Anal. Chem. 1995, 67,3879-3885
Use of Methyl Spacers in a Mixed Horizontally Polymerized Stationary Phase R. W. Peter Fairbank, Yang Xiang, and Mary J. Wirth* Deparlment of Chemistiy and Bkxhemistry, Universifyof Delaware, Newark, Delaware 19716
Studies of mixed horizontally polymerized monolayers of octadecyl- (cl8) and methyl- (CI) lrichlorosilanes show that CI groups are valuable as spacers in this type of chromatogmphicstationary phase. Molecular models are presented that predict C1 spacers to have less steric hindrance than propyl (Cs) spacers, which aids in the cross-linking of the siloxane monolayer. %i NMR measurements r e d significantly greater cross-linkingin the polymerization of the C1$C1 mixed monolayer compared to the C l d c 3 mixed monolayer. Contact angle measurements for a pure CI monolayer on a flat silica surface indicate that the methyl groups are predominantly directed away from the silica substrate. The chromatographic retention behavior of aniline shows that the CIS/ CI monolayer h a s significantly less silanol activity than does the c18/c3 monolayer. As a critical test of silanol activity, the retention behavior of a set of cationic peptide standards shows that the CI$CI monolayer has very low silanol activity and provides less peak asymmetry than does a monomeric phase made with the same high-quality silica gel (Zorbax-3OORX-sil). The baseline resolution of a m i v h w of three cytochrome e genetic variants establishes that the C1$C1 stationaryphase allows high column efficiency in addition to low silanol activity. The separation of organic bases presents a problem for chromatographers using reversephase HPIL because organic bases adsorb to unreacted silanols, leading to peak tailing.'-5 Silanol activity can be reduced using a variety of techniques. Silica of a higher purity can be used, or a lower grade silica can be pretreated to minimize isolated silanols.6.' The stationary phase can be exhaustively endcapped, or it can be synthesized from a silanizing reagent with bnky side groups on the silicon atom, resulting in a sterically protected surface.8.9 The mobile phase can be modified to reduce base adsorption by lowering the pH of the mobile phase with hinuoroacetic acid or by adding silanol blocking reagents, such as tetramethylammonium phosphate or tetrabutylammonium bisulfate.'0 The lower pH typically hydrc(1)Shapiro, I.: Koltoff, I. M.J. Am. Chm.Soe 1956. 72. 776. (2) Unger, K K Secker. N.; Roumeiiotir. P.J Chmnmtogr. 1976.125. 115. (3) Sadell P.: Cam. P.W.J. Chmmofoy. Sei 1983.21.314. (4)Landy, J. S.: Ward, J. L:Dorsey. I. G.J. Chmmfop. ki. 1983.21.49. (5)Tmshin. S.:Kever, J. I.:Vinopradova, L V.: Seienkii, B. C.J. Micmeolumi SeO. 1991.3,185. (6) Kohler. 1.: Chase. D. B.: Fariee. R D.: Vega. A, 1.; Kirkland. I. I. J.
Chmmotogr. 1986,352,275. (7)Kohler. J.: Kirkland. I. 1.1. Chmmofogr 1987,385,125. (8) Kirkland, J. J.: Ciajch. I. L; Farlee. R. D.And. Chmm. 1989.61.2. (9)Kirkland. J. J.; Dilts. C. H.; Henderson. J. E. LC-GC 1993,11,290 (IO) Paesen.J.;Claeys, P.: Roets. E.: Haogmartens.J.J Chmmatogr. 1993,630, 117.
Wo3-27W/9510367-3679$9.W/00 1995 American Chemical Society
Figure 1. Depiction of ideal horizontal polymerization of Cm and C , trifunctional silanes using space-filling models. The CIS groups are indicated lo constitute one-third of the monolayer. on the average.
