224Q
Anal. Chem. 1993, 65, 2249-2253
Effect of the Chain Mobility of Polymeric Reversed-Phase Stationary Phases on Polypeptide Retention Michael Hanson and Klaus K. Unger’ Institut fur Anorganische Chemie und Analytische Chemie, Johannes Gutenberg- Universittit, 6500 Mainz, Germany
Jutta Schmid, Klaus Albert, and E. Bayer Institut fur Organische Chemie, der Universitat Tubingen, 7400 Tubingen, Germany
The resolution of polypeptides in reversed-phase chromatography can be improved by inducing conformational changes in the solute. Protein unfolding can be achieved by an increased accessability of the hydrophobic stationary phase. We elucidated how far results from CP/MAS solidstate NMR relaxation measurements could be correlated with chromatographicdata in terms of protein and peptide retention. The retentivity and denaturation potential of reversed stationary phases is controlled not only by their hydrophobicity but also by their ligand mobility and steric properties. As a consequence, we developed a simplified model for protein retention on stationary phases with different dynamic properties:We used an n-octadecylphase (RPlS),a poly(octadecy1 methacrylate)phase (POMA),and a polybutadiene phase (PBD).
INTRODUCTION Contrary to the broad application of reversed-phase chromatography (RPC) there is still a lack of fundamental understanding of the retention mechanisms. The classical solvophobic theory of Horvath et al.1 has been refined by the work of Martire and Boehm2 and Dorsey and Dill.3 The key to the retention mechanism is assumed to be the transfer process of the analyte from the mobile phase into the stationary-phase environment. Increased transfer rates are consistant with an increased opening of cavities in the stationary phase. The number of cavities formed in the stationary phase is controlled by the spatial organization of the structural elements of the stationary phase. Almost all discussions on retention mechanisms refer to silanized silicas. Dorsey and Dill proposed an interphase behavior for n-alkyl chains in bonded phases. Due to the grafting of the n-alkyl chains to the silica surface a partial chain alignment of the alkyl chains takes place a t surface loads higher than 2.7 pmol/m2. Thus, lateral interactions between the “hydrophobic”ligands induce different kinds of molecular organization. One can observe that the disorder gradually increases from the chain segments next to the silica up to the chain ends of the ligands. Energy differences among the rotational isomers of n-alkyl chains are in the order of 17-21 kJ/mol; therefore, the interconversion of different alkylchain conformations should be easily possible. Because retention is dependent on the nature of both the stationary (1)Horvath, C.;Melander, W.; Molnar,I. J. Chromatogr. 1976,125, 129. (2)Martire, D.E.;Boehm, R. E. J.Phys. Chem. 1983,87, 1045. (3) Dorsey, J. D.; Dill, K. A. Chem. Rev. 1989, 89, 331. 0003-2700/93/0365-2249$04.00/0
phase and the modile phase, a changing mobile-phase composition together with the nature and size of the solute may induce changes in alkyl-chain conformations. In this interactive play, different molecular organization of the “hydrophobic” part in reversed-phase stationary phases should also cause changes in the three-dimensional arrangement of solutes, e.g., in polypeptides or in proteins. Thus, helix/coil transitions as well as folding/unfolding processes of proteins should be dependent upon the nature and the molecular organization of the stationary phase.
