Anal. Chem. 2000, 72, 71R-79R
Infrared Spectroscopy J. Kevin Gillie*
Applied Extrusion Technologies, 15 Read’s Way, New Castle, Delaware 19720 Jill Hochlowski
Abbott Laboratories,100 Abbott Park Road, Abbott Park, Illinois 60064-6101 Georgia A. Arbuckle-Keil
Rutgers UniversitysCamden, 315 Penn Street, Camden, New Jersey, 08102 Review Contents Instrumentation Two-Dimensional Correlation Spectroscopy Optorheological Applications Composition- and Concentration-Dependent Applications Temperature- and Pressure-Induced Applications Potential-Dependent (Electrochemical) Applications Raman Scattering Biopolymer (Protein) Applications Other Developments Combinatorial Chemistry Infrared Microscopy for Chemistry Validation Infrared Microscopy for Quantitative Analysis on Polystyrene Beads Infrared Microscopy for Analysis of Reaction Kinetics Library Encoding by Infrared Spectroscopy Applications of Raman Spectroscopy to Combinatorial Chemistry Understanding the System through Vibrational Spectroscopic Techniques Trends for Vibrational Spectroscopy in Combinatorial Chemistry Human Health Literature Cited
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This review on infrared spectroscopy highlights three areas in which infrared spectroscopy has become increasingly important during the last two years. These areas are two-dimensional infrared (2D IR) spectroscopy, combinatorial chemistry, and human health. This trend is quite evident in the abstracts examined in preparation for this article. No review article is complete without also including some discussion of the current developments in instrumentation. The review is focused on articles published during 1998 and 1999, but occasionally will include articles from earlier work if they provided additional information we thought pertinent. There is a wealth of other very interesting work that has been published and we encourage the reader to pursue them. As the reader follows the article, they should find that each area discussed impacts another area, which shows that the science of infrared spectroscopy is very exciting indeed. 10.1021/a1000006w CCC: $19.00 Published on Web 04/13/2000
© 2000 American Chemical Society
INSTRUMENTATION The techniques used to acquire and analyze infrared spectra continue to evolve. The advent of step-scan instruments has allowed for advances in the time and spatial resolution capabilities of infrared spectroscopy. The beam qualities associated with synchrotron light sources can also allow for improvements in spatial resolution beyond the current capabilities using standard sources and microscopes. Finally, IR imaging may make mapping chemical functionality easy and contribute to areas where spatial information has been ignored. Synchrotron IR light sources are now available at the National Synchrotron Light Source-Brookhaven National Laboratory; the Daresbury Synchrotron Radiation Source, Cheshire, England; the Advanced Light Source-Lawrence Berkeley National Laboratory; and UVSOR-Institute for Molecular Science, Okazaki, Japan. It is the beam attributes of low thermal noise, brightness, low divergence, and excellent signal-to-noise ratio that make these IR sources unique. These attributes make possible experiments where small aperaturing is important or sample scattering normally precludes IR spectroscopy. The beamlines and selected applications have been described for Drasebury (1), the Advanced Light Source (2), UVSOR (3), and the National Synchrotron Light Source (4, 5). The unique properties of IR synchrotron radiation are highlighted with applications to microspectroscopy and subnanosecond timeresolved spectroscopy (5). Synchrotron IR radiation has been used in high-pressure studies of natural and synthetic minerals (4), alkali halide thin films (3), reflection absorption spectroscopy of Cl atoms adsorbed on Ag(100) (1), and thin polymer laminates (1). Application to problems of geological interest include fluid inclusions (6), and inorganic-organic interaction at the bacterialmineral interface (7). An attenuated total reflection (ATR) sampling technique based on synchrotron IR radiation has been used to probe spatial surface heterogeneity in fibers (8). A crosssectional view of photochemical degradation in an acrylic polymer coating at 5-µm spatial resolution using a synchrotron IR source revealed the depth of photochemical degradation and the interdiffusion of adjacent clear and base coats (9). Single human hairs were also mapped at 5 µm using synchrotron-based FT-IR microspectroscopy (10). Spatial resolution for probing and mapping plant materials is enhanced using synchrotron-based FT-IR Analytical Chemistry, Vol. 72, No. 12, June 15, 2000 71R
microspectroscopy relative to standard FT-IR microspectroscopy where scattering is usually a difficult problem to overcome (11). Spatial chemical functionality information has been available through mapping techniques using motorized translation stages and infrared microspectroscopy. Although refinements continue to make this more accessible, it still has not broken into wide use. The advent of focal-plane array (FPA) detectors based on MCT or InSb offers the opportunity to advance this field and make it more widely utilized. Commercial instruments based on MCT offer a spectral range of 5000-900 cm-1 at 64 × 64 and 256 × 256 elements. The commercial InSb FPA detectors offer the spectral range of 10 000-2000 cm-1 with 256 × 256 and 640 × 512 elements. The cross section of a laminated polymer film was examined using a FPA detector (12). The technique was clearly able to identify the individual layers and even the individual pixels in the 7.5-µm-thick adhesive layer did not show interference from adjacent layers. This technique was also demonstrated for an epoxy-polyurethane sample (13) and human bone tissue (12). Couple the FPA detector with a step-scan FT-IR instrument and you have a powerful technique to resolve both spatial and temporal information such as the dissolution of a polymer (14). The commercial availability of step-scan technology is also beginning to impact IR spectroscopy. For example, step-scanning FT-IR photoacoustic spectroscopy has been used to perform depth profiling studies on polymeric multilayers (15, 16), polymer film laminations (17), latex films (18, 19), and a single particle and fiber (20). Time-resolved step-scan FT-IR spectroscopy with temporal resolution down to 15 ns has been used to study early and late M intermediates of bacteriorhodopsin (21, 22), and organic reactions and catalyst (22). Although these techniques are not in every laboratory yet, they show promise in enabling the IR spectroscopist to work on problems that were previously intractable. Their impact on material science, biological science, and chemical processes will increase as they are incorporated into solving research, industrial, and medical problems. TWO-DIMENSIONAL CORRELATION SPECTROSCOPY New applications of generalized 2D correlation spectroscopy continue to be reported. 