Retention Behavior on Alkyl Bonded Stationary Phases in Liquid Chromatography E. J. Kikta, Jr., and Eli Grushka" Deparfment of Chemistry, State University of New York at Buffalo, Buffalo, N. Y. 14214
The retention behavior on alkyl bonded statlonary phases for llquld chromatography has been studied as a function of chain length, surface coverage, solute type, moblle phase composition, and temperature. The data lndlcate that depending on the degree of surface coverage and the type of solute, mixed mechanlsms may play an important part in the retention behavior. Efficiencies and capacity ratios show an Increase as the bonded phase loading increases while asymmetry shows a decrease. Uslng a nonpolar moblle phase AH shows an odd-even effect as a function of the carbon number of the alkyl side chain in phenones. In a polar mobile phase AHseems to determine retention order except for the case of a heavlly loaded column. In general, the data lndlcate that alkyl bonded phase columns are most useful when the amount of bonded statlonary phase is maximized.
erage data were provided in the above paper, the results suggest that there may be a n optimum coverage for reverse phase liquid chromatography. Knox and Pryde (13) indicated that in reverse phase chromatography the nonaqueous modifier in the mobile phase is extracted by the bonded phase, and that solutes can partition between the mobile phase and the modified bonded phase. This paper reports the results of an investigation where the chain length of nonpolar bonded phases was changed and the variation in retention properties studied. In addition, surface coverages were varied and the chromatographic behavior was examined in detail. Particular care was taken to keep the conditions of the experiments as constant as possible in order to ensure the reliability of the results. In order to study the effect of the underlying surfaces, no attempt was made to eliminate residual OH groups.
EXPERIMENTAL Unabated interest has continued in the development of permanently bonded stationary phases for liquid chromatography. Though many such bonded phases continue to be prepared and applied to a great variety of separation problems with a high degree of success, few studies have been directed at providing a mechanistic description of their operation. Recent reviews (1-3) have aptly covered the current uses of bonded stationary phases in liquid chromatography. In a previous paper we have presented a detailed study on a polar bonded phase prepared by reacting Corasil 11 with l-trimethoxysilylchloromethylphenylethane ( 4 ) .Novotny and his co-workers ( 5 ) started with a similar material, but proceeded to hydrolyze the terminal chlorine to a hydroxyl group before studying its properties. Knox and Vasvari's (6) work on polymeric bonded phases presents one of the first systematic approaches of studying retention mechanism in bonded phases. Although much remains to be learned about the behavior of bonded phase systems, useful information has already been determined. Kirkland (7) has found that with polymeric phases adsorption on the polymer and interaction with stagnant pockets of mobile phases are important in determining chromatographic parameters. Little and his co-workers (8) have shown that in brush systems residual silanol groups play an important part in the separations. There are reasons t o believe that this also holds true for liquid chromatographic systems. Karger and Sibley (9) have shown that in gas chromatography, when employing a nonpolar bonded brush system, the type of bonded phase determines the retention characteristics of the system. A recent study by Majors and Hopper (10) suggests that in reverse phase systems the retention increases as the length of the bonded phase increases. They did not find, however, a correlation between surface coverage and retention time. Locke (11)has demonstrated that the retention order, in reverse phase chromatography, is a function of the solutes solubilities in the mobile phase. In a study of in situ bonded stationary phases, Gilpin and his co-workers (12)have shown that increasing the length of the bonded phase from ethyl t o octadecyl causes k' to increase. Even though no surface cov1098
ANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
Instrumentation. The liquid chromatograph employed has been described in detail elsewhere ( 4 ) .The copper solvent delivery tube was replaced by a 30 f t stainless steel tube for studies involving aqueous systems. Columns were 1.8 mm i.d. X 50 cm stainless steel for some preliminary studies, and 4 mm i.d. X 25 cm for all other studies. The 4 mm i.d. column was prepared by boring out 3.8 mm i.d. stainless steel tubing in a manner similar to that described by Asshauer and Halasz ( 1 4 ) . The same 4 mm i.d. column was used for all studies, except preliminary workups, thus eliminating one variable. Columns were thermostated by using a glass jacket through which water was circulated. Data were collected on either a Beckman Model 1005 or a Heath Schlumberger Model 255B chart recorder. Reagents. The spectroanalyzed hexane (with some methylcyclopentane impurity) and reagent grade methanol mobile phases were obtained from Fisher Scientific. The water in the mobile phase was distilled and passed through an Illinois Water Treatment Co. ion exchange column. Each mobile phase was stored in a single large jar to eliminate variability between different lots. Solutes were obtained