In its earliest form, liquid chromatography was used to separate the pigments of chlorophyll by a Russian botanist. Decades later, other chemists used the procedure for the study of carotins. Liquid chromatography was then used for the isolation of small molecules and organic compounds like amino acids, and most recently has been used in peptide and DNA research. Monolith columns have been instrumental in advancing the field of biomolecular research.
In recent trade shows and international meetings for HPLC, interest in column monoliths and biomolecular applications has grown steadily, and this correlation is no coincidence. Monoliths have been shown to possess great potential in the “omics” fields- genomics, proteomics, metabolomics, and pharmacogenomics, among others. The reductionist approach to understanding the chemical pathways of the body and reactions to different stimuli, like drugs, are essential to new waves of healthcare like personalized medicine.
Pharmacogenomics studies how responses to pharmaceutical products differ in efficacy and toxicity based on variations in the patient’s genome; it is a correlation of drug response to gene expression in a patient. Jeremy Nicholson of the Imperial College, London, used a postgenomic viewpoint to understand adverse drug reactions and the molecular basis of human disesase. His group studied gut microbial metabolic profiles and were able to see distinct differences in reactions to drug toxicity and metabolism even among various geographical distributions of the same race. Affinity monolith chromatography provides another approach to drug response measurements. David Hage at the University of Nebraska binds ligands to monolithic supports and measures the equilibrium phenomena of binding interactions between drugs and serum proteins. A monolith-based approach at the University of Bologna, Italy, is currently in use for high-speed screening of drug candidates in the treatment of Alzheimer’s. In 2003, Regnier and Liu of Purdue University described a multi-dimensional LC procedure for identifying single nucleotide polymorphisms (SNPs) in proteins. SNPs are alterations in the genetic code that can sometimes cause changes in protein conformation, as is the case with sickle cell anemia. Monoliths are particularly useful in these kinds of separations because of their superior mass transport capabilities, low backpressures coupled with faster flow rates, and relative ease of modification of the support surface.
Bioseparations on a production scale are enhanced by monolith column technologies as well. The fast separations and high resolving power of monoliths for large molecules means that real-time analysis on production fermentors is possible. Fermentation is well known for its use in making alcoholic beverages, but is also an essential step in the production of vaccines for rabies and other viruses. Real-time, on-line analysis is critical for monitoring of production conditions, and adjustments can be made if necessary. Boehringer Ingelheim Austria has validated a method with cGMP (commercial good manufacturing practices) for production of pharmaceutical-grade DNA plasmids. They are able to process 200L of fermentation broth on an 800mL monolith. At BIA Separations, processing time of the tomato mosaic virus decreased considerably from the standard five days of manually intensive work to equivalent purity and better recovery in only two hours with a monolith column. Other viruses have been purified on monoliths as well.
Another area of interest for HPLC is forensics. GC-MS (Gas Chromatography-Mass Spectroscopy) is generally considered the gold standard for forensic analysis. It is used in conjunction with online databases for rapid analysis of compounds in tests for blood alcohol, cause of death, street drugs, and food analysis, especially in poisoning cases. Analysis of buprenorphine, a heroin substitute, demonstrated the potential utility of multidimensional LC as a low-level detection method. HPLC methods can measure this compound at 40 ng/mL, compared to GC-MS at 0.5 ng/mL, but LC-MS-MS can detect buprenorphine at levels as low as 0.02 ng/mL. The sensitivity of multidimensional LC is therefore 2000 times greater than that of conventional HPLC.
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