2D correlation spectroscopy is based on a technique proposed by Noda in 1986 (23-25) which he extended in 1993 (26) to include spectral changes as a function of time or other physical variable. The advantages of this method are the ability to separate overlapping bands and investigate the time sequence of certain spectral events. Two-dimensional correlation analysis requires a series of perturbation-dependent spectra. The types of physical perturbations probed with this technique continue to expand and currently include the following: mechanical stress, applied potential, temperature, pressure, concentration, and composition. An overview of 2D IR spectroscopy was recently published (27). The theory of dynamic IR linear dichroism (DIRLD), which effectively combines dynamic mechanical analysis (DMA) and infrared dichroism spectroscopy, is discussed as well as the generation of synchronous and asynchronous 2D plots and the types of instrumentation needed to perform 2D IR spectroscopy. Optorheological Applications. The first applications of 2D correlation spectroscopy monitored the effect of a mechanical 72R
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perturbation or stress on the infrared spectral response from polymers such as polystyrene (28), poly(ethylene terephthalate) (29, 30), nylon 11 (31), polypropylene (32, 33), and a low-density polyethylene/perdeuterated high-density polyethylene blend (34). Polymer reorientation dynamics were investigated in this manner. For example, step-scan FT-IR studies of a composite film of isotactic polypropylene and poly(γ-benzyl-L-glutamate) (35) were able to distinguish overlapping amide bands that were not distinguishable by static infrared spectral analysis. The overlapping amide bands reoriented at different rates. Optorheological investigations of polymers enable researchers to probe the molecular dynamics of the polymer system. The relaxation kinetics of polystyrene and poly(vinyl methyl ether) blends have been examined (36). This study also analyzed the reorientation rates of two types of side chains in optically oriented copolymers. Dynamic IR spectroscopy has been used to correlate the reorientation movement of functional groups and the deformation of internal coordinates in poly(ethylene terephthalate) (PET) (37). The internal coordinate change depends on the morphology of the material and is important in describing the macroscopic deformation of oriented PET. A study of the submolecular motional behavior of uniaxially oriented polyamide 11 observed that the dynamic spectra depended upon the thermal treatment of the sample (38). Overlapping bands were resolved, and the hydrogen-bonded regions were more sensitive to the applied perturbation. In the investigations of uniaxially drawn polystyrene (39) in different crystalline forms, the dynamic response varied according to the conformation of the chains: trans-planar or helix. Shear-induced molecular alignment of confined fluids was measured in a new experimental design that applies step-scan time-resolved IR spectroscopy (40) to the study of a simple nematic liquid crystal, 5-cyanobiphenyl. This optorheological application of applied shear and electrical fields could be useful for studying other polymer and biopolymer samples. The orientation and relaxation behavior of binary blends of long deuterated chains and short undeuterated chains of uniaxially drawn films of poly(methyl methacrylate) (PMMA) (41) were analyzed in terms of their short-chain molecular weight dependence. A random polyester/polyurethane copolymer system (42) was studied using DIRLD. The infrared linear dichroism (IRLD) approach is discussed as well as the theory of DIRLD. The polyurethane segments align to form hard regions, while the polyester segments are more fluid and form soft regions. The observed dynamic response showed a larger contribution from hard segments relative to soft, indicating that the hard segments respond more to the dynamic perturbation. In another copolymer study, DIRLD was used to detect hydrogen bonds in prestretched polyester-polyurethane and unstretched nylon-6 films (43). A rheo-optical near-infrared (NIR) study of poly(dimethylsiloxane) (PDMS)/ polycarbonate (PC) block copolymers found different orientational and recovery phenomena for the hard (PC) and soft (PDMS) regions (44). Optorheological studies have been routinely applied to conventional polymers. Electrically conducting polymers usually display anisotropic conductivity upon uniaxial alignment. A DIRLD analysis of poly(p-phenylene vinylene) (PPV) (45) and PPV precursor found a correlation between the dynamic infrared
response and the viscoelastic behavior of the precursor or PPV. The precursor is more elastic while PPV is more viscous due to the increased conjugation on the backbone. A variable-temperature polymer stretcher has been used to analyze uniaxially drawn PET at five temperatures in the range of 30-150 °C (46). Changes in vibrational modes were observed above the glass transition temperature. This relates the molecular reponse of the polymer to its macroscopic mechanical properties. Diglycidyl ethers of bisphenol-A (DGEBA) epoxy resins were modified with R,ω-diols to allow a systematic variation of chain length and molar ratio of reactants. These epoxy networks were characterized in a temperature-controlled polymer stretcher (47). Uniaxial deformation was applied above and below the glass transition temperature of each resin. In the glassy state, the mechanical properties and the molecular orientation were less influenced by the diol chain length and the molar ratio than above the glass transition temperature. Isotropic and cold-drawn highdensity polyethylene (48) was analyzed as a function of temperature using DIRLD