James A. de Haseth

Phase Correction of Vibrational Circular Dichroic Features

Several sources of phase-correction-induced spectral anomalies in FT-IR vibrational circular dichroism (VCD) spectra have been investigated. Misidentification of the zero-phase retardation position in dichroic interferograms that exhibit no optical or electronic bias can produce spectral errors. Production of such errors is from the introduction of linear phase error into the phase curve. When the zero-phase retardation position is correctly identified, other spectral anomalies, such as “reflected peaks,” can appear in VCD spectra. These peaks are readily observed in quarterwave plate reference spectra. The anomalies are directly correlated to the arctangent function used to define the phase curve and result only from the nature of the VCD signal. VCD spectra can exhibit negative, as well as positive, peaks; consequently the phase correction must be designed to accommodate negative features. Both Mertz and Forman phase-correction algorithms have been modified to correct the phase of VCD interferograms without error. Such corrections are not necessary, or even desirable, for normal absorption spectrometry.

Evaluation of Particle Beam Fourier Transform Infrared Spectrometry for the Analysis of Globular Proteins: Conformation of β-Lactoglobulin and Lysozyme

The efficacy of the particle beam LC/FT-IR interface, in its development as a tool for the determination of dynamic protein structure from experiments such as HPLC separations and folding/refolding intermediate analysis, is presented here. The particle beam apparatus can be used to desolvate proteins rapidly in preparation of IR measurements. Several experiments have been designed to determine whether the operation of the particle beam apparatus causes alteration to the complex structural features of globular proteins, and whether it produces a solid-state spectrum representative of protein solution structure. It is shown here that the structural integrity of β-lactoglobulin is maintained when nebulized, desolvated from solution, and deposited onto the IR-substrate. Since enzyme activity is dependent upon the maintenance of higher-order structure, a complementary series of spectrophotometric-activity experiments with lysozyme collected from the particle beam were performed to determine the state of the tertiary and quaternary structures. The lysozyme particle beam deposit not only produced a secondary structure estimate similar to that of solution; it also retained its biological activity. It is demonstrated that the particle beam can induce structural changes in proteins with a carrier-liquid concentration gradient; this characteristic is useful for band assignment.

MAGIC-LC/FT-IR spectrometry

The development of a new high performance liquid chromatography (HPLC) interface with Fourier transform infrared (FT-IR) spectrometry is currently being studied. This interface removes HPLC solvents prior to infrared detection. The solvent elimination properties of this interface make it possible to obtain spectra from reverse phase chromatography with no interferences from residual solvent.

Glass nebulizer interface for capillary electrophoresis-Fourier transform infrared spectrometry.

A capillary electrophoresis system has been successfully interfaced to a Fourier transform infrared spectrometer. The design of the interface is a custom-designed glass microconcentric nebulizer. Typical deposit characteristics include reproducible circular deposits of uniform thickness that lack any splatter as found in earlier designs. Interface performance is demonstrated in that there is no loss of electrical current during operation and spectra of analytes can be readily produced. Furthermore, it has been shown that the interface maintains the plug flow characteristic of capillary electrophoresis.

Complete structure elucidation of a globular protein by particle beam liquid chromatography-Fourier transform infrared spectrometry and electrospray liquid chromatography-mass spectrometry sequence and conformation of β-lactoglobulin

The advantages to the use of both mass spectrometry (MS) and Fourier transform infrared spectrometry (FT-IR) in combination for the structural characterization of the tryptic digest of a model globular protein is demonstrated. HPLC has been interfaced to both spectroscopic techniques and has provided a high degree of structural detail for the target protein. beta-Lactoglobulins A and B were digested with trypsin and chromatographed with narrow-bore, reversed-phase HPLC. As determined by LC-FT-IR spectrometry, the conformation of each form of intact beta-lactoglobulin was randomized upon elution. The particle beam and the electrospray LC-MS interfaces enabled the acquisition of spectra for nanogram injection quantities. The primary structures were determined from the accurate molecular mass determinations of the digest fragments. Infrared spectra confirmed the presence of some amino acid functionalities.

