Chemistry, State University of New York at Buffalo, Buffalo, N. Y. 742 74
A study of the retention and zone broadening In a liquid chromatographic system composed of bonded stationary phase was carried out. The bonded phase used here, I-trimethoxysilyi-2-chioromethylphenylethane, can be both In the form of “brushes” and polymeric patches. The retention of the solutes on the bonded system was much lower than on pure Corasil (the support). Thus, surface modification is most likely the main contributor to retention, although other contributors might exist. Peak broadening seems to be a strong function of k‘, indicating mass transfer in stagnant mobile phase pockets. Temperature studies Indicate that AH of transition from the stationary to the mobile phase most likely determines the retentlon on the bonded phase. The plate height and peak asymmetry were functions of the temperature; both usually decrease with increasing T. 9Fluorenone was an exception to that observation.
The use of permanently bonded phases in modern high speed liquid chromatography is fast becoming one of the most important developments in separation science. Recent advances in this area were reviewed by Locke ( 1 ) .In general, there are two kinds of bonded phases: the so-called “Brush system” and the polymeric phase. In the former, first popularized in chromatography by Halasz (2), the bonded phase is essentially a monolayer of stationary phase molecules each anchored to the support. In the second case, the bonded phase is cross-polymerized on the support. It is no longer a monomolecular layer per se, although the polymeric phase can be rather thin. Some of the early work on polymeric phases in LC was discussed by Kirkland and DeStefano ( 3 )and Kirkland ( 4 ) . Since then, many other investigators have used various types of polymeric bonded phases [e.g. ( 5 )and references therein]. From the work published, however, it is clear that the retention mechanisms are not always understood. Little et al. (6) show that, used in GC, bonded high molecular weight Carbowaxes are relatively more polar than low molecular’ weight Carbowaxes. The opposite trend is found when these phases are coated conventionally. In addition, they found that n-octane bonded to Porasil behaves as a more polar system than bonded Carbowax 400 or bonded oxypropionitrile (OPN), and concluded that with bonded noctane residual Si-OH groups contribute to the retention. This interpretation can be studied and verified by changing the amount of the bonded phase or by modifying the unreacted support surface. Recently, Karger and Sibley (7) studied the characteristics of “brush” bonded phases in GC. They varied the activity of the underlying Si-OH groups of the support by silanization, and varied the degree
and type of the bonded phases, and found that for nonpolar bonded phases the degree of surface coverage and the type of bonded phase played a part in determining the retention. Horgan and Little (8) found that bonded systems generally were more efficient for the same solutes and the same capacity ratio than conventionally coated supports. Kirkland ( 4 ) found that with polymeric bonded phases, the factors involved in the separations are adsorption on the polymer phase and, in some cases, partitioning between the mobile phase and trapped pockets of the mobile phase (stagnant pockets of the mobile phase). The latter was particularly evident when a nonpolar mobile phase was modified with a polar substance. More recently, Knox and Vasvari (9) studied in detail the behavior of Permaphase E T H and ODS (polymeric bonded phases). With ETH, the mass transfer of the solute is slow, while asymmetric peaks result when using the ODS phase. Temperature studies also indicated differences between the two polymeric phases. The overall picture as to the role of the bonded phase in LC i still somewhat unclear, especially in the case of the “brush” type phases. To our knowledge, with the exception of Knox and Vasvari’s (9) paper, no other systematic study was carried out to examine specifically the effect of bonded phases on solute retention and on column efficiency. This communication describes experiments designed to investigate the effects of a different type of bonded phase. The permanently bonded stationary phase used in this study was obtained by reacting Corasil I1 