1032
Anal. Chem. 1987, 59, 1032-1039
Effect of Mobile Phase Composition and Highly Fluorinated Bonded Phases on the Apparent Free Energy of Transfer of Solute Functional Groups P a u l C. Sadek,*’ Peter
W.Carr, and Michael J. Ruggio
Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455
The purpose of the present work was to study in detail the chromatographic characterlstlcs of three types of highly fluorinated bonded-phase columns and one analogous hydrocarbonaceous phase in a variety of methanol/water mobile phases. The use of a 4bmember, multHunctlonaisolute set enabled the overall dlrect comparison of these four bonded phases. Partlcular attention was glven to the Characterization of the analogous fiuorlnated/hydrocarbon bonded phases. These two columns were studied with respect to thelr retention of members of an homologous serles (Le., through a methylene increment) and a wide variety of functional groups and their overall chromatographic stabllity in a strongly basic mobile phase (0.01 M sodium hydroxide in 50150 (v/v) methanoVwater).
Many nonchromatographic methods have been used to monitor the dynamics of reversed-phase HPLC supports and their interactions with solvents and solutes. These interactions have been probed chromatographically, primarily through the study of the retention of homologues (1-5). An objective of both types of studies is to interpret the effect of solvent composition on bonded-phase conformation and understand the influence of solvent-induced conformational changes on solute retention. The methylene increment, which is obtained by the study of a homologous series, is a very important parameter for comparing the relative hydrophobic character of various bonded phases. The methylene increment can discriminate the relative hydrophobicity of different bonded phases. The bonded phase is, of course, only one component of the packing material which can affect the overall retention process. Underivatized silanol groups are a second and very important contributor to the retention of hydrogen bond donor and acceptor solutes-in particular, amines. The complete quantitative deconvolution of the hydrophobic from the silanophilic retention effects has yet to be accomplished, but from studies of solutes with a wide range of functional groups (6, 7) it is a certainty that silanophilic interactions are important. Finally, the stationary phase modification via the adsorption of mobile phase components (or conversely, the solvation of the bonded-phase moiety) is also an important factor which can influence solute retention (8, 9). The purpose of the present work was to study in much greater detail than in our preliminary communication (see ref lo), under identical conditions, the chromatographic characteristics of three fluorinated phases (heptadecafluorodecyl, HFD; heptafluoroisopropoxypropyl,HFIPP; perfluorophenyl, PFP) and one hydrocarbonaceous analogue (decyl, Cl0) over a wide range of methanol/water compositions. Two homologous series (n-alkylbenzenes and n-alkylphenones) enabled To whom correspondence should be addressed: Department of Chemistry, Hope College, Holland, MI 49423.
us to study the resulting methylene increments in depth. In addition, a total of 42 solutes, representing the most commonly encountered functional groups, allowed for a more complete characterization of packing materials. The stability of the HFD and Clo columns was also studied in alkaline mobile phases. Surprisingly, it was found that the fluorinated phase was markedly less stable toward base, as indicated by the shorter contact times necessary to cause the sudden changes in the k ’values of benzene, toluene, pyridine, and nitrobenzene (as k’ plotted vs. solvent contact time). EXPERIMENTAL SECTION Chromegabond decyl (Clo)(10 pm, 60 A) and Chromegabond heptadecafluorodecyl (HFD) (10 pm, 60 A) packing materials were obtained from ES Industries (Marlton, NJ). Both packing materials were upward slurry packed into 5 cm X 4.6 mm column blanks; the Clo was packed from 100% methanol and the HFD from 100% tetrachloromethane. The HFIPP and PFP phases were prepared by the reaction of the appropriate dimethylchlorosilane obtained from Petrarch Systems (Levittown,PA) with LiChrosorb Si-60 10-pm silica from E. Merck (Darmstadt, FRG) in dry toluene. These were suspended in trichloromethane-isopropyl alcohol (9O:lO) and packed from 100% methanol. Methanol, 2-propanol, biphenyl, and uracil were obtained from MCB Manufacturing Chemists (Cincinnati, OH); N,N,N’,N’tetramethylethylenediaminewas from Chemical Dynamics (South Plainfield, NJ); anisole, iodobenzene, aniline, p-hydroxybenzoic acid, 1,4-dihydroxybenzene, p-cresol, o-cresol, m-cresol, pbromotoluene, p-nitrobenzyl chloride, 2-picoline,p-nitrobenzyl bromide, p-dichlorobenzene, benzyl alcohol, phenol, p-nitrotoluene, 2,2,2-trifluoroethanol, 1,1,1,3,3,3-hexafluoroisopropyl alcohol, benzonitrile, acetophenone, fluorobenzene,chlorobenzene, bromobenzene, pyridine, ethanol, anthracene, ethylbenzene, npropylbenzene, n-butylbenzene, tert-butylbenzene, and