Pharmaceutical Applications of Multidimensional Chromatography - Essay Example

Biomedical and pharmaceutical applications of multidimensional chromatography Biochemical separation techniques have long depended on chromatographic modes that evolved into newer and more advanced forms with complicated procedures for separation and analysis. In fact, chromatography as a whole has found its application in all fields of natural and biological science, with extensive use in biomedical and biopharmaceutical research. The chromatographic separation procedure of test molecules using a wide range of critically engineered stationary and mobile supports depends upon the nature and structure of the molecules. Though individual chromatographic techniques like the various forms of liquid chromatography (LC), thin layer chromatography (TLC), gas chromatography (GC) are there for preparation of homogenous groups of biomolecules required for further treatment, the recent most approaches involve combination of two or more different types of chromatographic separation columns for a finer and more efficient resolution. Hence, essentially speaking, one dimensional chromatographic separation is definitely not the last word for isolation and characterization of biomolecules, chiefly proteins. The limitation is due to its low resolving power in presenting a homogenous protein sample from a complex mixture of several other unwanted proteins, associated matrix components and metabolites that can otherwise be segregated in a much more efficient way by various types of matrix loaded multiple columns utilizing their differential properties and affinities. Moreover, combination of two or more analytical separation steps involve perfectly standardized pre-treatment processes that are to be coordinated in an automated way for a complete segregation of the test samples. (Mondello, P. 251) Multidimensional separation techniques were first devised with the implementation of two dimensional separation beds, in which two different parameters for migration corresponding to two distribution coefficients help to identify the separated components on the bed with more certainty and a high peak capacity than that achieved by one dimensional separation procedures. (Cortes, P. 14 - 15) A high value of peak density is an important requisite for effective separation process and it can easily be achieved by combining two or more chromatographic separation techniques. The greater the number of peaks that can be resolved effectively in a given multidimensional separation procedure within a fixed time frame greater is the overall efficiency of the process. Likewise with effective resolution of a higher number of peaks more and more complex samples can be duly segregated, characterized and analyzed for further investigation. In a simple two dimensional chromatographic process that is carried out by a combination of two different columns, the maximum peak capacity will be the product of the peak capacities of the individual columns, thereby generating a higher capacity of resolution for the overall analytical process. As for example, the peak capacities for each separation process being 100 and 200, the combined value of the maximum peak capacity of the two dimensional separation procedure becomes a product of the two, that is, 20000, thereby ensuring high resolution and larger separation between the collected fractions. (Chromatography Online.com, 1998 - 2008) Multidimensional chromatography, therefore, finds a maximum usage in biomedical research and especially in proteomics, where investigation of biological samples of protein mixtures requires prior separation and characterization in order to tag the target protein. In protein analysis research several novel methods of multidimensional chromatographic separations are being currently used in complementation with the 2-D polyacrylamide gel electrophoretic separation (2D-PAGE), for a much better resolution and greater separation space between analyzable eluents. Multidimensional approach involving multidimensional liquid chromatography (MDCL) used in unison with bio-mass spectrometry (MS) is not only giving speed and sensitivity but is also yielding very high resolution in the segregation procedures in biomedical research. Protein separations that are needed in both biomedical and pharmaceutical research for characterization, analysis and drug development are now carried out in novel ways using isolelectric focusing techniques combined with high performance liquid chromatography (IEF-HPLC), high performance liquid chromatography coupled with SDS- polyacrylamide gel electrophoresis (HPLC-SDS-PAGE) giving enhanced resolution. The MDCL is being substituted for further enhancement by the ion exchange chromatography (IEX) coupled with reversed phase liquid chromatography (RPCL) for characterization and analysis of protein samples. Additional dimensions are added to the combined separation procedures by affinity chromatography that helps in the functional analysis of protein samples. (Journal of Chromatography B, 2008) There are indeed a number of practical