| Research Summary |
|
|
Dr. Purdie's research emphasis is in the area of applied spectroscopy. The objectives are to develop rapid and convenient methods of analysis that are direct. In other words laborious separations are not part of the designed procedures. Methods of this kind are especially important in the clinical, forensic, food, and pharmaceutical sciences and in developing sensors for airborne pathogens, etc. Well-devised methods will be rapidly and readily amenable to routine, even robotics, handling. Analysis without separation puts a special premium on the need for enhanced selectivity in the method and/or the detector. In biologically important areas, selectivity is generally accomplished using enzymes. Enzymatic procedures are selective but not always specific so there can be interferences from structurally similar molecules. Other drawbacks to enzymatic methods are that the enzymes have limited shelf lives and they rely upon an auxiliary reaction that uses a product from the enzymatic reaction (e.g. H2O2) to generate the color necessary for absorbance or fluorescence detection in the visible or UV wavelength range. Successful enzymatic assays rely heavily on time consistent stoichiometric correlations between the enzymatic and color creation steps. In our work one answer has been to use circular dichroism (CD) spectropolarimetry rather than absorbance as the detector. To be CD active, the analyte must simultaneously absorb electromagnetic radiation and be optically active (chiral). Analytes that meet only one or other of these requirements are not detectable. The double molecular requirement is restrictive enough that many complex mixtures can be analyzed without separations, for example, alkaloids in unseparated solvent extracts of plant materials. Because of the chirality controlled specificity in binding drug molecules to receptors in the body, the preparation of enantiomeric forms of drug molecules is a rapidly growing industry, What our work can contribute to this industry are quality control assay procedures. Selectivity is enhanced even more by the combination of color derivatization and CD detection. Shifting the CD activity from the UV to the visible spectral range greatly reduces the interferences from other naturally occurring compounds that are strong UV absorbers and are extracted along with the analyte of interest. This has proved to the most important factor in developing a convenient procedure for the accurate determination of enantiomeric purity in pharmaceutical drug formulations. The color-inducing reagent we prefer is an optically active metal complex and the level of selectivity that is accomplished is unsurpassed. For instance the visible CD spectra for the isomeric forms R- and S-ephedrines and the R- and S-pseudoephedrines are so entirely different that any one can be positively identified. Another approach to achieving analytical selectivity is to find a reagent that will react directly with the analyte and in the process produce a colored derivative. Being able to do this eliminates the need for an auxiliary color reaction step and its associated limitations mentioned above. Colors from direct chemical reactions with target analytes for the most part are produced by the creation of multiply conjugated double bond systems in the products that are chromophores for the strong absorbance of UV and visible light. Examples of these that are used in clinical analyses are few in number and detection, as a rule, is limited to a single wavelength. One example of a direct chemical color derivatization reaction is the Liebermann-Burchard which through time became the CDC standard procedure for the direct measurement of serum cholesterol against which all other assays, including enzyme-based assays, are standardized. Direct chemical methods are usually more hazardous and therefore user unfriendly and difficult to automate. We have taken the application of direct chemical methods further by identifying an alternative to the Liebermann-Burchard reagent that is specific to only double and triple bonds in a host of molecules, but especially in steroids, lipids, and terpenes. Extra selectivity is added when absorbances are measured at multiple wavelengths, or even better, over the full visible spectral range. Differences in full spectrum patterns are often enough to easily discriminate among closely related structural molecules, and mixtures made from these molecules can be analyzed using mathematical algorithms that fit the whole spectrum from weighted aggregates of the spectra for the components. This has proved to be very effective in the direct measurement of the proportionate distribution of cholesterol over the three major lipoproteins (VLDL, LDL, and HDL) in human serum in a single color derivatization experiment. Most recently the reagent was found to discriminate among the unsaturated lipids in serum and plasma, which include cholesterol and the esters of the mono- and polyunsaturated fatty acids, oleic. linoleic, conjugated linoleic, linolenic, arachidonic, EPA, and DHA acids in a single routine assay. The ready diagnosis of lipids disease states are done quite simply by pattern recognition. If the amount for each component is desired this is simply done by a matrix solution of the over-determined system of linear equations for the full wavelength range. Recent Publications
|