MAGIC-LC/FT-IR: A viable interface for HPLC and FT-IR spectrometry☆

Abstract The interfacing of separation methods with selective detectors has been a long sought after goal. In some instances, such as gas chromatography interfaced with spectrometric methods such as mass spectrometry or Fourier transform infrared spectrometry, considerable success has been achieved. Interfaces with liquid chromatography have been far less successful due to the difficulty in eliminating the mobile phase. In the infrared, liquid mobile phases must be eliminated as they are opaque at all wavelengths at even short path lengths. The solutes are generally in such low concentration that long path lengths must be used to record their spectra in the presence of the solvent. Nonetheless, the solvent opacity precludes this option. Solvent elimination has been possible for high volatility nonpolar mobile phases, yet the majority of liquid chromatography is accomplished with polar (reverse phase) solvents. An interface has been developed that can accommodate both normal and reverse phase solvent systems, as well as gradient elution systems. This interface is the monodisperse aerosol generation interface for combining liquid chromatography with Fourier transform infrared spectrometry.

Flow Cell CCC/FT-IR Spectrometry

Abstract The development of flow cell LC/FT-IR spectrometry has been plagued by the fact that liquid chromatographic mobile phases absorb infrared radiation. This problem is exacerbated by low solute-to-solvent ratios in the eluates. High speed countercurrent chromatography (CCC) is used in this study to obtain high solute-to-solvent ratios that alleviate some of the problems of infrared absorption of the mobile phase. The high solute-to-solvent ratios allow the use of a simple flow cell interface which makes complex solvent removal procedures unnecessary. CCC/FT-IR (Countercurrent chromatography/Fourier transform infrared) spectrometry is useful for applications where the analyst is not limited by sample size, and high sample loadings are possible. CCC/FT-IR spectrometry is particularly useful to users of countercurrent chromatography to obtain useful structural information of mixture components with an FT-IR spectrometer.

Semiautomated Depositor for Infrared Microspectrometry

The measurement of minute samples is desirable in many areas of research and analysis, including biological, environmental, and forensic sciences. The use of manual solution direct deposition, combined with surface evaporation, is a very useful and convenient method for the transfer of many analytes to a spectroscopic sampling window. For Fourier transform infrared (FT-IR) spectrometric analysis, high quality attenuated total reflection (ATR) and transmission analyses are possible with suitable solution deposits. If the sample area is very small, placement of the analyte deposit on the active area of the sampling accessory must be done carefully. To achieve this, a novel direct deposition system has been developed. This system, a series of valves attached to a glass nebulizer, generates deposits that are reproducible, and placement of the deposits is precise. The valves allow the sample to be loaded, helium airflow to expel the sample, and the nebulizer to be cleaned after deposition. To help contain the sample to a small area once deposition has taken place, a vacuum line is attached to the nebulizer. This simple semiautomated deposition system allows for higher sensitivity and run-to-run reproducibility for minute sample analysis.

Preliminary studies for interfacing countercurrent chromatography (CCC) with Fourier transform infrared (FT-IR) spectrometry

Abstract High speed countercurrent chromatography (CCC) and its use as an interface to a Fourier transform infrared (FT-IR) spectrometer are described. In this preliminary study the high solute-to-solvent ratios attainable with CCC are investigated, and infrared spectra are recorded of CCC fractions of aromatic components. The resulting infrared spectra of mg/mL concentration CCC fractions have very high signal-to-noise ratios, and no interference bands from the stationary phase (water/methanol) can be seen. Chloroform was used as the mobile phase. This study clearly demonstrates that a CCC/FT-IR spectrometry interface is feasible.

Comparison of Columns for the Analytical High Speed Countercurrent Chromatograph

Abstract Use of a high speed countercurrent chromatograph (CCC) for analytical-scale separations is described. Separations are reported for columns of 0.85, 1.2, and 1.6 mm internal diameter. The effect of varying the experimental conditions was also investigated. It was found necessary to modify the CCC for the 0.85 and 1.2 mm i.d. columns, where no modification was necessary for the 1.6 mm i.d. columns. Carryover of the stationary phase frequently occurred with the 0.85 mm i.d. column, but carryover was not a problem with the 1.2 mm i.d. columns. The solute-to-solvent ratios of the eluates obtained with the 1.2 mm i.d. columns were higher than those obtained with the larger-bore columns. The 1.6 mm i.d. columns, however, proved to be better suited for preparative separations as faster flow rates were possible, which led to faster separations.