and the results compared with previous studies of polyethylene. Composition- and Concentration-Dependent Applications. A comprehensive series of analyses were performed on blends of atactic polystyrene and poly(2,6-dimethyl-1,4-phenylene ether) (PPE). Infrared (49), Raman (50), and NIR (51) correlation spectroscopy was applied to blends of different compositions: PS/ PPE ) 90/10, 70/30, 50/50, 30/70, 10/90. Composition was the spectral variation for this series of investigations. The 2D IR analysis explored the intermolecular interactions between the two polymers. For the Raman study, NIR excitation was used. The intermolecular interactions other than hydrogen bonding were analyzed. The NIR analysis demonstrated the advantages of 2D correlation analysis in separating overlapping NIR bands. Two-dimensional NIR correlation spectroscopy was also applied to the analysis of ethylene/vinyl acetate (EVA) copolymers (52). The composition dependence in 11 different copolymers was investigated. EVA copolymers are commonly used, and changes in the content of vinyl acetate cause variations in crystallinity and impact strength. The NIR bands due to the ethylene units were classified according to those originating from the amorphous phase and those from the crystalline phase. The usefulness of applying 2D correlation analysis along with chemometrics is discussed as well. Concentration-dependent correlation analysis was applied to electrolyte solutions (53). Spectral features from solute-solvent and solute-solute interactions were separated. An ATR cell was filled with each electrolyte concentration (0-1.0 M). The study demonstrated that 2D analysis is a powerful tool for extracting structural details from vibrational data. A heterogeneously catalyzed reaction was investigated by following the changes in concentration of reactants, intermediates, and products (54). Methanol synthesis from CO/CO2/H2 over a commercial Cu/ZnO/Al2O3 catalyst was monitored. The time dependence of reactant, intermediate, and product concentrations was monitored to obtain rate constants and to monitor the reaction pathway. Temperature- and Pressure-Induced Applications. The segmental mobility of a ferroelectric liquid-crystalline polymer (FLCP) was studied at different temperatures and field strengths
(55). An increase in temperature increases the rate of reorientation of the FLCP due to a decrease in the rotational viscosity. No field dependence was observed for the relaxation times. Temperatureinduced conformational changes were observed in nylon 12 (56). Both IR and NIR spectra were recorded from 25 to 200 °C. Conformational changes were found to first occur in the hydrocarbon chains and then the hydrogen bonds were subsequently broken. Heterospectral correlations between NIR and IR regions were useful in making band assignments in the NIR region. Pressure-modulated dynamic ATR IR spectroscopy was applied to samples of isotactic polypropylene and linear low-density polyethylene (57). A commercial high-pressure diamond ATR microaccessory was modified with a piezoelectric pressure transducer to apply sinusoidal pressure modulations. The results were similar to those found in optorheological studies. The advantages are that a wider variety of materials can be analyzed (various thicknesses) and films of sufficient strength to withstand the dynamic strain of a rheometer are not necessary. In a related study, an ATR cell with temperature and pressure control was used. Spectra were collected as a function of pressure (58). Several temperatures were studied as well. It was observed that linear low-density polyethylene (LLDPE) is strongly affected by the application of pressure. Crystal lamellae of LLDPE reorient, and further compression leads to disintegration of crystals due to partial melting. Temperature-dependent NIR spectral analyses of n- and tertbutyl alcohols were performed in the temperature ranges of 2085 and 25-75 °C (59). The thermal dynamics of hydrogen bonding in the alcohols were evaluated. Using 2D NIR correlation spectra, the first overtone of the OH stretching modes of the monomer, cyclic dimer and linear hydrogen bonds in the polymers were assigned. The first and second overtones of the OH stretching vibration were also analyzed in decan-1-ol (60) in the temperature range of 15-76 °C. Potential-Dependent (Electrochemical) Applications. Twodimensional correlation spectroscopy was applied to analyze the potential-dependent reorientation of a water layer at the interface using surface-enhanced IR absorption spectroscopy (SEIRAS) (61). The spectrum changes in intensity and frequency with applied potential. 2D IR was used to confirm the presence of a very broad band at 3200 cm-1 that changes intensity independently of the band at 3505 cm-1. The orientations deduced from IR spectra were in agreement with molecular dynamics calculations. SEIRAS is useful in monitoring the electrochemical processes at the surface of an electrode. Two-dimensional correlation spectroscopy is useful in analyzing the time-dependent data (62) and provides information that is not easily discovered in onedimensional IR spectra. In a new approach to studying electrode dynamics, SEIRAS, ATR, and dynamic IR spectroscopy using a step-scan interferometer were combined (63). The electrode potential was sinusoidally modulated at a particular frequency while the in-phase and quadrature spectra were collected. The system was demonstrated using a self-assembled monolayer of 4-mercaptopyridine on a gold electrode. The charge-transfer rate was estimated. Two-dimensional correlation spectroscopy was applied to the analysis of corrosion products on copper in contact with air containing SO2 and water (64). From the time-resolved IR spectra, Analytical Chemistry, Vol. 72, No. 12, June 15, 2000