from various mwufacturers and were found to be of sufficientpurity for the present study. Corasil I1 was obtained from Water Associates. All the Corasil I1 used for preparing columns was from the same lot. The silane reagents, trimethoxysilylmethane,1-triethoxysilylnonane and 1-trimethoxysilyloctadecanewere obtained from Union Carbide Corp. and used without further purification. Procedure. The permanently bonded stationary phases were prepared by reacting Corasil I1 with the appropriate silane compound. The Corasil I1 was first washed with 100 cm3 of 25% nitric acid in sulfuric acid for 2 h. It was then washed with distilled deionized water, methanol, and chloroform and dried in a vacuum oven for 24 hat 120 "C, p = 0.5 mm. To prepare each bonded phase, 3 g of the treated Corasil I1 and 0.002 M of a silane compound were allowed to react in 15 cm3of dry benzene with constant shaking. Reaction time was varied from 2 to 100 h to obtain various surface coverages. After the appropriate reaction time, the supports were washed with benzene and chloroform and dried over a nitrogen steam. They were further dried in a vacuum oven for 22 h at 84 O C , p = 0.5 mm. Columns were drypacked using conventional techniques. Injections of solutes [l to 5 Mglpl of the appropriate mobile phase] were made with a 10 pl Glenco microliter syringe through a homemade injection head fabricated from Swagelok % or 1/4 in. union-T's specially modified to eliminate dead volume. Two mobile phases were used: (1) hexane and (2) a 50%/50% water-methanol mixture. For the thermodynamicsstudied the following temperature ranges used were: (1)with hexane as the mobile phase, 25-60 "C in 5 or 10 "C increments; (2) with the water-methanol mixture, 30-59 "C in 5 "C increments. All measurements were made in, at least, triplicate.
RESULTS AND DISCUSSION The surface coverages of the various supports used in this study are described in Table I. Surface coverages were estimated by (a) knowing the amount of material bonded, (b) assuming that four OH groups per 100 A* of the surface are available for reaction with the moiety being bonded (15),and (c) knowing the surface area of Corasil 11. The percent carbon bonded was obtained from elemental analysis. It was estimated that on the average, two of the alkoxy groups in the silane compound reacted with the surface. The results of Gilpin and Burke (16) suggest that the maximum surface coverage attainable with short phases, such as C1, is between 70 and 80%of a monolayer on silica. Under the mild bonding conditions used in the present work, the above figures seem to be an upper limit. Hexane Mobile Phase Studies. Initial studies used hexane as the mobile phase. Although it may seem odd to study bonded alkanes using hexane as the mobile phase, in actuality, a greater understanding of retention behaviors can result. In reverse phase systems it is frequently observed that the efficiencies are poorer than those in normal phase systems of similar particle size and packing methods. If a bonded alkane
Table I. Analysis of Surface Coverage for Columns Used in This Study Column No.
Column type
%C
% coverage
c1
0.44 0.36 1.16 0.28 0.77 0.18 0.44
70 10.3 19 44.5
I I1 I11 IV
v
VI VI1
c9 ClS
c1 c9 c9 c9
22 5.1
13
system shows higher efficiencies in a nonpolar phase environment, it may indicate that the bonded reverse phase is poorly wetted by polar mobile phases, leading to a high interfacial mass transfer term which can dominate the zone broadening processes. The use of alkane mobile phase also allows studying the effect of the unbonded support surface. I t is now accepted that residual SiOH groups can result in inferior columns. The magnitude of the effect of these groups needs to be examined. In addition using hexane as mobile phase allows comparison with previous work which dealt with a polar bonded phase (4). Column I was 1.8 mm X 50 cm packed with a C1 bonded support. It seems that the bonded phase deactivated the Corasil 11. Capacity ratios for most compounds were usually much less than 1.This result is expected and is consistent with the observation of Gilpin and co-workers (12). The small bonded nonpolar methyl groups do not interact strongly with the solutes, and, in addition, the high coverage shields the underlying silica from any appreciable interaction with any polar functionality on the solute. These two factors give rise to the observed low k’ values. Table I1 lists capacity ratios obtained on columns I1 and I11 a t two temperatures using hexane as the mobile phase. Benzene is assumed to be the unretained solute in all cases. The data in Table I1 show some interesting trends. T h e magnitudes of the retention times, and the order of elutions, clearly indicate that in this LC system the presence of unreacted surface OH groups is of prime importance. All the capacity ratios on column 111,with the exception of nitrobenzene and benzaldehyde, are smaller than on column 11.This agrees with the results of Majors and Hopper (10) and Gilpin et al. (12). Table I shows that the surface coverage of column I11 is larger than that of column 11. It is possible, therefore, that the higher density of the CIS bonded phase and the bulkiness of that phase prevent the solutes from reaching and interacting with the underlying silica gel surface. It is to be noted, however, that
Table 11. k’for Various Compounds Used in This Study ( 0= 1 cm/s, Mobile Phase is Hexane) Column I1 C9 Column I11 C13 Compd