The spacings between ClS groups would ideally be random. lyzes stationary phases. Various means have been introduced to increase hydrolyticstability, including the use of chlorodiisopropyk and chlorodiisobutylalkylsilanes for steric protection of the surface,@ formation of si-C bonds to the surface through Si-CI bond formation, followed by a Grignard reaction to attach the organic ligand,",lZ and formation of Si-H bonds, followed by addition of a terminal ~lefin.'~Despite these advances, there is not yet a satisfactory stationary phase with adequately high hydrolytic stability and low silanol activity for the most demanding applications. Recently, a new method has been introduced for potentially reducing the silanol activity while increasing the hydrolytic stability: horizontal polymerization of mixed trichlorosilanes into dense monolayers."-l8 Ideal horizontal polymerization would have highly dense bonding among reagent groups, forming an exoskeletal mesh to protect the unreacted silanols from base adsorption. F i e 1illustrates what is intended for the structure of a mixed monolayer of Cl&, where the C e functional group constitutes no more than onethird of the monolayer This cross sectional depiction shows the hydrocarbon groups directed away from the substrate and the siloxane backbone overlayingthe silica substrate. The role of the short spacer groups is to control the coverage of the C18groups while providing a barrier over the silica (11)Kocke. D. C.; Schmermund. I. T.: Banner. B. Anal. Chem. 1972.44,90. (12) Pesek. I. I.: Swedberg, S. Al. Chmnmtogr. 1989,361,2067. (13) Montes. M. C.: van Amen, C.; Pesek. J. I.: Sandoval. I. E.J Chmnmtw. A 1994,688.31. (14)FaNnmbi. H.0.; Wirlh, M. I. AnolChm. 1993.65.822. (15)FaNnmbi. H.0.; Wirlh, M. 1. AnoLChm. 1992.64,2783. (16) FaNnmbi. H.0.: Wirih, M. 1. US. Wtent Appl. W.215,June 17.1992. (17)Wirth, M. I.: FaNnmbi. H.0. In ChnnicollyMod$ied Su&€s: Pesek. I. I.. Leigh. 1. E.. Eds.; Royal Society of Chemistry: Cambridge. U.K. 1994. (18)Wirth, M.J. LC4X 1994,Z2. 656.
Analytical Chemisty, Vol. 67,No. 21,November 1. 1995 3879
substrate. *C NMR studies of a mixed CI& monolayer showed that the CISchains were randomly interspersed when the CIS chains constituted approximately one-third of the monolayer.Ig Chromatographic studies are consistent with the random distribution of Clx chains: the mixed horizontally polymerized phase behaves chromatographically like a conventional monomeric stationary phase of the same Clx c~verage.'~ Improved hydrolytic stability over monomeric phases has also been demonstrated.15 presumably owing to multiple bonding and high density at the surface. The spacefillig view depicted in Figure 1 suggests the possibility that horizontal polymerization would provide very low silanol activity, because access to the silica would be blocked by the dense, two-dimensionalsiloxane polymer. However, previous work with CldC3 mixed horizontally polymerized phases did not bear out this expectati~n?~ Comparing the CIS/Csphase with a Conventional monomeric CISphase synthesized on the Same type of silica gel, aniline exhibited signiicantly longer retention on the CI& phase: its capacity factor was nearly &fold larger.17 No C d C 3 phase has been reported to have efficiencycomparable to that of monomeric CISphases for organic bases. Quantitative %i NMR data revealed that the C,& monolayers are cross-linked only 20% as much as an ideal two-dimensionalsiloxane monolayer would be.'9 These results indicate that the synthesized Cn/C3 monolayers are too far from the ideal two-dimensionalmonolayer of F i r e 1to be advantageous for separations of organic bases. The marked nonideality of the monolayer might he intrinsic to the CISand CSfunctionalgroups because the Si-0-Si distance is not sufficiently large to accommodate long alkyl chains on adjacent Si atoms for a planar monolayer. While the actual structures of the bifunctional silane monolayers are not known, the densest, most completely cross-linked monolayer would be a lattice of 12 membered rings alternating in Si and 0. A top view of a small section of such a monolayer is illustrated in F i r e 2a for the case of the pure C1 bifunctional silane. This structure was drawn using Hyperchem, with the siloxane bonds initially placed nearly in-plane. Molecular mechanics (MM+) was then used to optimize the geometry. Edge effects are evident, but the center of the structure shows that two-dimensional polymerization is sterically possible. The case of a Cp monolayer is illustrated in F i r e 2b. The MM+ optimization of the geometry quickly moves the silicon atoms out of plane to reduce the repulsive interactions among the ethyl chains. These models illustrate that any alkyl group longer than one carbon atom cannot be accommodated sterically in a completely cross-linked planar monolayer; however, methyl groups alleviate the steric restriction. Consequently, using CI groups as spacers in mixed monolayers might be advantageous chromatographically over C3 spacers. In a mixed Cl~/Clmonolayer, the second carbon of the CIa chain could be accommodated sterically if its three neighbors were CI groups. Thus, in principle, a mixed C&I monolayer could be planar and fully cross-linked if the ratio of C18 to CI were no more than 13. The relaxation of the steric restrictionsfor CI groups makes the use of these spacers worth exploringfor mixed hifunctional siloxane monolayers. The use of CI as spacer groups has not previously been explored. The purpose of this work is to investigate horizontally polymerized monolayers of C1S/Cl with regard to structure and chromatographic performance. The extent of cross-linking is investigated with %i N M R Given the small size of methyl (19) Fahmmbi. H.0.: Bruch. M.