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it was found that (a) the components of the surface layer can be discriminated, (b) sulfite and sulfate grow on the surface, and (c) Chevreul’s salt is the main component of sulfite. Raman Scattering. A new technique, two-dimensional Raman scattering (2D Raman) was introduced (65). It was applied to the study of the microstructural dynamics of a liquid crystal in the presence of an electric field. In this technique, the Raman anisotropy is used to monitor the submolecular motions of the liquid crystal. The two-dimensional correlation analysis of the Raman anisotropy provides information about the reorientation between different segments of the liquid crystal. In this study, a photoelastic modulator (PEM) introduced sinusoidal perturbations in order to induce orientational changes. The motions of both the rigid core and the flexible tail were monitored, and a mechanism for the electric-field induced reorientation was proposed. The investigators further demonstrated this technique (66) in a mixed, binary liquid crystal system. The studies indicated that the flexible parts of the liquid crystal molecule can reorient independently of the rigid core. Biopolymer (Protein) Applications. Some applications of two-dimensional correlation spectroscopy to biopolymers are an extension of the types of methods described above for a different system. The DIRLD study of cellulose (67) clearly separates OH vibrations. The NIR spectra of milk were analyzed with regard to fat concentration dependence (68), and pH-dependent conformational changes were analyzed in cytochrome c (69). In many ways, two-dimensional correlation spectroscopy is especially useful in analyzing biopolymers because they exhibit complicated spectra with peaks from both the peptide backbone and the amino acid side chains. Deconvolution by two-dimensional correlation analysis was used to monitor the effects of chain length and temperature on helix-forming peptides (70). Two-dimensional correlation spectroscopy is becoming a powerful method for studying the secondary structure of proteins. The conformation of proteins can be determined with this technique. The hydrogen-deuterium exchange in myoglobin (71) demonstrated that the amide I band is composed of at least four different conformations. Ribonuclease was used as a model protein and both mid-IR and near-IR spectral regions were recorded (72). Information about folding and unfolding of the β-sheet structure in ribonuclease was determined. Temperature-dependent correlations were used to follow the heat-induced denaturation of ovalbumin (73) in the NIR region, and hydrogen-deuterium exhange in streptavidin (74) was used to monitor changes in the β-sheet structure. A different method of studying the secondary structure involved analyzing the pressure-induced changes in proteins (75) because pressure causes alterations in the spatial structure of the protein and can cause the loss of secondary structure accompanied by denaturation. The solvent effect on aggregational properties of β-amyloid polypeptides (76) involved monitoring spectra as a function of the composition of the solvent mixture. A novel application of two-dimensional correlation spectroscopy involved using fractional secondary protein structure as the perturbation (77). Spectral regions were identified by R-helix- and β-sheet-based correlation maps. A companion paper (78) extended this to heterospectral applications by correlating Raman versus circular dichroism and Raman versus IR. 74R
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Other Developments. Applications of two-dimensional correlation spectroscopy will continue to expand to an ever-widening array of systems. Physical chemistry examples include the following: the kinetics of the photodecomposition of Mo(CO)6 (79) and chemisorption of nitric oxide on Pt(100) (80). Concerns about normalization of the dynamic spectra (81) have been voiced as well as complications due to baseline fluctuations, bandwidth changes, and random noise (82). Routinely step-scan interferometers in the frequency domain are used, but examples of timedomain DIRLD (83) have been demonstrated as well as the use of rapid-scan spectrometers (84, 85). A different type of 2-D IR has recently been reported. Two ultrafast IR laser pulses are used to examine the structure of biopolymers (86). This was highlighted in Chemical and Engineering News (87), which compared multidimensional IR techniques to multidimensional NMR techniques. Although the spectra are displayed as 2D IR plots, the principle differs from that introduced by Noda (26) in that a pump pulse and a probe pulse is used and the resultant spectra are nonlinear. The basic concept of two-dimensional correlation spectroscopy continues to be extended to new areas of investigation. It is clearly a useful technique in discriminating overlapping peaks and observing relationships that would not be discernible by onedimensional IR analysis. COMBINATORIAL CHEMISTRY The application of combinatorial chemistry (88, 89) has exploded during the past decade to become an integral component of nearly every drug discovery effort. Combinatorial libraries are generally synthesized on a solid support, polystyrene beads being the most commonly used medium. These polystyrene beads can be as small as 50 µm in diameter and may contain as little as 200 pmol of compound per bead. As infrared and Raman spectroscopies have each been shown to be applicable to the analysis of compounds on a solid support, vibrational spectroscopy has found increasing use in the arsenal of analytical methods available to the combinatorial chemist. Applications have included the validation of synthetic reactions, quantitative analysis of yield, the kinetics of reaction on solid support, and investigations of solidsupported reaction mechanisms, as well as in library encoding strategies. Although photoacoustic (90), transmission (as KBr pellet) (91), and diffuse reflectance (92) infrared spectroscopic techniques have found application for validation of chemistry on bulk resin samples, it is generally more desirable to acquire spectra for compounds attached to an individual bead. This allows for the analysis of unique structures or library encoding data when synthesized in the 1-bead-1-compound mix and split (93) format, as well allowing analysis of bead-to-bead variability for an individual compound synthesized on a pool of beads. Single-bead analysis is accomplished by employing infrared microscopy, where spectra may be acquired via either transmittance or attenuated reflectance modalities or Raman microscopy. Infrared Microscopy for Chemistry Validation. Since IR excels at confirming the presence of specific functional groups within a structure, chemistry validation is generally performed by looking for the presence or disappearance of a particular functionality formed or destroyed during the reaction sequence.