40 O
C
50 “C
Benzene Acetophenone Propiophenone n-Butyrophenone Isobut yrophenone Valerophenone Isovalerophenone Hexaphenone Heptaphenone Octaphenone Nonaphenone Decaphenone Myristophenone Benzophenone 9-Fluorenone Benzaldehyde Salicylaldehyde 2-Chlorobenzaldehyde
0
4.13 2.55 1.62
1.11
2,4-Dichlorobenzaldehyde
1.11
0.784
4-Chlorobenzaldehyde 4-Methylbenzalde hyde 4-Methoxybenzaldehyde 4-Bromobenzaldehyde Nitrobenzene Chlorobenzene Anisole p-Dichlorobenzene
3.39 5.59 36.7 3.24 0.901
7.91 4.25 3.65 3.36 3.33 3.14 2.91 2.69 2.50 2.28 2.19 1.78 7.07
0
2.78
1.96
5.55
0
2.46 0
0.542 0
0
40 O
C
0 6.02 2.09 1.93 1.69 1.52 1.21 0.857 0.843 0.677 0.526 0.432 0.213 4.09 5.14 4.46 2.46 1.16 0.720 2.31 4.12 27.3 1.81 0.925 0 0.464 0
50 OC
40 OC h’IIlk111
0
1.26
0.575
0.830 0.534
1.21
1.31 2.03 1.89 1.99 2.19 2.60 2.29 3.19 3.69 4.33 5.07 8.37 1.73 1.08 0.926 1.04 1.40 1.54
1.47 1.36 1.34 1.79 0.974
0
1.17 0
ANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
1099
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Flgure 3. H, k’, and asymmetry for hexaphenone, column 111, mobile phase is hexane. (0)HETP 40 OC; (A) HETP 50 OC; (0)k‘ 40 OC; (V)k‘, 50 OC; (0) asymmetry, 40 OC; ( 0 )asymmetry, 50 OC
1
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Figure 2. HETP plots for benzene, mobile phase is hexane. Column II: (V)40 OC; (01) 50 OC.Column 111: (0)40 OC; (A) 50 OC
some of the smaller solutes were retained to about the same extent on both columns. The behavior of the phenones is particularly interesting. As length of the alkyl side chain increases, the capacity decreases. The decrease in k’ may be due to the fact that the bulkier phenones are sterically hindered from interacting with the underlying silanol groups on the Corasil surface. Alternatively the larger molecules can have a greater affinity for the mobile phase. It should be emphasized that although the bonded phases and the mobile phase are alkanes, the former presents, most likely, a different environment by the virtue of being bonded. The last column of Table I1 gives ratios of k’ on the two columns. As the side chain of the phenone increases, so does the ratio h’II/k’III;that is the relative change in retention with increasing carbon number is greater on the CIS column. This is shown in Figure 1,which is a plot of In k’vs. the side chain length. Except for the acetophenone, the points lie on a straight line. Acetophenone having the smallest possible alkyl side chain may interact with the bonded phase quite differently than the other phenones. The slope of the line for column I11 is much steeper (-0.235 vs. -0.0853). Figure 1and the data in Table I1 seem to indicate that the larger degree of surface coverage in the CIS column is, most likely, the pre1100
ANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
Figure 4. HETP vs. velocity. 50 OC column 111, mobile phase is hexane: (1) propiophenone, (2) hexaphenone, (3) 4-bromobenzaldehyde, (4) 2-chlorobenzaldehyde, (5) 2,4-dichlorobenzaldehyde
dominating reason for the low k’ of the long-chain phenones on that column. For compounds other than phenones, over a k’ range of 36, the ratio k’11/k’111varies by less than a factor of 2. On the other hand, the phenones with a k’ range of less than 8 show a k’II/ k’III ratio that varies by about a factor of 6. From this observation two possible conclusions can be drawn. (a) For compounds without significant alkyl residues, the bonded phase acts only as a surface deactivator. Compounds which contain significant alkyl side chains are more likely to interact with the alkyl bonded phase. (b) The size of the solutes affects their penetration to the support’s surface. Figure 2 shows H E T P plots obtained for benzene (k’ = 0) on column I1 (C,) and column I11 (CIS) a t 40 and 50 “C. Note that even if benzene is retained slightly, the error introduced will be fairly small. The efficiency on the ($8 is somewhat superior. This is similar to the data of Gilpin et al. (12)which show poorer efficiencies for shorter bonded alkyl supports. The poorer efficiencies may be due to poorer packing characteristics of that column. This point will have to be examined further. Figure 2 shows that for an unretained solute H is independent of T over a narrow temperature range. Figure 3 shows plots of H , k’, and asymmetry for hexaphenone on column 111.Trends similar to these plots are typical of all the data on this bonded phase. I t should be noted that asymmetry is roughly independent of temperature, but that
Table 111. Thermodynamic Data for Columns I1 and I11 (Mobile Phase is Hexane) Column I1 C9 Column I11 CIS Compd Anisole 2,4-Dichlorobenzaldehyde 2-Chlorobenzaldehyde Myristophenone Hexaphenone 4-Bromobenzaldehyde Valerophenone n-Butyrophenone Propiophenone AcetoDhenone
4
AH,kcal AS,eu AH,kcal AS,eu 5.99 6.41
15.8 15.2
6.96 8.06
16.4 19.8
7.82
19.8
7.22 7.38 7.79 7.15
16.6 16.8
9.40
25.5
18.3
15.7
9.75 8.26
25.7 20.6
7.73
17.2
8.89
21.7
8.57
19.6
N