D.;Wuth, M.1.A d ahen. 1993.65.2048.
3880 Analytical Chemisl!y, Vol. 67, No. 21. November 1, 1995
b
Figure 2. Molecular models for nearly two-dimensional siloxane polymers of maximum density: (a) pure C, and (b) pure CP.
groups, it is possible that they will not orient in the desired way that is depicted in Fires 1and 2, and this question is investigated by measurements of contact angles. For the chromatographic studies, Zorbax 300Rx-sil is chosen as the silica substrate because, in the case of the conventional monomeric phase, low silanol activity is expected without endcapping?" The chromatographic performance of the C1a/C1phase is tested by three tvpes of organic bases, and comparisons are made to a conventional monomeric CIS phase. The organic bases include aniline, a set of cationic peptide standards, and a mixture of cytochrome c genetic variants. EXPERIMENTAL SECTION Chemicals and Sample Preparation. n-Octadecylhichlo-
rosilane, methyltrichlorosilane.and dimethyloctadecylchlorosilane were purchased from Hiils America (Piscataway, NJ) and were used as received. Aniline was purchased from Aldrich (Milwaukee, Wl). Cationic peptide standards were purchased from Alberta Peptides (Edmonton, Canada). The cytochrome c mixture contained a combination of samples from the hearts of canines, bovines, and equines and was purchased from Sigma (St. Louis, MO). The silica used in these experiments was Zorbax 300RXsil(5.3 fim diameter, 300 A pore size, 45 mz/g surface area). The synthesis of the horizontally polymerized phase was the same as described previously" and is summarized briefly here. The silica was boiled in concentrated nitric acid for 24 h to remove any atmospheric contaminates adsorbed to the silica surface. The silica was then rinsed with pure water until the pH of the filtrate reached neutrality and was dried at 100 "C under a continuous flow of NI using a Sybron Thermolyne Type 21100 tube furnace. The dried silica was placed in a humidification chamber at room temperature, where moist nitrogen at 50% relative humidity was
Table 1. Experlmental Condltionr for Each of the Samples Run on the HPLC
stop time
sample mobile phase gradient aniline A, 85%ACN+H20 none cationic peptides A, HzO + 0.1%TF& B, ACN + 0.1%TFA initial, 0%B; increase 1%B/min cytochrome c genetic variants A, HzO + 0.1%TF& B, ACN + 0.1%TFA initial: 25%B; increase 1%B/min
flowed through the silica gel until the humidity of the effluent reached 50%. It has been shown that a reproducible amount of water on the order of a monolayer adsorbs to silica surfaces at this humidity level.2O In preparation for derivahtion, n-heptane was passed through a dried silica column to remove polar surfactants which might impede the horizontal polymerization. Under a nitrogen blanket, 4 mL of n-octadecyltrichlorosilaneand 1 mL of methyltrichlorosilane were added to 50 mL of filtered n-heptane. This composition was shown to provide a 1:3 ratio of C18/C3 in the mixed m~nolayer.~~Jg After mixing, the solution was poured into a flask containing the humidified silica and a small stirring bar. The hydrolysis of the silanizing reagents was observed by the immediate evolution of HCl gas from the flask. The reaction was allowed to continue for 24 h at room temperature with stirring. The derivatized silica was then rinsed with 200 mL each of heptane, toluene, tetrahydrofuran, methylene chloride, and acetone and dried for 2 h at 120 "C. The pure C1 monolayer was made on a flat silica plate (Esco Products). The silica plate was pretreated in the same way as the silica gel and then exposed to nitrogen at 50% relative humidity and finally immersed in n-heptane. Under a nitrogen blanket, the methyltrichlorosilane reagent was added to the container holding the n-heptane and silica plate. The reaction was allowed to proceed for 24 h. No sign of a film due to excess water was observed. The plate was cleaned with the same types of solvents as were used for cleaning the chromatographic silica sample. For preparation of the monomeric C18 phase, the silica was pretreated the same way as it was for the horizontally polymerized phase, except that it was not humidified. A 5 g silica sample was refluxed with 2 mL of dimethyloctadecylchlorosilane and 50 mL of toluene, with 1mL of pyridine added as a catalyst. After 24 h, the silica was filtered with 200 mL of fresh heptane, toluene, and acetone and then dried for 2 h at 120 "C. Each chromatographic silica sample was packed into a 15 cm x 4.6 mm column using a Haskel pump, Model MCP 110. Silica (2.5 g) was added to a 5050 mixture of cyclohexanol and acetone for