3^rd International Conference and Exhibition on Advances in Chromatography & HPLC Techniques July 13-14, 2017 Berlin, Germany

High Performance Liquid Chromatography is a non-destructive procedure for resolving a complex mixture into its individual fractions or compounds. It is based on differential migration of solutes with the solvents. The solutes in a mobile phase go through a stationary phase. Those solutes with a high affinity for the mobile phase will spend more time in this phase than the solutes that prefer the stationary phase. As the solute rise up through the stationary phase they separate. The process is called chromatographic development. The fraction with greater affinity to stationary layer travels slower and shorter distance while that with less affinity travels faster and longer.

HPLC is a popular method of analysis for natural products because of its high accuracy, precision and is not differed by the stability or the volatility of the compounds. HPLC combined with diode array detector (HPLC-DAD), mass spectrometer (HPLC-MS) have been successfully utilized for the qualitative and quantitative determination of various types of phytoconstituents like alkaloids, glycosides, tannins, tri-terpenes, flavonoids etc. HPLC methods are used readily for the determination of drug in biological fluids and pharmaceutical dosage forms. HPLC determination with spectroscopic detection is useful for routine quality control of drugs in pharmaceutical dosage forms and stability studies. HPLC columns are usually packed with pellicular or porous particles. A chromatographic detector is capable of establishing both the identity and concentration of eluting components in the mobile phase stream. A broad range of detectors are available to meet different sample requirements. Detectors respond to a particular compound only and the response is independent of mobile phase composition and the response of bulk property detectors is dependent on collective changes in composition of sample and mobile phase.

The hyphenated technique is developed from the coupling of a separation technique and an on-line spectroscopic detection technology. Several remarkable improvements in hyphenated analytical methods over the last two decades have significantly broadened their applications in the analysis of biomaterials, especially natural products, pre-isolation analyses of crude extracts or fraction from various natural sources, isolation and detection of natural products, chemical fingerprinting, and metabolomics. Techniques like HPLC coupled to NMR (Nuclear Magnetic Resonance) or electrospray ionization tandem mass spectrometry (ESI-MS-MS) have been proven to be extremely powerful tools in natural product analysis, as they aid in the fast screening of crude natural product extracts or fractions for detailed information about metabolic profiles, with minimum quantity of material. The application of various hyphenated techniques even allows the discovery of new molecules, including complete and conclusive structure elucidation, and relative configurations as compared to time-consuming and costly isolation and purification processes. Hyphenated HPLC techniques include HPLC-MS, HPLC-ESI-MS, HPLC-IC-MS, HPLC-NMR-MS, HPLC-DAD, HPLC-CE-MS, HPLC-UV, Coupling LC and MALDI-TOF. MALDI (Matrix Assisted Laser Desorption Ionization) is a very sensitive technique for determining the mass of proteins, polymers and peptides. MALDI basically used in protein identification. MALDI sample preparation is fast and easy and therefore a primary choice in proteomics.

This includes a micro fabricated separation device. The availability of the fused-silica capillary marked a significant breakthrough for gas chromatography and all gas chromatographs manufactured were equipped to use fused silica capillary columns. Fused-silica capillaries have a huge contribution to the developments of other micro separation technologies like supercritical fluid chromatography. The success of one separation technique relies on sample introduction technologies, separation column and sensitive detectors that can preserve chromatographic fidelity of high resolution chromatographic peaks, as is evident from the many injectors and detectors optimized and available for open tubular GC. A particle packed column is comprised of a nano litre enrichment column and a micron or sub-micron separation column packed with suitable grade of C18. The HPLC-Chip is made from a biocompatible polyimide and the functionality of this chip is equivalent to conventional nanospray LC/MS.  Monoliths consist of a single rod of porous material with several unique features in terms of permeability and efficiency. Micro-fabricated column based on pillar-arrays were formed by arrays of nonporous silicon pillars with a diameter of approximately 4.3μm. The pillars were covalenty coated with a monolayer of hydrophobic C8-chains to enable reversed-phase LC separations.

