Anal. Chem. 1991, 63, 1861-1867
1881
Synthesis and Characterization of Dibutyltetramethyldisilazane-Bonded Silica Phases for Reversed-Phase High-Performance Liquid Chromatography Renen Zhang,' Zemin Xie, Rui Zhao, Xiaohua Li? and Guoquan Liu Institute of Chemistry, Academia Sinica, Beijing, People's Republic of China Marie-Isabel Aguilar and Milton T. W. Hearn* Department of Biochemistry, Monash University, Clayton, Victoria 3168,Australia
A new silane, dl-n-butyttetramethylne (DBTMDS), has been synthesized. Sllanlzatlons with DBTMDS of a narrowpore and a wlde-pore silka yielded relatlvely high concentratknr d bonded n-butyi ligands, of 4.22 and 4.09 pmWm*, respectively. The chromatographk properties of the n-butyl bonded phase (C,-NH), synthedzed with DBTMDS, were compared with n-butyl bonded phases (C,-CL) synthesized wlth n ~ h y 3 c ) r k a n (BDMCS) e and Vydac-C, under the same chromatographk conditlorw. The adsorption of basic compounds Including N,Ndlethylanlllne (N,N-DEA), dansylarglnhe (Dns-Arg), and (phenyWo)hydatoln-arginhe (PlH-Atg), as well as several and m a l proteins Including anglotendn I, anglotensin 11, and lysozyme, was shown to be legs for the C4-NH sorbents than for C4-CL and Vydac-C, sorbents. Whlk the S value for the basic protein lysozyme was slgnlflcantly lower on the wlde-pore C4-NH sorbent, the S and log k , values for a range of selected proteins were comparable to those obtalned wlth the VydacC, sorbent. The tests of hydrolytk stability of the bonded phases showed dgniflcantly greater stability for the C4-NH sorbents than for the C44L sorbents under lrocratk mobilephase condltlons of 10% and 40% acetonitrlle wlth 0.1% TFA.
INTRODUCTION Silica-based reversed-phasehigh-performance liquid chromatography (RP-HPLC) has become the method of choice for a large proportion (about three-quarters) of all high-performance liquid chromatographic separations. Many of the benefits of RP-HPLC can be attributed to the silica itself, its excellent physical characteristics and chemical reactivity for easy modifcation of the surface silanol groups. Unfortunately, this reactivity, although the key to the success of silica supports, is also in part the source of its limitation. Owing to steric considerations even under optimal experimental conditions, only partial derivatization of the silanols is accomplished, leaving an excess of unreacted silanol groups. This can lead to undesirable characteristics such as unpredictable retention order changes, tailing of certain polar solutes, and low recovery of basic solutes (1-5). Stationary-phasestability is of great concem to the end user of chromatographic columns, particularly in preparative ap*Author to whom correspondence should be addressed. Visiting Professor, Department of Biochemistry, Monash Univemit , Clayton, Victoria 3168,Australia. Present addreas: Institute for $rotein Research, Osaka University, 3-2, Yamadaoka, Suita, Osaka 565, Ja an. *Present axdress: De artment of Chemistry, Beijing Normal University, Beijing, Peopbs Republic of China. 0003-2700/91/0383-1861$02.50/0
plications for the purifcation of biotechnological produds used as drugs, where column degradation products and loss of resolution can adulterate the fractions to be isolated. Recent research has shown that, in addition to the type of silica used and the rehydroxylation procedure (6),the degree of n-alkylsilane coverage of the matrix also significantly influences the stability of the bonded phase (7,8). In the development of column packing materials consisting of silica chemically modified with n-alkylsies, there are two widely employed bonding strategies. One approach involves the reaction of an n-alkylsilane of the type RSiX3 or &Six2 (X = chloro or alkoxy) with silica. Silica modified by &Six2 and RSiX, silanes exhibits a high multifunctional surface pattern. This strategy often leads to the generation of a polymeric layer that stabilizes the silica to hydrolysis and that appears to be at least partially effective in masking certain undesirable properties of the residual acidic silanols present on the silica surface. However, there me some strong disadvantages to polymeric phases. These materials can be more difficult to reproduce, and variation in the total organic coverage is often observed from batch to batch. In addition, the relatively thick polymeric layer can produce slow masstransfer kinetics, leading to reduced column efficiency compared to the corresponding monolayer, bonded-phase, packing materials. The second popular approach uses the reaction of n-alkyldimethylchlorosilanes or trialkylchlorosilanes of the type RMe2SiX or R3SiX (X = chloro). The advantages of this reaction are that it is reproducible and convenient; the resulting surface of monolayer coverage exhibits excellent mass-transferproperties that produce high column efficiency for most solutes. However, very dense coverage of the surface of silica by n-alkylmonochlorosilanesmay not be achieved as with di- or trichlorosilanea. In order to overcome the relatively low reactivity of monochlorosilanes, the reaction conditions have been studied in detail (9,101.Various approaches have been to used, including more reactive silanes (9-14)and