a total volume of 30 mL. The resulting slurry was sonicated for 20 min and then poured into the slurry chamber. Methanol was used as the packing liquid. The columns were repeatedly repacked after chromatographic runs to ensure that differences in the column efficiency were not due to irreproducible packing irregularities. For preparation of the cationic peptide standards, 0.5 mL of HPLC grade water was added to the sample vial. To prepare the cytochrome c sample, 5 mg of the bovine, canine, and equine genetic variants were added to an HPLC vial and dissolved in 1% acetic acid in water. These samples were kept in a -20 "C freezer to prevent degradation. A M solution of aniline was prepared fresh on the day of use in 85%acetonitrile in water. (20) Gee, M. L;Healy, T. W.; White, L. R J. Colloid Interface Sci. 1990, 40, 450.
(min)
post time NA 5 5
3 30 20
wave- injection volume
length b i n ) (nm)
P
10 10 3
r
a a
0- i-0
"\IV\
I.
CUL)
210 210 220
.
. . . io. . . . h. . . . . . . . . . . . ~. . ..". -h %
. . .-io'
I ,
. . .&-
Figure 3. %i NMR spectra of the horizontally polymerized packing materials. The peaks corresponding to unterminated (R-Si-03), terminated (RSi(02)0H), and doubly terminated (RSi(O)(OH)z) reagent groups are labeled on the spectra: (a) c1&3 and (b) CI&.
Equipment. A Hewlett Packard 1090 HPLC was used in these experiments. Table 1 contains information regarding initial conditions, use of gradients, mobile phases, and stop times for each sample. The flow of the mobile phase was 1 mL/min, and the detector response time was 1ms. Each sample was injected three times to confirm reproducibility. A Bruker 300 ML NMR spectrometer was used to obtain the ?3i NMR spectrum. As in previous reports, cross-polarization and magic angle spinning techniques were used to obtain the spectra reported.lg A contact time of 5 ms was used in all experiments. A Mattson G a l w 5020 Fourier transform infrared spectrometer,equipped with a mermrycadmium-telluride detector cooled by liquid Nz, was used to obtain infrared spectra for the silica plates. The molar absorptivity of the methyl stretch was determined using tetramethylsilanein CCL. Contact angle measurements were made on an apparatus built in-house. RESULTS AND DISCUSSION
NMR Spectroscopy. I3CNMR spectroscopy confirmed that the ratio of CI$Ci was less than 1:3. zgSiNMR spectroscopy was used to investigate the siloxane bonding of the CpJC1 monolayer, and the spectra for the horizontally polymerized c18/c3 and the CJCi phases are shown in Figure 3. For the Cl$CB case, which had been detailed previously, the spectrum shows that there are three types of reagent silicon atoms bonded to the surface.1gThe peaks at -58 and -50 ppm correspond to reagent silicon atoms Analytical Chemistty, Vol. 67, No. 21, November 1, 1995
3881
having one terminal hydroxy group and two terminal hydroxy groups, respectively. Both of these peaks are considered to be due to defects in the monolayer because these groups terminate rather than propagate the two-dimensional polymer. The peak at -68 ppm is due to reagent silicon atoms having no terminal hydroxy groups. This type of silicon atom would be the only type in the spectrum if there were ideal two-dimensional polymerization, with all silicon atoms attached through oxygens to other silicon atoms. Previous work revealed that the c18/c3 monolayer is structured as linear polymer chains with most reagent silicon atoms attached to a terminal hydroxy group, and only 20%of the reagent silicon atoms cross-linked to reagent silicon atoms in adjacent chains.Ig Since these terminal hydroxy sites amount to defects, it is likely that aniline tailed when eluted from this phase, because the silica substrate is exposed between the polymer chains. For the c18/c1 case, the -68 ppm peak, due to the unterminated reagent silicon atoms, is much larger than either of the other two peaks. Qualitatively, this is consistent with the idea that C1 spacers allow two-dimensional polymerization. Quantitatively,one must account for occasional bonding of the reagent silicon atoms to the silica substrate, which would be spectrally indistinguishable. Analogous to the c18/c3 study,lg the 29SiNMR spectrum of the underivatized silica gel was obtained to account for the number of reacted surface silanol groups. Quantitative results were obtained as detailed before,Igwhere the buildup and decay of the magnetization from the cross-polarizationwas measured for each type of silicon atom. The primary source of error was the slow relaxation of the Si atoms of the bare silica gel. The results revealed that no