Anal. Chem. 1991, 63, 2849-2852
2849
Studies of the Surface Composition of Phenyl and Cyanopropyl Bonded Phases under Reversed-Phase Liquid Chromatographic Conditions Using Alkanoate and Perfluoroalkanoate Esters R. K. Gilpin,* Mieczyslaw Jaroniec, and Shujywan Lin Department of Chemistry, Kent State University, Kent, Ohio 44242
Two polar bonded phases, phenyl-sllica and cyanopropyldllca, have been studied under reversed-phase conditions uslng homologues of akanoate and perfluoroalkanoate esters as test solutes. For comparative purposes a shllarly prepared n-butyl phase also has been studied under equivalent conditlons. Retention measurements have been carried out as a function of mobllephase compoeltlonfor water-methand and water-acetonltrlle and Incremental methylene (-CH,-) and perfluoromethylene (-CF,-) selectivities calculated. These latter data have been used to estlmate the thermodynamic constant K,, which describes the sorption equillbrlum of solvents between mobile and stationary phases assumlng Ideality of these phases. For water-methanol, differences In composltlon between the mobile and surface phases due to preferential Intercalationare small. However, the data indlcate a significant sorptlon excess of acetonitrile by the bonded phases. This preferential sorptlon of acetonitrile is greatest for alkyl phases and decreases wlth Increasing POlarity of the bonded phase (Le., cyano-silica < phenyl-dilca < alkyl-silica).
with differing hydroorganic mixtures (12, 13, 21-25). Unfortunately, agreement between published data is often poor even for commonly used binary hydroorganic eluents such as methanol-water and acetonitrile-water. The shapes of the sorption excess curves vary dramatically for a given type of reversed-phase material, and it is not uncommon to find reported excesses for each of the individual components under the same binary conditions (12,13,21-25). Previously, we have studied the surface-phase composition of octyl- and octadecyl-modified silica in contact with three different binary hydroorganic eluents (12,13). This was carried out chromatographically by measuring the incremental methylene (-CH,-) and perfluoromethylene (-CF,-) selectivities using alkanoate and perfluoroalkanoate ester homologues. In order to extend thiswork, the current investigation focuses on two commonly used polar bonded phases, cyanopropyl-silica and phenyl-silica. Values of the nonspecific -CH2- and -CF2- selectivities again have been determined using the same ester homologues a t varying compositions of water-methanol and water-acetonitrile. The resulting data have been used to estimate the thermodynamic constant, K, which describes the sorption equilibrium of solvents between mobile and stationary phases assuming ideality of these phases.
INTRODUCTION The role of stationary-phase effects in reversed-phase liquid chromatography (RPLC) has been debated often in the past, and a range of differing opinions have emerged about its importance. In some of the earliest retention models, such as those based on interaction indices, the mobile phase has been considered to be the key element controlling elution and the bonded phase treated as a passive acceptor of the solute (1-4).In other models, molecular interactions in the stationary phase have been recognized as important contributing factors (5-8). However, adequate descriptions of how these interactions are affected by intercalated solvents are often missing. Similarly, although the dependence of retention and selectivity on the chain length of alkyl bonded phases has been studied extensively (see refs 9-11 and the citations in ref 9),again the influence of intercalated solvents has been neglected. Recently, more quantitative treatments have been suggested (12-14)which consider both the competitive interactions that arise between the solute and solvents for a chemically modified solid as well as the solute-solvent and solvent-solvent interactions that occur in both the mobile and stationary phases (15-20). In doing this an accurate description of the stationary phase’s composition is needed since it may be significantly different than that of the eluent (12,13,Zl-25). Differences in composition between the mobile and stationary phases can be determined directly by measuring sorption isotherms (21,22,25)or indirectly from chromatographic measurements of nonspecific selectivity (26) as a function the eluent’s composition (12, 13). Both of these techniques have been used to study the intercalated solvent composition of octyl and octadecyl bonded phases in contact