attributes of multidimensional chromatography. The most significant are peak capacity dispersion, relative speeds of separation between the coupled processes and perfect compatibility between multiple modes applied for the overall procedure. Coupled column fractionation followed by solvent extraction step is also a novel multidimensional segregation procedure employed in a wide variety of biomedical and pharmaceutical research aiming to isolate homogenous samples of proteins with appreciable therapeutic value. Ideally, two dimensional chromatography, is known to be used in various modes, like, multiphase chromatography, coupled column chromatography, sequential analysis, column switching technique, boxcar chromatography and it is considered to be the simplest perfect example of multidimensional chromatography usually used to efficiently single out small amount of samples from complex heterogeneous mixtures. One of the most significant aims of multidimensional chromatographic separation is the isolation of individual target proteins in pharmaceutical research. These protein molecules are known to have therapeutic value and are isolated from complex mixture of host cell proteins after being cloned by recombinant DNA technology in bacterial cells. (Access Excellence, 1999) The fraction from the primary columns corresponding to the highest peak is subsequently loaded to the secondary column for clean fractionation without any possible overlapping contaminants. The process can be repeated by subjecting the eluent with the highest peak to subsequent tertiary and quaternary dimensions for a more homogenous sample having the required purity to be used for drug development in pharmaceutical research. One of the most widely used multidimensional procedures for individual protein isolation in biopharmaceutical research is liquid chromatography that usually involves a size exclusion separation followed treatment by a tailor made affinity column loaded with matrix containing certain receptor molecules or ligands having the capacity to selectively bind with the specific protein of interest. (Avis, P. 66) Usually protein separations used for designing therapeutic drugs in pharmaceutical procedures that require a thorough knowledge as to how the proteins function and how they are affected within the cell system under diseased condition, involve high resolution separation techniques. This definitely demands for much advanced chromatographic techniques that involve complete identification of each and every protein molecule within the analyzable fractions. Hence, the analyzable mixture is to be characterized completely in order to know the extent to which the proteins are functional and affected in cells. This necessitates the use of comprehensive multidimensional chromatography, first introduced at the University of Utah, Salt Lake City, by which each and every fraction from the primary separation is subjected to further separation irrespective of the peak capacity, in order to separate and subsequently characterize the complete set of protein molecules within the mixture, either through mass spectroscopy or similar detection technique. (Journal of High Resolution Chromatography, 1987) The most widely used technique in pharmaceutical separation procedures is the highly efficient comprehensive two dimensional gas chromatography, often abbreviated as GC x GC, due to the enhanced level of sensitivity, high ionization selectivity and increased response at even low concentrations of test materials. Targeted multidimensional gas chromatography is used to screen drugs and metabolites in biological fluids like urine. Using a 50% phenyl methyl polysilphenylene column a complex sample mixture having highly related drugs and a metabolite, prolintane, in canine urine were efficiently resolved using GC x GC procedure. (Journal of Chromatography A, 2003) Such a technique involves complete retention of test sample even in the second dimension and thereby improves the sensitivity and the efficiency of the overall analytical separation process. There are indeed a number of clinical and pharmaceutical applications of targeted two dimensional GC. The process has appeared fruitful for the isolation and characterization of individual drug samples like, amphetamines, inhalation anesthetics, tri-cyclic antidepressants, and antiepileptic drugs from complex mixtures of contaminants, in order to find out their pharmacological properties and study their effects in the cellular environment. (Grob, P. 741) Gas Chromatography coupled with Mass Spectroscopy has been used for the qualitative and quantitative analysis and preparation of pure samples of pharmacological drugs used in pharmaceutical investigations for elucidating their structure and mechanism of action in treatable cells. Inhalation anesthetics, like, Desflurane, Sevoflurane, Enflurane, and Halothane exhibiting very close retention times can be efficiently eluted using the GC x GC separation procedure with considerable efficiency. Tri-cyclic antidepressants, like Imipramine, Amitryptyline, Trimipramine, and Chlorimipramine can be