more than 15%of the peak intensity at -68 ppm is due to attachment to the surface. Accounting for the small intensity of the peak at -60 ppm, at least 60% of the reagent silicon atoms are cross-linked, which is three times higher than for the c18/c3 case. The C d C I monolayer thus approaches the twodimensional monolayer more closely than does the (&/&monolayer. This agrees with the predictions from the molecular models that the C1 spacers allow formation of a denser barrier monolayer over the silica substrate. Contact Angle Measurements. The C1 spacers allow for extensive cross-linking, but their sizes may not be large enough to force them to orient away from the substrate. The orientations of the methyl groups in the c18/c1 monolayer are important for assessing its prospects in chromatography, and these orientations can be inferred from measurements of contact angles. The hydrophobicity of the surface is expected to be higher if the methyl groups are oriented away from the substrate. Surface hydrophobicity is fundamentally related to the interfacial tension between the surface and the water droplet, ySl. The contact angle, 8,between a surface and a drop of water is related to ysl through Young's equation, Ylv
cos e = Y s v - Ysl
(1)
The terms ysv and ylv are the interfacial tensions of the surfacevapor and liquid-vapor interfaces. In general, the contact angle is large (~90")for a hydrophobic surface and small (moo) for a hydrophilic surface. For the flat silica plate, infrared spectroscopy revealed a coverage of 11f 2 pmol/mz of CI groups, which is in agreement with the model of Figure 2a that predicts 10pmol/mz. The contact 3882 Analytical Chemistry, Vol. 67, No. 21,November 1, 1995
angle was measured to be 77" & lo,which is in agreement with a previous report.z1 By way of comparison, for methanethiol on gold, it is known that the methyl groups are oriented away from the substrate, because a gold-sulfur bond is formed. The contact angle for methanethiol on gold was reported to be 78",22which is virtually the same as that for the CI monolayer on silica. The methyl coverages are comparable for the two surfaces; therefore, the similarity in contact angles suggests that the methyl groups are oriented away from the substrate for the silica case. Contact angles for cl8 were reported to be 110" for both octadecyltrichlorosilane on silicaz1 and octadecanethiol on The c18 monolayer is expected to have a higher contact angle because the chain lengths are longer. The contact angle data are thus consistent with the methyl groups being oriented away from the substrate for C1 on silica. As a check, it would be valuable to know what the contact angle would be if the methyl groups were directed toward the substrate and the siloxane backbone was in contact with the water droplet. To approximate this case, a clean, bare silica plate (contact angle, 0") was heated at 600 "C for 2 h. This is s d c i e n t to dehydrate 70% of the surface SiOH groups, forming siloxane (Si-0-Si) leaving an SiOH concentration of 1.5pmol/ mz. This is lower than the estimated 2.5 pmol/mz SiOH concentration in the pure CI monolayer, based on 25% OH groups in a 10 pmol/mz monolayer. However, the bare siloxane surface was measured to have a contact angle of only 21" f lo,which is significantly more hydrophilic than the surface coated with the C1 monolayer. The contact angle data thus support strongly the notion that the methyl groups are predominantly directed away from the substrate, and this is promising for the use of CI spacers in chromatographic stationary phases. It may at first seem surprising that the methyl groups orient away from the substrate, because methyl groups are expected to be too short to self-assemble at room temperature, based on investigations of longer chain lengths.24 Rather than being due to a cooperative affect, such as self-assembly, the orientation is likely due to orientation of the reactants at the heptane/water interface during the reaction. The heptane/water interface is created by the thin layer of water adsorbed to the silica surface upon humidhcation, and this water layer is in contact with the heptane that is used as the solvent for the horizontal polymerization. The methyl groups would tend to orient toward the heptane at this interface, resulting in an oriented monolayer without requiring the cooperativity of alkyl chains that is involved in self-assembly. Chromatography. While the structural features of the CIS/ C1 monolayer, as revealed by the NMR and contact angle measurements, portend a favorable chromatographicphase, the chromatographic study of base retention is required as a h a l test. For each chromatographic measurement, the CIS/CIphase was compared with a monomeric phase prepared on the same silica gel. The monomeric CIS phase synthesized for this work on Zorbax-3OORX-si1was determined by microanalysis to have a c18 coverage of 3.3 f 0.1 pmoVm2, which is the same as the coverage for the commercial phase. However, this monomeric phase is not the same as the commercial product, Zorbax-RX-C18,because (21) Wasserman, S. R; Tao, Y.