degree and types of interactions of the analyte molecules (i.e., solutes) with the two phases. In the case of LC the two phases can also affect each other to a significant degree. Hence, information in terms of these cooperative effects is also needed. For example, for chemically bonded phases the solvent can cause a change in the orientation and mobility of the anchored chains (I). Likewise, the chains and underlying unreacted silanol groups can cause enhancement of the solvent structure at or near the surface (2).Both result in changes in the surface's affinity for the solute and, hence, alteration in selectivity among different solutes. In LC it is the surface-mobile phase cooperative effects that account for experimental observations such as longer than expected retention times based on changes in mobile-phase polarity, inconsistent measurements of chromatographie void volume with alteration in composition and polarity of the mobile phase, and enhanced selectivity when certain mobile-phase modifiers are used (3-5). Likewise it is the cooperative effects that often are hardest to measure and interpret. In the past half dozen years a host of novel approaches has been used to derive fundamental information about various aspects of separation processes. The integration of chromatographic and spectrometric methods has been especially helpful in answering questiona ahout the chemical and structural nature of the surface, homogeneity and interaction of attached layers, overall molecular and segmental chain motion for immobilized groups, and in the case of LC, substantial cooperative effects. NMR, IR, and luminescence techniques have been widely used in these studies; a discussion of how each of these techniques can be used to provide information about chromatographic processes involving chemically modified phases is presented in the remainder Of this REPORT. NMRstudias NMR studies fall into two broad classes: those carried out under drystate conditions (6, 7)and those carried out under solvated conditions (2, 5,8).The experimental counterparts of these are, respectively, solid-state NMR involving cross-polarization and magic-angle spinning (CP-MAS) techniques and conventional solution NMR experiments. The CP-MAS results are acquired under experimental conditions that mimic those of GC, whereas the conventional solution NMR results are acquired under conditions similar to those of LC. In an operational sense, solid-state NMR techniques are especially attrac tive because the sample is run in the 1466 A
. .. I '
.
Figure 1. Inversion-recovery spectra for the terminal methylchlorwllane-m~ifiedsilica
.
Labeling: 1% enridlmenl 100% at terminal position. Solvmt: DMSO. Puiae delay (in seconds): (a) 0.3.(b) 0.6. (e) 0.9. (d) 1.2, (e) 1.5, (f) 1.8, (g) 2.1
dry state, requiring no special pretreatment or handling. By rapidly spinning the sample at an angle of 54.T with respect to the field (i.e., the magic angle) the resulting resonance signals are narrowed via reduction in chemical shift anisotropy. Because of experimental limitations, rotor speeds of 3-5 kHz are used. A g o d discussion of solid-state NMR can be found in Reference 9. To further enhance the weak signals arising from the low natural ahundance of carbon, and because of carbon's lower magnetic moment and longer spin system recovery times, proton-carhon cross-polarization (CP) techniques are also used. For chromatographic materials that are run in their native state CP becomes especially important, as most surfaces are modified at levels from about 5% to 15%depending on the immobilized ligand's size and shape, the degree of reactivity of the modifying monomers,
* ANALYTICAL CHEMISTRY, VOL. 57, NO. 14, DECEMBER 1985
and the surface area of the base silica material. For conventional solution NMR, as in the case of solid-state NMR, a major problem encountered is signal intensity. The experiment is complicated further by multiple line coalescence arising from extremely broad resonance signals with similar chemical shifts. These disadvantages can he overcome synthetically by isotopic enrichment at a single point within the surface-immohilized moiety (I, 8). This is illustrated hy work carried out on chemically immobilized alkyl chains, which represent the most widely used type of surface in liquid chromatography. Selective labeling along the chain has been used to study changes in motion as a function of distance from the surface. Signals arising from carhons labeled near the site of attachment are broad and asymmetrical, resulting from reduced mobility as well as heterogeneous surface-bonding effects (&IO). In the case of the terminal methyl carbon the dominant motion contributing to relaxation is carbon-carbon rotation (8).Similar results have been reported for the drystate counterpart (6).A typical inversion-recovery sequence for a terminally labeled dodecyl chain immobilized to silica and placed in contact with DMSO is shown in Figure 1. Good signal to noise was obtained with only 250 pulses per time point. From a plot of these and similar data, it is possible to calculate the spin-lattice relaxation time (7'1) for the la-
