High Performance Liquid Chromatography Journal Art Essay

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Journal Article ReviewHIGH PERFORMANCE LIQUID CHROMATOGRAPHY Journal Title: Journal of Forensic Science 1996 ; 41(5) : 804-811 Article Title: Identification and Quantitation of source from Hemoglobin of Blood and Blood Mixtures by High Performance Liquid Chromatography Authors: Edgard O. Espinoza, Dr. P.H., Mark A. Kirms, Ph.D., and Maureen S. Filipek, B.S. Introduction: The high frequency of occurrence of bloodstains at crime scenes has resulted in an increased need for sensitive identification methods. In the case of wildlife forensic evidence, this translates to specific techniques capable of identifying the source species (animal) of a bloodstain. Standard techniques include immunodiffusion, immunoelectrophoresis, and isoelectric focusing. Each of these techniques has certain limitations. The application of another analytical technique, High Performance Liquid Chromatography (HPLC), apparently overcomes many of these inherent limitations, to provide a rapid, efficient and sensitive method for the determination of origin of bloodstains. Article Summary : Blood evidence is regularly examined in the forensic wildlife community. As such, sensitive and accurate techniques must be employed. This article examines the techniques currently being used and their limitations, and offers an alternative analytical technique, High Performance Liquid Chromatography. Use of this technique avoids the necessity for specific antisera, required for previously employed immunological methods. In this report, the analysis of 275 animal samples (262 individual samples and 13 mixtures) by HPLC is considered, and the benefits explained. The hemoglobin of 22 discrete families comprising 38 genera and approximately 50 different species was collected, from both wet and dry samples. A Hewlett Packard 1090 Series II HPLC with a diode-array detector set to detect the various components of hemoglobin was used. Solvents were applied using the gradient method, enabling high resolution separation of the specific components. Analysis by HPLC of these different animal hemoglobin samples resulted in species specific retention times for the a-globin, b-globin and heme components of the hemoglobin. “The unique chromatographic retention times produced by the globin chain signals from species to species serve as the profiles to which an unknown species is compared and identified.” The chromatographic results obtained using HPLC were reproducible, with the heme signal for all samples appearing at the same retention time (8.22 min + 0.18 min), and samples as small as 1.2 mg of hemoglobin were able to be analysed. Reasonably good determination (quantitation) of hemoglobin quantities was also possible using this method, as was identification of blood mixtures. On a whole, the technique of HPLC provided a rapid, sensitive and reproducible method for the identification and quantitation of animal blood samples. Analytical Methods: 1) Immunodiffusion Precipitating antibody is often produced during the normal sequence of events in an infection. When rabbits are injected with the blood from another animal, antibodies are formed that react with the invading animal blood to neutralise its presence. These antibodies can be isolated, and used in further analysis, as they will react specifically with antigens of that particular animal species. The process of immunodiffusion takes advantage of the fact that antibodies and antigens will diffuse towards each other on an agar gel-coated plate. Antiserum and the extracted blood evidence are placed in separate holes on the gel. Eventually, antibodies from the antiserum and antigens meet in the gel. If the antibodies for the specific antigens exist in the antiserum, they will bind with that antigen. Agglutination occurs if the antigen is recognized, possible as a result of the antibodies being bivarient. This agglutination results in a visible line between the two wells to which the antiserum and the recognized antigen were originally added. The major limitation of this technique is the necessity for known antiserum. While there are a number of animal antiserums widely available, they are not obtainable for all wildlife families. 2) Immunoelectrophoresis Immunoelectrophoresis combines the process of serum electrophoresis and an antigen-antibody interaction. Electrophoresis is a separation technique that is based on the mobility of ions in an applied electric field. Positively charged ions migrate towards a negative electrode and negatively-charged ions migrate toward a positive electrode. The ions have different migration rates depending on their total charge, size, and shape, and can therefore be separated. In this case, the blood serum is placed on a barbitone impregnated gel and exposed to an electric current, upon which the various proteins (antigens) migrate to form bands, or zones. Antisera is then added to a trough in the electrophoretogram, and subsequently diffuses toward the antigen zones. A reaction occurs upon contact, resulting in the precipitation of antigen-antibody complexes. The presence of a precipitin band indicates that the antigen is present for specific antiserum used. Once again, this procedure is limited to analysis with those readily available antisera. 3) Isoelectric Focusing Isoelectric focusing is a well-established technique for the separation of amphoteric molecules such as proteins. Separation is carried out on gels on which a stable pH gradient ha