EXPERIMENTAL SECTION Chemicals and Materials. LiChrospher Si 300 (mean diameter of 10 pm), Monospher (mean diameter of 2.1 pm), and all solvents used in this work were obtained from E. Merck (Darmstadt, Germany). Polybutadiene and dicumyl peroxide were supplied by Aldrich Chemie (Steinheim, Germany). Proteins were supplied by Sigma (Deisenhofen, Germany). Water was deionized with a Milli-Qsystem (Millipore-Waters,Eschborn, Germany). Coating Procedure. The coating procedure was carried out as described by Hanson et al.‘ We used a 1 % (w/w) loading on the nonporous silica (Monospher) and a 10% (w/w) loading on the porous silica (LiChrospher Si 300). While the nonporous packings were used for the chromatographic experiments, the porous packings provided a higher sensitivityfor the solid-state NMR studies, due to their higher surface area. Silanization. The octadecylsilanemodification of the silicas was performed as described in ref 4. Chromatography. The chromatographic experiments were performed with a LC unit consisting of an Autochrom 300 benchtop gradient controller system/terminal, two Model A2200 LC pumps (Bischoff,Leonberg, Germany) and a Rheodyne 7410 injector. A Shimadzu SPD-6VA UV detector and a Shimadzu C-R3A integrator were also used. The modified nonporous silicas used in the chromatographic experimentswere suspendedin 1.4-dioxane/toluene/cyclohexanol (l:l:l,v/v/v) and packed by the downward flow method into stainlesssteel columns (34X 4.6 mm i.d.) (Bischoff)at a packing pressure of 250 bar, using methanol as a packing solvent. For the protein and peptide separations we used 6-min gradientsfrom water/0.05 % TFA (A) to acetonitrile/O.OS%TFA (B). The gradient stepness was increased gradually in the dwelltime variation experiment. Solid-state NMR Spectra. For the NMR experiments we used polymer-coated or silanized LiChrospher Si 300 (E. Merck) with a nominal pore diameter of 30 nm because of its higher surface area and, hence, higher signal intensity during spectrosCOPY. Solid-state NMR spectra were obtained on a MSL 200 NMR spectrometer (Bruker,Karlsruhe, Germany)at 4.7T withsamples of 200-300 mg in double-airbearing rotors of ZrOz. Magic angle spinning was carried out at a spinning rate of 3700 Hz. Temperature control was performed by the Bruker BVT 100 temperature unit. CP/MAS NMR spectra were recorded with (4) Hanson, M.; Unger, K. K.; Schomburg, G. J. Chromatogr. 1990, 517, 269.
0 1993 American Chemlcal Society
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 17, SEPTEMBER 1, 1993
-b
E RP18
POMA
E PED
0
1.0
,M
MIN
210nrn
210nrn
Ln
J Un ” L
400nm
0
400nrn 5 0
W r
400 nrn
5 0
MIN
MI N Flguro 1. Chromatograms of the protein mixture on the RP18 (a), POMA (b) and PBD phase (c): lysozyme (L), cytochrome c (C), (apo) myoglobin (M), heme (H). MIN
MIN
1.0
0 5
-,>-
E
a proton 90° pulse length of 5 ps and a repetition time of 2 s. Proton spin-lattice relaxation times in the rotating frame T l p ~ were measured as described by Schaefer and StejskaL6 T r p ~ v a l u ~ were calculated using a least-squaresanalysis (Bruker program SIMFIT). All NMR spectra were externally referenced to liquid tetramethylsilane.