-T.; Whitesides, G. M. Langmuir 1989,5,1074. (22) B i n , C. D.;Troughton, E. B.; Tao, Y.-T.; Evall, J.; Whitesides, G. M.; Nuzzo, R G. J. Am. Chem. SOC. 1989,111, 321. (23)Zhuravlev, L. T. Langmuir 1987,3, 316. (24) Brzoska, J. B.; Shahidzadeh, N.; Rondelez, F. Nature 1992,360,719.
Table 2. Chromatographic Data: (a) Horizontally Polymerized Phase and (b) Monomeric Phase
analyte
hexanophenone
(a) (b)
aniline
cationic peptide @e& 3)
(a) (b)
(a) (b)
a
to (min)
tr (min)
N
asymmetry
1.2 1.2 1.3 1.3 1.1 1.6
2.09 1.98 1.52 1.82 21.4 26.4
1500 1680 1728 876 71 190 41 190
2.0 2.3 2.1 3.8 1.9 3.5
both the pretreatment of the silica and the silanhtion are not the same as the proprietary treatments used for the commercial product. These treatments can affect silanol activity. Results for the commercial product are excluded from this report to avoid codicts. The silica gel was pretreated the same way for the monomeric CISand the horizontally polymerized C d C I to allow fair comparison. Chromatograms of aniline at neutral pH are shown in Figure 4 on an expanded scale for both the horizontally polymerized CIS/ C1 and the monomeric CISphases. Both phases provide good performance, despite the absence of trifluoroacetic acid. The asymmetry factor was calculated as the ratio of trailing to leading half-widths at 10% above the baseline. Efficiency,N, was calculated from the Jeansonne and Foley which applies to asymmetric bands. Table 2 shows that the retention time of aniline is 15%less, the efficiency is twice as high, and the asymmetry factor is half as much for the horizontally polymerized phase compared to the monomeric phase. For the case of CIS/C~,the capacity factor of aniline was measured to be 4fold larger than that obtained here for Cls/C1.15 The C3 spacers thus resulted in a stationary phase that was uncompetitive with the monomeric phase. The superior performance of the Cls/C1 phase over the c18/c3 phase agrees with the expectations from the structural models and the NMR spectra: polymerization of C1 groups forms a better barrier over the silica substrate than does that of C3 groups. The comparison between the CIS/CI phase and the conventional monomeric phase is complicated by the fact that aniline is a very early eluting compound for both columns, which enables effects other than adsorption to silanols to contribute to both peak width and asymmetry. Hexanophenone, which is an early eluting compound that is inert toward silanols, was studied to assess broadening effects other than adsorption to silanols. The chromatographic data for hexanophenone are presented in Table 2. The low column efficiency and peak asymmetry for this early eluting compound are likely due to extracolumn broadening. The essential information is that the retention times, column efficiencies, and asymmetry factors of this non-silanol-active analyte, hexanophenone,are comparable for the horizontally polymerized and monomeric phases. The differences for aniline are thus attributed to lesser interaction with silanols in the case of the horizontally polymerized phase. While the elution behavior of aniline shows that the C1 horizontally polymerized phase is quite competitive with the monomeric c18 phase, compounds having stronger interactions with silanols are required to judge base elution more critically. A set of cationic peptides has been reported to be a very sensitive
1.s
min
'
mAu
b
2
40-
35-
33-
25
-
20-
15
-
10
-
I
-
5-
0
LL
-/ I .s
i
min
Figure 4. Chromatograms of aniline at neutral pH using (a) the horizontally polymerized CI$CI stationary phase and (b) the conventional CIS stationary phase.
probe of silanol activity by virtue of strong coulombic interactions between protonated lysine groups and the surface Si-0- g r o u p ~ . ~ g The four cationic peptides are retained in order of increasing number of lysine groups, which increases from 1 to 4. The chromatograms for the cationic peptides at pH 2 are shown in F i i r e 5. The chromatograms obtained for both phases are (26) Mant, C. T.;Hodges, R
(25) Jeansonne, M. S.; Foley, J. P. J Chromatog. Sci. 1991,29, 258.