naphthalene were from Aldrich (Milwaukee, WI); benzylamine, nitrobenzene, p-nitrobenzoic acid, p-aminobenzoic acid, p-nitrophenol, p-aminophenol, o-nitrotoluene, and p-hydroxybenzaldehyde were from Eastman Kodak (Rochester, NY); benzaldehyde was from Mallinckrodt (Paris, NY); benzene and p ethylphenol were from Baker (Phillipsburg,NJ); benzoic acid (No. 428443-p) was from the National Bureau of Standards (Washington, DC); p-chlorobenzoic acid was from BDH (Poole, U.K.); and a series of n-alkylphenones were from Pierce Chemical (Rockford, IL). Support degradation studies were performed on the HFD and Cl0 packing materials. A 50/50 methanol/water (v/v) mobile phase was made alkaline by the addition of 0.01 M sodium hydroxide (pH* 12) to accelerate column breakdown. The test solutes (benzene, toluene, nitrobenzene, and pyridine) used in this study were injected periodically and their capacity factors determined in order to monitor the time-dependent changes in the retentivity of the columns. Uracil was used throughout as a void volume marker. RESULTS AND DISCUSSION Stationary Phase: Methylene Increment Studies. In order to define the methylene increment, a series of compounds in which strong silanophilic, strong hydrogen bonding, and strong dipole-dipole or induced dipole interactions are excluded should be examined. The mobile phase composition
0003-2700/87/0359-1032$01.50/0CZ 1987 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 7, APRIL 1, 1987
Table I. Least-Squares Analyses of the Plots of In
k ~ l ~ y l ~ nvs. zsn Carbon sl
1033
Number
-RT In kLh MeOH”
nb
70 60 50 40 30
5 5 5 5 5
70 60 50 40 30
Cld
Pe
Arf
0.18 0.33 0.39 0.48 0.56
0.02 0.01 0.03 0.03 0.04
0.998 0.999 0.999 0.999 0.999
-107 -194 -229 -283 -329
5 5 5 5 5
0.245 0.33 0.44 0.56 0.68
0.01 0.01 0.02 0.03 0.04
0.999 0.995 0.999 0.999 0.999
-145 -197 -261 -329 -400
70 60 50 40 30
5 5 5 5 5
0.32 0.44 0.55 0.68 0.79
0.01 0.02 0.02 0.03 0.15
0.999 0.999 0.999 0.999 0.989
70 60 50 40
5 5 5 3
0.46 0.59 0.70 0.81
0.02 0.02 0.02 0.01
0.999 0.999 0.999 0.999
mc
A
B
cl
y intg
f9 f5 f16 f16 f23
-1.92 -1.33 -0.80 -0.75 -0.49
+1134 +788 +474 +444 +292
f23 f12 f40 f40 f55
+1143 +782 +477 +455 +277
f6 f6 f12 f20 f21
-1.33 -0.74 -0.16 +0.23 +0.73
+787 +438 +97 -141 -429
f15 f16 f28 f48 f53
+791 +431 +91 -156 -444
-189 -259 -326 -404 -465
f5 f9 fll f17 f91
-0.78 -0.01 +0.58 +1.10 +1.65
+459 +8 -330 -648 -975
f12 f22 f26 f42 f224
+460 +3 -349 -662 -994
ClO -274 -349 -416 -480
f9 A10 f9 15
+0.01 +0.76 f1.44 +1.91
-5 -452 -853 -1175
f24 f25 f24 f7
0 -452 -848 -1176
PFP
HFIPP
HFD
“Percent methanol in the mobile phase; flow rate, 1 mL/min. bTotal number of data points used in generating the line. ‘Slope of the least-squares line. dConfidencelimits at the 90% level. e Correlation coefficient. f Apparent free energy of transfer for a methylene group (cal/mol) (see eq 1). gThis is the regression calculated value for In k;, where qi is benzene. hThis is the free energy of transfer for benzene. A is calculated from the y intercept and B is the chromatographically observed value. was fixed, since in reversed-phase chromatography it has a dominant effect on retention. Two homologous series of solutes were chosen: the n-alkylbenzenes and the n-alkylphenones. These solutes allow the measurement of an important property of a reversed-phase support, namely, the methylene increment, which is the ability of the chromatographic system to discriminate between molecules which differ by a single methylene group. This property is also referred to as the relative hydrophobicity or hydrophobic selectivity of the column. In order to quantify the effect of the addition of a methylene group on solute retention, a substituent factor was defined as follows:
AF = -RT In [ k : + , / i t : ]
I
,
eC’O,e
HFD
(1)
where R is the gas constant (cal/(mol K)) and T i s the temand k: refer to the capacity perature (K). The terms factors of adjacent members of a homologous series. This equation was written by analogy to the relationship between k’ and the free energy of transfer of a solute In k ’ = In q5 - AGo/RT
0.9
(2)
The notation A r was used to reflect the fact that eq 2 is strictly valid only when a single process governs solute retention. As shown below, the A r value for all adjacent pairs of solutes is essentially constant (statistically) and therefore can be used to represent the average effect of addition of a methylene group to the free energy of transfer of this group. The retention volumes of a series of n-alkylbenzenes were examined in this fashion on all four packing materials under identical mobile phase conditions. The results are summarized in Table I. (Figure 1 shows the composite methylene increment plot vs. mobile phase.) These plots are reasonably linear; the lowest correlation coefficient obtained was 0.9897. The AF values are decidedly different in the strong eluents, but tend toward a similar value as the mobile phase becomes
70
60 50 40 % methanol
3C
Flgure 1. Plot of d[ln k’]/dncH2vs. % methanol in the mobile phase for the four bonded phases.