RESULTS AND DISCUSSION Three proteins [lysozyme (L),cytochrome c (C),myoglobin (M)] and different peptides (reduced and oxidized tropomyosin analogs, T m W T M L m , TmA230JTmA23A were used as analytes in this study. The retention behavior of these solutes was examined on three different reversed-phase stationary phases: (i) octadecyl-bonded silica; (ii) polymeric phase without side chains, polybutadiene (PBD); (iii) polymeric phase with side chains, poly(octadecy1 methacrylate) (POMA). Columns with these stationary phases have been tested under gradient conditions. Previous studies on the retention of the three proteins (L, C, M) on polymethacrylates with different hydrophobicity have shown that the elution order is strongly dependent on the denaturation potential of the stationary phasess The peak of the most stable protein lysozyme shifts in the elution pattern according to its conformational state, whereby cytochrome c and myoglobin act as so-calledmarker proteins. However, the question occurred as to whether the denaturation potential of a stationary phase is only dependent on its hydrophobicity (or hydrophylicity) or whether there is a dependence on additional parameters. We applied the test mixture to the packings examined in this study, PBD, POMA, and RP18, and we observed that the unfolding of lysozyme under the given conditions does not correlate with the polarity of the stationary phase as expected. On the nonpolar, purely hydrocarboneous polybutadiene phase we observe lysozyme to be eluted in its unfolded and folded form (Figure IC). Obviously, the denaturation potential of PBD is less than for RP18 and POMA despite its higher hydrophobicity. Differences between the octadecylsilane phase and the octadecyl methacrylate phase cannot be seen for lack of a sufficient dwell time of the proteins on the stationary phase (Figure la,b). However, a dwell time variation leads to the appearence of strong kinetic effects. While the elution pattern of the test mixture does not change on the RP18 phase, even for very fast separations in the range of less than 30 s, changes for lysozyme can be obseved on POMA. By reducing the ,(5) Schaefer, J.; Stejskal, E. 0. J. Am. Chem. SOC.1976,98, 1031. (6) Hanson,M.;Unger,K.K.;Mant,C.T.;Hodges,R.S.J.Chromotogr. 1992,599,65.
A 1.0
210 MIN
Flguro 2. Chromatogramsof the LCM protein mlxture on POMA: dwell time variatlon.
Table I. Peptide Retention on Different Stationary Phases. retention K’ RP18POMAPBDpeptide Monospher Monospher Monospher neurotensin 5.1 4.5 4.0 TmLo, 8.7 7.9 6.3 TmLFlEd 9.5 9.1 8.1 TmA230, TmA23~
8.7 9.0
7.9 8.6
7.0 8.1
“TrnLk and TmLw are oxidized or reduced tropomyosin analogues as described in ref 2. TmA23 denotes TmL analogues with alanine substitution at position 23. dwell time of lysozyme on the POMA phase it gradually remains folded,and as a consequence it is eluted earlier (Figure 2). The denaturation potential of the studied stationary phases follows in this case the sequence RP18 > POMA > PBD. Moreover, this observation is underlined by the retention and capacity factors of a set of peptides collected in Table I. It should be emphasized that the carbon load of all three stationary phases is essentially the same. In order to understand the gradation in retention and in the denaturation potential of the peptides and proteins it is not sufficient only to use the hydrophobicity of the stationary phase. It appears that steric and dynamic behavior of the hydrocarboneous ligands might lead to these differences. The organization of hydrocarboneous ligands and their mobility has been intensively studied on silanized silicas by applying flourescence spectroscopy, solid-state NMR spectroscopy7 with relaxation time measurements, FTIR spectroscopy? and others. None of these techniques has yet been applied to the characterization of polymeric stationary phases. Also correlations between the dynamic behavior of hydro(7) Albert, K.; Bayer, E. J. Chromatogr.1991,544,345. (8)Sander, L. C.; Callis, J. B.; Field, L. R. Anul. Chem.1983,55,1068.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 17, SEPTEMBER 1, 1993