new bulky ligands that provide more effective steric protection to the Si-0-Si bond (15). Lork et al. (11)have summarized the reactivities under comparable conditions of some silanes as fOUOWS: C,N(CH), > C8OCOCF3 > CsCl>>C80H = C8OCH3 >> C8OCp As alternative synthetic procedures to the use of n-alkylmonochlorosilanes,the reaction of trialkyl(dimethy1amino)silaneswith Cabosil-M5 (commercial fume silica) has been studied in detail (16),while the C8-enolate silanization of Nucleosils has also been extensively investigated (17). In this paper, the synthesis of a new silane, di-n-butylt e t r a m e t h y l d i s i i e (DBTMDS), is described. By using this silane, two C4-bondedsilica packing materials with different pore sizes have been prepared and compared to the corresponding n-butyldimethylchlorosilane(BDMCS) derived sorbents. The reactivity of both silanes with silica has been 0 1991 American Chemical Society
1882
ANALYTICAL CHEMISTRY, VOL. 63, NO. 17, SEPTEMBER 1, 1991
examined, and the resulting n-butylsilica sorbents have been compared with a commercially available Vydac-C4sorbent. EXPERIMENTAL SECTION Apparatus. The following instrumentation was employed: Hitachi 635 liquid chromatograph,Hitachi ultraviolet absorption detector, Shimadsu HP 3390A integrator, and Hitachi recorder; Du Pont Instruments 8800 system (quaternary chromatographic pump; Series 8800 gradient controller; column oven), Waters Model 450 variable-wavelength detector, and Perkin-Elmer LC1-100 laboratory computing integrator; Chemco 124 and Shandon column packers. Chemicals. The chemicals used in preparing the bonded phases and the solutes used in the chromatographicexperiments were the highest purity commercially available or repurified. A narrow-pore QDHY silica (mean particle diameter, dp, 5 pm) with a specific surface area of 300 m2/g and mean pore diameter (pd) of ca. 90 A was obtained from the Chemical Works, Qing Dao, China. The wide-pore silica, Nucleosil300-5 (dp 5 pm, pd 300 A), with a specific surface area of 97 m2/g,was purchased from Macherey-Nagel,FGR. The Vydac-C4sorbent (dp 5 pm, pd 300 A with specific surface area of ca. 100 m2/g) was provided by K. Harrison, Separation Group, Hesperia, CA. Preparation of Reagents. Purification of Reagents. Tetrahydrofuran (THF) was gently refluxed with sodium for 3 h to remove water and then fractionated by distillation, and a fraction boiling at 65.5-66.5 "C was collected. Dimethyldichlorosilanewas distilled and a fraction boiling at 68-70 "C collected. Preparation of BuMgCl Grignard Reagent. The BuMgCl Grignard reagent was prepared by standard protocols. In brief, 102 mL of BuCl was dissolved in 250 mL of purified THF. Then 0.5 mL of dibromoethanewas added and the solution stirred under nitrogen. The solution was added under p i t i v e nitrogen pressure to a flask containing 25 g of magnesium,and the reaction mixture was gently refluxed for 2 h after the end of the reaction. Preparation of BuMeaiCl. Me2SiC12(3.5 mol 450 g) and 300 mL of purified THF were added into a three-necked flask, under a stream of dry nitrogen. BuMgCl (3 mol) was then added dropwise with stirring into the three-necked flask. The mixture was refluxed for 3 h. After cooling, the mixture was distilled to remove most of the THF. After cooling again, lo00 mL of hexane was added, the mixture refluxed, and the reaction solution then vacuum-filteredto remove MgC12. The fiitered solutionwas stored overnight and vacuum distilled to remove hexane. Finally, the reaction solution was fractionated and the fraction boiling at 138 OC, giving 206 g of pure BuMGiCl, collected. The refractive index was 1.4220, the chlorine content was 24% (w/w) by elemental analysis (theory 23.56%), and the yield was 62%. Preparation of ( B U M ~ ~ S ~ )To ~ N124 H .g of BuMezSiCland 700 mL of petroleum ether, dried with sodium, in a three-necked flask was passed a stream of ammonia gas at 0 "C with stirring. When the ammonia gas was no longer adsorbed, the addition was continued for 2 h to ensure the reaction was completed. The byproduct, solid NH4C1,was removed by vacuum filtration, and most of the petroleum ether was removed by distillation. Finally, the reaction solution was fractionated under vacuum, and the fraction boiling at 96-98 "C/6-7 mmHg was collected. The yield was 84%, refractive index, n = 1.4404. Elemental Anal. Calcd for ( B U M ~ ~ S ~ ) C, ~ N57.93; H : H, 11.56; N, 5.65. Found C, 57.96; H, 12.65; N, 5.75. Preparation of C4Bonded Packings. Treatment of Silicas. Silica (10 g) was added to 300 mL of 20% (w/v) hydrochloricacid solution (6 M),refluxed at 90 "C for 2 h, allowed to stand 1day, and then washed with water until the washings showed neutral pH. The silica was heated at 100 "C for 2 h in air and then at 200 "C for 6 h under vacuum (0.1 mmHg) and cooled before use in the preparation of the bonded phases. Preparation of Narrow-Pore C4Bonded Phase with BuMe$iCl. Toluene (100 mL) and 10 mL of pyridine, both dried over 5-A molecular sieves, and 12 g of BuMezSiClwere added to 10 g of dry QDHY silica in a 250-mL three-necked flask. The slurry was refluxed with stirring for 15 h. At the conclusion of the reaction, the silica product was filtered, washed with toluene 4 times, and extracted with toluene for 15 h. The bonded phase was washed consecutively with dry acetone, ethanol/water (1:1), water to remove the pyridineHC1 salt, and ethanol. Finally, the modified