weaker (i.e., water rich). We believe that this is a direct consequence of the increasingly dominant role played by the mobile phase in establishing solute retention. Relative to the hydrocarbonaceous column, the three fluorocarbon columns have lower selectivity toward a methylene group (see slopes in Table I). Consequently, the apparent free energy of transfer for a methylene group is more positive (less favored) for all of the fluorinated phases relative to the Clo phase. Inspection of Table I shows that the slopes of plots of log K’vs. C, are strikingly similar for the Clo, HFD, and HFIPP columns relative to the PFP column, which has a distinctly lower slope. The similarity of the first three slopes and the lower slope of the PFP column persists at other eluent
1034
ANALYTICAL CHEMISTRY, VOL. 59, NO. 7, APRIL 1, 1987
Table 11. Least-Squares Analyses of In k’vs. C, Plots for n-Alkyiphenones 90 methanol”
n
slope
90 80 70 60 50 40
9 9 9 8 6 6
0.151 0.229 0.328 0.445 0.578 0.683
100 90 80 70 60 50 40
9 9 9 8 8 6 5
0.172 0.266 0.369 0.480 0.610 0.752 0.870
Clb
PC
Ard
Clb
0.999 0.999 0.999 0.999 0.999 0.999
-89 -135 -194 -263 -342 -404
*I
0.999 0.999 0.999 0.999 0.999 0.999 0.999
-102 -158 -218 -284 -361 -445 -515
HFD column 0.002 0.002 0.002 0.003 0.011 0.010
fl fl f 2 *7 i6
Clo Column 0.003 0.003 0.003 0.005 0.009 0.012 0.019
i 2 12 i 2 f3
14 f7 ill
“Percent methanol in water/methanol mixture; flow, 1 mL/min. free energy of transfer for a methylene group; cal/mol from eq 1.
Confidence limits at 90% level. Correlation coefficient. dApparent
compositions. For many compositions, the three columns have the same slope within the 90% confidence limits. This is to some extent due to the statistical computation of the uncertainty in the slope. The average A r values, whose confidence limits are tighter, do reveal that these three columns do not have the same methylene increment. There are real differences between the columns, the sequence of columns in order of decreasing methylene group selectivity being Clo > HFD > HFIPP >> PFP. The fact that the four columns have different methylene group selectivities is supported by the data of Table 11,which was measured on the Clo and HFD columns. These data were obtained from log k’ values for a minimum of five and a maximum of nine n-alkylphenones. The larger number of solutes and the concomitant larger range in k ’values ensured a more precise measurement of the slope. The slopes in Table I1 are in good agreement with those listed in Table I; i.e., both the n-alkylbenzenes and the n-alkylphenones yield the same methylene increment on the same column. Now, a statistically significant (at the 90% confidence interval) difference in the apparent free energies of transfer for the methylene groups can be seen for the Clo and the HFD columns, even for the strongest solvent employed in the data of Table I1 (90/10 methanol/ water). When the A r c H , values for the HFD column were plotted vs. the for the Clo column, the variable being the methanol composition of the mobile phase, a straight line resulted whose least-squares slope was 0.89. This indicates that the apparent free energy of transfer of a methylene group on the HFD phase is 89% of the value obtained for the C,,, irrespective of the eluent composition. In order to more rigorously compare k ’values on two different stationary phases in the same mobile phase, a less intuitive approach is adopted below. We consider that two chemically and physically different columns are to be compared by examining the retention of a number of test solutes on both columns. For a reference solute (such as benzene), its distribution coefficient can be related to the difference in its chemical potential in the mobile and stationary phases as follows:
We now consider the effect of addition of a substituent (e.g., -CH3) to the reference solute. The standard-state chemical potential of the substituted reference solute (SRS) in the stationary phase can be written as
Keq = exp(-AM/RT) Mm
R
where ps* is the standard-state chemical potential of the SRS and u is the magnitude of the “perturbation”factor generated by the substituent. A similar equation can be written for the solute’s mobile-phase standard-state chemical potential Pm
* = PmR +
(g)g
where F ~ *is the mobile phase SRS chemical potential. Consequently, eq 4 can be rewritten through the use of eq 5 and 6
Equation 7 will describe any column/solvent/solute system. The numerical subscripts 1 and 2 used in the remainder of this section correspond to the Clo and HFD columns, respectively. Substitution of eq 3 and 7 into eq 1 and 2 yields
k,’ =
(3)
where
& = PsR -
(5)
(9)
(4)
here gsRand pmRare the standard-state chemical potentials of the reference solute in the stationary and mobile phases, respectively.
