18th International Conference on World HPLC & Separation Techniques August 29-30, 2018 Toronto, Ontario, Canada

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.

Analytical testing capabilities for these disciplines of Veterinary drug analysis have evolved significantly. So the scope of Analytical Techniques is playing a major role in the current Veterinary Medicine analysis. It majorly utilizes Chromatography and Mass Spectrometry to provide an array of toxicology and drug tests. The instrumentation majorly involved in drug testing are Gas Chromatography–Mass Spectrometry (GC/MS), Tandem Liquid Chromatography–Mass Spectrometry (LC/MS/MS), High-Resolution Accurate Mass Spectrometry (HRAMS), High Performance Liquid Chromatography (HPLC), Inductively Coupled Plasma – Mass Spectrometry (ICP-MS), Atomic Absorption (AA).

Chromatography basically is a method of separation of compounds from a mixture. This technique is both analytical as well as preparative and is employed widely in industries and also in laboratories. Chemical analysis is done mostly all over the world with chromatography or any other various related to chromatography techniques. Chromatography is a physical technique and has a vast application in chemical field starting from basic organic chemistry to forensic science. Some common applications include lead in water, detection of cocaine in urine, PCB’s in fish and alcohol in blood. Various types of chromatography techniques are- paper chromatography, displacement chromatography, thin layer chromatography, supercritical chromatography, column fluid chromatography, gas chromatography, expanded bed adsorption chromatography, liquid chromatography in various combinations.

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 passes over a stationary phase. Those high affinity solutes of the mobile phase will spend more time in this phase than the solutes that prefer the stationary phase. As the solute rises up through the stationary phase they gets separated. This 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.

From the beginning of the reasearch on Agriculture, analytical chemistry has played a major supporting role and still continuing the same till this generation. However, many highly interesting changes in the objectives and contributions of analytical chemistry have been implemented. One of the main involvements of analytical chemistry in the agriculture research has always been to protect farmers from fraud when purchasing the fertilizers and many other auxiliary agricultural products.The scope of Analytical Chemistry is very high from the past, till the present in the Agricultural Researches.

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.

HPLC/UHPLC chromatography is a commonly used separation mode in Reversed phase. Its retention of compounds possessing hydrophobic and organic functionality are provided dynamically. Combination of both hydrophobic and van der Waals type interactions between all the target compounds including both the stationary and mobile phases enables by reversed phase retention of these compounds.

HPLC can be used in the following applications:

• Purification of Water
• Pre-concentration for components of trace amount
• Ligand-exchange chromatography
• For Proteins, Ion-exchange chromatography
• For carbohydrates and oligosaccharides, High-pH anion-exchange chromatography

Quality can be designed to process 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.

• Chromatography instrumentation Controls and automates virtually
• Provides information regarding data management, security features, instrument validation, and reports.
• Powerful and malleable
• Increases productivity by managing from sampling till reporting results.
• Less Expensive

Chromatography systems are often defined by their pressure characteristics. Common acronyms like LP or HPLC are used to refer to low pressure and high-pressure liquid chromatography respectively. Systems that operate at pressures <50 psi (~3 bar) are categorized as low-pressure chromatography systems. Low pressure chromatography is often used for simple protein separations that do not require high resolution. Low pressure systems range from basic to semi-automated and can be equipped with gradient capabilities, detectors, valves, and fraction collectors.

Applications:

• Low pressure systems are ideal for the purification of recombinant tagged proteins and work with most pre-packed column.
• Low pressure systems requires a sample pump and are often equipped with fraction collectors, gradient capabilities and detectors to monitor

Mass spectrometry using electric and magnetic fields measures mass-to-charge ratio of molecules. Several ionization methods like electron impact, chemical ionization, electrospray, fast atom bombardment, matrix assisted laser desorption ionization, and others are involved in Mass Spectrometry. Mass spectrometry is also categorized by approaches of mass analyzers like magnetic-sector, quadrupole mass analyzer, quadrupole ion trap, time-of-flight, Fourier transform ion cyclotron resonance, and many other types are involved in Mass Spectrometry.In order to measure the characteristics of individual molecules, a mass spectrometer converts them to ions so that they can be moved about and manipulated by external electric and magnetic fields.

The Mass Spectrometry (MS) methodology is applied for the analysis of biological samples which helps in the possibility for the identification of many metabolites. In vector, 100 chromatograms are integrated. Subsequent dimensional multivariate analysis helps in simplifying and how to understand the two-dimensional pseudo spectrum which is plotted continuously. For validation of this process, High Pressure liquid chromatography-Mass Spectrometry is used for analysing the samples collected from the embryo of couple of human beings. Based on the particular proteins and peptides many microorganisms have their own similar mass spectral signatures that are present in the cells. Identifying the unknown peaks with Gas Chromatography is challenging in the discovery of metabolomics which may leads to discovery of novel or unexpected metabolites which may cause allergic diseases. These allergic diseases processes furtherly allow us to understand and helps in understanding how genotypes are related to phenotypes. High mass accuracy, high resolution, high sensitivity analyte detection by reporting on a Gas Chromatography/Quadrupoles- Orbitrap mass spectrometry.