silica was dried for 10 h at 120 "C in vacuo and sampled for elemental analysis. Preparation of Narrow-Pore C4 Bonded Phase with (BuMe2Si).JVH. Toluene (100mL) dried over the 5-Amolecular sieve and 10.5 g of ( B U M ~ ~ S ~ were ) ~ N added H to 10 g of dry QDHY silica in a 250-mL three-necked flask. The slurry was refluxed with stirring for 15 h. The bonded silica was then washed, extracted, washed, and dried as above. Preparation of Wide-Pore C4 Bonded Phases with (BuMe#i)&H and BuMe2SiC1. Nucleosil300-5with a specific surface area, by BET measurement, of 97 m2/g and average pore size of 300 A was used for the bonded phases. Silica (10 g) was rehydroxylated in 80 mL of b o i i g water that contained 100 ppm hydrofluoric acid for 70 h and dehydrated under vacuum at a temperature of 200 "C for 6.5 h. The bonding procedure for widepore silica was the same as for the narrow pore silica except the reaction time was 20 h and the amount of silica used was about 3 g for each bonding procedure. RESULTS AND DISCUSSION Organoaminosilanes as Silylation Agents. It was well-known with chemically modified silicas that, during formation of the bonded monolayer, the conversion is limited by the maximum number of active sites that are accessible to the silanizing reagent. Silanization may thus be regarded as a self-exclusion process, with the maximum conversion occurring when the bonded silica reaches the state of complete size exclusion of further silane modifiers, regardless of their concentration and reactivity. Diffuse reflectance infrared Fourier transform spectroscopy (DRIFT), %Sicross-polarization magic angle spinning NMR (%i CP-MASNMR), and other methods of spectral and dye binding analysis have indicated that there are at least two different types of silanol groups on the surface of silica (18-20): e.g., (i) silanol groups that are associated or hydrogen-bonded and (ii) those with isolated hydroxyl groups. The less adsorptive type B silica materials contain higher concentrations of hydrogen-bonded or associated SiOH groups compared to the unbonded, acidic SiOH surface groups of the more adsorptive type A silica materials. Further, type B silicas contain ca. 32% geminal (Si(OH1.J and ca. 68% single (SiOH) groups. Upon reaction with silanes, the geminal silanol groups are totally modified, as assessed by ?Si CP-MAS NMR. According to a simple model in which the silica surface is occupied with geminal silanols,about one-half of the silanols is believed to react easily with silylation agents, while the reactivity of the other half is decreased by steric hindrance. Buszewski et al. (14) treated thissituation as consecutivefirsborder reactions and estimated that the ratio of the rate constants was k,/lt2 > 4. Clearly, silanization reagents that enable maximal coverage, rapid reaction kinetics, and effective modification (or masking) of residual silanol groups are preferred. Recent studies have shown that organoaminosilanes satisfy many of these requirements. For example, it has previously been shown by Erard et al. (16)that organosiloxy layers of maximum density could be prepared by the use of organo(dimethy1amino)silanes as silylating agents. Under the reaction conditions of treatment of silica with pure organo(dimethy1amino)silanes at 180 OC/lOO h, the intrinsic reactivity of the n-alkyl(dimethylamino)silanes resulted in limiting ligand densities of 4.78 pmol/m2 for the trimethylsilyl group and 4.20 pmol/m2 for the propyldimethylsilyl group (16).Moreover, Bien-Vogelsang et al. (17) have described a high-vacuum technique for the silanization of silicas such as Nucleosil with some organo(dimethy1amino)silanes so that similar concentrations of bonded ligands can be achieved a t ambient temperatures in approximately 4 h. Table I documents the surface coverage of alkylsilyl bonded phases using various organo(dimethy1amino)silanes as silylating reagents as reported recently. These data show that all of these reagents reach or exceed the limit of ligand coverage of silica
ANALYTICAL CHEMISTRY, VOL. 63, NO. 17, SEPTEMBER 1, 1QQl
Table 1. Surface Coverages of n-Alkylsilyl Bonded Phases Prepared from Organoaminosilanes
organoaminosilane (trimethy1amino)silane propyldimethyl(dimethy1amino)silane
N-octyldimethyl(dimethy1amino)silane decyldimethyl(dimethy1amino)silane decosyldimethyl(dimethy1amino)-
spec surf. ligand area, density, mz/g
amino)silane
Scheme I. Proposed Mechanism for the Reaction of DBTMDS with Silica ?SI-OH
+
BuMe,Si.
NH
-+
&i--O-SiMe,Bu
+ BuMe,SiNH,
+
Si-O-SiMe,Bu
+ NH,
BuMe,Si'
mol/m2 ref
207 207 297
4.78' 4.20'' 4.10
16 16 10
207 207
4.31' 4.12"
16 16
silane Natadecyldimethyl(dimethy1-
1863
360
4.23b
16
&--OH
+
,OH
= S!
BuMe,SiNH,
BuMe,Si,
+ OH
,OSiMe,Bu
NH
,
--t
=s;I
+
BuMe,SiNH,
OH
BuMe,Si'
1
Treatment with pure organo(dimethy1amino)silane at 180 "C/ 100 h. The silanization reaction was carried out in a glaea ampule in the absence of solvents and end capped with a 5% solution of
,OSiMe,Bu
= S!
+ NH,
OSiMe,Bu
hexamethvldisilazane.