It should be noted that pLm,lR= pL,,zRand dFm,lR/du = dpm,zR/duin all cases since the mobile phases are identical. In addition for convenience dks,lR/du= as,l,dps,2R/du as,2, and dpm,2R/du= = am,2.
ANALYTICAL CHEMISTRY, VOL. 59, NO. 7, APRIL 1, 1987
Now consider the case where the phase ratios d1 # 4, and R # p,,,?, but a,,l may or may not equal as,2. With these assumptions and writing the logarithmic form of eq 8 and 9, we obtain
o.81
1035
0 0
.
a 0
a
0.7
0 0
The first two terms are constant and independent of u. Now allow a,,, to equal a,,,. Substitution of eq 10 into eq 12 yields C; when a,,l = as,2 (12) In k,’ = 1 In k,’
+
or
Itl’ = Ak,’ + 0 Therefore, a plot of k,’ vs. k,’ will be linear with a slope other
than unity, but the plot will have a zero intercept. A linear plot of k,’ vs. k2/ will be obtained as the substituent varies only if the substituent induced change in the standard state chemical potential of the solute in the two stationary phases is the same. This does not mean, however, that the addition of a substituent has no effect on k’. When all three parameters assume different values, i.e., 41 R , and as,l # as,2,eq 10 and 11can be rewritten # 42, I . L ~ # ,I~ as In Itl’ = AI (a,,l - am)a (14)
+
In k,’ = Az Solving eq 15 for In k,’ = A , -
(
u
+ (as,2- a,)u
(15)
and substituting into eq 14 give
=as,^) A-, am
or In k,’ = Al
+ (e) as,, - am In k,’ (16) + BA, + C In k,’
(17)
Now consider the solute pair benzene and toluene where CI = -CH3. For reversed-phase HPLC, one expects the change in the standard-state chemical potential of the reference solute created by the addition of the substituent to be much greater in the mobile phase than in the nonpolar stationary phase, or as,,. If this is the case, the term C will i.e., a , >> approach unity. Therefore, when introduction of a substituent into a reference solute causes differentially insignificant changes in the stationary phase chemical potential of that solute, plots of k i vs. k,’ MUST BE LINEAR since C MUST BE UNITY. Thus linearity in a plot of k,’ vs. kl’ does not require that a,,l = as,,. These stationary phase interaction terms need only be small relative to the mobile phase interaction terms in order for a plot of k,’ vs. 12,’ to be linear. The fact that plots of log k \+,/k \ vs. volume percent water on the above three columns are similar (albeit not equal), whereas the PFP column is definitely smaller, can be explained most simply by assuming the a , is considerably larger than a, for the Cl0, HFD, and HFIPP phases, but the interaction coefficient in the stationary phase (a,) is on the same order of magnitude as the interaction coefficient in the mobile phase (a,) for the PFP stationary phase. The issue then is whether or not a, is large compared to a,. Clearly, the absolute value of the slope of plots of log k,’ vs. log k,’ cannot be used to ascertain the relative size of a, and a , since this slope is a consequence of three parameters (a,,,, as,,, and a,)-not just two parameters. However, we can estimate the relative magnitudes of a, to a,,l or a,,, by examining the trend in the term C in eq 17 as the mobile phase is gradually made weaker (i.e., more water is added). When mobile phase interactions
I
a
o
alkylbenzenes alkylphenones
‘1a
0.5
40
20
60
%ti20
Flgure 2. Plot of the slopes generaged by In kTHFD vs. In ktclo(Le., C) vs. % H,O in the mobile phase. Flow rate is 1 mL/min.
