Robert J. Markovich

Fourier transform infrared assay of membrane lipids immobilized to silica: Leaching and stability of immobilized artificial membrane-bonded phases

A nondestructive, sensitive assay to monitor the hydrocarbon content of silica-based chromatography particles has been developed. The assay requires a microscope accessory interfaced with a Fourier transform infrared (FTIR) spectrometer. For determining hydrocarbon content, undiluted alkyl-silica-bonded phases were pressed into a thin wafer. Hydrocarbon content was quantitated using the integrated hydrocarbon band intensity between 2995 and 2825 cm-1 [i.e., band area C-H] and the integrated silica oxide band intensity between 1945 and 1780 cm-1 [i.e., band area Si-O]. Plotting the [band area C-H]/[band area Si-O] ratio vs the carbon content determined by elemental analysis gave a correlation coefficient of r = 0.997. The FTIR assay was validated on 5-, 7-, and 12-microns silica particles using three different immobilized artificial membrane (IAM) silica-bonded phases. The utility of the FTIR assay in determining hydrocarbon content was demonstrated by evaluating hydrocarbon leaching from IAM phases exposed to mobile-phase solvents. The ability of organic solvents to leach hydrocarbon from IAM phases containing phosphatidylcholine (PC) as the immobilized ligand was chloroform greater than ethanol approximately methanol greater than ethyl acetate greater than methylene chloride greater than acetonitrile greater than acetone. Acetone and acetonitrile cause very little hydrocarbon leaching from HPLC-IAM.PC columns. When challenged with different mobile phases, IAM.PC columns perfused with mobile phase are more stable than IAM.PC-bonded phases stirred in mobile phases. IAM.PC contains lecithin linked to silica by amide bonds.(ABSTRACT TRUNCATED AT 250 WORDS)

Fourier transform infrared assay of liposomal lipids.

Quantitating the lipid content in organic lipid solutions and extracted membrane preparations is described. Fourier transform infrared analysis of thin films using perdeuterated nonadecane as an internal standard permitted quantitation with greater than 95% accuracy.

Introduction to Fourier Transform Infrared Spectroscopy and Applications in the Pharmaceutical Sciences

The applications of infrared spectroscopy to pharmaceutical sciences is small compared to the applications of infrared spectroscopy to the fields of chemistry, biology, and biochemistry. This is unfortunate because modern routine infrared spectrometers are excellent research tools that provide very high signal-to-noise, high resolution, and extensive data-manipulation computer software packages. This review summarizes basic principles of infrared spectrometers and the use of Fourier self-deconvolution.

Formation of the antiplanar-antiplanar phosphate conformation of dilauroylphosphatidylcholine bilayers

Infrared spectroscopy was used to investigate lipid conformational changes that occur in dilauroylphosphatidylcholine (diC12PC) bilayers with and without fatty-acid-amino-acids as guest molecules in the membrane. Incorporating 2.5 mole% N-decanoylglycine (decgly) into diC12PC liposomes caused formation of the antiplanar-antiplanar (ap-ap) phosphodiester conformation which was stable in room temperature IR spectra. Several other fatty-acid-amino-acids incorporated into diC12PC bilayers were found to also elicit the ap-ap phosphodiester conformation. Unlike these diC12PC/fatty-acid-amino-acid mixed bilayers, pure diC12PC bilayers would form the ap-ap phosphodiester conformation only under low temperature incubation conditions. Dry diC12PC films incubated at 5 degrees C for 0.5 h (brief incubation) or 16 h (prolonged incubation), and then rapidly hydrated (i.e., vortexed at 25 degrees C in D2O), caused the ap-ap phosphodiester conformation to persist in the diC12PC liposomes equilibrated to room temperature. Slow hydration for 16 h at 5 degrees C in both buffered and non-buffered D2O of diC12PC lipid films also produced the ap-ap phosphodiester conformation. In contrast, slow hydration for 16 h at 5 degrees C in PBS/D2O of diC12PC/decgly mixed films caused the greatest number of ap-ap phosphodiester conformers. Using pure diC12PC bilayers, infrared data indicate that incubation of diC12PC films causes the headgroup phosphodiester conformation to change from gauche-gauche (g-g) conformation to the ap-ap conformation. Under all liposome formation conditions examined, no changes in hydration of either the phosphate group or the carbonyl ester group were detected and in addition, no trans/gauche conformational changes in the acyl chain were observed.