EXPERIMENTAL SECTION Reagents. The ethyl alkanoate [CH&H2),COOC2Hs for m = 0-41 and methyl perfluoroalkanoate [CF3(CF2),,,COOCH3 for m = 0-21 esters were purchased from the Aldrich Chemical Co. (Milwaukee,WI) as were the (3-cyanopropy1)dimethylchlorosilane and phenyldimethylchlorosilane.The n-butyldimethylchlorosilane and HPLC grade solvents, methanol and acetonitrile, were obtained respectively from Huls America (Piscataway, NJ) and Fisher Scientific (Pittsburgh, PA). The HPLC grade water was purified in-house using a Millipore (Milford,MA) Model Mill-Q deionization syatem. All mobile phases were prepared from these solvents on a v/v basis and degassed before use. Chromatographic Packings. The chromatographic packings were synthesized as follows. LiChrosorb Si-60 lo-” silica (2 g) with the specific surface area equal to 550 m2/g (E. Merck, Cherry Hill, NJ) was rinsed with deionized water and dried at 383 K for at least 4 h. The resulting material was placed in a specially designed reaction vessel and allowed to equilibrate overnight with 200 mL of water-saturated toluene, and the toluene removed. Subsequently, 50 mL of dry toluene and 10 mL of either (3-
0003-2700/91/0363-2849$02.50/0
cyanopropyl)dimethylchlorosilane, phenyldimethylchlorosilane, or n-butyldimethylchlorosilanewere added to the reaction vessel and its contents refluxed for 12 h. During the reaction, the solution
was mixed and outgassed with a stream of dry nitrogen. The modified silica was washed sequentially with dry toluene, water-saturated toluene, and diethyl ether, and then it was dried at 383 K for 4 h. The three different phases were packed in upward fashion into 15 cm x 4.6 mm i.d. stainless steel column blanks by using a dynamic slurry procedure and a Haskel (Burbank, CA) Model DSTV-52C air-driven fluid pump to pressurize the system (12). The suspension solvent was 2-propanol, and the delivery solvent was methanol. In the case of the butyl phase, an additional column, 5 cm x 4.6 mm i.d., was prepared. 0 1991 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 63,NO. 24, DECEMBER 15, 1991
Table I. Surface Coverage for the Chemically Bonded Phases surface
code
butyl octyl octadecyl phenyl cyanopropyl a
c4
C8 Cl8
Ph CN
total % C
normalized % C”
4.30 6.80 11.13 4.51 5.86
0.72 0.68 0.56 0.56 0.96
Percent carbon/number of carbon atoms in bonded silane.
Chromatographic Measurements. All chromatographic measurements were carried out with a Laboratory Data Control (Riviera Beach, FL) Model ConstMetric I pump, a Rheodyne (Burbank, CA) Model 7120 injection valve fitted with a 20-pL loop, and an IBM Instruments (Danbury, CT) Model LC/9525 refractive index detector. Retention data were recorded and processed on an IBM Instruments Model 9OOO data system. The column temperature was maintained at 303 K in a water bath using a Fisher Scientific Model 730 controller. Samples of the test solutes were prepared in pure methanol at an approximate concentration of 1mg/mL and their retention volumes measured on each column using varying compositions of water-methanol and water-acetonitrile as mobile phases at a flow rate of 1.0 mL/min. Measurements were made for 4; = 0.3-0.6 for the cyano and phenyl phases and 4; = 0.1-0.8for the butyl phase. The reported capacity factors, k’,,,which are averages from at least duplicate injections, were calculated using either DzO or methanol to determine the void volume.