are entirely dominant, the term C must approach unity. Figure 2 shows the variation in the slope of plots of log k hFd vs. log k ~ ,as, the water content of the mobile phase is varied. The database is comprised of the retention factors for both the n-alkylbenzenes and the n-alkylphenones. There is no question that the slope increases as the solvent is weakened. At 60% water, the C term has achieved a value of 0.8 and is still rising. Due to the very considerable curvature in the plot shown in Figure 2, it is not readily possible to estimate the asymptotic value of C. It is clear that the monotonic increase in C with an increase in the water content of the mobile phase indicates that the mobile phase is becoming increasingly dominant in controlling retention. However, the fact that the slopes are definitely less than unity at all eluent compositions unambiguously indicates that solute interactions with the stationary phases are very important factors controlling retention in reversed-phase chromatography. We can be assured that this is true because if the terms u , , ~and us,,were small compared to a,, then C would have to be unity at all mobile phase compositions. The data of Figure 2 clearly demonstrate that this is not the case. Although the comparison of two bonded-phase columns carried out above does not allow a quantitative evaluation of the contribution of the mobile and stationary phase contributions to retention, it does support the concept that solute-stationary phase interactions are very important in reversed-phase HPLC. Clearly, the stationary phase plays an active role, the extent of which changes substantially with mobile phase composition. These comments pertain to retention and not necessarily to selectivity. Selectivity differences can be detected when highly fluorinated phases are compared to hydrocarbonaceous phases due to the very significant differences in the chemical and physical properties of these ligands. Differences in selectivity will be much smaller when both stationary phases are hydrocarbonaceous, e.g., C8 vs. CU. Stationary Phase: Functional G r o u p Analysis. In order to examine the effect of a substituent on retention, a substituent factor similar to that described for the methylene increment in eq 1 was employed:
A r x = -RT In ( k : / k $ )
(18)
where k r X and k b are the capacity factors of a substituted
1036
ANALYTICAL CHEMISTRY, VOL. 59, NO. 7, APRIL 1, 1987
Table 111. Apparent Free Energy of Transfer for Substituents on Clo Column with Percent Methanol in Mobile Phase" Xb
3
-I -CH3
-CH,-c1 -Br -F
70% MeOH
60% MeOH
-798 (1,a)
-952 (1,a)
-1337 (1,a)
-1503 (1,a)
-544 (2,a)
-615 (2,a)
-839 (2,a)
-982 (2,a)
-475 -255 -169 -168 -142
-597 -325 -219 -219 +42
-769 (3,a) -371 (4,a) -261 (6,a) -283 (5,a) +55 (9,d) -54 (7,d) +57 (10,c) -25 (8,b) +479 (11,d) +484 (12,d) +789 (13,d) +924 (14,d) -82 4.0 x 105 -1151
-909 (3,a) -421 (4,a) -315 (5,a) -362 (5,a) +230 (10,d) -56 (7,d) +9 (9,d) -163 (7,a) +415 (11,d) +396 (12,d) +728 (13,d) +940 (14,d)
(3,a) (4,a)
(5,a) (6,a)
-NO1
-C(=O)CH,
(7,a)
+433 (11,d) +459 (12,d) +720 (13.d) +801 (14,di +1 2.2 x 105
-C(=O)H -CN
-OH Arc
s?d
p r
(4,a)
(5,a) (6,a) (10,c) -15 (8,d) +33 (9,b) -89 (7,a) +454 (12,a) +343 (11,d) +745 (13,d) +849 (14,d)
+11 (8,d) +53 (9,c) +80 (10,a)
-OCH3
(3,a)
-34 2.7 x 105 -474
+6
50% MeOH
40% MeOH
-142 4.6 x 105
-1993
"All data were obtained through the use of eq 18. Column was 5 cm X 4.6 mm, flow rate was 1 mL/min. Units are in cal/mol. *Functionalgroup. "Average A r value for all X. dTotal variance from the column mean.
+1100
1
+goo'
'
0
+
0 +
I F)+700*
e
0
8
oclo
e
:HFD HFIPP
1
+ 300
I 30
b
50 %H20
70 %H20
Figure 3. Plot of Ar (cal/mol) for the hydroxy and phenyl functional groups vs. % H,O in the mobile phase. Flow rate is 1 mL/min. Columns are denoted as follows: PFP (X): HFIPP (0):HFD (+): C,, (0).