Yan et al. (94) reported the first application of FT-IR microscopy in the transmission mode to the analysis of individual bead-bound combinatorial chemistry library members. The attachment of a terminal acetylene moiety was confirmed by the appearance in the infrared spectrum of bands at 2120 and 3288 cm-1 for the triple bond and terminal alkyne C-H stretches, respectively. Conversion of an acid chloride to an amide carbonyl functionality was confirmed by the simultaneous disappearance and appearance of bands at 1800 and 1670 cm-1. This report also probed beadto-bead homogeneity of a synthetic reaction on polystyrene by the comparison of infrared spectra of multiple beads from a resin pool. An improvement in the spectral quality obtained for this technique was achieved by the physical action of preflattening individual beads to present to the incident infrared beam a shorter and more uniform path length (95). The resultant higher resolution allowed the authors to easily distinguish a reactant carbonyl frequency at 1720 cm-1 from the product carbonyl band at 1670 cm-1. This flattened-bead technique was applied (96) in conjunction with MAS NMR, to the validation of a multistep reaction sequence monitoring the appearance of carbonyl stretch, alkene bend, and N-H stretch bands. Optimization of three consecutive steps of a multistep reaction sequence (97) on polystyrene was monitored by FT-IR microscopy relying upon the appearance of bands such as C-O-C skeletal vibrations at 1280 and 1100 cm-1 and a sequence of carbonyl conversions. The ability to simultaneously distinguish multiple carbonyl moieties within the structure was particularly useful. Attenuated total reflectance FT-IR microscopy has also been used (98) to acquire the spectra of combinatorial chemistry library members on single polystyrene beads. An ATR objective used in conjunction with a microscope, with bead pressed against the objective to acquire spectra, differs from transmission mode microscopy in that the compound on the surface of a bead is selectively analyzed. Five of the steps in a six-step synthesis lent themselves to validation by observing the infrared bands appearing when functional groups are formed, including the following: a thiouronium at 1645 cm-1, an ester at 1729 cm-1, a carboxylate at 1616 and 1408 cm-1, an amide at 1678 cm-1, and a sulfonyl at 1325 and 1125 cm-1. ATR microscopy has also been applied (99) to the analysis of library compounds generated in another combinatorial chemistry format known as “crowns”, which consist of an array of polyethylene pins coated with a polymer upon which the library compound is synthesized. An ATR objective pressed to the surface of a crown confirmed the removal of a Fmoc protecting group as evidenced by loss of characteristic bands within the infrared spectrum. Infrared Microscopy for Quantitative Analysis on Polystyrene Beads. The application of ATR microscopy has been used for the quantitative analysis (98) of library compounds on individual polystyrene beads. By normalizing the peak area for a carbonyl chromophore characteristic of a library compound against a prominent polystyrene band, the authors demonstrated a 5% standard deviation for loading across 40 individual beads sampled. Quantitative data have also been acquired in transmission mode FT-IR microscopy (100) by employing a specially designed
flow-through cell for the analysis of single, solvent-swelled beads. Spectra were acquired on individual beads trapped within a flow cell, and the yield of a reaction in progress was determined by providing a fixed path length and chromophore standardization, thus giving an accurate quantitative assessment of material on individual beads at any time point. This method was employed for the calculation of yield in an amide coupling reaction. Infrared Microscopy for Analysis of Reaction Kinetics. Infrared spectroscopy offers an easy way to determine reaction kinetics by monitoring the formation with time of a specific band assignable to a functional group being generated during synthesis. The infrared flow cell described by Pivonka et al. (100) was used to investigate the kinetics of a coupling reaction. They monitored the attachment to resin of a deuterium-containing reactant as starting material flowed in situ over a single polystyrene bead. Analysis of the C-D stretch band at 2299 cm-1 determined firstorder kinetics and the half-life for the coupling reaction resultant in deuterium incorporation. This same setup was used to study the kinetics of a photocleavage reaction from resin (101). In this instance, the reduction with time of a CN band as the nitrilecontaining moiety cleaved from the bead was monitored. The analysis of first-derivative data gave excellent kinetics data in situ for this reaction. The ability to acquire single-bead infrared spectra by transmission microscopy has also allowed reaction kinetics monitoring wherein individual beads are extracted from a reaction mixture at fixed time points (94), thus halting the progression of reaction. The disappearance of a characteristic 5-oxazolidinone carbonyl band at 1800 cm-1 was used to study (102) the kinetics of ring opening by amines for this functional group. Similarly, single-bead transmission spectra record the kinetics (103) by simultaneously monitoring the disappearance of an aldehyde band and the appearance of an N-H band during a reaction on a solid phase. Also studied have been reactions involving the oxidation of alcohol to aldehyde (104) and the esterification of an alcohol (105). A determination of reaction kinetics, generating data for a wide range of compounds from a series of acid-labile linkers using transmission microscopy, was reported by Yan et al. (106). These studies allowed the authors to investigate relative cleavage