i t increases sharply at low velocities. Capacity ratios tend to increase slightly at low velocity. Possible explanations for that behavior were given elsewhere ( 4 ) .Data obtained on column I1 showed that h’ was constant, but the asymmetry decreased slightly over the velocity range used. Figure 4 shows typical H E T P data, for retained solutes on column I11 a t 50 OC. For pictorial clarity the actual experimental points are omitted and only fitted lines [tb the Snyder equation (17)]are given. No attempts were made to fit the data to Knox’s equation (6). However, for the sake of completeness, the reduced plate height, h, and reduced velocity, u, are shown in the figure. For a given chemical class, e.g., phenones or halogenated benzaldehydes, as k’ increases, H increases. This was observed by other workers (e.g., ref 18). It should be noted that for equal K’, the H values of the phenones are higher than those of benzaldehydes. This may be attributed to the mixed retention mechanism. Long side chain phenones, unlike the other solutes, can interact not only with the underlying silica but with the bonded aliphatic groups. Although the efficiencies are rather poor, especially a t velocities higher than 0.5 cm/s, they are in agreement with, or better than, those obtained by other workers (viz., 10 and 12). H and k’ decreased as the temperature increased. This is in agreement with observations of other workers (4,5,19,20). The H vs. velocity plots on column I1 are similar to those on column 111, with the exception that the efficiencies for the same solutes are lower in the latter column. Figure 2 seems to indicate that the columns were packed relatively well (for the size particle used in the present study), although the Cg one is somewhat inferior. Figure 4 and other data obtained (although not given here) in this study show that H increases with k’. This behavior might indicate the existence of mobile phase stagnant pockets, either in the pores of the Corasil or in patches of polymerized bonded phase. With the silanes used in this study, cross-linking of monomeric units is possible. However, it is felt that the extent of polymerization was small. Kirkland (7) described the retention behavior in polymerized bonded phases where the solvent is entrapped in the polymers. Such stagnant pockets result in slow mass transfer and low efficiencies. In order to gain greater insight into the nature of the interactions in this system, enthalpies and entropies of transfer from the stationary bonded phase to the mobile phase were determined. AH was obtained by plotting In k’ vs. 1/T, while A S was calculated in a manner suggested by Knox (6). Weights of the bonded phases were estimated from elemental analysis. The calculated A S values are only “order of magni-
Figure 5. AH vs. carbon number for phenones C6H5CO(CH2)N--1CH3, mobile phase is hexane, column 111 (0),column II (A)
tude” values, whose main purpose is to indicate trends. The precision in the measurements is about 5-10% relative error. The results are shown in Table 111. Given the fact that the bonded phases and the mobile phase are alkanes, the high values of AH are somewhat surprising. The reasons might be due to adsorption on the silica surface. Similarly high AH values were obtained by Diez-Cascon et al. (21). However, unlike these workers, we did find an increase in AH when the bonded chain length is increased. With the exception of the phenones, k’ seems to follow A H ; Le., a high k’ value is associated with a high AH value. The phenones, on the other hand, exhibited a peculiar behavior shown in Figure 5. On both the Cg and CIS columns, AH (and AS) shows an “odd-even” dependence on the length of the side chain. Similar “odd-even’’ effects are observed in other physical and chemical properties such as melting points and solubilities of compounds within a homologous family. This experiment was repeated several times and in all cases the odd-even effect persisted. Figure 5 indicates that AH does not determine the retention order and that entropy effects are quite important. The reason for the odd-even phenomenon may be due to steric or to solubility effects. I t can be speculated that the bonded phase, by the virtue of being anchored, is in a relatively high state of molecular order, somewhat resembling liquid crystals. Odd-even effects in such classes of compounds are very well known, and they are usually attributed to entropy effects. More work is needed to fully explain the observed AH behavior. It is interesting to note that on the CIScolumn, AH and A S are higher than on column 11.Whether this is due to the longer bonded phase or to the fact that column I11 is covered to a greater extent is not clear. As mentioned, the bonded alkane is not identical in nature with the alkane mobile phase. This fact might account for the observed trend in AH as the length of the bonded phase increases. Additional work is needed to ascertain the significance of the results given here. Locke (22) suggests that one should measure AH of sorption (i.e., AH of AHof solution in mobile phase) and relate it, transfer hopefully in a quantitative manner, to the percent surface coverage. From the extent of surface coverage one can then calculate the AH of adsorption due to unreacted SiOH, and that due to the bonded phase. Such a study, which is now being pursued, should yield important information regarding the nature of the bonded phase. Reverse Phase Studies. It was decided to examine the effect of surface coverage, while holding the chain length constant, on the chromatographic behavior in typical reverse phase systems. The columns used and data obtained are summarized in Table IV. It should be noted again in striving to eliminate as many variables affecting the systems as possible, the same column was used for all the studies. In the process of repackirlg the column many of the old supports were lost, making further studies with these packings impossible.