benzene and benzene, respectively. Tables 111-VI list the Ar, values for various mobile phase compositions. The functional groups are listed in order of decreasing favorability of transfer from the mobile phase to the stationary phase (relative to benzene). The uncertainty in Ar, for a given substituent can be estimated through the equations of error propagation. For the values used, the standard deviation in ArXvaries from 130 for the least retained species (AT, = 940) to 2 for the most retained species (Ar, = -1500). The effect of mobile phase composition on the free energy of transfer of a substituent is remarkable and most dramatically shown in the APx values for the hydroxy and phenyl
functional groups (Figure 3). The phenyl group substituent effects are extremely dependent on the mobile phase composition, as evidenced by the very steep positive slope obtained with all four columns. In contrast, the A r of the hydroxy group is virtually independent of the mobile phase composition. Apparently, the increase in the concentration of hydrogen bond donors in the solvent (water) and the increase in cavity formation energy in the eluent are reflected in the very positive ATI-OH values, as well as in the nearly constant values per se. The numerical indexes to the right of the AT, values in Tables 111-VI denote the order of decreasing favorability of transfer in 70130 methanol/water. Juxtapositions in the order
ANALYTICAL CHEMISTRY, VOL. 59, NO. 7, APRIL 1, 1987
1037
Table IV. Apparent Free Energy of Transfer for Substituent on HFD Column with Percent Methanol in Mobile Phasea X
3
-I -CH3 -CH2-
-c1
-Br -F -0CH3 -NO2 -C(=O)H3 -C(=O)H -CN -OH
E
S2
Dr
70% MeOH
60% MeOH
50% MeOH
-336 (1,~)
-527 (1,b)
-778 (1,b)
-141 ( 3 , ~ )
-247 ( 2 , ~ )
-493 (2,c)
-40 ( 7 , ~ ) -215 (2,b) -96 (6,b) -141 (4,b) +77 (8,d) -106 (5,a) +112 (10,d) +81 (9,b) +251 (11,~) +308 (12,b) +436 (13,c) +752 (14,c) +67 8.4 x 104 +942
-153 ( 5 , ~ ) -250 (2,b) -103 (7,b) -190 (4,c) +80 (9,d) -119 (6,a) +127 (10,d) +54 (8,c) +242 (11,~) +362 (12,c) +505 (13,~) +849 (14,c) +45 1.3 x 105 +630
-278 (4,d) -303 (3,b) -138 (7,b) -273 (5,b) +41 (9,c) -146 (6,a) +97 (10,d) +38 (8,d) +232 (11,~) +306 (12,c) +487 (13,c) +853 ( 1 4 , ~ ) -25 1.7 x 105 -355
40% MeOH -1034 (1,b) -617 ( 2 , ~ ) -463 ( 3 , ~ ) -379 (5,b) -210 (6,b) -381 (4,b) -136 ( 3 , ~ ) -205 (7,a) -1 (10,b) -15 (9,d) +75 (11,c) +241 (12,c) +411 (13,~) +878 (14,c) -131 2.2 x 105 -1836
30% MeOH -1440 (1) -913 (2) -604 (3) -527 (4) -275 (6) -472 (5) -236 (8) -267 (7) -63 (10) -128 (9) +63 (11) +87 (12) +316 (13) +927 (14) -252 3.1 x 105 -3532
"All conditions and abbreviations are the same as in Table 111. The only difference is the bonded phase itself: here it is HFD. Table V. Apparent Free Energy of Transfer for Substituent on HFIPP Column with Percent Methanol in Mobile Phasea X
-I -CH3 -CH2-
-c1
-Br
-F -OCH3 -NO2 -C(=O)H3 -C (=O) H -CN
-OH Ar S2
Car
70% MeOH
60% MeOH
50% MeOH
-285 (1,d)
-491 (1,d)
-690 (1,d)
-933 (1,d)
-1184 (1)
-129 (3,d)
-235 (2,d)
-412 (2,d)
-564 (2,d)
-749 (2)
-40 (7,d) -161 ( 2 , ~ ) -70 ( 5 , ~ ) -83 (4,d) +69 (9,c) -44 (6,b) +50 (8,a) +128 (10,c) +198 (11,b) +429 (13,b) +320 (12,c) +713 (14,b) +78 7.0 x 104 1095
-133 (5,d) -209 ( 3 , ~ ) -101 ( 6 , ~ ) -137 (4,d) +10 (8,b) -66 ( 7 , ~ ) +63 (9,c) +69 (10,d) +161 (11,b) +262 (12,b) +338 (13,b) +725 (14,b) +18 8.7 x 104 +256
-281 ( 3 , ~ ) -251 ( 4 , ~ ) -135 ( 6 , ~ ) -187 (5,d) -72 (8,b) -113 (7,b) +3 (9,b) +26 (10,~) +114 (11,b) +163 (12,b) +310 (13,b) +695 (14,b) -59 1.1 x 105 -830
+
40% MeOH
-421 (3,d) -285 (4,d) -180 ( 6 , ~ ) -227 (5,d) -155 (7,b) -141 (8,b) -50 (9,b) -16 (10,~) +2 (11,b) +lo2 (12,b) +184 (13,b) +704 (14,b) -138 1.4 x 105 -2684
30% MeOH
-534 (3) -323 (4) -223 (6) -282 (5) -190 (7) -152 (8) -118 (10) -96 (11) -122 (9) +7 (12) +154 (13) +705 (14) -222 1.9 x 105 -3107
"All conditions and abbreviations are the same as in Table 111. The only difference is the bonded phase itself; here it is HFIPP.