phobic stationary phases and the retention of analytes have been performed mainly for low molecular weight analytes and very few for peptides.9 Moreover the organization and mobilityof the structuralelements of a hydrophobicstationary phase is not only dependent on the chemical structure, but also on the changes of the mobile-phasecompositionas carried out in reversed-phase gradient elution. For silanized silicas a fully extended and a fully collapsed conformation has been proposed, depending on the content of organic solvent in the mobile phase.10 In addition, the induction of conformational changes to the structure of proteins by mobile-phase effects is the same for all proteins studied since the chromatographic conditions remained the same for the different packings. Therefore, possible changes should arise from stationaryphase effects. We have used solid-state NMR relaxation measurements to elucidate differences in the mobility of the structural elements of the hydrophobic stationary phases and tried to correlate the solid-state relaxation parameters with the chromatographic retention of analytes. The strongest arguments against this approach are (i) that we do not monitor the mobility of the stationary phase in situ, i.e., in the solvated state, and (ii) that we receive only information on the stationary phase but not on the mobility or configurational changes of the analytes, e.g., proteins, during surface/solute interactions. This, however, is a general problem in many areas when high-resolution physiochemical methods cannot be applied to a system in situ. Recent results from microcalorimetry indicate that an in situ observation of surface/ solute adsorption with a nonchromatographic system is possible; however, in this case steric and dynamic effects cannot be sufficiently described.'l We will first review the advantages and disadvantages of solid-state and suspended-state NMR spectroscopy and, second, discuss the merits of the CPIMAS technique in the structure and mobility elucidation of hydrophobic stationary phases used in this study. We have found, in comparative measurements, that the mobility changes caused by the disorder gradient of n-alkyl chains bonded to silica can be monitored by either suspendedstate or solid-state NMR spectroscopy. The results reveal, however, that the basic molecular chain organization can only be characterized by solid-state relaxation parameters. For instance the existence of a critical chain length of six to eight methylene groups in the n-alkyl chain as well as proof of the critical ligand concentration of 2.7 pmol/m2 is derived from solid-state NMR measurements. Moreover, a correlation between RPC retention times and solid-state relaxation parameters is even possible without considering the influence of the mobile phase. On the other hand, changesthe retention behavior due to different mobile-phase compositionscan only be monitored under real chromatographic conditions by suspended-state and NMR imaging experiments. For deducing basic differences in molecular organization of hydrocarboneous surfaces, 13C solid-state NMR spectroscopy is the technique of choice. High-resolution solid-state NMR spectra are usually recorded by combining the cross polarization technique with magic angle spinning. In CP/MAS experiments the buildup of heteronuclei magnetization (13C or 29Si) is determined by the cross-relaxation constant TCH.The decay of l3C magnetization is characterized by the proton spin-lattice relax. time variation ation time in the rotating frame T l p ~Contact (9)Lork, K.-D.; Unger, K. K.; Brfickner, H.; Hearn, M. T. W. J. Chromatogr. 1989,476, 37. (10) Lochmfiller. C. H.; Wilder, D. R. J. Chrornatogr. Sci. 1979, 17, 574. . (11) Hanson, M.;Unger, K. K.;Rouquerol, J.; Denoyel, R. J.Biochern. Biophya. Method., in preparation.
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Table 11. T l pValues ~ of Methylene and Methyl Carbons of Different Stationary Phases with n-Alkyl Ligands tip^ (ma) tip^ (nu) species RP18 POMA species RP18 POMA Ci-Ci6
28.9
7.1
Cl6
52.1
9.4