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ANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
1101
Table IV. Ir’ Various Compounds Used with Mobile Phase 50% Water/5O% Methanol ( T = 40 “C) Compd Acetophenone Propiophenone n -Butyrophenone Isobutyrophenone Valerophenone Isovalerophenone Hexaphenone Heptaphenone Octaphendne Nonaphenone Decaphenone Myristophenone Benzenesulfonic acid
Column IV C1(44.5%)
Column VI c~(5.1%)
Column I1 Cg(10.3%)
Column VI1 cg(13%)
Column V c9(22%)
0.075 0.20 0.31
0.17 0.20 0.28 0.26 0.31 0.29 0.49 0.72 1.22 2.01 3.28 2.96 0
0.544 0.700 0.918 0.789 1.24
0.608 0.828 0.962 0.941 1.46 1.23 2.07 3.29 5.45 8.90 15.0
0.658
0.44 0.53 0.75 1.03 1.50 3.00 3.00 0
Usually the amount of a particular packing prepared was enough to fill only one column. For that reason, some of the bonded phases investigated in the earlier section were not used here. The only solutes used in this study were the phenones and benzenesulfonic acid. The latter was used to investigate the homogeneity of the packed bed. The inert peak was obtained by injecting pure methanol and observing changes in the baseline due to the refractive index differences. With the exception of some of the phenones on column VI, the k’ values are lowest on column IV where the surface coverage (with C,) is largest. This is consistent with the results of Gilpin et al. (12) which showed that very short chain lengths make poor separation media. The first column studied was column I1 which was cycled to a 50% water/50% methanol mobile phase using a series of solvents suggested by Scott and Kucera (23). As expected, the retention order for the phenones is inverted from that in the “pseudo” normal phase system. The more nonpolar the solute, that is the longer aliphatic side chain, the higher the capacity ratio. The data in Table IV show that, in general, k’ increases with increasing Cg surface coverage. Due to the very long retention of myristophenone, its capacity ratio was not determined. The behavior shown in Table IV indicates increasing interaction between the solute and the bonded phase as the amount of the latter increases. Two possible explanations are as follows: (a) The methanol solvates the alkane bonded phase better than the water, hence a methanol layer is formed on the support. The larger the coverage of the Cg, the more methanol is entrapped on the surface. The solute then partitions between the mobile phase and the Cg-methanol phase. Higher amounts of methanol cause the longer retention times. This explanation is consistent with the observation of Knox and Pryde (23). Telepchak (24), on the other hand, suggested that in a reverse phase, the adsorption mechanism is responsible for the magnitude of the retention. The data in Table IV are compatible with both possibilities. (b) The water preferentially solvates the residual Si-OH group on all the columns. With column VI, the low loaded column, this represents a relatively low energy surface, similar to that in column IV. This might be the reason for the similar k’ values on both these columns. As the surface coverage increases, the residual Si-OH groups are still covered with water, but the solute molecules can interact with more Cg-bonded phase, and k’ increases. More studies are needed to resolve thi? point. A possible direction for future investigation lies in the examination of similar system with (1)deactivated Si-OH groups and (2) fully cross-polymerized bonded supports. Locke ( 2 1 ) suggested that in a reverse phase system, the retention order within a class of compounds is solely a function 1102
ANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
1.14
2.00 3.00 5.10 9.32 16.9 0.231
0.225
1.21 2.27
1.99 4.05 3.55 7.18 12.17 19.8 38.64 77.67 0.055
of solute solubilities in the mobile phase. In general, the present results do indicate ,that as the solubility of the phenones decreases, the retention time increases. However, the data in Table IV show additional trends. If the retention was governed only by solubilities, then plots of relative retention vs. carbon number would give striight lines, having the same slopes, for all the columns. Calculation has shown that this is not the case here, and that the slopes depend on the surface coverage. This may indicate that the higher loaded alkyl columns are capable of selective interactions with the solutes. Much can be learned from the plate height dependence on surface coverage. For illustration purposes, Figures 6 and 7 show H E T P vs. velocity (and h vs. v) for columns VI (5.1%) and V (22%),respectively. In general, it was found that for a given k‘ a t a given velocity H improved as the loading increased. For example, for k’ = 3 a t 1cmh, the plate height was 4.1,4.9, 2.2, and 1.5 mm respectively on columns VI, 11, VII, and V. The H values, although poor, are in agreement with, or better than, values reported by Majors et al. (20)for similar reverse phase systems. The efficiencies on columns VI and I1 were poor for all compounds except for benzenesulfonic acid. The latter compound gave nearly the same H value on all the columns (Hvalues