RESULTS AND DISCUSSION The surface coverages (i.e., percent carbon and percent normalized carbon) of the cyanopropyl, phenyl, and butyl phases are given in Table I along with comparative data previously reported for octyl and octadecyl phases (12). The cyanopropyl surface had the highest relative coverage whereas the coverage of the butyl and phenyl surfaces were similar to those of the octyl and octadecyl phases. As in ref 1 2 the dependence of In k’,,vs nc, the number of carbon atoms in the chain portion of the alkanoate and perfluoroalkanoate esters, is linear for the butyl, cyanopropyl, and phenyl bonded phases with both water-acetonitrile and water-methanol eluents. These linear relationships observed for In k’,, vs nc are consistent with those reported in other investigations (12, 13,26,27), and they provide a means of evaluating the nonspecific (hydrophobic) selectivity, ac (26): s = In ac = In (k’n+l/k’,,) (1) where k’n+l and k’,, are respectively the capacity factors for homologous solutes with n 1 and n carbon atoms in their chain portions. Representative plots of s vs &, the volume fraction of water in the mobile phase, are given in Figure 1 for both the methylene (-CH,-) and perfluoromethylene (-CF,-)selectivities. These were extrapolated to 4; = 1using a linear or second-order polynomial fit in order to obtain estimates of the nonspecific selectivity in the pure water, s, (12, 13,27). Summarized in Table I1 are the values of s, for the two polar surfaces (i.e., CN and P h phases) as well as similar information for the nonpolar alkyl surfaces. The values of s, for the Ph and CN phases are similar to each other, but they are smaller than the nonspecific selectivities for the C4, Cs, and CI8phases. Thus in 100% water, the methylene and perfluoromethylene groups have a higher affinity for the alkyl bonded phases than either the phenyl or cyanopropyl phases. Although these results are consistent with what might be expected from polarity arguments, s, provides a more quantitative basis for comparison of these interactions. For the two polar bonded phases, s, values were consistently greater for the perfluoromethylene group (denoted in Table 11 by s,(~)) than for the methylene group (s,(H)). The ratio of these values, f = s,(~)/s(H) as well as those for the alkyl
+
-0.5 0.0
0.2
0.4
0.6
1 .o
0.8
Volume Fraction, $;
Figure 1. The natural logarithm of the methylene (solid line) or perfluoromethylene (dashedline) selectivity, s,plotted agalnst the volume fraction 4; of water in the water-methanol (triangles) and wateracetonltrile (circles)mobile phases on the C, column at 303 K. The solid points represent the extrapolated values of s to 4; = 1 (pure water). Table 11. Equilibrium Constants K,, for Water-Organic Mixtures on Various Bonded Phasesa Water-Methanol Clad
Cad C4
Ph CN
1.06 1.02 0.98 1.06 1.07
1.10 1.06 0.95 1.01 1.02
0.37 0.38 0.41 0.69 0.70
0.32 0.42 0.40 0.51 0.85
1.08 1.04 0.97 1.03 1.04
1.19 1.18 1.10 0.91 0.83
1.73 1.75 1.74 1.33 1.31
1.45 1.48 1.59 1.46 1.58
1.97 1.73 1.81 1.33 1.31
1.40 1.30 1.55 1.46 1.58
Water-Acetonitrile Clad Cad C4
Ph CN
0.34 0.40 0.40 0.60 0.77
1.41 1.33 1.17 0.91 0.83
“The values of the correlation coefficient for all linear dependences (eq 2) studied were at least 0.99 or better. *The values of K,, obtained on the basis of retention data for ethyl alkanoate esters. CK,, obtained on the basis of data for methyl perfluoroalkanoate esters. dKwovalues for the water-methanol and wateracetonitrile mixtures on the C8 and CSI columns were evaluated also by means of eq 2 using the selectivity data published previously (12). These values differ from those reported in Table I of ref 12 because different equations were used to represent the surface composition; in ref 12 a semiempirical equation was employed to analyze the nonspecific selectivity data, whereas in the current paper a thermodynamic relationship (2) wa8 utilized to evaluate Kwn.