experiments allow measurement of the dependence of the amplitude of 13C signals on the applied contact time and therefore enable determination of the parameter TCHfrom the rise and a rough estimation of the tip^ value from the falloff of the resulting curve. An accurate determination of T l p is~ possible by inserting a relaxation period 7 between the beginning of the lH spin lock condition and the beginning of the contact time. This pulse sequence is used in our measurements. In n-alkyl bonded silicas the high mobility of the n-alkyl chain results in a liquid-like behavior of the hydrophobic ligands, even in the solid state. Therefore changes in the spin dynamics derived from solid-state NMR measurements can basically be correlated with the retention behavior of analytes in HPLC. Also, effects of the surface and chemical structure of the parent silica as well as the nature, chain length, and density of the hydrophobic ligands can be studied. The influence of the mobile-phase composition upon the conformational behavior of alkyl chains can be investigated by suspended-state NMR spectroscopy. Because of the apparently higher spectral resolution in the solid state in comparison to suspended-state measurements, a deeper insight into the dynamic behavior of different hydrophobic ligands is possible.'* The motional behavior of a distinct environment of the hydrophobic ligand in the kilohertz range is described by the lH and 13C spin-lattice relaxation parameters in the rotating frame Tp. Since Tlpc values may be strongly influenced by spin-spin interactions, an interpretation may be ambiguous. Only T l p is~ left for probing spin dynamics. In cross-linked polymers [e.g., poly(methy1methacrylates)] the T l p values ~ of the carbons of different polymers are compromised to one value due to spin diffusion. In very flexible molecules or in polymer side chains, spin diffusion is quenched. In the case of n-alkyl chains grafted to silica, different T l pvalues ~ for each resolved carbon atom can be observed. Thus, the dynamic behavior of all resolved carbon atoms of the hydrophobic ligand can be determined from high-resolution solid-state 13C NMR spectroscopy without introducing any labeling. The high-resolution solid-state 1% NMR spectrum of the investigated POMA material is given in Figure 3. Besides the backbone signals (C, 45.2 ppm, CH2 52.7 ppm, and CH3 17.8 ppm), the CHZsignal of Cz-C16 at 29.8 ppm is clearly visible. The signal of the terminal methyl group C coincides with the resonance of the backbone methyl group. Therefore, the dynamic behavior of the CH groups of the octadecyl chain of POMA is characterized by the tip^ values. Table I1 shows T l p values ~ of RP packings containing n-octadecyl chains either chemically bonded to silica via an n-alkylsilane or coated onto the silica using an n-alkyl methacrylate copolymer. All investigated systems are characterized by small correlation times. In the case of the POMA and the n-octadecyl stationary phase, this is proven by temperature-dependent measurements of T l p ~In . the case of PBD systems (Table 111),this is concluded in analogy from block copolymer measurements of polybutadiene and polystyrene. In both systems no direct chemical linkage between the flexible polybutadiene and stiff polystyrene (PS) or silica domain is formed. PBD in PBD/PS block copolymers shows (12) Pfleiderer, B.; Albert, K.; Bayer, E.; Lork, K. D.; Unger, K. K.; Brmkner, H. Angew. Chern., Int. Ed. Engl. 1989,28,327.
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ANALYTICAL CHEMISTRY, VOL. 65. NO. 17. SEFTEMBER 1. 1993 C-2 - C-16
0 I
C-l
CH, I
c-2 - c-16 (CHJ,, I
c-17 CH, I
C-18 CH,
A
roo
$50
PP"
e
0
F@um9. CPlMAS NMR s p e m m of POMA.
b
3:O MIN
Flpura 4. SimpiiRed surface mdei of C,rmodified silica.
Polymer main chain Sh.
SYrIaSe
Flpum 5. Simplified suface model of POMA-coated silica.
Table 111. Results of the Contact Time Variation Experiments for PBD-Coated Silica chemical shift chemical shift (ppm) T I ~(Hm ~ ) (ppm) TI- (ms) 130 40
6.8 4.4
31
4.6
the typical elastomeric behavior. With increasing temperature all relaxation parameters exhibit higher values. Thus, in all investigated systems, increasing T l pvalues ~ correlate to an increasing mobility of the species of interest.