between 0.5 and 1mm). We cannot explain, a t this point, the reasons for the poorer efficiencies on columns VI and 11. It is felt that poor packing is not one of the reasons, since the H values for benzenesulfonic acid are quite acceptable for CoraGl columns. It is possible that with the low surface coverage, solute molecules which have low solubility in the mobile phase interact more favorably with the silica surface. The kinetics and thermodynamic behavior of such systems may lead t o excessive band broadening. As the surface coverage increases, interaction between solutes and the bonded phase is more likely t o occur. This interaction may have faster mass transfer characteristics. Note that on the highly loaded column, Figure 7, the plate height is only a weak function of the mobile phase velocity. Asymmetry as a function of k‘ is shown in Figure 8. The two lightly loaded columns show increasing asymmetry as a function of capacity ratio. On the heavier loaded columns, the asymmetry seems to exhibit a maxima. The behavior shown in Figure 8 is quite interesting. Unlike the results discussed before, and in an earlier paper ( 4 ) , the asymmetry is not a function of the velocity. Rather it seems to be a function of surface coverage. It is conceivable that at low surface coverage, surface adsorption and thermodynamic factors associated with it play a major part in determining the peak shape, i.e., the solutes isotherms. As the amount of Cg increases, the underlying surface is not as accessible to solute molecules, especially the bulkier ones. On the other hand, as the side chain of the phenone increases, the solubility in the mobile
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vs. velocity. Column V, 40 ‘C, mobile phase is 50% methanol/bO% water: (1) heptaphenone, (2) valerophenone, (3) nbutyrophenone, (4) propiophenone, (5)acetophenone, (6) benzenesulfonic acid Figure 7. HETP
U (CM/SEC)
vs. velocity. Column VI, mobile phase is 50% methanol/50 % water, 40 ‘C: (1) decaphenone, (2) nonaphenone, (3) octaphenone, (4) heptaphenone, (5)valerophenone, (6) benzenesulfonic acid Figure 6. HETP
20 1.8
phase decreases and, as speculated before, the solute partitions into the methanol on the surface. The partition isotherms of the long chain phenones are more linear than the adsorption isotherms of the smaller phenones; hence the maximum is the plot for column V. Alternatively, Figure 8 may indicate that as the aliphatic portion of the phenone becomes larger and the amount of Cg increases, the interaction is basically that of an aliphatic solute partitioning between a hydrocarbon bonded phase and the polar mobile phase. The importance of the interaction of the more polar aromaticcarbonyl portion of the solute decreases. This reduces the chances of mixed retention mechanisms, leading to more symmetric peaks associated with a simple partitioning mechanism. Further studies are needed to elucidate the asymmetry behavior, and one possible set of experiments is the dependence of the peak shape on the amount of solute injected. Thermodynamic data for the four Cg columns are given in Table V. The data for columns I1 and VII, whose C g surface coverages are about the same, are quite similar, indicating possibly that the thermodynamic properties of the surfaces are close in nature. The behavior of A H for columns 11,VI, and VI1 as a function of the carbon number of the side chain (or the k’ value) indicates that the enthalpy governs the retention order. In the case of column V, AH goes through a maximum, perhaps indicating that enthalpy alone does not govern the retention order.
CONCLUSION The retention mechanism on bonded aliphatic phases in liquid chromatography has been shown to be a complex phe-
1.6 Asvm
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k‘
Asymmetry vs. k‘, T = 40 ‘C: (A)column I1 (10.3%),(v) column V (22%), (0)column VI (5.1%), (0)column VI1 (13%) Figure 8.
nomenon depending on several factors. AH may or may not determine the order of retention depending on the type of mobile phase and the extent of surface coverage. Mixed retention mechanisms seem to play a part in systems with small amounts of bonded phase, since the underlying silica is still available to solute molecules for interaction. The efficiency and k’ increase as loading is increased while asymmetry seems to decrease as a function of increased loading. This study indicates that a column is of maximum usefulness when the amount of bonded stationary phase is increased. For solutes with small or nonexistent alkyl portions, the bonded alkyl groups serve mainly to deactivate the underlying silica surface. The rate of change or retention of compounds with increasing alkyl portions can verify this speculation. T h e nature of the retention mechanism is then a function of the solute, solvent, type of aliphatic bonded phase (cHain length), and amount of bonded aliphatic phase. Further investigations should be carried out to determine if optimum bonded chain lengths and loading levels exist for various separation problems where reverse phase chromatography is needed.