phases is about 1.5. This value is consistent with nonchromatographic measurements of the hydrophobicity of a -CH2group relative to that of the -CH2- group (12). Figure 1also illustrates differences in chromatographic behavior of the alkyl and perfluorinated series. For all bonded phases studied, the methylene and perfluoromethylene selectivities are almost identical in organic rich mobile phase (above 50%) and their difference begins to increase with the water content. A second aspect of the current work has been to study differences in composition between the mobile and stationary phases due to preferential intercalation of one of the solvents. It has been demonstrated previously (13) that this incorporation process can be characterized by the thermodynamic constant K,,, which describes the sorption equilibrium between the stationary and mobile phases for water and organic solvent molecules. Combining this sorption model for the surface phase with a partition model to describe a solute’s distribution between the stationary and mobile phases leads to a simple linear equation: (s,
- s)-1 = (s,
-sop
+ Kwo(s,- s0)-1$&/q5:
(2)
ANALYTICAL CHEMISTRY, VOL. 63, NO. 24, DECEMBER 15, 1991
I
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4.0
-
I
3.0
I
L v
2.0 1.o
0.04 0.0
0.04
1 .o
2.0
4.0
3.0
I Volume Fraction Rotio, 0 ,
/ 0oI
Figure 2. Dependence of (s, - s)-' on I # J ; / ~ ; for ethyl alkanoate esters (open symbols) and methyl perfluoroalkanoate esters (closed symbols) chromatographed in the water-acetonitrile mobile phase on the C, column at 303 K.
:
:
:
:
:
:
~
0.2 0.4 0.6 0.8 1.0 1.2 1.4
5.0
:
:
1.6 1.8 2.0 2.2 I Volume Fmction Ratio, 9: / 9,
: '
2.4
Flgure 4. Dependence of (s, - s)-' on &/4; for the water acetonitrile mobile phase. Other information is as in Figure 3. ,
;L O +
I
0.0
0.0
0.2
0.4
0.6
0.8
1.0
Volume Fmction Ratio,
1.2
1.4
Volume Fraction, 9;
1.6
I eW / 0,I
Figure 3. Dependence of (s, - s)-' on $;/4; for ethyl alkanoate esters (open symbols) and methyl perfluoroalkanoate esters (closed symbols) chromatographed in the water-methanol mobile phase on the Ph (clrcles)and CN (triangles) columns at 303 K. In eq 2, 6: = 1- r#J& is the volume fraction of organic solvent in the mobile phase and so is the nonspecific selectivity in the pure organic solvent. A plot of (s, - s)-l vs the volume fraction ratio, &/4:, is linear with a slope which is proportional to the equilibrium constant K,,. Equation 2 has been employed previously to analyze hydrophobic selectivity data and to calculate K,, for octyl and octadecyl phases as a function of varying compositions of water-methanol, water-acetonitrile, and water-tetrahydrofuran (13). Similarly, this same relationship has been used to analyze data from the C4, Ph, and CN columns. Shown in Figure 2 are representative plots of (s, - s1-l vs 4&/4: for alkanoate (open symbols) and perfluoroalkanoate (closed symbols) esters chromatographed on the C4 column with water-methanol and water-acetonitrile mobile phases. The linearity of these plots demonstrates that eq 2 is a good mathematical description of hydrophobic selectivity as a function of the mobile-phase composition. Figures 3 and 4 are analogous to Figure 2 except they were obtained respectively on the Ph and CN columns. In all cases the correlation coefficients for the linear fits in Figures 2-4 were at least 0.99 or better. The plots in Figures 2-4 were used to calculate the values of K,, which are summarized in Table I1 for both the alkanoate and perfluoroalkanoate esters. Under ideal conditions (i.e., infinite dilution of the solute) K,, should be independent of the nature of the solute. Most of the results in Table I1 satisfy this condition within a few percent spread except for the water-acetonitrile mobile phase on the CN and Ph columns. In these latter cases the estimated values of Kwovaried 10% and E%,respectively. It is unclear whether these larger differences are due to the extrapolation error which arises from