beled methyl group. In DMSO, 7’1 is 2.2 8. This value varies depending on solvent viscosity and class, as discussed below. Although little experimental work has been carried out to date, the terminal carbon is a t an ideal position to function as a surface-bound probe to study interfacial phenomena (2).This is the result of the rapid rotational component of the end methyl group; the fact that the rotational motion is affected little as a function of hydrocarbon spacer distance; and, assuming the spacer arm is several units long, the absence of heterogeneous surface effects. In Figure 2, the distance that the labeled methyl group is extended into the interfacial region can be altered by varying the spacer arm length (Le., distance L from the surface). Because of heterogeneous effects the minimum distance is probably around 4-6 carbons (8,10,II).It is uncertain what the maximum distance might be before steric limitations, such as chain folding, occur. However, initial experiments have been carried out in our own laboratory to investigate the interfacial region using immobilized terminally labeled dodecyl groups (2). The data suggest a significantly greater degree of solvent structuring near the surface for certain contact liquids (see Example 3 below). NMR measurements can be made either on the sorbent or the sorbate. To date most studies have been carried out at the surface. These studies have been useful in answering fundamental questions about chemically bonded phases especially in terms of cooperative effecta in liquid chromatography. Three examples are considered below. Example 1. Recently, spin-lattice relaxation measurements have been reported for several linear hydrocarbon phases as a function of paition of labeling, surface attachment chemistry, and extent of modification (8). Tl was constant a t lower surface coverages. However, a significant reduction was observed a t higher concentrations of the immobilized groups, indicative of increased steric interaction between the alkyl chains. Interestingly, these l488A
-
observations were found to correlate well with chain interaction models previously proposed to explain chromatographic data (12). Example 2. For reversed-phase chromatography carried out in nearly 1 M water, incremental additions of organic solvents to the mobile phase can result in increases in solute retention (5).This behavior is opposite to that expected if only polarity changes in the mobile phase are considered. Likewise, apparent inconsistencies in the measurement of column void volume have been reported with variations in the contact solvent (3).In this latter instance the validity of information derived from peak position experiments is directly dependent on the ability to assess void volume accurately. In GC this is not a problem; however, in LC a correct measurement is often extremely difficult. Both of the above can be explained in terms of solvent-induced changes in the chemically attached layer. Correlations of NMR line shape (5) and spin-lattice relaxation time (8) with variations in the properties of the contact solvent (such as composition, viscosity, and polarity) have provided additional evidence for preferential surface solvation models. The organic component of mixed mobile-phase systems is selectively extracted into the immobilized chains, which results in structural, dimensional, and polarity changes of the microenvironment.
I
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Recently a “breathing” surface model has been proposed to account for differences in solute retention and selectivity resulting from shrinking or swelling of the bonded layer with alterations in the mobile phase (4). Example 3. The earliest surface models for reversed-phase materials involved rigid descriptions of the chains. The extremes were the “bristle” and the “collaped” configurations ( I ) . These were easy to understand and lent themselves to modeling, as the surface was assumed to have a more or less static character. Hence, variations in solute retention were explained almost totally by changes in the mobile phase. Recently, segmental and total chain mobility have been studied using NMR techniques (2,8,10).The resulting data show that the chain’s dynamics are affected by changes in solvent composition and type. In neat organic solvents spin-lattice relaxation is proportional to the inverse of solvent Viscosity ( q ) and fits different linear relationships (2).Theoretically for small unbound molecules or molecules with little or no segmental motion, a slope of unity is expected. The slope is reduced as the importance of segmental to total motion increases. Hence, by measuring 7’1 vs. 1/q it is possible to study cooperative effecta between the solvent and the immobilized chains. Interestingly, both methanol and acetonitrile, the two most widely used organic components of binary reversed-phase chromatographic systems, have been found to fitthe same relationship and potentially confer to the surface similar effects in terms of orientation and mobility of the immobilized chains (2). Lumllreocencas h u h As in the case of NMR the major luminescence studies fit into two categories: the chemical immobilization of luminescent molecules as a means of studying the surface (23,14) and the use of luminescent molecules as mobile-phase additives to investigate sorption and secondary equilibria. Luminescence studies have been used to w e s s the homogeneity and distribution of bonded groups on the surface. These studies can be snbdivided further based on the relative amount of the probe molecule used. For example, 5-(dimethylamino)-lnaphthalenesulfonamide has been chemically incorporated (13)into linear hydrocarbon phases in an effort to w e s s the microenvironment surrounding the probe as well as to study changes induced in the microenvironment as a result of liquid in contact with the chemically attached layer. For these studies to measure the surface effectivelya small amount of a luminescence prohe is incorporated into
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ANALYTICAL CHEMISTRY, VOL. 57,
NO. 14,
DECEMBER 1985