UHPLC (Ultra-HPLC) or UPLC (Ultra Performance Liquid Chromatography) is now being adopted in industrial labs, especially the pharmaceutical industry due to its high speed, high resolution and solvent saving.  A UHPLC method uses a sub-2micron column as it reduces the analysis time by 80% and save the mobile phase consumption by a huge amount compared to the conventional HPLC.Micro and Nano HPLC ensure high levels of flow rate flexibility and reproducibility.

Hydrophilic interaction chromatography or hydrophilic interaction liquid chromatography (HILIC) is a variant of normal phase liquid chromatography that partly overlaps with other chromatographic applications such as ion chromatography and reversed phase liquid chromatography. It uses hydrophilic stationary phases with reversed-phase type eluents.

Quality can be designed to processes through systematic implementation of an optimization strategy to establish a thorough understanding of the response of the system quality to given variables, and the use of control strategies to ensure quality. The concept of method development includes modelling of the influence of values of variables on quality, design of experiments, and simplification of processes as information is collected. The extension of QbD (Quality by Design) philosophies is now applied to the development of manufacturing processes and analytical methods. The ability of a chromatographic method to successfully separate, identify and quantitate species is determined by a powerful factor called experimental design. Automation of a process is one of the keys for increasing the productivity of a research group. Scaling-up a compound separation performed on an analytical system to a preparative liquid chromatography system requires an optimization step on the analytical column. This step concerns the development of the gradient method for the isolation of the target compound with the best balance between its purity, data throughput, and analysis time.

HPLC can be used in both qualitative and quantitative applications that are for both compound quantification and identification. Normal phase HPLC is rarely used now, almost all HPLC separation can be performed in reverse phase. Reverse phase HPLC (RPLC) is ineffective in for only a few separation types. HPLC is applied for molecular weight determination, in analytical chemistry, pharmaceutical and drug science, clinical sciences, food technology, and consumer products, combinatorial chemistry, polymer chemistry, environmental chemistry and green chemistry.

HPLC is a very common method for metabolomics analysis. With the invention of electrospray ionization, HPLC is coupled to mass spectroscopy. HPLC has lower chromatographic resolution, requires no derivation for polar molecules and separates molecules in the liquid phase. Relevant to proteomics, due to the complex structure and nature of proteins, instrumentation and methods development for sample clean-up, pre-concentration, fractionation, chromatographic separation and detection becomes an immediate requirement for the identification of peptides and proteins. Latest techniques and equipment for separation and detection include nano-HPLC and multidimensional HPLC for protein and peptide separation. HPLC is considered as most reliable and most sensitive technique in genomics used to determine DNA methylation. HPLC finds applications in glycomics and lipidomics. In case of glycolipids, they can be analysed directly without separation of the lipid component. HPLC has a wide application in lipidomics to separate lipids prior to mass spectrometry. Separation can be achieved by either reverse-phase (RP) HPLC or normal-phase (NP) HPLC.

HPLC techniques are applied for purification and separation of various biological samples. The analysed samples are subjected to sequencing studies either manually or using various softwares. HPLC is also used for characterization of various metabolites.

The HPLC methodology applied to the analysis of biological samples makes it possible for the identification of many metabolites. Samples from two human embryos culture medium were analysed by high-pressure liquid chromatography–mass spectrometry (HPLC–MS). They work on the principle that many microorganisms have their own unique mass spectral signature based on the particular proteins and peptides that are present in the cells. Identification of unknown peaks in gas chromatography (GC/MS)-based discovery metabolomics is challenging, and remains necessary to permit discovery of novel or unexpected metabolites that may allergic diseases  processes and/or further our understanding of how genotypes relate to phenotypes. Here, we introduce two new technologies and advances in pharmaceutical analytical methods that can facilitate the identification of unknown peaks.

The HPLC industry is showing rapid growth, with value expected to hit 228 billion dollars up from 164 US billion dollars by 2016, with an annual growth of nearly 7%, according to a recent industrial marketing research report. Geographically, global separation technique market has been segmented into four areas namely, North America, some parts of European region, Asia-Pacific and Rest of the World.