with l-trimethoxysilyl-2-chloromethylphenylethane, [(CHSO)Si(CH~)~-CGH&H~C This ~ ] . compound has been used by Parr and Grohmann (10) as the initial bonded group which provided the chlorine for the attachment of amino acids in solid phase peptide synthesis. Grushka and Scott ( 1 1 ) have used this group for synthesizing the initial link when using permanently bonded peptides as stationary phases in liquid chromatography. Novotny et al. (12, 13) have used similar compounds for preparing polar bonded stationary phases, but they replaced the chloride with other functional groups before making any studies. In addition, they polymerized the bonded phase on the surface. Although they did point out briefly some mechanistic features of such polar bonded phases, they were more interested in showing the potential of the phases. Since this silane compound binds easily to silica gel, we too thought that it might have use in liquid chromatography. Comparisons of retention on the bonded phase with retention on a pure Corasil I1 column were made. Surface coverage was varied. Solutes were varied in a systematic way so that variations in size and steric configuration could shed insight on the mechanism of
D. C. Locke. J. Chromatogr. Sci., 11, 120(1973). I. Halasz and I. Sebastian, Agnew. Chem., int. Ed. Engi., 8,453 (1969). J. J. Kirkland and J. J. DeStefano, J. Chromatogr. Sci., 8,309 (1970). J. J. Kirkland, J. Chromatogr. Sci., 9, 206 (1971). (5) W. A. Aue, S. Kaplai, and C. R . Hastings, J. Chromatogr., 73, 99 (1972). (6) J. N. Little, W. A. Dark, P. W. Farlinger. and K. J. Bombaugh, J. Chromatogr. Sci., 8,647 (1970). (7) B. L. Karger and G. Sibley, Anal. Chem., 45, 740 (1973).
D. F. Horgan and J. N. Little, J. Chromatogr. Sci., I O , 76 (1972). J. H. Knox and G. Vasvari, J. Chromatogr., 83, 181 (1973). W.Parr and K. Grohmann, Tetrahedron Lett., 28, 2633 (1971). E. Grushka and R. P. W. Scott, Anal. Chem., 45, 1626 (1973). M. Novotny, S . L. Bektesh, K. B. Denson, K. Grohmann, and W. Prr, Anal. Chem., 45, 971 (1973). (13) M. Novotny. S. L. Bektesh, and K. Grohmann, J. Chromatogr. 83, 25 (1973).
(1) (2) (3) (4)
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(8) (9) (10) (11) (12)
ANALYTICAL CHEMISTRY, VOL. 46, NO. 11, SEPTEMBER 1974
separation with this polar phase. Effects of temperature were also studied. Further work is now being carried out t o determine the effect of mobile phase composition and the effect of the chain length of the bonded phases on the retention mechanism.
EXPERIMENTAL Instrumentation. The liquid chromatographic system consisted of a gas displacement apparatus similar to that of Karger and Berry (14). The solvent tank with a gas ballast is connected in parallel with the reciprocating piston type pump. The gas regulator is adjusted to a pressure equalling the pump output. The gas ballast system provides excellent pulse damping, and a greater reproducibility of flow rates than a conventional reciprocating piston pump. A Milton Roy Minipump, maximum pressure 1000 psi, was employed. Alternately, a mobile phase delivery system made of 150 ft of Ih-in. 0.d. copper tubing connected to a N:! gas tank was used. The nitrogen pressure supplied the need pressure drop. The tubing was the solvent reservoir. Using such long tubing ensured bubble-free operation for at least 6 hours at a flow rate of 100 cm3/ hour. After the delivery of about 360 cm3 of solvent, the reservoir was depressurized and refilled. This system produced an extremely constant flow rate and a steady detector base line. An LDC UV detector having an 8-microliter cell, and operated at 254 nm, was used in conjunction with a Beckman 10-inch strip chart recorder. The column was a 30-cm X 2.8-mm precision bore glass column. All connections, with the exception of those between the gas supply and solvent tank, were made with %G-in. Teflon tubing. The liquid pressure gauge was an Acco Hellicoid Gage (01500 psi). Injections were made with a 10-microliter Glenco Syringe through a Chromatronix injection port. The column was thermostated by using a glass jacket through which water was circulated. The temperature of the water was controlled by a Thermotrol (Hallikainen Instruments). The temperature was monitored at the top and bottom of the glass jacket and the difference was undetectable (120)t 23.1 (25)‘ ... 0.03 2.89 0 .oo ...