I
0
I
3.0 MIN
Major differences in the TI+,H value of the POMA and n-odsdecyl bonded silica are observed. TUXvalues of the protons associated with each resolved carbon atom of the
ANALYTICAL CHEMISTRY, VOL. 65, NO. 17, SEPTEMBER 1, 1993
n-octadecyl chain are much lower in the case of the C18 side chain of POMA. This can be explained by the completely different structures of both packings. Conventional RP packings belong to the class of interphases; the alkyl chains are aligned more or less perpendicular to the surface and easily undergo concerted motions. Starting from the C 4 4 5 bond, there is a high propability of gauche interactions. Through lateral interactions between the n-alkyl chains from C4 to Cl8, two major environments of n-alkyl-chain conformations exist: one ordered organization with more all-trans conformation and one more disordered organization with a significant population of gauche bonds. Both environments easily undergo converted motions, which results in an overall segmental motion and a high T~,,Hvalue of 28.9 ms. For graphic representation in Figure 3, only the all-trans conformation with chains perpendicular to the surface are shown. In the POMA packing the polymeric chain can be quite disordered and/or folded, so there are a large number of orientations of the n-alkyl chainspossible. This inducessteric constraints, thereby confiningthe motions to a lower extent, which are reflected by the low T ~ value H of 7.1 ms. Finally, the highly cross-linked polybutadiene phase without long side chains shows the lowest mobility. The T~,,H value amounts to 4.6 ms (Table 111). Taking the dynamic measurements into consideration, we developed a simplified model for the hydrophobic surfaces which also elucidates their accessibility for peptides and proteins (Figures 4-6). According to the molecular size of proteins of about 2.6-8.2 nm for molecular masses of 100lo00 kDa,lS one can conclude that they are not able to completely penetrate the stationary phase. However, the n-octadecyl ligands of the bonded silica allow easy access of the proteins to the stationary phase due to their high mobility. According to the model of Dorsey and Dill? an increased mass-transfer rate is possible. The increased dwell time of the protein at the n-alkyl bonded phase induces van der Waals interactions between the alkyl groups and the protein core and leads to its unfolding. The elution profile of the protein mixture on the POMA phase does not show significant differences from the RP18 phase under the same conditions. Only the heme of myoglobin is retained differently. Although the retention mechanism in both cases is mainly determined by the n-octadecyl ligands, a similar chromatographic behavior seems to be contradictory, since the POMA side chains are sterically hindered in their mobility. An answer to this question can be given by the dwell time variation of the proteins on the two stationary phase. For the RP18-phase we could not observe any dwell time effect. However, the chromatograhic runs of lysozyme on the POMA phase show (13) Guiochon, G.; Martin, M. J. Chromatogr. 1985,326, 3. (14) Haneon, M.; Unger, K. K.; Mant, C. T.; Hodges, R. S. J. Chromatogr. 1992,599, 77.
2259
decreasing denaturation potential for decreasing dwell time (Figure 2). Thus, the octadecylligandsshowdifferent kinetics in protein denaturation. One might concludethat there must be a conherence between the mobility of the stationary phase and its denaturation kinetics. In the case of lysozyme, a total unfolding cannot be induced even during long chromatographic runs on the PBD phase. The low mobility and the decreased contact area of the protein with the polybutadiene phase due to the lack of side chains promote a mild chromatographic environment and compete with the hydrophobicity of the polymer. In addition, we observed dwell time effects of proteins, for instance, papain, on the polybutadiene phase (Figure 7A). This behavior correlates with the other results since the lack of side chains was expected to have a higher impact on the kinetics compared to the sterically hindered n-octadecyl side chains of POMA. A further dwell time effect for POMA is shown for papain in Figure 7b. However, in both cases, for POMA and RP18, a total shift to folded papain could not be observed, probably due to the higher stability of papain compared to lysozyme. In previous studies we have described a method to classify the denaturation potential of stationary phases by using model peptides of defined stability." A further aspect of phase characterization is described by the peptide retention. Table I shows the capacity factors of some peptides on the different stationary phases. The observed retention power is RP18 > POMA > PBD and correlates with the proposed grade of denaturation potential.
CONCLUSIONS It has been demonstrated that polypeptide retention in RPC is controlled not only by the hydrophobicity or polarity of the stationary phase but also by the mobility of the ligands and sterical effects. Especiallyin cases where conformational changes on proteins are induced, the motion dynamics of the stationary phase appear to have a major impact on the unfolding kinetics. Furthermore, smaller molecules such as peptides can penetrate a stationary phase with highly mobile ligands and generate a larger contact area between the analyte and the retentive site, which results in a strong retention. In cases where the penetration of analytes is sterically hindered or mobile side chains do not exist, we observed a decreasing peptide retention due to the lower contact area between peptide and stationary phase.
ACKNOWLEDGMENT M.H. thanks the Deutache Forschungsgemeinschaft(DFG) for the support. RECEIVEDfor review November 4, 1992. Accepted April 26, 1993.