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the surface layer. This is shown in Figure 3a. Further, it is assumed that the bound layer will behave the same with the probe molecule as it does without the probe molecule. In a gross sense this seems reasonable if the probe concentration is low compared with the bonded-phase coverage. However, in terms of the microenvironment directly in contact with the bound luminescent molecule the assumption is probably incorrect. Hence, information derived by this method is related both to the bulk phase ligands (e.g., linear hydrocarbons) and the probe molecules. The second subset of the chemically anchored type of study is carried out by bonding relatively high amounts of the luminescent molecule to the surface. This is shown in Figure 3h. Experimentally it is necessary to dilute the sample with the unreacted base material to avoid inner-filtering effects. An example of this type of study is the use of (3-(3-pyrenyl)propyl) dimethylchlorosilane (3PPS) to measure
reaction distribution. Observation of both monomer and excimer emissions from chemically altered 3PPS surfaces has led investigators to suggest the existence of both high- and low-density regions (14). These results are further evidence of the complexity of the stationary phase. On a macro basis, synthetic procedures are such that statistically about one quarter to one half of the available silanol groups are in-
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Figure 3. Plctoriai view of varlous Woes of luminescence studies (a) imnwbiiizedpmba In a low w i n g density ?.My showing inwrpumed and isolated moi8wi86. (b) im mobillzed pmbe in a high bonding density study showlng w m e r and excimer emlsabons. X , = di% fw excimer emissim. (c) Honwpsnews bonding model y8. nmhomagenews bonding modst de. rived from hl@ bondingdensity Ivninesmna) studies. (d) Robe used as mobilephase additive lo nudy saption and semndq equilibriumeffects 1470A
ANALYTICAL CHEMISTRY, VOL. 57. NO. 14, DECEMBER 1985
volved in the reactions. On an actual microchemical basis the extent and uniformity of coverage may be much more complicated (Figure 3c). These ideas are consistent with NMR observations (8)and IR results (15,16). The second major experimental approach is to use the luminescent molecule as a mobile-phase additive to study surface sorption and secondary equilihrium effects. This is illustrated in Figure 3d. The wavelength of the emission maximum serves as an indicator of the relative polarity of the probe’s microenvironment. For example, 8-aniline-1-naphthalenesulfonic acid (ANS) fluorescence maximum varies about 50 nm in nonpolar vs. polar solvents. In organic aliphatic and aromatic hydrocarbon solvents the emission is between 440 and 455 nm, whereas in methanol it occurs a t 490 nm. Recently, reversed-phase surfactant systems have been studied using the ammonium salt of ANS as a probe (17). Under totally aqueous mobile-phase conditions high concentrations of surfactant (i.e., tetrabutylanmonium ion) have been found to cause a rearrangement of the surface-immobilized chains. Using another experimental approach we have observed similar effects with other surfactants. The ANS results help demonstrate that simple ion pair and dynamic ion exchange models are gross oversimplifications of a much more complex mechanism when surfactants are used as mobile-phase additives.
infraredstwlles In an operational sense IR experiments can be carried out in several modes including transmission, reflectance, and photoacoustic. To date transmission has been used the most to study chromatographic materials; however, with the increasing availability of modern Fourier transform spectrometers and diffuse-reflectance and photoacoustic attachments these latter techniques, especially diffuse reflectance, seem appealing. Regardless of the collection mode, chromatographic materials are difficult to study by IR because of their chemical and structural features. Two major problems typically encountered are.reduced spectral throughput and limited IR transparency. These result, respectively, from the physical characteristics and backbone and surface groups of the base chromatographic support material. For example, for modern HPLC packings that are prepared via derivatization of porous silica, much of the spectral window is obscured by large bands resulting from adsorbed surface water and the backbone Si-Osi stretching. The problem is further complicated by the highly porous nature of the support and re-
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grouping and is concerned either with monitoring the concentration of functional groups on the surface before and after modification or with investigating the extent of chemical derivatization of the surface. A majority of the measurements l i a v 4 made ~ ~ directly on the groups of interest; however, a few workers have used indirect means to monitor surface reactivity. For example, in this latter case, the relative intensity of the OH bending bands of water (1620 em-9 adsorbed on the underivatized surface silanol groups has been used to determine the extent of modification (18). Within the past few years, IR techniques have been used to study orientational and conformational effects (15,16,19,20). The data derived from these studies are important a~ they provide additional insights about basic mechanisms involved in the seua-
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1473A
bonded in high- and low-density regions. In spectrum b in Figure 4, the high-frequency band arises from ligands that can hydrogen bond with unreacted silanols, and the lower frequency band arises from cyanoalkyl groups hindered from such interaction. Also shown in Figure 4 for comparative purposes is spectrum c obtained from the corresponding stabilized monomer 3-cyanopropyl- trimethylsilane. When spectra were acquired from these same surfaces in contact with a polar mobile-phase modifier, such as 1-butanol, the nitrile bands were broad but symmetrical. These latter results are indicative of displacement of the ligands from the surface and subsequent ligand-solvent interaction.