3.91 3.14 1.32
1.09 0.68 0.37 0.32 0.56 0.26 0.59 2.32 0.70 2.17 5 .03
+
, . .
..
.. ..
...
.. ,..
... (
.
.
,..
0 .oo
+
0.00
0.39 0 .00
2.55
CS = Corasil 11; percentage indicates amount of carbon in the bonded material. Retention too long for a reasonable study, only one run taken. Adsorption in the UV weak, accurate measurement impossible.
‘
bonded phase described here is in a “brush” or in a polymeric configuration. T h e methoxy groups on the Si can be hydrolyzed easily. Even though the bonding reaction was carried out in dry benzene, the water adsorped on the silica gel surface of the Corasil is enough to convert the methoxy groups to silanol groups. Both Lynn (16) and Marsden (17) believe that the bonded phase on the surface is partially polymerized so that both the “brush” and the polymeric configuration exists. It should be noted that doubling the reagents did not double the surface coverage (as indicated by the carbon analysis which was repeated twice). An explanation might lie in the kinetics and mechanism of the condensation reaction. We did not investigate this reaction. Change in the retention of the solutes, however, was clearly a function of the surface coverage as discussed below. Retention Studies. Table I gives the values of capacity ratios obtained for compounds used in this study. All capacity ratios were determined relative to benzene as the unretained solute. T h e assumption that benzene is unretained may not be strictly correct, since weak interaction with Corasil I1 may occur, but benzene was our best choice for an “unretained” reference molecule, since we were limited by a UV detector. The reproducibility of the capacity ratios is estimated to be f 2 % for columns I and 111, and &5% for column 11. Looking at Table I, the highest capacity ratios for any compounds are obtained on pure Corasil 11-column I. This contrasts with the results of Novotny e t al. (12, 13) who found that with bonded phases k‘ values were usually larger. The interaction in the case of pure Corasil is an adsorptive one with the silica hydroxyl groups acting as Lewis Acids and the carbonyl groups as Lewis Bases. A second factor in retention on this column is the aliphatic nature of (16) M. Lynn, Corning Glass Works, Corning, N.Y., 1973. (17)J. Marsden, Union Carbide, Tarrytown. N.Y.. 1973.
private communication, private communication,
A N A L Y T I C A L CHEMISTRY, VOL. 46, NO. 11, SEPTEMBER 1974
1371
t
‘:e
-I
\
OC
0‘
[
A
r
\
- I 5/-
0
k-
r -I 8
0
\
,
‘T?,
2
4
6
CARBON
NUMBER
,
I
1
vs. carbon number of the side chain for various compounds on column Ill. 0 = 4 (C=O)(CH2)&H3, carbon number n 1; A = is0 analog
Figure 1. In
+
-
the mobile phase us. the aliphatic nature of the solute increases, the capacity ratios on column I decrease as expected. The height equivalent to theoretical plate for various compounds run on column I were higher by a factor of 4 to 10 than those found on the other columns a t the same mobile phase velocity. The largest capacity ratio values are for column I--i.e., pure Corasil 11. The capacity ratios tend to be lower on column I11 with a higher surface coverage of organic groups than on column 11. It is possible that on column 111,a greater portion of the bonded phase is in a polymeric form, and thus the major contribution of the organic groups is evidently the deactivation of the underlying.si1ica; interaction with the bonded phase is not very strong. This is as expected, and similar conclusions were reached by Karger and Sibley (7) in the case of GC. On closer examination of Table I, several reversals of retention become evident. The most notable reversal is that between benzophenone and g-fluorenone. On column I, pure Corasil 11, the capacity ratio of benzophenone is almost three times that of 9-fluorenone. On columns I1 and 111,9-fluorenone is retained to a greater extent than benzophenone. Several possible phenomena may contribute to this reversal. The surface of the Corasil I1 is modified by the silane bonded phase. On pure Corasil 11, the geometry of the solute molecule plays an important role in determining the retention. Benzophenone, being sterically less hindered, can interact more strongly with the silica surface than the more rigid 9-fluorenone. With the bonded phase, the access of the benzophenone to the surface is reduced. Interaction between the phenyl group of the bonded silane and the two solutes in question can occur. In this case, the rigid structure of the 9-fluorenone can allow for a greater T complexation with the bonded phase. Adsorption to the bonded phase is also possible. These facts (plus some others such as wettability of the bonded phase by the mobile phase as compared to the bare support) can cause the retention reversal. A reversal of retention can also be seen for the isovalerophenone and valerophenone on column I and 111. Several effects could, either individually or combined, explain this phenomenon. The modification of the silica gel surface by the bonded phase may hinder the adsorption of isovalerophenone on the Corasil I1 surface. Another possible explanation is the difference in size of the two solute molecules. 1372