kinetics of diverse compounds from specific linkers, as well as relative cleavage kinetics from different linkers of a single compound. A typical cleavage kinetics experiment entailed acquiring data on six individual beads at different time points, which allowed the synthetic chemist an analytical tool to rapidly optimize reaction conditions and necessitated the use of very little material. ATR has also been used to study reaction kinetics (107). ATR microscopy acquires data on a sample presented to the objective only a short way into the material whereas transmission microscopy records the entire sample. This distinction allowed the authors an opportunity to compare reaction kinetics on the surface and interior portion of an individual bead, demonstrating that no bias for surface against interior was in effect. Library Encoding by Infrared Spectroscopy. Large combinatorial libraries generated in a mix and split synthetic scheme and screened in high-throughput format pose a challenge in that the structure of an individual library member selected as interesting must be identified from among tens of thousands or more possibilities. One method for dealing with this deconvolution Analytical Chemistry, Vol. 72, No. 12, June 15, 2000
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problem has been to employ library encoding strategies wherein some easily determined property of an individual bead can be determined and correlated to the actual library compound structure. An infrared-based strategy (108, 109) was used wherein nitrile-containing compounds, with nine independently distinguishable frequency bands, used in varying combinations and ratios, were employed to encode for first-position monomers in large combinatorial libraries. These nitrile-containing compounds were attached orthogonally to resin via a lysine linker and remained on individual beads after the library compound had been cleaved for biological assay. This leaves a barcode-like chemical signature readable by infrared transmission microscopy. Applications of Raman Spectroscopy to Combinatorial Chemistry. Raman spectroscopy offers a potential adjunct to infrared analysis as the functional group detection at which the two excel is somewhat complementary. Raman has more recently than infrared been brought to bear on the analysis of combinatorial chemistry library compounds. In a pattern similar to that seen for infrared spectroscopy, Raman spectroscopy was first used (110) for the analysis of bulk resin samples. Amide III bands in the Raman spectra of resin-bound peptides demonstrated the Fmoc deprotection of different amino acids analyzed. Single-bead Raman spectra (111), acquired in the microscope mode, were used for the validation of compounds containing Raman-active chromophores. Acetylene- and nitrile-containing compounds attached to resin showed unique bands at 2232 and 2120 cm-1 respectively. An automated system for the acquisition of spectra by singlebead Raman microscopy has been described (112) with the ability to acquire 99 spectra in an unattended, overnight run. This system was employed for the validation of solid-phase reactions for a large series of heterocyclic and aromatic-containing compounds. Deconvolution for large mix and split libraries was also achieved by the direct detection of library compound monomers, evidenced by their Raman chromophores. To accomplish this, spectra were recorded for each monomer used in a library synthesis and mathematically compared to the spectra of full library compounds. Maximal overlap was generally observed by virtue of common Raman chromophores for the spectra of a library compound and those of the corresponding individual monomers present within the structure. A good review (113) on the complementary capabilities and applicable modalities for infrared and Raman spectroscopy details the relative merits of these systems. Understanding the System through Vibrational Spectroscopic Techniques. The ability to understand the mechanics or behavior of materials during solid-phase synthesis has also been examined by vibrational spectroscopic techniques. The efficiency of different mixing methods employed in solid-phase synthesis was monitored by single-bead infrared microscopy (114). Acquiring infrared spectra on several random beads synthesized using different mixing techniques, the simultaneous loss of an aldehyde band and formation of a hydrazone band during reaction indicated the relative uniformity of reaction completion for a variety of mixing protocols. With an infrared flow cell trapping single beads for microscopic examination, an understanding of mobility and partitioning effects inside polystyrene beads was achieved (115). Deuterated solvents 76R
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were used to determine diffusion and partitioning information into polystyrene beads for a series of analytes in different solvents. Infrared transmission microscopy has also been used to gain insight into the nature of site interactions for compounds bound to polystyrene beads (116). The interactions between compounds bound to the resin could be observed by noting the intensity of bonded versus unbonded hydroxyl stretch bands at 3420 and 3580 cm-1, respectively, . The effects of variables such as resin type, linker type, and specific structural features of compounds attached were each studied. Raman microscopy allows the analysis of library compounds attached to (112, 117) and diffusing from beads into an agar matrix as a gel diffusion assay is carried out for large mix and split combinatorial libraries. Structural elucidation via direct diversomer detection for individual compounds by spectral comparison is achievable in this format, allowing the potential for assay in situ structure-activity