treated with trimethylchlorosilane (TMS) (11,151. Synthesis of DBTMDS and Reaction Mechanism with Silica. Organoaminosilanes are generally accessible by the reaction of halosilanes with ammonia or &ea in the presence of an inert solvent. Steric hindrance can make reactions of this type more difficult; on the other hand, the condensability of the silylamines, which is analogous to that of the silanols, makes the isolation of certain organoaminosilanes impossible. Generally, the stability of the organosilylamines depends on the size of organic substituents on the Si atom. Thus, hexamethyldisilazane, (Me3Si)zNH, is the sole organosilicon product of the reaction between trimethylsilyl chloride and ammonia (21). The stability of the triorganosilylamines increases with increasing size of the organic group; thus triethylsilylamine is the main product of the reaction between triethylsilyl chloride and ammonia, though some hexaethyldisilazane is also formed (22). The results of the organic elemental analysis of the product from the reaction of n-butyldimethylchlorosilanewith ammonia show that, due to the condensability of the n-butyl(dimethylamino)silane,its dimer, DBTMDS, was formed during the synthesis process. The product presumably arose as a two-stage reaction, namely, 2BuMe2SiCl 4NH3 2BuMe2SiNH2 2NH4C1 2BuMe2SiNHz ( B U M ~ ~ S ~ ) ~NH3 NH As noted below, DBTMDS readily reacts with silicas to generate a bonded high coverage monolayer. In this investigation, the precursor to DBTMDS, n-butyldimethylchlorosilane,was synthesised in 62% yield via the Grignard reaction of nBuMgCl with M@iC12. The same monwhlomilane precursor can also by synthesised via a hydrosilanization reaction between Me2SiHCl and l-butene. The mechanism shown in Scheme I can be proposed for the reaction of (BuMe#iI2NH with silica. In the case of isolated silanols, the silanization reaction with DBTMDS is expected to give rise to the transient product n-butyl(dimethy1amino)silane. When two or more isolated or free silanols are cloee standing (i.e., separated only by a short distance), the generation of this highly reactive species will result in high levels of the n-alkyl ligand being bonded to the hydroxylated surface due to the small diffusional distances involved and reduced steric requirements (11). In the case of geminal silanols, the reaction could conceivably occur via a similar stepwise process (although at a reduced rate due to steric considerations) or alternatively through a six-membered-ring intermediate, whereby both n-butyldimethylsilyl groups are consecutively introduced. Although other types of organoaminosilanes such as alkyldimethyl(dimethy1amino)silanes of the type RMe2SiN(CH3) have previously been used by Erard et al. (16) and Bien-Vogelsang et al. the main difference between (n-BuMe2Si)2NHand
+
(In,
--
+ +
t
H,
,OH = s,' +
BuMe,Si
OH
BuMe,Si
,
NH
'
&------Si
,Me,Bu
I +
\
=Si
NH
\
0 ...___...si
H'
/ 'Me,Bu
Table 11. Bonded Phases for Surface Coverages of C, Sorbents' elem. anal.
ligand density,"
column packing
spec surf. area: m2/g
of C,' % w/w
pmol/m2
C4-NH-1 C,-CL-l C4-NH-2 C4-CL-2
300 300 97 97
7.96 6.74 2.73 1.61
4.22 3.50 4.09 2.37
'C4-NH sorbent prepared with di-n-butyltetramethyldisilazane. C4-CL sorbent prepared with n-butyldimethylchlorosilane. bSpecific surface area was measured by the BET method using nitrogen adsorption. The carbon content was determined by using a Model 1102 elemental analyzer (Italy Carlo Erba). Elemental analysis data were corrected for background carbon in the unbonded silica. dThe density of the C4 ligand was calculated accordingly Berendsen and de Galan (29). these other types of organoaminosilanes reagents is that DBTMDS involves both less steric hindrance and the bifunctional potential for consecutive reaction modifications of accessible silanols within the pore structure of the silica particle. Chromatographic Characterization of Bonded Phases. In the present study, DBTMDS and BDMCS were used to prepare C4bonded phases hereafter called the C4-NHand the C4-CLsorbents, respectively. The results are shown in Table 11. The surface coverages of the bonded phases synthesized with DBTMDS were 4.22 and 4.09 pmol/m2 for the narrowpore QDHY silica and the Nucleosil 300-5wide-pore silica bonded phases, respectively. Differences in the ligand density of bonded phases are reflected in the capacity factors for neutral solutes. Table 111lists the capacity factors for some aromatic compounds on the column packings synthesized with DBTMDS and BDMCS with different pore size silicas. In agreement with the ligand density results, the capacity factors show the same trends, i.e., the capacity factors of aromatic compounds obtained with the C4-NHsorbent are larger than with the C4-CL sorbent. Residual silanol groups present on the surface of silanized silica can influence the chromatographic behavior of polar
1864
ANALYTICAL CHEMISTRY, VOL. 63, NO. 17, SEPTEMBER 1, 1991 C4-NH
Table 111. Retention Characteristics of Aromatic Compounds on C4 Columnsa column
benzene toluene
1 C4-NH-1 k'
k'
2 C4-CL-1
1.64 1.49 1.53 1.47 1.46 1.68 0.46 1.64
1.10 1.00 1.04 1.00 0.87 1.00 0.28 1.00
re1 retent re1 retent
3 C4-NH-2 k' re1 retent 4 Cd-CL-2 k '
log
naphthalene
biphenyl
1.95 1.77 1.83 1.76 1.89 2.17 0.61 2.17
3.01 2.74 2.84 2.73 4.23 4.86 1.37 4.89
re1 retent Relative retention of benzene = 1.00. Chromatographic conditions: columns 1 and 2 (C4 phase bonded to the QDHY narrowpore silica), 4 X 150 mm; mobile phase 70% methanol; W 254 nm; columns 3 and 4 (C4 phase bonded to the Nucleosil 300-5 widepore silica), 2 X 150 mm; mobile phase 35% acetonitrile; UV 280 nm. Additional details on the C4-sorbentcharacteristics are given
kl
I O
a
in Tnhle
TI.
Table IV. Retention Characteristics of Dansylamino Acids on C4Columnsa column
C4-NH-1 C4-CL-1
Dns-Gln
Dns-Arg
Dns-Ser
2.76 1.00 2.46 1.00
7.24 2.62 7.50 3.05
7.79 2.82 7.24 2.94
k' re1 retentb k' re1 retentb
a Chromatographic conditione: C4-NH column and C&L column 4 X 150 mm; mobile phase 11.5% 2-propanol containing 50 mM NaH2P04,pH 6.0; UV 254 nm. Details on the C4-sorbent characteristics are given in Tables I1 and 111. bDns-Gln retention
= 1.00.