The bulkier isovalerophenone may not be able to penetrate the bonded phase to the adsorbing surface as easily as the valerophenone. This was also the observation of Karger and Sibley (7). In support of this, the retention of the two compounds on the less covered surface is nearly identical. In other words, the “forest is thinner” on column 11. Also, it should be noted that the smaller butyrophenone and isobutyrophenone do not show retention reversal. Interactions with, and adsorption on, the bonded phase can not be excluded. The role of the mobile phase cannot be neglected. This can be interpreted from the fact that, in general, as the side chain length of the phenones increases, k’ decreases. In fact, Figure l shows a plot of In k’ obtained on column I11 us. the carbon number of the aliphatic side chain. As might be expected, a linear relationship exists, indicating additive contributions of CH2 groups to the change in free energy, AG”. Also shown in that figure are the two points of the is0 compounds. Several other interesting trends are seen on column I11 with the more highly substituted compounds. Chlorobenzene has a capacity ratio of 0.00. Yet all the substituted chlorobenzaldehydes have smaller capacity ratios than benzaldehyde; 2,4-dichlorbenzaldehyde < o-chlorobenzaldehyde < 4-chlorobenzaldehyde < benzaldehyde. Though the same retention order is obtained on column I, the change in capacity ratios from column I to I11 for the chlorinated compounds is not the same. The k‘&‘III are: ochlorobenzaldehyde, 19.0; 2,4-dichlorobenzaldehyde,17.6; and 4-~hlorobenzaldehyde,32.0. The greatest decrease is shown for the chlorinated compound that most closely resembles the bonded organic phase. These data indicate that the nature and position of functional groups have an important influence on the retention. To get the incremental contribution of the C1 atoms, a plot of In k’ us. the number of C1 atoms can be made. The 2,4-dimethyoxybenzaldehyde and p-methoxybenzaldehyde show the opposite retention trends on column 111. In addition to other possible mechanisms, the methoxy group itself probably plays a strong role in the retention, and increasing the number of methoxy groups increases the compound’s affinity for the cplumn. This again illustrates the complexity of multiple interactions. p-Nitrobenzaldehyde’s large retention on both columns I and I11 can also be a result of such multiple interactions. The retention of salicylaldehyde is less than that of benzaldehyde on both columns I and 111. The ortho-hydroxyl group probably weakly intramolecularly bonds to the carbonyl, thus providing less electron density to the silica hydroxyls. The 4-methylbenzaldehyde is retained to a greater extent on column I and I11 than benzaldehyde. At a first glance, one would think that the methyl group would give the compounds a greater aliphatic character and decreased retention. However, the methyl group is an electron donating group. This could enhance the basicity of the carbonyl, with possible increases in the adsorption to the unreacted Corasil. A small reversal of retention is shown for 4-chlorobenzaldehyde and 4-bromobenzaldehyde on columns I and 111. As indicated, the bonded phase is most likely present as areas of “brush” and areas of polymers. The retention data and the k’ values on columns I1 and 111, as compared to column I, might indicate that the “brush” areas are more abundant. But, a t this stage of the research, this is only speculation. In fact, the efficiency studies, presented next, can be interpreted to mean that the polymeric areas are more important, at least as far as the mass transfer processes in the column are concerned. Efficiency Studies. Several solutes were selected to study the plate height on column 111. The compounds were
ANALYTICAL CHEMISTRY, VOL. 4 6 , NO. 11, SEPTEMBER 1974
W
3
z 0 z
31 2
I
w z
W S
24 I
62 4
1
1
560
I
w
N
z
2
m
0 4
r
I
i
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n
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r
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6 0
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0
0 K L
1
0 02
-. 1 u
c z
00
1
04
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0 8
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Figure 3. HETP vs. mobile phase velocity for various compounds on column 111.