relationships to be rapidly determined. Trends for Vibrational Spectroscopy in Combinatorial Chemistry. With the approximate ten years of experience in hand of screening combinatorial chemistry libraries, generated in both the large mix and split format and the smaller focused or parallel formats, controversy (118) exists as to the relative benefit of continued screening of the larger libraries. This debate probably has bearing only on the necessity of library encoding strategies. Whether any given laboratory effort synthesizes large or several (relatively) small numbered compound collections, there will always be the necessity to acquire analytical data on these compounds in a rapid, high-throughput manner. Vibrational spectroscopy has played a key role to date in the analysis of compounds generated by solid-phase organic synthesis, and the trend toward generating more automated systems is to be expected in this area. Automation of Raman spectral acquisition has been previously described (112). Haap et al. (119) have similarly reported a high-throughput infrared transmission microscopy setup with an x-y stage, allowing mapping across hundreds of immobilized polystyrene beads. A multispectral imaging spectrophotometer with near-IR camera capable of recording spectra across an array of 320 × 240 pixels can simultaneously acquire spectra on multiple beads (120). These and other automated systems are likely to have more importance in the future of vibrational spectroscopy in its application to combinatorial library analysis. HUMAN HEALTH The application of infrared spectroscopy to problems in human health is increasing. This is logical if one considers that the strength of IR is in probing chemical functionality and the environment in which it resides. Carbonyl groups, amide groups, and hydroxyl groups, for example, all have distinct absorptions that are modified by hydrogen bonding from the surrounding matrix. Although the unaided human eye may not be able to distinguish subtle differences in protein spectra, the use of standard mathematical treatments has exposed the richness of information available in each spectrum. This application is highlighted because it is increasingly important in studies of diseases, cancer, metabolism, drugs, and drug delivery. This trend is emphasized by recent conferences
such as the symposium at SPIE that highlighted infrared spectroscopy as a new tool in medicine (121). Upcoming conferences such as the International Conference on Fourier Transform Spectroscopy and Spectroscopy 2000 also feature symposia focused on application of vibrational spectroscopy to health issues. Shaw and Mantsch have authored an excellent review of vibrational biospectroscopy which provides a number of interesting examples illustrating applications to plants, animals, and humans (122). Noninvasive spectroscopic techniques for clinical chemical analysis are of considerable interest. Since blood is the primary metabolic transport system in the body (123), spectroscopic determination of metabolites in blood would be advantageous. For example, the blood glucose level measured by utilizing the spectral region between 1062 and 997 cm-1, the most specific region identified for glucose in dried blood sera, showed a high correlation to that determined by a glucose oxidase method (124). Similarly, glucose was determined in whole blood using partial least-squares models and the spectral range between 1500 and 900 cm-1 (125). Additional blood components such as albumin, triglycerides, cholesterol, total proteins, and hemocrit have also been analyzed by mid-IR and strong correlations to standard measurement techniques demonstrated (126- 128). The challenge will be to move these correlations to noninvasive techniques (123). The infrared spectrum is unique for each molecule, which, of course, is its strength. Is there a similar uniqueness associated with healthy and diseased tissue? Can infrared spectroscopy detect relatively small pathophysiological changes associated with diseased tissue? Disease pattern recognition (DPR) using infrared spectroscopy is being explored (129). By examining more than 2000 spectra from “healthy” and “diseased” individuals using various mathematical algorithms, Werner et al. demonstrated that clear distinctions can be found in infrared spectra of ‘healthy” and “diseased” individuals (129). Use of infrared spectroscopy for DPR is supported by other studies demonstrating spectral differences between normal and diseased tissue on the cellular level. The infrared spectra of exfoliated cervical cells from patients with cervical cancer differ from those of cells from noncancerous women (130). By examining over 2000 individuals’ cells, the authors were able to demonstrate a continuum of changes paralleling the transition from normalcy to malignancy (130). Differences in the protein secondary structure and protein conformation were found in the infrared spectra of human benign and malignant astrocytomas (131). The application of IR spectroscopy to the analysis of cultured tumor cells and grading breast cancer sections is discussed with potential sources of errors outlined (132). Use of fiber optics to examine various tumor tissues at different stages of development is expected to lead to better diagnostic aids such as endoscopic examination (133). Imaging of thin section of human colon carcinoma by infrared microspectroscopy using a x,y stage and alternatively a MCT focal plane array is being used to develop knowledge of the structural alternations that occur upon carcinogenesis (134). Diagnosing thyroid neoplasms using multivariate approaches to study the IR spectra is possible. Four different spectral patterns were observed corresponding to colloid goiter, adenoma, carcinomas, and negative disease (135).