Table V. Comparison of the Retention of (Pheny1thio)hydantoinaminoAcids on C4 Columns"
PTH-Ser PTH-Gln PTH-Arg PTH-Ala
column 1 C4-NH-2 k'
re1 retentb 2 C4-CL-2
k' re1 retentb
1.88 0.40 0.16 0.39
2.66 0.60 0.21 0.51
2.51 0.56 0.56 1.36
4.47 1.00 0.41 1.00
aColumns 1 and 2, 2 X 150 mm; mobile phase column 1 10% acetonitrile containing 50 m M NaHzP04,pH 6.0; mobile phase column 2 5% acetonitrile containing 50 m M NaHzP04,pH 6.0. Details on the C4sorbent characteristics are given in Tables I1 and 111. bPTH-Ala retention = 1.00.
solutes, particularly basic compounds, in reversed-phase liquid chromatography. Therefore, we can use the chromatographic characteristics (capacity factor and peak shape) to examine the performance in this regard of the bonded phases. Tables IV and V show the capacity factors of several dansylamino acids and (pheny1thio)hydantoinamino acids (PTH) with the C4-NH and C4-CL sorbents of different pore sizes. Of the amino acid derivatives, the arginine (Arg) derivatives are basic compounds, and their retention behavior will reflect the amount of residual silanol groups on the surface of the bonded phase. With the narrow-pore QDHY silica bonded phases, dansylarginine (Dns-Arg) was eluted before dansylserine (Dns-Ser) on the C4-NH column but after Dns-Ser on the C4-CLcolumn; on the wide-pore silica bonded phases, (pheny1thio)hydantoin-arginine(PTH-Arg) was eluted before the (pheny1thio)hydantoin-glutamine(PTH-Gln) on the C4-NH column, while inversion of elution order was observed for the C4-CL column. In all cases with the C4-NH sorbent, the asymmetry factor of the eluted peak of these analytes was less than 1.1. Bij et al. (23)reported that dibenzo crown ethers and some peptides with unprotected groups exhibit nonlinear retention
0
0.1 0.2 0.3 0.4 0.5 0.6 volume fraction of water in methanol
0.7
Figure 1. log k'as a function the eluent composition for dibenzo18-crown-6 ethers. Solid and open symbds represent the C 4 C l and CrNH sorbents derived from the narrow-pore W H Y silica.
dependencies, according to their log k 'vs methanol concentration plots, as a result of silanophilic interactions between the analyte and residual silanol groups of the stationary phase, and introduced the "dual retention mechanism" concept. We used dibenzo-18-crown-6 (DB18C6) as a probe to test the C4 bonded phases with the narrow-pore QDHY silica using mobile phases with different volume fractions of water in methanol. Plots of the logarithmic capacity factor of DB18C6 obtained on C4-NH and C4-CL columns against the composition of methanol-water eluent are shown in Figure 1. Each plot passes through a minimum when the aqueous eluent contains about 20% water for the C4-NHsorbent and about 40% water for the C4-CL sorbent, respectively. In RP-HPLC with nalkylsilicas, a common feature of the experimental results is increasing retention of analytes with decreasing water concentration. This phenomenon is consistent with an interpretation that the solute retention with n-alkylsilicas is due to silanophilic interaction at low water concentrations (H-B), whereas water 'masks" silanophilic sites at higher water concentrations so that retention is primarily due to hydrophobic interactions under these conditions. The difference in volume fraction of water in methanol a t the minima with the C4-NH and C4-CLsorbents presumably reflects the difference in the number and accessibility of silanophilic sites; i.e., the silanophilic sites on the C4-CLbonded phase are more abundant and accessible than on the C4-NH bonded phase. Comparison of Chromatographic Characteristics of the C4-NH Column with the Vydac-C4 Column. The chemistry of the support surface, together with the nature of the mobile phase, determines the thermodynamic phase equilibrium of a solute and thus its retention time and peak shape. According Kohler and Kirkland (3,the Nucleosil300-5 used in this investigation and Vydac both belong to the less acidic type B family of silicas with surface silanol concentrations of at least 7 Hmol/m2. In order to compare the bonded phases of these two silicas under the same chromatographic condition, we have used the strongly basic compound, N,Ndiethylaniline (N,N-DEA),and several peptides and proteins, angiotensin I, angiotensin 11, and lysozyme, as basic probes. The interaction between basic solutes and the residual silanol groups at the surface of bonded silica phases often results in increased retention time of solutes and peak asymmetry. The results given in Table VI show N,N-DEA is isocratically eluted before biphenyl with the C4-NH sorbent, while the elution order of these solutes is reversed for the Vydac-C4 sorbent. The gradient elution separations of angiotensin I, angiotensin 11, and lysozyme with the C,-NH sorbent and Vydac-C4sor-
ANALYTICAL CHEMISTRY, VOL. 63, NO. 17, SEPTEMBER 1, 1991 Table VI. Comparison of the Retention of NJV-Diethylaniline (N,N-DEA) on Wide-Pore C, Sorbents"
column
naphthalene
biphenyl
NJV-DEA
1 C4-NH-2
k' 2.55 4.57 3.91 re1 retentb 0.56 1.00 0.86 2 C4-CL-2 k' 0.49 0.62 1.95 re1 retentb 0.80 1.00 3.17 3 Vydac-C1 k' 3.73 6.22 15.6 re1 retentb 0.60 1.00 2.51 "Size of columns 1 and 2 as in Table V and column 3 4.6 X 75 mm; mobile phase 35% acetonitrile. Details on the sorbent characteristics are given in Tables I1 and 111. bBiphenyl retention = 1.00.