(1) 2,4dimethoxybenzaldehyde, (2) pmethoxybenzaldehyde, (3) 9-fluorenone, (4) benzophenone, (5) 2,4dichlorobenzaldehyde, (6) benzene, ( 7 ) isobutyrophenone, (8) 4-chiorobenzaldehyde
Table 11. T h e Empirical Coefficients of Equations 2 and 3
60
50
40 MlNU TES
Figure 2. Separation of a Flow rate 1.60 c m 3 / h r
30
k’
Compound
mixture of various phenones on column Ill
chosen to cover a range of capacity ratios and molecular sizes. Direct comparison of column I11 with column I (pure Corasil 11) was not possible since the same solute had different k’ values on the two columns, and the use of different solutes to get the same k’ values could be misleading. However, in general, at a given mobile phase velocity, the efficiency on the pure Corasil I1 column was much lower. Thus, the separation shown in Figure 2 for several phenones on column I11 could not be achieved on column I because of the larger H E T P value on the latter column. Undoubtedly, changing the nature of the mobile phase could have improved the separation on column I. This, however, was not attempted. The work of Horgan and Little (8) seems to indicate that, for equal k’ values, smaller plate heights are obtained on bonded systems. They attributed the improved efficiency to the fact that the bonded phase can be more evenly distributed, and to the elimination of possible liquid phase “pooling.” Another explanation might be that the bonded phase used in their study, which is of a “brush” configuration, blocks the access of the solute molecules to some pores which contain stagnant mobile phase. All these factors improve mass transfer. Mass transfer in the polymer bonded phases is probably slow, possibly due to stagnant pockets of mobile phase [uiz.( 9 ) ] . Our first attempt was to fit our data to the equation proposed by Knox (9, 18, 19) for reduced height equivalent t o theoretical plate. h -k 2y/v -k Avn -k CY
(1)
Here h is the reduced H E T P and u is the reduced velocity. The quantity y is the obstructive factor for logitudinal molecular diffusion. A describes nonequilibrium in the flowing part of the mobile phase, or the “goodness” of packing. C (18) G. J. Kennedy and J. H. Knox, J. Chromatogr. Sci., 10, 549 (1972) (19) J. N. Done and J. H. Knox, J. Chromatogr. Sci., 10, 606 (1972).