1
Flgure 5. Sorption excesses of acetonitrile from water calculated according to eq 4 for the bonded phases studied. an inadequate number of experimental points or reflect some degree of nonideality. The values of K,, in Table I1 are the average of the individual values of K,, estimated on the basis of the selectivity data for alkanoate and perfluoroalkanoate esters. Since the thermodynamic constant K,, describes the sorption equilibrium for solvents between the mobile and stationary phases assuming ideality of both (13),its application is limited to mobile phases where nonideality effects are not significant. Under ideal conditions, K , can be used to evaluate the volume fraction of water in the bonded phase, + ,; where (13)
(3) When K,, = 1,eq 3 predicts that the compositions of solvents in the stationary and mobile phases are identical (i.e., 4; =
49. Since eq 3 was derived for ideal phases, it will not predict an azeotropic point on the excess sorption isotherm, i.e., the value of the volume fraction, &, at which 4; = 4;. The probability of an azeotropic point is greater for systems with I?, close to unity and has been reported for alkyl bonded phases in contact with water-methanol (12,221. In the current investigation all of the water-methanol data (Table 11) are close to unity and thus indicate that there is a little difference between mobile- and surface-phase compositions. However, in the case of water-acetonitrile the values are significantly smaller than unity and indicate a preferential sorption of acetonitrile by all of the bonded phases. This preferential sorption of acetonitrile is greatest for the alkyl phases and decreases with increasing polarity of the bonded phase. The relative order in terms of preferentially sorbed acetonitrile is CN < Ph < C4and CB< Cle. The curves in Figure 5, which are the sorption excesses, 4:, for acetonitrile in the various
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Anal. Chem. 1991. 63, 2852-2857
bonded phases, were calculated as follows: (4) The curves show that for water-acetonitrile the intercalated solvent composition of the alkyl bonded phases differs significantly from that of the mobile phase. Although this effect decreases with increasing polarity of the bonded phase, it is not negligible even in the case of the cyanopropyl phase.
CONCLUSION It has been shown that changes in nonspecific selectivity as a function of the mobile-phase composition are useful to study hydrophobic interactions in the bonded phases of different polarity and to estimate the surface-phase composition. Analysis of the nonspecific selectivity data show that the preferential intercalation of one of the organic components of hydroorganic mobile phases is more significant with acetonitrile than with methanol. Further studies of other solvents and surfaces are needed in order to formulate more general and quantitative information about this phenomena and its influence on reversed-phase retention mechanisms. Such studies of the surface-phase composition for reversed-phase systems must be concerned not only with the chemical nature of the mobile and surface phases but also with the structure and conformation of the bonded phase, which may change as a function of the eluent. Registry No. Acetonitrile, 75-05-8; methanol, 67-56-1.
LITERATURE CITED (1) Jandera, P. J . Chromatcgr. 1984, 374, 13. (2) Jandera, P.; Colin, H.; Guiochon, G. Anal. Chem. 1982, 54, 435.