1885
Table VIII. Physical Properties of Peptides and Proteins
solute
MW
angiotensin I1 angiotensin I insulin cytochrome c lysozyme bovine serum albumin
1050
PI
1300 6 500 12400 14300 68OOO
5.3 9.4 11.0 4.9
C4-NH
2.0
,
I
LYS ~~~
~
Table VII. Retention Characteristics and Asymmetry Factors of Angiotensin I, Angiotensin 11, and Lysozyme for Wide-Pore C4-NH and Vydac-C4 Sorbentsa
column
compound
retent time, min
k'
Mymre1 metry retent* factorc
C4-NH-2 angiotensin I1 16.2 15.7 1.00 1.48 angiotensin I 19.5 18.9 1.20 1.27 lysozyme 31.1 30.1 1.92 1.37 Vydac-C4 angiotensin I1 15.6 17.3 1.00 2.66 angiotensin I 19.3 21.4 1.21 3.46 lysozyme 30.7 34.1 1.97 2.97 "C4-NH-2 column and Vydac-C, column 4.6 X 75 mm; mobile phase gradient elution (A) 10% acetonitrile-0.1% TFA, (B)50% acetonitrile-0.1% TFA for 30 min. bAngiotensin I1 retention = 1.00. 'Chromatographic peak asymmetry factors calculated by using the method described by Kirkland et al. (27). Details of the sorbent characteristics are eiven in Tables I1 and 111.
1.0
1
'y: *A
I
A
0.5 I 0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.5
0.6
a WDAC-C4
2.0 (b)
e 0.0
0.1
0.2
0.3
0.4
3 Figure 3. Plots of log k vs $ for peptides and proteins separated on (a) Nucleosil300-5 C,NH and (b) VydacC, wide-pore packing materials. Retention data were derived wlth gradient times ranging between 30 and 90 min at a flow rate of 1 mL/mln. Solutes used were A-I = angiotensin I, A-I1 = angiotensin 11, INS = insulin, CYT = cytochrome c, LYS = lysozyme, and BSA = bovine serum albumin.
4 0
10
20
30
40 ( m i n )
0
10
20
30
40 (min)
Flgure 2. Chromatography of angiotensin I1 (l), angbtensin I (2), and lysozyme (3) on differentwidapore C4 sorbents. Chromatographic conditions: (A) C,-NH, column 4.6 X 75 mm, gradient elution 10% acetonttrlie containing 0.1% TFA to 50% acetonibile containing 0.1% TFA in 30 min, flow rate 1.0 mL/mln, UV 215 nm. (B) Vydac-C, column 4.6 X 75 mm. other conditions as in A.
bent are shown in Figure 2. Although the selectivities of these two wide pore sorbents are very similar with these two basic peptides and lysozyme, the respective asymmetry factors of the gradient eluted peaks (Table VII) were smaller with the C4-NHsorbent than with the Vydac-C4sorbent when packed under identical conditions into columns of the same dimensions. Pearson et al. (28) have shown that when n-octyldimethylsiica groups are bonded onto the 5-pm Vydac TP silica and the resultant sorbent is compared to other n-odyldimethyl bonded silicas, resolution of peptides and proteins was much more a function of relative selectivity than the theoretical plate number, or asymmetry, of the eluted peaks. Similar results
are also evident in our studies. Evaluation of Peptide and Protein Retention Properties Eluted on the C4Sorbents. The surface characteristics of the C4-NH sorbents were further assessed through the analysis of the gradient elution retention characteristics of selected peptides and proteins, which are listed in Table VIII. The retention of proteins in gradient elution reversed-phase chromatography can be evaluated according to the linear solvent strength theory (29) in terms of the following equation: log R = log ko - StJ where k is the median capacity factor and $ is the corresponding mole fraction of organic solvent. The parameters S and log ko,which can be determined from linear regression analysis of plots of log k vs $, are related to the surface area of the solute that is in contact with the stationary phase and the affinity of this contact area for the stationary phase in the absence of organic modifier. The derivation of these parameters allows the suitability of the wide-pore C4-NH packing to be compared with the commercially available Vydac-C4sorbent. Figure 3 shows plots of log k vs $ for the peptides and proteins listed in Table VIII. The S and log ko values derived from these plots are listed in Table IX. It was found for all
1888
ANALYTICAL CHEMISTRY, VOL. 63, NO. 17, SEPTEMBER 1.1991
Table IX. Gradient Retention Parameters for Solutes Eluted on C4-NHand Vydac-C4Columns" C4-NH slolute angiotensin I1 angiotensin I insulin cytochrome c lysozyme bovine serum albumin
S
1% ko
6.98 0.68 7.33 0.74 8.27 1.11 10.70 t 1.15 4.34 1.13 13.27 2.14
1.66 0.05 2.02 f 0.09 2.75 i 0.22 3.45 0.26 3.19 0.55 5.01 0.66
** *
*
* *
Vydac-C4 S log ko
*
6.40 0.15 6.53 0.37 10.46 i 1.10 9.65 & 1.23 9.58 1.77 10.81 2.16
*
*
1.68 0.01 1.99 0.04 3.14 f 0.22 3.22 0.28 3.55 0.45 4.18 0.64
* * *
"Experimentaldata was acquired with the wide-pore Nucleosil300-5 C4-NH and Vydac-C4 sorbents. Chromatographic conditions are detailed in the legend to Table VII.