Benzene Isobut yrophenone
D
0
0.26 2,4-Dichlorobenzaldehyde 0.26 4-Chlorobenzaldehyde 0.56 Benzophenone 0.68 9-Fluorenone 1.09 p-Methoxybenzaldehyde 2.17 2,4-Dimethoxybenzaldehyde 5.03
t
S
0.068 0.064
0.066 0.061 0.078
0.084 0 ,092 0.122
)
n
3.8 2.06 2 .73 1.65 2.97 1.68 1. 4 3
0.25 0.36 0.31 0.39 0.33 0.45 0.51
5.2
0.31
describes nonequilibrium between the flowing and stationary (both stagnant mobile phase and the stationary phase) parts of the column. The initial least squares fit was made using n = 0.33 and y = 0.6 as suggested by Knox. This led to reasonable values of A (about 2) but to negative C values for several compounds. We then extended our program to fit n and y, but we still obtained negative C values for some compounds. Since a negative value of C is meaningless, it was decided that Equation 1 did not properly describe our system, perhaps because we did not operate over a large range of velocities. We then fitted our data to the empirical equation proposed by Snyder (20)
H
= Dun
and to its reduced equivalent h = 6un
(2)
(3)
D, 6 , and n are empirical constants for a given system. Experimental values of H are estimated to be accurate to f5%. Values of D, 6, and n are given in Table 11. Figure 3 shows the curves generated, for the eight compounds studied, from the coefficients given in Table 11. Experimental points are not shown on Figure 3 for clarity of presentation. The fits were quite good (about 98% confidence level) though the data did show a small scatter. Our H us. u (or h us. v ) plots in Figure 3 fall in the same range as those of and Knox and Vasvari (9). It should Horgan and Little (8), be mentioned that we also studied the behavior of valerophenone. In this case, however, we got an n value of 0.13. (20) L. R. Snyder, J. Chromatogr. Sci., 7, 352 (1969).
A N A L Y T I C A L CHEMISTRY, VOL. 4 6 , NO. 11, SEPTEMBER 1974
1373
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I
I
”I-’,, 30
50
40
312 0
I
592 8
I
I
- IO 0 ,
,
,
1
I
6C
T ?
Figure 4. Effect of temperature. 0 = I(; A = H (mm), = asymmetry. -- acetophenone; - - - propiophenone. The velocity was kept constant at 0.37 cm/sec
Since such a small n value is indicative of some difficulties, the valerophenone results are not discussed. A rough correlation is seen between the k’ values and the D values. As k’ increases, so does D. This relation was observed by many other workers [see for example reference (21) for a discussion on this topic]. No simple correlation could be found between the behavior of k’ and 6. If it is desirable, however, one could (and perhaps should) obtain an average “n” from the values of the eight solutes. The fit of Equations 2-3 will still be good and 6 will follow the same trend as D. While a t low velocities the H E T P curves in Figure 3 are close in magnitude, at high velocities compounds with high k’ value give higher H values. This could indicate that a significant (and perhaps the dominant) contribution to H is the poor mass transfer in the Corasil I1 pores or the presence of stagnant mobile phase pockets. The dependence of H on k’ might suggest that the polymeric areas of the bonded phases are rather important in governing the overall efficiency of the column. Alternatively, one can speculate that the “brush” areas do not prevent access of solute molecules to pores on the Corasil which are filled with mobile phase. Slow mass transfer on Corasil was also observed by Knox and coworkers (18, 22). As observed by many other workers, the increase in H (or h ) with velocity (or u ) is greater as k’ increases (18,221. Temperature Studies. These studies were done with hexanes from a different batch. Methylcyclopentane is present in different amounts in hexanes of different lots. The percentage of methylcyclopentane determines the k’ values of the various solutes. Thus, even though individual solutes had capacity ratios different from those previously obtained, the overall trend is consistent. Temperature studies can help in understanding the thermodynamic and kinetic processes which occur in the column. For that purpose we investigated five solutes: acetophenone, propiophenone, benzophenone, 9-fluorenone, and benzaldehyde. In agreement with the observation of Schmit et al. (23) and Novotny (12), the plate height de(21) L. R. Snyder, in “Gas Chromatography 1970,” R, Stock and s. G. Perry, Ed. Eisever Publishing Co., London, 1971, pp 81-1 11. (22) J. N. Done, G. J. Kennedy, and J. H. Knox, “Gas Chromatography 1972,” S.G. Perry and E. R. Adlard, Ed., Applied Science Pub., Essex, England, 1973, p 145. (23) J. A Schmit. R. A. Henry, R. C. Williams, and J. F. Dieckman, J. Chrornatogr. Sci., 9, 645 (1971).