lxmchmh The three techniques considered in this REPORT are not the only ones that have been used or that can be used. Furthermore, there are a number of variations on the three techniques presented that potentially expand their scope, for example, broadline quadrupole splitting NMR measurements and time correlation IR measurements. Perhaps the next major step in the development of new chromatographic surfaces will be the use of new base materials such as po-
rous carbon, in which case electrochemical techniques will be available as additional tools to study these surfaces. References (1) Gilpin, R. K. J. Chromatogr. Sei. 1984, 22,371.
(2) Gilpin, R. K.; Gangoda. M. E. J. Magn. Res. 1985.64.408. (3) McCormick, R. M.; Karger. B. L. Anal. Chem. l980.52,2249. (4) Martire, D. E.;Boehm, R. E. J. Phys. Chem. 1983.87,1045. ( 5 ) Gilpin, R: K.; Gangoda, M. E. J . Chromatogr. Scr. l983,21,352. (6) Sindorf,D. W.; M a d , G. E. J. Am. Chem. Soc. 1983.105.1848. (7) Sindorf, D.W.; Maeiel, G. E. J. Am.
Suffolk,B. R.; Gilpin, R. K., submitted for publication in Anal. Chim. Acto. (17) Dowling. S . D.; Seitz, W. R. Anal. (16)
Chem. 1984,57,602.
(18) Majors, R. E.;
Hopper. M. J. J . Chro-
matogr. Sei. 1974,12, 767.
Sander, L. C.; Callis. J. B.; Field, L.R. Anal. Chem. 1983.55,1068. (20) Leyden, D. E.: Kendall, D. S.; Burgraff,L. W.; Pern, F. J.; DeBellow, M. Anal. Chem. 1982.54.101. (19)
Chem. Sac. 1983,105,3767.
(8) Gilpin, R. K.; Gangoda, M. E. Anal. Chem. 1984,56.1470. (9) O’Donnell, D. J. In “NMR and Maero-
molecules: Sequence. Dynamic, and Do-
main Structure”; ACS Symposium Series 247: Randall, J. C., Ed.; American
Chemical Society: Washington, D.C.,
1984; pp. 21-41, (10) Gangoda, M. E.;
Gilpin, R. K.
J. Magn. Res. 1983.53,140. Palmer. A. R.: Maciel. G. E. Anal. Chem. 1982,54,2194. (12) Gilpin, R. K.; Squires, J. A. J . Chrornologr. Sei. 1981,19.195. (13) Lochmfiller,C. H.; Marshall, D. B.; Wilder, D. R. Anal. Chim. Acta 1981,
(11)
130, 31.
(14) Lochmtiller, C. H.;Colborn, A. S.; Hunnieutt, M. L.;Harris. J. M. Anal. Chem. 1983.55.1344.
Suffolk,B. R.; Gilpin, R. K. Anal. Chem. 1985.57.596.
(15)
Roger Cilpin reit,ri,ed his R.S. degree in 1969from Indiona S f a l e Uniuersit ) and his Ph.1). depree in 197~?from f h e L’niuersifyof Arizona. In 1978. h e joined f h e f a c u l t yat Kent S t a t e Unii3ersiry. u here he ts now associate professor ofchemrsfry. His research i n . terests are in f u n d a m m t a l and a p plied CC. LC. and TLC; chromatographic. IR. and N M R sfudies of chemically modified surfaces: and pharmaceutical analysis.
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