(3) Colin, H.; Gulochon, 0.; Jandera, P. Anal. Chem. 1983, 55, 442. (4) Colin, H.; Guiochon, G.; Jandera, P. Chmmafopaphk 1983. 17, 83. (5) Colln. H.; Krstulovic, A.; Guiochon, G.; Yun, 2 . J . Chromafop. 1983, 255, 295. (6) Colin, H.; Krstulovic, A.; Gonnord, M. F.; Guiochon. 0.; Yun, 2 . ; Jandera, P. Chromatopapble 1983, 77, 9. (7) Melander, W.; Stoveken, J.; Horvath. Cs. J . Chromfcgr. 1980, 799, 35. (8) Sadek, P. C.; Carr, P. W.; Ruggio. M. J. Anal. Chem. 1987, 59, 1032. (9) Lork, K. D.; Unger, K. K. Chromatograph& 1988, 26, 115. (10) Sentell, K. 8.; Dorsey, J. G. Anal. Chem. 1989. 67, 930. (11) Issaq,H. J.; Jaroniec, M. J . LiquMChromafogr. 1989, 72, 2067. (12) Gilpin, R. K.; Jaroniec, M.; Lin, S . Anal. Chem. 1990, 62. 2092. (13) Gilpin, R. K.; Jaroniec, M.; Lin, S. Chromatopaphle 1990, 30, 393. (14) Schantz, M.; Barman, B. N.; Martire, D. E. J . Res. Natl. Bur. Stand. 1988, 93, 161. (15) Martire. D. E.; Boehm, R. E. J . Phys. Chem. 1987, 97, 2433. (16) Martire, D. E.; Boehm, R. E. J . phvs. Chem. 1983, 87, 1045. (17) Jaroniec, M.; Martire, D. E. J . Chromatcgr. 1986, 357,1. (18) Jaroniec, M.; Martire, D. E. J . Chromatcgr. 1987, 387, 55. (19) Martire, D. E.; Jaronlec. M. J . Li9. Chromafop. 1985, 8 , 1363. 111,K. A. Chem. Rev. 1989. 89, 331. (20) Dorsey, J. G.; 0 (21) McCormick, R. M.; Karger, B. L. Anal. Chem. 1980, 52, 2249. (22) Koch, C. S.; Koster. F.; Findenegg, G. H. J. Chromafcgr. 1987, 406, 257. (23) Le Ha, N.; Ungvaral, J.; sz. Kovats, E. Anal. Chem. 1982, 5 4 , 2410. (24) Slaats, E. H.; Markovski, W.; Fekete, J.; Pome, H. J . Chromtwr. .. 1981, 207,299. (25) Yonker, C. R.; Zwler, T. A,; Burke, M. F. J. Chromatogr. 1982, 247, 257: l a m 247. 269. (26) Johnson;-B.-P.; 'Khaki, M. G.; Dorsey, J. G. J . Chromatogr. 1987, 384, 221. (27) Colin, H.; OUiOChOn. G.; Yun, 2.; Diez-Mas, J. C.; J. Jandera; J. Chromafcgr. Sci. lg83, 27, 179.
RECEIVED for review May 23,1991. Accepted September 12, 1991. Support from PRIME Grant N00014-86K-0766 is acknowledged.
Capillary Isoelectric Focusing of Proteins in Uncoated Fused-Silica Capillaries Using Polymeric Additives Jeff R. Mazzeo and Ira S. Krull* Northeastern University, The Barnett Institute 341 MU, Department of Chemistry, 360 Huntington Avenue, Boston, Massachusetts 02115
Isoelectrfc focuslng performed In the caplllary format (CIEF) represents a powerful separatlon technlque for the analysls of protelns. To date, all CIEF separatlons have been performed In coated caplllarles, whlch essentially ellmlnate electroendosmotlc flow. Thus, focused protein zones were eluted wlth a technlque called salt moblllzatlon. Here we demonstrate the abllHy to perform CIEF In uncoated caplllarks through the use of polymerlc addltlves In the sample/ ampholyte mlxture. These addlthres can then act as dynamlc coatings of the slllca wall, thereby coverlng many of the posslble adsorptlon sltes to which protelns may adsorb. We also demonstrate that through the Judlckuschoke of addntve concentration, electroendosmotk flow may be controlled, but not totally ellmlnated, obvlatlng the need for performlng salt moblllzatlon of focused proteln zones. That Is, malntalnlng some flow allows for the moblllratlon of focused zones past the UV detectlon window during the focusing step. The developed methodology Is shown applicable to basic, neutral, and acldlc protelns. The main dlsadvantage Is broadened peaks for very acldlc protelns. The flnal optbnlzed condltlons are evaluated In terms of reproduclblllty and qualltatlve capablllty. 0003-2700/91/0363-2852$02.50/0
INTRODUCTION Isoelectric focusing (IEF) is one of the most powerful separation modes available for resolving proteins. A pH gradient is responsible for the resolving power of IEF. The protein(s) to be focused is (are) mixed with a solution of ampholytes, synthetic polyamino, polycarboxyl compounds having various isoelectric points (PIS).The mixture is then added to a separation medium and an electric field applied, with an acidic buffer at the anode and a basic buffer a t the cathode. Due to the action of the field and the incoming protons and hydroxyl ions, a pH gradient is established, stretching from the PI of the most acidic ampholyte at the anode to the PI of the most basic ampholyte at the cathode. Proteins move to the position in the gradient where pH = PI and stop, thereby leading to separation by PI. Through the use of immobilized pH gradients, separations of proteins differing in PI by
