11^th International Conference & Expo on Chromatography Techniques April 22-24, 2019 Dublin, Ireland

Chromatography-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 phyto-constituents 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.

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. Specific detectors are UV-VIS, Photo diode array, fluorescence, and mass spectroscopic detectors. Bulk Property detectors include refractive index, electrochemical and light scattering detectors.

Liquid chromatography is a fundamental separation technique in the life sciences and related fields of chemistry. Unlike gas chromatography, which is unsuitable for non-volatile and thermally fragile molecules, liquid chromatography can safely separate a very wide range of organic compounds, from small-molecule drug metabolites to peptides and proteins. Traditional detectors for liquid chromatography include refractive index, electrochemical, fluorescence, and ultraviolet-visible (UV-Vis) detectors. Some of these generate two-dimensional data; that is, data representing signal strength as a function of time. Others, including fluorescence and diode array UV-Vis detectors, generate three dimensional data. Three-dimensional data include not only signal strength but spectral data for each point in time.

Mass spectrometers also generate three-dimensional data. In addition to signal strength, they generate mass spectral data that can provide valuable information about the molecular weight, structure, identity, quantity, and purity of a sample. Mass spectral data add specificity that increases confidence in the results of both qualitative and quantitative analyses.

One of the most characteristic features of the development in the methodology of pharmaceutical and biomedical analysis is that HPLC became undoubtedly the most important analytical method for identification and quantification of drugs, either in their active pharmaceutical ingredient or in their formulations during the process of their discovery, development and manufacturing.

Drug development starts with the discovery of a molecule with a therapeutic value. This can be done by high throughput screening during which separations by fast or ultra-fast HPLC are performed. At the discovery stage there can be also characterizing synthetic or natural products. Drug metabolism and pharmacokinetics (DMPK) is the step where the candidate compounds for drug are tested for their metabolism and pharmacokinetics. The studies involve use of LC-MS or LC-MS/MS. The goal in the discovery stage of drug development is to discover a new, safe and active chemical entity (NCE) that will become medication for diseases. During the last decade parallel synthesis of potential lead compounds, using combinatorial chemistry has been done. Due to its high sensitivity and selectivity, HPLC coupled with tandem mass spectrometry, HPLC-MS/MS has become the predominant method in bioassays, and pharmacokinetic and metabolic studies.

Chromatography and spectroscopy are orthogonal techniques, i.e. their types of information are very different and are specific. Chromatography is a separation method and spectroscopy is a technique which yields a ‘fingerprint’ of individual or from mixture of molecules. HPLC is a technique for separation, quantification and identification of components in a mixture. It is especially suitable for compounds which are not easily volatilized, thermally unstable and have high molecular weights. The advantage of UV method over HPLC method is that the UV method does not require the elaborate treatment and procedures usually associated with chromatographic method. It is less time consuming and economical. The HPLC and UV spectrometry methods are adequate methods to quantify a drug in pure form and its dosage form. Since these methods are simple, specific, rapid, precise and accurate, they may be successfully and conveniently adopted for routine quality control analysis of drugs in bulk and pharmaceutical dosage form.

With modern technology and facilities, our food supply is more diverse and more highly processed than ever before. To ensure the safety and nutritional quality of our food many countries and organization have promulgated regulations that stipulate acceptable levels for individual chemical additives, residues and contaminants in food products. Other regulations require food packaging to list ingredients relating to nutritional content, such as preservative, artificial chemicals, unsaturated and saturated fat. Food manufacturers and processors themselves must be able to assess product quality. Meeting all of these requirements is the function of food analysis.

Increasingly, food analysis methods are built around high-performance liquid chromatography (HPLC), which has proven to be an optimal technology for detecting and/or quantifying the vast majority of food analytes. These methods employ a stepwise approach while separating and analysis the sample, it first removes the sample matrix, then isolates the analytes of interest and individually resolves them on a chromatographic column. The efficiency of the separation depends on, among other things, the differential interaction of analytes of interest with both mobile and column stationary phases during the separation. It is important to classify food analytes according to their relative volatility and polarity are factors that must be considered when selecting an appropriate analytical method for their determination.

Gas chromatography (GC) is widely used in applications involving food analysis. High-performance liquid chromatography (HPLC) and to mention state-of-the-art GC techniques used in the major applications pertaining to the quantitative and/or qualitative analysis of food composition, natural products, food additives, flavour and aroma components, a variety of transformation products, and contaminants, such as environmental pollutants, pesticides, fumigants, natural toxins, veterinary drugs, and packaging materials. Among the several new qualitative and quantitative techniques being developed in food analysis applications, fast-GC/mass spectrometry (MS) will have the most impact in the next decade. Three approaches to fast-GC/MS include low-pressure GC/MS, GC/time-of-flight (TOF)-MS and GC/supersonic molecular beam (SMB)-MS.

Chromatography-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. HPLC has the advantage of much wider range of analytes measurements with a higher sensitivity than gas chromatographic methods. 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. The nucleosides and nucleotides of DNA are separated and quantified by HPLC-UV method. HPLC finds applications in glycomics and lipidomics where glycan part is cleaved either enzymatically or chemically from the target and subjected to analysis. 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.