solutes except lysozyme that both the S and log ko values were similar on both columns. This result further indicates that the selectivity of these peptides and proteins on the C4-NH column is comparable to the Vydac-C4column. While the log ko value for lysozyme was similar on both columns, the S value was, however, significantly lower with the C4-NHcolumn. As discussed previously, lysozyme is an extremely basic protein with a PI equal to 11.0. The high level of surface positive charges would enhance the likelihood of this protein interacting strongly with any accessible, residual silanol groups. The lower S value observed with the C4-NH sorbent is consistent with the results in the previous sections that demonstrated a diminished retention and peak tailing for lysozyme on this packing material. Overall, therefore, the wide-pore C4-NH packing material exhibits favorable retention characteristics compared to the Vydac-C, packing and has equivalent and, in the case of lysozyme, superior properties for the separation of basic peptides and proteins. Stability of Synthesized Bonded Phases. The extent of surface coverage of a bonded phase has a significant impact on stability for a given ligand and silica. The stabilities of the narrow-pore QDHY silica C4-NHand C4-CLsorbents were examined by using extensive isocratic elution with mobile phases composed of different concentrations of organic solvent-water containing 0.1% TFA. The acid hydrolysis of the silanes is a reversible reaction. Consequently, under gradient elution conditions, the relative stability of these C4-NHand C4-CLsorbents would be anticipated to be lower since gradient operation is more likely to remove hydrolyzed products from the silica surface prior to recombination occurring. Figure 4 and Table X present the results of these isocratic experiments and indicate that the C4-NHbonded phase with high coverage (4.22 pmol/m2) possessed the higher resistance to hydrolysis under these conditions, as compared to the C4-CL bonded phase with less coverage (3.50 bmol/m2). With the C4-NH sorbent, the capacity factor for naphthalene was reduced 3% after elution with 3000 column volumes of 40% acetonitrile in water with 0.1% TFA. The capacity factor for naphthalene was unchanged by 2000 column volumes passed over the C4-NHsorbent. The capacity factor for naphthalene was reduced 8.7% after 3000 column volumes of 40% acetonitrile in water with 0.1% TFA for the C4-CL sorbent. However, the acid-catalyzed hydrolysis of bonded phases
60
I
loo0
200 C a b " volumes
3000
4000
Figure 4. Loss of capacity factor (k') with n-butyl ODHY silica as a function of column volume of mobile phase passed through the COC umns in the different lsocratk elution testings. (0)C 4 4 H column; (X) C4CL column; (---) 40 % acetonitrile-water with 0.1 % TFA, k' measured for naphthalene; (--) 10% acetonltrk-water wlth 0.1 F A , k' measured for PTH-Ala; column at room temperature (18-20 OC); flow rate 1.0 mL/mln.
became significant when the mobile phase was richer in water with 0.1% TFA. In this case, the capacities for (phenylthio)hydantoin-alanine (PTH-Ala) were reduced 31.6 and 48.6% respectively for the C4-NH and C4-CLsorbents after elution with 3500 column volumes of 10% acetonitrile containing 0.1% TFA. The results of elemental analysis for column packing materials after passage of 3000 column volumes of 40% of acetonitrile in water with 0.1% TFA and 3500 column volumes of 10% acetonitrile in water with 0.1% TFA show that the contents of the carbon-bonded phase were reduced 23.5 and 49.3% for C4-NH and C4-CL columns, respectively. These results confirm that the C4-NH-bonded phase possesses greater resistance against acid hydrolysis than does the C4-CL sorbent and indicate that the improved bonding method can serve to increase ligand stability.
CONCLUSIONS In this work, a new silane, di-n-butyltetramethyldisilazane, has been synthesized. The main characteristic of this silane is that is pmesses both high reactivity with silica and reduced steric hindrance with vicinal or geminal silanols for the bonded reaction. The silanizations of two different pore size silicas with this silane yielded relatively high concentrations of bonded n-butyl ligands (4.22 and 4.09 pmol/m2, respectively, for narrow-pore and wide-pore silicas). The bonded phase synthesized with DBTMDS exhibited lower retention factors for basic compounds than the corresponding bonded phase synthesized with n-butyldimethylchlorosilaneand the commercial bonded phase, Vydac-C1. The DBTMDS bonded phase also exhibited significantly higher hydrolytic stability than the bonded phase synthesized with BDMCS.
Table X. Characteristics of C,-NR and C4-CL Bonded Phases in the Stability Study column
initial
C4-NH-1 c,-CL-1
5.72 4.72
capacity factop final reduction, %* 5.55 4.31
3.0 8.7
initial 28.0 8.02
capacity factorb final reduction, %** 19.2 4.12
31.6 48.6
initiald 7.96 6.47
elemental anal. C% finale reduction (%) 6.06 3.28
23.5 49.3
a Naphthalene. *PTH-Ala, chromatographic conditions as in Figure 4. CFinalI C values on the phases. Percentage reduction in k'after passage of (*) 3000 column volumes of 40% acetonitrile in water with 0.1% TFA and (**) 3500 column volume of 10% acetonitrile in water with 0.1% TFA, respectively. dInitial %C values on the sorbents after synthesis.
Anal. Chem. 1991, 63, 1887-1874
ACKNOWLEDGMENT Special thanks are given to Guanzi Qu of the Institute of Chemistry, Academia Sinica, for the elemental analysis of C4 bonded phases, to Xiguang Li of Department of Chemical Engineering, Monash University, for providing the specific surface area, and to Philip Holt of the Center for Bioprocess Technology, Monash University, for providing assistance in packing columns. LITERATURE CITED (1) (2) (3) (4) (5) (8) (7) (8) (9) (10) (11) (12) (13) (14) (15)
Sander, L. C.; Wise, S. A. J . Chromatogr. 1984, 376, 183-181. Nahum, A.; Horveth, Cs. J . chrometog. 1981, 203, 53-83. N. H. C.; Olsen, K. J . Chromatog. Scl. 1980, 18, 512-524. Nawrockl. J.; Busrewskl, 8. J . chrometog.1988, 449, 25-38. Sander, L. C. J . chrometoby. W . 1988, 26, 318-327. Nondek, L.; Vyskocll, V. J . Chnwnetog. 1981, 206, 581-585. Kohler. J.; Kirkland, J. J. J . Chrornetw. 1987, 385, 125-150. Sagllano, N.; Floyd. T. R., Jr.; Hartwick, R. A.; Dlbussob, J. M.; Mllier, N. T. J . Chromatoga. 1988, 443, 155-172. Klnkel, J. N.; Unger, K. K. J . Chromatog.1984, 376. 193-200. Chew. W.; McCown, M. J . c l w o m e m .1985. 378, 173-185. Lork, K. 0.: Unger, K. K.; Klnkel. J. N. J . Chromatoga. 1986, 352, 199-211. Webch, T.: Frank, H. J . Chromatogr. 1989, 267, 39. Wing, R. 0.: Barry, A. J.; Bulke. M. F. J . Chromatog. 1987, 384, 105-1 18. Buszewskl, B.; Nondek. L.; Jurasek, A.; Berek, 0. Chromatogpaphle 1987, 23, 442-448. Kkland, J. J.; Qlajch, J. L.; Farlee, R. 0. Anal. Chem. 1989, 67, 2-11.