Table V. AH (kcal) and A S (eu) for Several PhenonesCs Columns (Mobile Phase 50% Water/SO% Methanol) Column VI (5.1%) Compd Acetophenone Propiophenone n-Butyrophenone Isobutyrophenone Valerophenone Isovalerophenone Hexaphenone Heptaphenone Octaphenone Nonaphenone Decaphenone Benzenesulfonic acid
AH
6S
Column I1 (10.3%)
Column VI1 (13%)
Column V (22%)
AH
AS
AH
AS
AH
AS
1.52 1.76 2.05
1.99 2.29 2.63
2.92 3.27 3.08 3.01 3.58 3.38 4.03 4.30 5.21 5.40 6.34 3.54
12.54 13.27 12.20 12.06 13.16 12.68 13.77 13.67 15.66 15.26 17.19 16.37
2.54 4.76 6.10
10.12 16.03 18.97
6.59
19.39
6.74 5.22 4.75
18.75 13.03 10.49
1.99
12.91
2.78
4.20
3.38 4.45 5.57 6.03 6.82
17.04 19.63 21.60 22.09 23.64
3.46 3.83 4.77
5.41 5.61 7.34
ANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
1103
LITERATURE CITED (1) D.C. Locke, J. Cbromatogr. Sci., 11, 120(1973). (2) A. Pryde. J. Chromatogr. Sci., 12, 486 (1974). (3) E. Grushka, Ed., "Bonded Stationary Phases in Chromatography", Ann Arbor Science, Ann Arbor, Mich. 1974. (4) E. Grushka and E. J. Kikta, Jr., Anal. Chem., 46, 1370 (1974). (5) M. Novotny, S. L. Bektosh, K. B. Denson, K. Grohmann, and W. Parr, Anal. Chem., 45, 971 (1973). (6) J. H. Knox and G. Vasvari, J. Chromafogr., 83, 181 (1973). (7) J. J. Kirkland, J. Chromatogr. Sci., 9, 206 (1971). (8) J. N. Little, W. A. Dark, P. W. Farlinger, and K. J. Bonbaugh, J. Chromafogr. Sci., 8 , 647 (1970). (9) 6. L. Karger and G. Sibley, Anal. Chem., 45, 740 (1973). (IO) R. E. Majors and M. J. Hopper, J. Chromatogr. Sci., 12, 767 (1974). (11) D.C. Locke, J. Cbromatogr. Sci., 12, 433 (1974). (12) R. K. Gilpin, J. A. Korpi, andG. A. Janicki, Anal. Chem., 47, 1498(1975). (13) J. H. Knoxand A. Pryde, J. Chromatogr., 112, 171 (1975). (14) J. Asshauer and I. Halasz, J. Chromatogr. Sci., 12, 139 (1974).
(15) K. Unger, Angew. Chem., lnt. Ed. Engl., 11, 267 (1972). (16) R . K. Gilpin and M. F. Burke, Anal. Chem., 45, 1383 (1973). (17) L. R. Snyder, J. Chromatogr. Sci., 7, 352 (1969). (18) L. R. Snyder, "Gas Chromatography 1970", A. Stock and S. G. Perry, Ed., Elsevier. London 1971, pp 81-111. (19) J. A. Schmitt, R. Altenny, R. C. Williams, and J. F. Diekman J. Chromatogr. Sci., 9, 645 (1971). (20) R . E. Majors, ref 3, p 139. (21) A. Diez-Cascon, A. Serra, J. Pascual, M. Gassiat, and J. Albaiges, J. Chromatogf. Sci., 12, 559 (1974). (22) D. C. Locke, Queens College, New York. N.Y., private communication, 1976. (23) R. P. W. Scott and P. Kucera, J. Chromatogr. Sci., 12, 473 (1974). (24) M. J. Telepchak, Chromatographia, 6, 234 (1973).
RECEIVEDfor review December 1,1975. Accepted March 19, 1976.
High Pressure Liquid Chromatography of p-Methoxyanilides of Fatty Acids Norman E. Hoffman* and John C. Liao Todd Wehr Chemistry Building, Marquette University, Milwaukee, Wis. 53233
Saturated and unsaturated fatty acids were quantitatively converted to p-methoxyanilides to enhance their uv detectability. Rapid methods for the synthesis of the anilides with triarylphosphine reagents were developed. The p-methoxyanilides were separated by reverse phase chromatography using I O + partlcles packed in a 30-cm long column. Wateracetonitrile and water-methanol were used in hyperbolic gradient elution.