efficiently eluted at proximal retention times ranging from 8.90 in case of Amitryptyline to 32.3 minutes for Chlorimipramine. (Grob, P. 751) Targeted multidimensional gas chromatography has also proved effective in producing fairly high concentration of eluents from complex test mixtures of antiepileptic drugs. The percent recoveries of the antiepileptic drugs undergoing effective separation process by targeted multidimensional gas chromatography reveal a cent percent recovery for Phenytoin, 88 % for Carbamazepina, 75 % for Primidone, 96 % for Phenobarbital and 85.1 % for Valproic Acid. (Grob, P. 754) Quite a number of pharmaceutical procedures demand proper analysis and quantification of each and every component in blood alcohol. The individual characterization of the volatile organic components in a complex sample is possible by GC x GC even with very similar retention times at appreciably low detection limits. With increasing spells of drug abuse multidimensional chromatographic techniques are being used for the separation, characterization and analysis of amphetamines, cannabinoids, cocaine, barbiturates, arylcyclohexylamines, and lysergic acid diethylamine (LSD). Certain steroid based drugs can be separated and effectively characterized by the novel multidimensional segregation procedures at practically very similar retention times yielding perfect peak densities. These steroids can be effectively quantified from biological fluids like urine samples by ion chromatograms coupled with mass spectral analysis. Originating from packed capillary column chromatography, unified chromatography slowly made its way, where the separation techniques used in super critical fluid chromatography (SFC), gas chromatography (GC) and high performance liquid chromatography (HPLC) were combined to efficiently segregate and characterize complex polymers, biological fluid samples and proteins used in biomedical research. Targeted multidimensional liquid chromatography is still in its nascence for exhibiting its prospects and applicative value in comparison with a GC x GC approach. However, many functional variations of liquid chromatography are being coupled with mass spectrometry for identification of amino acid sequences in polypeptide chains, mapping of the genes responsible in post-translational modification of functionally significant proteins along with characterization, quantification and analysis of protein sequences for biomedical research. Recent researches have used multidimensional liquid chromatography coupled with mass spectrometry for the characterization, identification and analytical separation of peptides and phospho-peptides. Multidimensional liquid chromatography used in the investigation involved strong cation exchange and strong anion exchange along with reversed phase liquid chromatography for effecting the required separation. (Journal of Proteome Research, 2007) Workshops are organized and proteomics facilities are being set up for the development of two dimensional electrophoretic methods along with MDCL techniques as future protein research tools, in order to minimize the time requirement and maximize the efficiency of both online and offline LC X LC + MS separation procedures for further applications in biomedical and pharmaceutical investigations. References 1. Cortes, Hernan J. Multidimensional Chromatography. Boca Raton: CRC Press, 1990. 2. Majors, Ronald E. "Multidimensional and Comprehensive Liquid Chromatography." 1 October 2005. Chromatography online.com. 1998 - 2008 retrieved from: http://chromatographyonline.findpharma.com/lcgc/article/articleDetail.jspid=201272&pageID=1&sk=&date= on November 30, 2008. 3. McKay, Patrick. "An Introduction to Chromatography." 1999. Access Excellence @ the National Health Museum. 1994 - 2008 retrieved from: http://www.accessexcellence.org/LC/SS/chromatography_background.php on November 30, 2008. 4. Avis, Kenneth E. and Vincent L. Wu. Biotechnology and Biopharmaceutical Manufacturing, Processing and Preservation. Boca Raton: CRC Press, 1996. 5. Dai, Jie; Wen-Hai, Jin; Quan-Hu, Sheng; Chia-Hui, Shieh; Jia-Rui, Wu and Zeng, Rong. "Protein phosphorylation and expression profiling by yin-yang multidimensional liquid chromatography (yin-yang MDLC) mass spectrometry." Journal of Proteome Research 6 (2007): 250 - 262. 6. Tang, Jia; Gao, Mingxia; Deng, Chunhui and Zhang, Xiangming. "Recent development of multi-dimensional chromatography strategies in proteome research." Journal of Chromatography B 866 (2008): 123 - 132. 7. Mondello, Luigi; Lewis, Alastair C. and Keith D. Bartle. Multidimensional Chromatography. Hoboken: John Wiley and Sons, 2002. 8. Grob, Robert Lee and Eugene F. Barry. Modern Practice of Gas Chromatography. Hoboken: Wiley IEEE, 2004. 9. Kueh, A. J.; Marriott, P. J.; Wynne, P.M. and Vine, J. H. "Application of comprehensive two-dimensional gas chromatography to drugs analysis in doping control." Journal of Chromatography A 1000 (2003): 109 - 124. 10. Giddings, J. C. "Concepts and comparisons in multidimensional separation." Journal of High Resolution Chromatography 10 (1987): 319-323. Read More