1374
$
creased with increasing temperature. Figure 4 shows a plot of H, k’, and the peak asymmetry for acetophenone and propiophenone as a function of the temperature at a constant velocity of 0.37 cm/sec. All three quantities decrease with increasing temperature. The asymmetry of propiophenone disappears at high temperatures. Also, the curve of H for that solute seems to approach an asymptote. As suggested by Knox and Vasvari (9),the decrease in the asymmetry might indicate the linearization of the partition isotherm with increasing T. Benzophenone and benzaldehyde showed a temperature dependence similar to that of acetophenone and propiophenone as shown in Figure 4. With 9fluorenone, the asymmetry first decreased, as expected, but a t high temperature the peak “fronted.” The H us. T showed a slight minimum a t about 37 “C. However, the minimum was so shallow that to a first approximation, one could assume H to be independent of temperature. The capacity ratio decreased continuously with increasing T . The reasons for the behavior of the asymmetry of that solute are not clear, but probably reflect the thermodynamic contribution to the peak width. Figure 5 shows H ( h ) us. u ( u ) for acetophenone at two temperatures. Also shown in the Figure are the k’ values at the two temperatures and the asymmetry at the hi’gh temperature only. The behavior of the k’ plots are especially noteworthy. In this particular Corasil I1 bonded phase system, we found that with all solutes, and a t all temperatures, the capacity ratio decreased a t low velocities; (the retention volume of benzene, the reference solute, remained constant throughout the velocity range). Knox suggested (24), and later found experimentally (9),that at low velocity, thermodynamic tailing resulting from nonlinear isotherm is observed. Indeed, in Figure 5, the peak asymmetry increased to a certain constant value, as the velocity is decreased. This can explain the reduction in the k’ values. The reasons for the peculiar shape of the asymmetry plot are not clear. It should be emphasized here the k’ values were obtained from peak maxima. No attempt was made to calculate k’ from the first moment. It should be noted also that Kirkland found ( 4 ) that k’ can increase with velocity in certain cases. In order to elucidate the k’ dependence on the velocity, static measurement of k’ should be determined and compared to the chromatographically observed value. The decrease in H with T can be due to the decrease in (24) J. H. Knox, Department of Chemistry, University of Edinburgh, private communication, Oct. 4, 1973.
A N A L Y T i C A L CHEMISTRY, VOL. 46, NO. 11, SEPTEMBER 1 9 7 4
Table 111. AH Values for Several C o m p o u n d s Compound
Propiophenone Benzophenone Benzaldehyde Acetophenone 9-Fluorenone f
=
IS-R 1% f”
k‘ (at 40 “C)
(kcallmole)
0.52 0.72
1.08
14.9
1.10
14.5
1.44 1.34
18.4 16.6
2.23
27.8
0.83
1.09 1.25
AH
ratio of the volumesof the stationary to mobile phase.
tionary phase as did Knox and Vasvari (9). Consequently, we could not estimate AS. However, the trend seems to be the same as with AH. In general, AH seems to be rather small (being 2.23 kcal/mole for k’ of 1.25). The discrepancy in the benzaldehyde results might be attributed to the fact that it is not a phenone as are the rest of the compounds. This point should be further investigated. Nonetheless, following the argument of Knox (9), one can interpret the results in Table I11 to mean that AH determines the retention in our system.