S. Chromatogmphia 1987,24, 805. (27)Sander, L. C. 1.Chmmafogz Sci. 1988,26,380. Analytical Chemistry, Vol. 67, No. 21, November 1, 1995
3883
a
b
4 0 + . . . . , 0 5
, ' . ' 10
15
20
'
25
'
'
Figure 5. Chromatograms of cationic peptide standards with trifluoroacetic acid in the mobile phase for (a) the horizontally polymerized CI$ C1 stationary phase and (b) the conventional C18 stationary phase.
considerably better than those reported earlier in the literature for CISp h a ~ e s . 2It~is~evident ~~ that the horizontally polymerized phase has higher column efficiency than the monomeric phase. Figure 6 shows the third peak on an expanded scale, and Table 2 lists the retention times and asymmetry factor for the third peak. The retention time is 20% shorter for the horizontally polymerized phase, indicating decreased interaction with Si-0- groups. The column efficiency is 2-fold higher, and the peak asymmetry is almost half as much, further indicating less interaction with silanols. These results are consistent with the expectation from the molecular models and the %i NMR spectra: CI spacers enable an effective barrier to be formed between the mobile phase and the substrate silanols. A fundamental issue in assessing the prospects for practical use of horizontally polymerized phases is column efficiency,which is critical for the most demanding separations. Heterogeneous 3884 Analytical Chemistry, Vol. 67, No. 27, November 7, 1995
polymerization over the silica gel sample would constitute another source of band broadening. Alteration of the pore structure from excess polymer would make the separation of proteins difficult. To test these aspects of column efficiency, the genetic variants of cytochrome c are used, which are separable only with wide-pore silica for columns having high efficiency and low silanol activity. Figure 7 shows chromatograms of the protein mixture for the two stationary phases. Baseline resolution is achieved for the horizontally polymerized C1& phase, while tailing is evident for the monomeric phase. Since none of the peaks is isolated for the monomeric case, the asymmetry factor was not calculated. The shorter retention times and lesser tailing are again indicative of lower silanol activity for the horizontally polymerized phase. The baseline resolution indicates that the pore structure of the silica remains intact after polymerization and that the stationary phase is homogeneous.
a
b
Figure 0. Chromatograms on an expanded scale for the third peak of the cationic peptides: (a) the horizontally polymerized C&1 stationary phase and (b) the conventional Cle stationary phase.
In conclusion, for the elution of organic bases, the use of methyltrichlorosilane (Cl) as a spacer group appears to allow the dense, two-dimensional horizontal polymerization needed for blocking access from the mobile phase to the substrate silanol groups. As a result, mixed horizontally polymerized monolayers of C ~ Cprovide I improved separationsof aniline, cationic peptide standards, and cytochrome c genetic variants, as indicated by short retention times, high efficiency, and low asymmetry factors.
Figure 7. Chromatograms of cytochrome c genetic variants with trifluoroacetic acid in the mobile phase for (a) the horizontally polymerized CI$C1 stationary phase and (b) the conventional CIS stationary phase.
spectra, and to Dr. Fajl Ahmed of Phenomenex, Inc., for suggesting the peptide standards and genetic variants of cytochrome c. This work was supported by Dow Chemical Co., and by the National Science Foundation under Grant CHE9113544. Received for review May 23, 1995. Accepted August 4,
ACKNOWLEDGMENT We are grateful to Dr. Joseph J. DeStefano of Rockland Technologies for generously donating the silica gel, to Dr. Martha D. Bruch of the University of Delaware for obtaining the %i NMR
1995.a
AC9504934 @Abstractpublished in Advance ACS Abstracts, October 1, 1995.
Analytical Chemistry, Vol. 67, No. 21, November 1, 1995
3885