Recently, there has been interest in developing high pressure liquid chromatographic (HPLC) methods for the determination of long chain fatty acids. Durst et al. ( 1 ) have thoroughly reviewed these recent developments and pointed out the need to derivatize these acids in order t o enhance their detectability. Amides have not been used as derivatives. p-Methoxyanilides of fatty acids have a maximum in their uv spectrum a t 254 nm. These anilides also have high absorptivity a t this wavelength used in most uv detectors for HPLC. The purpose of this paper is to report a study of the preparation and chromatography of a large number of long chain saturated and unsaturated fatty acid p-methoxyanilides. Two rapid methods were developed to convert these acids to pmethoxyanilides, one using triphenylphosphine (2, 3 ) and carbon tetrachloride to prepare an intermediate acyl chloride and one using carbon tetrachloride and a reagent containing a diphenyl phosphino group bonded to a polymer ( 4 ) .Gradient elution HPLC of these anilides was studied with two solvent combinations, water and acetonitrile or methanol.
EXPERIMENTAL Apparatus. A model ALC 202 liquid chromatograph equipped with a model 660 solvent programmer, model 6000 pumps, and a model 400 loop injector (Waters Associates, Milford, Mass.) was used. This chromatograph has a uv detector that was operated at 254 nm. Throughout all the work reported here, the chromatographic solvent flow rate was 1ml/min. A %-in.0.d. by 30 cm K-Bondapak CIS column (Waters Associates) was used. The column's plate number was 3000 measured with p-methoxylauranilide and a solvent of 83% acetonitrile and 17% water. 1104
ANALYTICAL CHEMISTRY, VOL. 48, NO. 8, JULY 1976
Gradient elution was started at 100%water and continued to 100% organic solvent (acetonitrile or methanol) in a hyperbolic manner. "Curve 2" of the Waters programmer was used. The time setting for the program was 40 min. Reagents. All reagents were used as purchased without further purification. Saturated straight chain fatty acids c14, CIS, c16, CIS, C20, C22, C24, and unsaturated fatty acids palmitoleic (Clc:~),oleic CIS:^), linolenic (C18:3),arachidonic (CZO:~), erucic ( C l ~ l )linoleic , (CZZ:~), 4,7,10,13,16,19-docoshexaenoic (C22:6),nervonic (c24:1)acids were purchased from Applied Science Laboratories, State College, Pa. Straight chain saturated fatty acids c6, cs, (210, Cl2, carbon tetrachloride, chloroform,ethyl acetate, triphenylphosphine, chlorodiphenylphosphine, p-methoxyaniline, N,N'-dicyclohexylcarbodiimide, spectrophotometric grade methanol and acetonitrile were obtained from Aldrich Chemical Co., Milwaukee, Wis. Polystyryldiphenylphosphine reagent was synthesized ( 5 ) from cross-linked polystyrene (2% divinylbenzene, 200-400 mesh) obtained from Bio-Rad Laboratories, Richmond, Calif. Phosphorus analysis showed 79% of the phenyl rings of the polymer were substituted with diphenylphosphino groups. Carbon tetrachloride and ethyl acetate were dried over Linde Type 4A molecular sieves (ALFA Products, Beverly, Mass.). Derivative Formation. a.Triphenylphosphine Method. A mixture of fatty acid, 5-10 mg of each (about 0.7 mmol total acids), 0.5 g (1.9 mmol) triphenylphosphine and 2 ml dry carbon tetrachloride were placed in a 15-ml vial. The vial was sealed with a Teflon-lined screwcap and placed in an 80 "C oil bath for 5 min. Cloudiness appears when the reaction is complete. After withdrawing and cooling to room temperature, the vial contents were mixed with 0.5 g (4.1 mmol) of p-methoxyaniline dissolved in 8 ml of dry ethyl acetate. The vial was returned to the oil bath (80 "C) for 1h. An insoluble oil appears a t this point but, because it is insoluble, it has no effect on chromatography. If the temperature of the bath is raised to 140 "C, the first step can be completed in 2 min and the second step in 20 min. b. Polystyryl-Diphenylphosphine Method. A mixture of fatty acids as in a.,1.4 g (4.4 mmol phosphorus) of polystyryl-diphenylphosphine reagent, and 2 ml of carbon tetrachloride was placed in a 15-ml vial sealed with a Teflon-lined screwcap. The vial was heated for 5 min at 80 OC in an oil bath with gentle shaking. The vial was withdrawn from the oil bath, cooled to room temperature, and a solution of 0.5 g (4.1mmol) of p-methoxyaniline in 8 ml of ethyl acetate was then added. The vial was returned to the oil bath (80 "C) for an additional 10 min of heating. If the temperature of the bath is raised to 140 OC, the first step can be completed in 2 min and the second step in 3 min. c. N,N'-Dicyclohexylcarbodiimide(DCC)Method. This method was used to prepare the anilides for identification purposes. Because it is an established method, the products were used to compare