SUMMARY viscosity of the mobile phase or to changes in the kinetic processes which occur in the column. Knox and Vasvari (9) found, for their system, the reduced plate height h a t a constant reduced velocity to be temperature independent. This would indicate that the change in the diffusion coefficient of the solute is responsible for the change in H as the temperature is varied. We did not attempt to plot reduced plate height a t a constant reduced velocity as a function of T. However, the fact that the two H plots in Figure 5 seem to converge a t low velocity and that they are not parallel at high velocities might indicate that perhaps kinetic factors and the mobile phase viscosity are of importance in determining H. This speculation, however, must be studied in more detail since, as was discussed before, not all solutes show a large change in the plate height with temperature. From the relation log k’ us. 1/T (Van’t Hoff plots), one can obtain the heat of transfer from the stationary to the mobile phase. The intercept of such plots is related to the entropy of transfer. Table I11 shows AH‘S and a function related to the intercept, namely, AS-R log f, for several compounds as obtained on column 111. The factor f is the ratio of the volumes of the stationary to mobile phases. Except for benzaldehyde AH increases with k’, as was found by Knox and Vasvari (9) for Permophase ODS system. We did not try to approximate the ratio of the mobile to sta-
The results show that the retention behavior on bonded phases can be due to several contributions; among them, surface modification, and interaction with the bonded phase and with the mobile phase, as indicated by retention reversal of several solutes. Temperature studies indicate that the order of retention on the present bonded phase is determined mainly by the heat of transition from the stationary to the mobile phase. Studies of peak broadening indicate that stagnant pockets of mobile phase contribute significantly to the rate of mass transfer. It still remains to be determined what the phrase “stationary phase” really means. Indications are that the bonded phase used here, and perhaps by other workers, can exist in patches of “brush” and polymeric areas. A study is needed in which the degree of polymerization on the support surface is carefully controlled. In addition, the effect of the chain length of the bonded phase on the chromatographic behavior of various solutes should be investigated. Such studies are now in progress in our laboratory. Varying the surface coverage of the bonded phase should be also be examined since, as was demonstrated here, it can modify the retention behavior in certain cases.
RECEIVEDfor review January 23, 1974. Accepted April 29, 1974.
Method for Prediction of Partition Coefficients in Liquid-Liquid Chromatography Paul Menheere, Claude Devillez, Claude Eon, and Georges Guiochon Laboratoire de Chirnie Analytique Physique, €cole Polytechnique. 7 7, rue Descartes, 75005-Paris
The potential value of ternary systems in liquid-liquid chromatography is explored and it is shown that in the absence of secondary effects, variations in selectivity due to changes in eluent composition can be determined from the changes in interfacial tension. Furthermore, the free energy of the partition process can be predicted with meaningful precision.
The interfacial tension between the two phases in a liquid-liquid chromatographic system is the master parameter for prediction of the potential selectivity, as the interfacial tension between the two liquids is dependent upon the differences in their physical nature ( I ) . A quasi linear relationship between the free energy of partition and the interfacial tension was established for (1) C Eon, B (1973)
Novosel, and G
Guiochon, J
Chromatogr. 8 3 , 77
those case where the two phases were mutually insoluble; however, such a relationship is fallible as soon as mutual solubility becomes a significant factor. The same arguments apply when two immiscible phases of a ternary mixture are the “eluents” phases and indeed Huber et al. (2-4) have already suggested the considerable potential of such systems. The investigation of these systems is therefore mandatory, and the present availability of reliable partition coefficients of numerous steroids in a series of ternary mixtures of water, ethanol, and isooctane ( 4 ) gave impetus to this work. Under defined conditions, it is possible to predict chromatographic behavior that is concomitant with the solu( 2 ) J. F K . Huber, J. Chrornatogr. Sci.. 9, 72 (1971). (3) J. F. K . Huber, C. A. M . Meijers, and J. A. R J Hulsman, Anal. Chem., 44, 111 (1972). ( 4 ) J. F. K . Huber. E. T Alderlieste. H Harren, and H Pope, “Advances in Chromatography-1973,” A Zlatkis. Ed.. Department of Chemistry, University of Houston, Texas, 1973, p 327
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