of these indexes show that the elution sequence changes with mobile phase composition. Changes in elution order could be quite beneficial when a separation is optimized on a given stationary phase. In order to classify variations in retention of a substituent with respect to the bonded phase, a letter index was assigned: the most retained (a) through the least retained (d). For nonpolar substituents, the Clo bonded phase is most retentive, followed in general by the PFP, HFD, and finally the HFIPP bonded phase. In contrast, highly polar groups invariably have least affinity for the Clo phase. The PFP phase is usually the most retentive for the more polar substituents. Overall, the PFP phase is quite retentive toward both nonpolar and polar functional groups. This conclusion is substantiated by the very negative sum of the Ar, values (ZAI'). However, based on the total variance (9) in the AI', values from each column mean, the Clo column is in general the most selective column. Although the retention of solutes with extensive a systems (e.g., biphenyl, naphthalene, anthracene) is relatively impervious to the presence of silanophilic groups, Tables I11 and VI show that both the Clo and PFP columns exhibit quite strong retention of both phenyl and fused ring substituent
solutes. The retention mechanisms on these phases are likely quite different. These multiring solutes cannot completely overlap with a single perfluorophenyl moiety since the solute is larger than the bonded group. Consequently, strong T-T interactions may play a major role in retention on the PFP bonded phase. Increased retention has been found for highly conjugated aromatic solutes when phenyl and naphthyl bonded phases were studied (11). Packing Material Stability Study. The vulnerability of reversed-phase supports to attack by bases is a well-documented liability of silica-based liquid chromatographic materials (12, 13). It has been shown that the solubility of silica gel in water increases by more than ZOO-fold in the pH range 5 C pH C 7 (14). The presence of salts in the eluent also leads to a decrease in column lifetime (15). Wehrli et al. (12),studied the rate of dissolution of silica as determined from the Si concentration (via atomic absorption spectroscopy). They found that the nonderivatized silica dissolved a t a far greater rate than did bonded-phase material. The length of the bonded-phase silane ligand was also quite significant, e.g., C,, bonded silica dissolved much less rapidly than did C8 bonded silica. Their experiments were
1038
ANALYTICAL CHEMISTRY, VOL. 59, NO. 7, APRIL 1, 1987
Table VI. Apparent Free Energy of Transfer for Substituent on PFP Column with Percent Methanol in Mobile Phase”
x
1-
I* -I
70% MeOH
60% MeOH
SO’% MeOH
-399 (1.b)
-520 (1,c)
-761 ( 1 , ~ )
-221 (2.b)
-426 (2,b)
(3,b) (5,d)
-169 -131 -27 -117
-CH3
-CH2-
-c1
--Br -F -OCH, -NO*
(3,b)
-320 -174 -72 -218
(4,d) (6,d) (5,~)
(4,b) -128 (6,a) -108 (7,b)
+11 (8,b) -24 (7,c)
+5l (9,b)
-89 (8,a) 0 (10,b) +l9 (l1,a)
+139 (11,d) +82 (10,a) +236 (12,a) +245 (13,a) +457 (14,a) +10 4.8 x 104 +143
--C(=O) H,
-C(=O)H -CN S1
>3 AI’
(9,d)
-106 (12,a) +212 (13,a) +473 (14,a) -49 6.5x i o 4 -1246
40% MeOH
30% MeOH
-1019 (1,c)
-1234 (1)
-585 (2,b)
-731 (2,b)
-875 (2)
-418 (3,b) -220 (5,d)
-554 (3,b) -288 ( 5 , ~ )
-661 (3) -321 ( 5 )
-104 (7,d)
-131 (9,d)
-262 -169 -91 -102 -37 -16
-332 ( 4 , ~ ) -243 (6,a) -138 (8,b) -149 (7,a)
(4,~)
(6,a) (9,c) (8,a) (10,a)
(11,a) +75 (12,a) +179 (13,a) +475 (14,a) -145 9.5x 104 -2036
-164 (10) -414 (4) -301 (6) -148 (11) -164 (9) -178 (8) -242 (7) -39 (12) +38 (13) +479 (14) -302 1.7x 105 -4221
-120 (11,b) -130 (10,a) +5 (12,a)
+112 (13,a) +477 (14,a) -231 1.3 x 105 -3241
‘All conditions and abbreviations are the same as in Table 111. The onlv difference is the bonded ahase itself: here it is PFP. HFD 6
A&
50lM 8 .