Fingerprinting is a quality control model that builds upon spectroscopic and chromatographic technology. It is different from the traditional quality control model in the sense that fingerprinting looks at the “complete information” or comprehensiveness of the chromatograph, and displays integrated quality information. Since the secondary metabolites, which are chemical components of medicinal herbs, are inherently unstable, the fingerprints of these chemicals possess a fuzziness that cannot be precisely measured, just like the fuzzy phenomenon in our daily lives. Comprehensiveness and fuzziness are the two basic traits of a fingerprint. The similarity of fingerprints is established through these basic traits. Fingerprint analysis focuses on accurate identification (of similar peaks), and not on precise calculation. The comparison of fingerprints emphasizes similarity and the fingerprints compared do not need to be exactly the same. When it is impossible to find out all the complex components of a traditional medicine, fingerprints can be used to check the stability of the intrinsic quality of the medicine.

HPLC techniques are applied for purification and separation of various biological samples. The analysed samples are subjected to sequencing studies either manually or using different software’s. This is studied as Data mining and sequence analysis. HPLC is also used for characterization of various metabolites.

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.

Chromatography-HPLC is the most versatile of all chromatography methods but also the most complex. It was first made available in the laboratory during the 1970s and is currently used for the analysis of amino acids, peptides, proteins, carbohydrates, lipids, nucleic acids and related compounds, vitamins, hormones, metabolites, and drugs. HPLC can be coupled to various detectors such as UV, fluorescence or mass spectrometry (LC/MS and LC/MS/MS) and is routinely used for quantitative analysis in biological samples such as blood, urine and other body fluids. HPLC consists of using a liquid mobile phase to pass under high pressure a mixture of analytes extracted from the sample through a column containing the stationary phase. Analyte separation is based on differences in interaction with both the mobile phase and the stationary phase.

HPLC is a proven method for isolating analytes of interest in complex matrices such as biological fluids. Its use in the clinical laboratory has steadily increased over the past decades as its unmatched analytical performance and versatility allows for testing of many different types of clinically relevant analytes. With the recent advances in detection technology such as mass spectrometry and sample preparation techniques such as bio-affinity chromatography and online automation, HPLC based methods will likely remain the gold standard of clinical testing for many of the current but also future biomarkers and therapeutic drugs.

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, testing of herbal products, de-replication of natural products, and metabolomics.

Rapid identification and characterization of known and new natural products directly from plant and marine sources without the necessity of isolation and purification can be achieved by various modern hyphenated techniques. 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. 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.

High Performance Liquid Chromatography (HPLC) 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.

In normal-phase chromatography, the stationary phase is polar and the mobile phase is nonpolar. In reversed phase the stationary phase is nonpolar and the mobile phase is polar.

Flash Column Chromatography (FCC) or Flash Chromatography is a quickest and the easiest way to separate complex mixtures of compounds. It uses compressed air to push the solvent through the column. This provides better separation and reduces the amount of time required to run a column.

Ion exchange chromatography (IEC) uses an ion exchange mechanism to separate analytes based charge difference. It is performed in columns but can also be useful in planar mode. It uses a charged stationary phase to separate compounds including cations, anions, amino acids, proteins, lipids and peptides. Loading samples in buffers of low ionic strength makes ion exchange chromatography an excellent purification step after HIC.

Affinity chromatography is based on selective non-covalent interaction between an analyte and sample molecules. It is highly specific, but not robust method. It is used in biochemistry in the purification of proteins bound to tags. Fusion proteins are labelled with compounds such as antigens or biotin which bind to the stationary phase specifically. Later after purification, some of these tags are removed and the pure protein is obtained. Immobilized Metal Affinity Chromatography is used to separate molecules based on the relative affinity for the metal. Often these columns can be loaded with different metals to create a column with a targeted affinity.

Chiral chromatography includes the separation of stereoisomers. Enantiomers have no chemical or physical differences apart from being three-dimensional mirror images. For chiral separations to happen, either the mobile phase or the stationary phase must themselves be made chiral by inducing different affinities between the analytes.

Size-exclusion chromatography (SEC) also known as gel filtration chromatography, separates molecules according to the size or more accurately according to the hydrodynamic diameter or the hydrodynamic volume. Small molecules enter the pores of the media where they are trapped and removed from the flow of the mobile phase. Mean residence time in the pores depends upon the effective size of the analyte molecules. It is a low-resolution chromatography technique and thus it is used for determining the tertiary structure and quaternary structure of purified proteins.

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. In addition, the much shorter run time significantly reduces UHPLC method development scouting time. HPLC method development principles can be applied to UHPLC method development. 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.

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.

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 Nano spray 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 covalently coated with a monolayer of hydrophobic C8-chains to enable reversed-phase LC separations.

The global chromatography instrumentation market is segmented on the basis of systems, consumables, applications, and regions. The report studies the global chromatography instruments market for the forecast period of 2015 to 2020. The market is expected to reach USD 9.223 Billion by 2020 from USD 7.062 Billion in 2015, at a CAGR of 5.5%.

It is anticipated that North America and Europe will continue to lead the market over the next five years; the chromatography market in Asia will expand and increase its market share. The drivers behind the expansion are two-fold: first the expansion of local companies in Asia and secondly, Western Pharma outsourcing its research and manufacturing operations to Asia, particularly China and India.