Healthy and diseased tissue can be distinguished because IR spectroscopy can probe protein structural and matrix changes. For example, the amide I band can be used to monitor secondary structural changes in proteins, the amide II band can be used to study protein dynamics through hydrogen-deuterium exchange, and changes caused by temperature can also be monitored (136). IR spectroscopy, especially polarized IR, has revealed valuable information on the orientation of functional groups and substructures within membranes (137). Physiological changes that are either characteristic of a disease, lead to a disease, or are part of the pathology of a disease are being explored with infrared spectroscopy. The aggregation of β-amyloid peptides is characteristic of Alzheimer’s disease. The aggregation of amyloid analogues has been followed in CCl4 solutions (138) and in hexafluoropropanol solutions (139). It is suggested that these analogues are suitable models for studying amyloid aggregation (138) and that infrared spectroscopy can be used to standardize the aggregation grade of β-amyloid structures. Protein changes associated with cataractous human lens have been outlined with the importance of myopia and diabetes (140) on maturing lens studied. The changes in the protein secondary structures markedly increased the total β-type and random coil structures and decreased the R-helical conformation (140). Chemical analysis of multiple sclerosis lesions by IR microspectroscopy reveals that lipids and proteins could be oxidized at active MS lesion sites, suggesting a role for free radicals in the pathogenesis of MS (141). Medical microbiology can be impacted by infrared spectroscopy. The fingerprint-like spectral signatures of intact microbial cells appear to discriminate microbial species and strains, detect in situ intracellular components, and characterize growth-dependent phenomena or cell-drug interactions (142). Mathematical treatment of the data including resolution enhancement techniques, difference spectroscopy, and pattern recognition methods is often required (142). Neural networks combined with IR spectroscopy can provide a rapid and accurate method to discriminate antibiotic susceptibility as demonstrated on methicillin-resistant and methicillin-susceptible Staphylococcus aureus (143). Controlled drug delivery is dependent on many factors including the structure and interaction of the drug in the delivery system and the penetration of the drug through membranes, e.g., epidermis. Infrared spectroscopy suggests that the structure of recombinant human growth hormone is modified during encapsulation within biodegradable polymers (144). IR spectroscopy was used to study the effects of chemical penetration enhancers on the in vitro percutaneous absorption of tamoxifen through porcine epidermis (145). The enhancement of the permeability of tamoxifen by eugenol and D-limonene is due to lipid extraction and improved partitioning into the stratum corneum. Only lipid extraction is important for mentone-enhanced permeability. Drug penetration studies have also been performed using step-scan FTIR photoacoustic spectroscopy (146). In this example, the diffusion coefficient for clotrimazole penetration into membranes was determined. The nondestructive nature of this technique is valuable. Advanced IR techniques are coming into use for human health issues. Synchrotron infrared microspectroscopy was used to Analytical Chemistry, Vol. 72, No. 12, June 15, 2000
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noninvasively assess the in situ human cell responses to metal surfaces of the type generally used in metal implants (147). Subtle changes in the IR spectra are believed caused by material-induced mutagenesis. Biomedical imaging using high-resolution IR spectroscopy and high-definition digital imaging available through focal plane arrays is being explored. The ability of this system to generate spectroscopic signatures and images from single human breast cells is demonstrated (148). J. Kevin Gillie is a Research Scientist in the Research and Development group at AET Films. His current responsibilities include directing the analytical sciences lab at AET Films. He received a B.S. in chemistry from James Madison University in 1983 and a Ph.D. in physical chemistry from Iowa State University in 1989. Prior to joining AET Films, he worked in the Analytical Sciences Laboratory for The Dow Chemical Co. He has ten years of experience applying vibrational spectroscopy to solve complex industrial and materials problems. Since joining AET Films, he has focused on applying a variety of analytical techniques to the complex development, manufacturing, and application of oriented polypropylene films. Jill Hochlowski received a Ph.D. in chemistry in 1983 from the University of California, San Diego, with a thesis in Marine Natural Products from Scripps Institution of Oceanography. She spent 13 years at Abbott Laboratories conducting research on bioactive microbial metabolites in the Pharmaceutical Products Division. The next four years in the Combinatorial Chemistry group at Abbott were spent developing library encoding methodologies and structural validation techniques for compounds synthesized on solid supports. She is currently the group leader for a high-throughput purification group, which solves isolation and validation problems for medicinal and combinatorial chemistry clients. Vibrational spectroscopy has played a role throughout her career in research on natural products, combinatorial, and purification/validation chemistries. Georgia A. Arbuckle-Keil is an Associate Professor of Chemistry at Rutgers University-Camden. She received a B.A. in chemistry from Rutgers University in 1983 and a Ph.D. in chemistry from the University of Pennsylvania in 1987. Prior to joining the faculty at Rutgers University in 1989, she was a postdoctoral assistant at Princeton University. Her research interests include the synthesis and characterization of electroactive polymers using thermal analysis and vibrational spectroscopy. She routinely applies TGA-IR and DIRLD to the analysis of conducting polymers. She is a member of the Society for Applied Spectroscopy and the American Chemical Society. She is chair-elect 2000 of the Philadelphia Section of the ACS.
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