B- C C -
Imllmln A- melhanol E--- 50150 methanollwater C-..E+OOl E NaOH
mathanol/wstsr 001 NaOH toluene
7-
toluene 15
I
k’
rene
’tL ‘0
pyrldlne I
.
5
10
.
15
. .
20
25
I
30
.
35
.
40
o d = .
45
50
. 5S
time (hrl
0
I
0 1 0 1 0
10
5
15
20
time ( h r l
Figure 4. Plot of k’ vs. time after initiation of 0.0 1 M NaOH in 5060 methanoVwater pumping through the column. Flow rate is 1 mL/min.
Figure 5. Plot of k ’ v s . time after initiation of solvent A, B, or C pumping through the column. Flow rate is 1 mL/min.
conducted over times which ranged from 2 days to several weeks, but the first datum was not obtained until 25 h after exposure of the support to base. Unquestionably, their results reflect the qualitative results of the effect of base on the support. However, static studies of the type reported in their work underestimate the rate of base attack which will be observed in a dynamic study, Le., one in which the supernatant is continuously replenished. The present study of the stability of the HFD column was undertaken in part due to our expectation that these materials would be far more stable than hydrocarbon columns in view of the extremely low solubility of highly fluorinated nonpolar materials in water (16). Figure 4 shows a plot of the k’of a series of solutes vs. time of contact between the mobile phase and the bonded phase. Prior to exposure to the 50/50 methanol/water 0.01 M sodium hydroxide, the column had been equilibrated with methanol. The slow equilibration of the packing upon decrease in eluent strength is reflected in the very rapid rise in k’ at the origin of the plot. The first injection of solute was made less than 1 min after solvent change over. The slow decrease in k’continued for 42 h (more than 3600 column volumes). After this time, the k ’ decreased more rapidly. This was accompanied by significant pressure fluctuations with each pump stroke. These phenomena indicate
that both a structural breakdown of the support and modification of retention occur as the column deteriorates. Inspection of the column revealed that the first 0.25 cm of the packing material had dissolved. The uppermost portion of the remaining support material developed a hard crust. Apparently, the dissolved silica was swept downstream, and some reprecipitated on the support. Figure 5 shows a similar plot for a new HFD column. The first two hours (sections A and B) represent equilibration of the column with methanol and 50/50 methanol/water, respectively. The section denoted C indicates the introduction of 5 0 / 5 0 methanol/water-0.01 M sodium hydroxide into the column. Note the same general initial increase in k’ (see Figure 4) between the first injection and the later injections. Since both columns (Clo and HFD) show the same behavior, it is probable that this is a solvent effect which reflects the initial nonequilibrium of the column with the new mobile phase. Although the k ’ of pyridine remains roughly constant on the HFD column, the other solutes show a decrease in k’ during section C. Kohler and his co-workers observed a similar phenomenon when silanophilic solutes were compared t o nonsilanophilic solutes as a column was exposed to strong base (19). It may be that the k’ values of the nonpolar solutes decrease as bonded phase is stripped from the column, but the diminution in the k’of a silanophilic solute which should
Anal. Chem. 1987, 59, 1039-1043
occur is approximately compensated by an increase in silanophilic interactions as more bare silica becomes available. At about 18 h the k’appears to decrease more abruptly in coincidence with a rapid increase in column back pressure and pressure fluctuation with each pump cycle. This is a considerably shorter time than with the hydrocarbon analogue column. This difference in stability likely results from one of two factors. First, the HFD column has a lower carbon load (8.6%) than the Clo column (10%). Thus on average there is more bare silica open to attack by base. Second, the HFD ligand is far more rigid than the hydrocarbon ligand and it is very probable that it assumes a fully extended conformation. In contrast, the hydrocarbon may be collapsed, thereby forming a protective blanket over the surface.
CONCLUSIONS It is apparent that fluorinated bonded phases are much less retentive than the analogous hydrocarbon materials. In view of the great differences in chemical and physical properties of hydrocarbons and highly fluorinated materials (17), it is remarkable that the columns, particularly the Clo and HFD columns, are so similar and have very analogous chromatographic selectivities and that, as shown in other work, the solute parameters of most significance are their size and hydrogen bond basicity (18). We had anticipated that differences in dispersive interactions would be more significant, or at least more readily observable. The fluorinated phase retentivity increases in the sequence PFP < HFIPP