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(16) Erard, J. F.; Nagy, L.; Kovats, s2.E. coHo#s Surfaces 1984, 0 , 109-132. (17) Bien-Vogelsang. V.; Deege. A.: Flgge, H.; Kohler, J.; Schomburg, 0. Chrometogrephle 1984, 10, 170-179. (18) Kohler, J.; Chase, D. B.; Farlee, R. D.; Vega, A. J.; Kirkland, J. J. J . Chromatogr. 1986, 352. 275-298. (19) MaCD0~lld,R. S. J . Php. chem.1918, 62, 1168-1178. (20) Boonstra, B. B.: Cochrane, H.; Dannenberg, E. M. Rubber Chem. Technd. 1975, 48. 558-578. (21) Osthoff. R. C.; Kantor, S. W. Inorg. Synlh.1975, 5, 55-83. (22) Balky, D. L.; Sommer, L. H.; Whitmore, F. C. J . Am. (2”. Soc. 1984. 70, 435-436. (23) Bij, K. E.; Horvath, Cs.; Melander. W. R.; Nahum, A. J . Chromatogr. 1981, 203, 85-84. (24) Heam, M. T. W.; &ego, B. J . Chromatogr. 1981, 278, 497-507. (25) barn, M. T. W.; &ego, B. J . Chmmtogr. 1989, 255, 125-136. (26) barn, M. T. W.; Agullar, M. I . In Modern Physlcel Methods In 8kchembby; Neuberger, A., Van Deenen, L. L. M.. Eds.; Elsevler ScC ence Publishers B.V.: Amsterdam, 1988; Part B, pp 107-142. (27) Klkland, J. J.; Yau, W. W.; Stoklosa, H. J.; Dllks. C. D., Jr. J . Chromtogr. SCI. 1977, 75.303-318. (28) Pearson, J. 0.; Lln, N. T.; Regnk, F. R. Anal. 8bchem. 1982, 724, 217-231. (29) Berendsen, 0. E.; de Galan, L. J . LIq. Chromafogr. 1978. 7 , 561-588.
RECEIVED for review February 25, 1991. Accepted June 5, 1991. This work was supported by the Chinese National Foundational Committee of Natural Science and the Foundation of Academia Sinica,the Australian Academy of Science, and the Australian Research Council.
Theory for Electrostatic Interaction Chromatography of Proteins J a n StHhlberg* Astra Pharmaceutical Production AB, Quality Control, 151 85 Sodertalje, Sweden
Bengt Jonsson Division of Physical Chemistry 1, Chemical Center, University of Lund, 221 00 Lund, Sweden Csaba HorvQth Department of Chemical Engineering, Yale University, New Haven, Connecticut 06520 A dmpie thwretkai framework for the effect of the eluting salt concentration on the retention factor of proteins in ionexchange chromatography under conditions of linear eiutbn is described. It Is based on the solution of the linearized Polsson-Boltrmann equatbn for two opporltely charged pianar surfaces in contact with a salt dutlon. The theory predick a thew rdatkn between the bgarlthmk retfactor and the reciprocal square root of the ionic strength of the eiwnt in the salt concentration range used in linear elutlon chromatography. A large body of retention data obtained in lon-exchange chromatography of proteins over a*wkle range of experimental conditions was plotted as in k’ VI V d I , where k’ and I are the retention factor and ionic strength, respectively. The plots are linear or nearly so,as predicted for a moderate salt concentration range by the theory. From the slope of such plots the characteristlc charges of the proteins were estimated by uslng only fundamental physicochemical constants. The chromatographlcaiiy measured protein charges compare well to those obtained from tltrimetric experlments at the same pH, although certaln devlations are noted. The theoretical approach presented here offers a more realistic treatment of the ion-exchange chromatography of proteins than the stolchkmetrlc dbplacement model and can serve as a convenient framework for the analysis of retentlon data. 0003-2700/91/0383-1887$02.50/0
INTRODUCTION Ion-exchange chromatographyfor preparative and analytical separation of proteins has been a standard technique for many years. Its importance has further increased during the last decade with the rapid developments in biotechnology. In ion-exchangechromatography of proteins, the stationary phase is a sorbent having a hydrophilic surface with fixed charges and the mobile phase is usually a buffer solution containing an eluting salt. In most cases the separation is performed by gradient elution with increasing salt concentration. The type and concentration of salt depends on the particular separation problem at hand. The understanding of the influence of the concentration and type of salt on the retention behavior of a protein is therefore of fundamental interest. The traditional theory for ion-exchange chromatography of proteins ( 1 ) is based on a stoichiometric ion-exchange process and was introduced by Boardman and Partridge (2). One of several proposed formulations (3) for this process is PCNc NS8 s P NSS NcC (1)
+
+
+
where P is the protein in the mobile phase, C is the counterion D the protein, which is assumed here to be monovalent, and jk represents the coions bound to the stationary phase. N, and N , are the number of salt ions involved in the exchange process. Overbars indicate that the species are bound to the stationary phase. 0 1991 American Chemlcal Society