Metabolomics, the study of the endogenously synthesized small molecules repertoire (nonproteinaceous), is of great relevance for establishing a wide view of cell physiology at specific moments, linking metabolic profiles to phenotypes and genotypes. To better understand biological systems, such as helminths life cycle, helminthic infection, and host-parasite interaction, metabolomics studies are crucial. For that, mass spectrometry-based metabolomics is the most popular strategy. Nontargeted metabolomics allows researchers to profile entire metabolomes present in cells, tissues, biofluids, or even samples as complex as stools. Through different mass spectrometric techniques, it is possible to unveil chemical markers for helminths, such as Schistosoma mansoni (a trematode) and Ascaris lumbricoides (a nematode), in addition to study mechanisms of action for different drugs, which targets parasites. Therefore, Mass Spectrometry allows designing biochemical pathways that may clarify the processes of parasite life cycle, helminthic infection, and host-parasite interaction, providing targets to further interference for parasite control or even infection treatment.

Mass spectrometry (MS) is one of the major analytical techniques which ionizes chemical species and sorts out based on their mass-to-charge ratio of the ions. In general it can be expressed as, a mass spectrum which measures the masses within a sample. This spectrometry is used in many other different fields which are applied to pure samples along with complex mixtures. Some of the new approaches in Mass Spectrometry are:

• Materializing separation Technologies
• Hybrid Mass Spectrometry
• Paths in glycoproteins and glycans
• Tom Probe Tomography
• Protein Phosphorylation and Non-Covalent interaction
• Overture in isolation, enrichment and separation
• Structural proteomics and genomics
• Lipidomic, metabolomics and ultra-trace analysis
• Complementary Multi technique Access
• Mass spectrometry in the field of food science
• New MS technologies in Metabolomics/Lipidomics
• Biomolecular, Carbohydrates, and microbe analysis
• Nano scale and microfluidic separations

Mass spectrometry involves the measurement of the mass-to-charge ratio of ions. It has become an essential analytical tool in biological research and can be used to characterize a wide variety of biomolecules such as sugars, proteins, and oligonucleotides. it is an instrument that can ionize a sample and measure the mass-to-charge ratio of the resulting ions. However, the versatility of this function has allowed it to become a vital tool in a wide range of fields, including biological research. This versatility arises from the fact that mass spectrometers can give qualitative and quantitative information on the elemental, isotopic, and molecular composition of organic and inorganic samples. Furthermore, samples can be analyzed from the gas, liquid, or solid state, and the masses that can be studied range from single atoms (several Da) to proteins (over 300,000 Da). Most of the different types of analysis can be done based on their state and reliability of the particular technique. 

High-temperature mass spectrometry is the most versatile method for the elucidation of vaporization processes. It is shown that by using the mass spectrometers of inorganic solid analysis various non-thermal vaporization processes, not included in the original program of HTMS, can be studied. Comparative investigations are especially useful for elucidating the mechanisms of the rather complex processes in question, opening at the same time new approaches to some basic problems of high temperature science. The investigations proposed, including measurement and analysis of distribution patterns of various related species, stable isotope tracer studies, time resolution studies and quantum chemical calculations, are demonstrated with two examples: the vaporization of alumina and graphite under varied conditions.

The focus of the proteomics section is for the development and application of new tools for the identification and quantification of ensemble of proteins. Researches include shotgun organellar proteomics of Tetrahymena thermophila and the investigation of myogenesis-related proteins and their posttranslational modifications. In the current post-genomic era of large scale ‘omic analyses, Proteomics occupies a central position due to the vast diversity of functional and structural roles for proteins. To understand both normal and dysfunctional physiological states necessitates a quantitative understanding of protein alterations, both across the proteome as well as to individual proteins. Indeed, in addition to the breadth of functional and regulatory capacity introduced by splice variants and isoforms, the quantity, location, and functional states of proteins are continuously fine-tuned by myriad potential post-translational modifications (PTM). Understanding variations in both quantity and protein species or proteoforms, is thus central to understanding much of biology. Accordingly, we use the terms proteoform and protein species interchangeably here in an effort to avoid any semantic ambiguities

NMR is the renowned technique for determining the structure of organic compounds. When compared to all types of spectroscopic methods, it is the only process through which it is normally expected to complete analysis and interpretation of the entire spectrum. Despite, larger amounts of sample are needed for mass spectroscopy, samples weighing less than a milligram also yields good data which may be obtained with modern instruments, as NMR is non-destructive. Recent progress in magnet design has led to compact permanent NMR magnets with a great performance, opening new perspectives for analytical applications of benchtop NMR. Nowadays, it is possible to measure multi-nuclear and multidimensional NMR spectra on the workbench of the chemical laboratory. The purpose of the conference is to present recent technological and methodological developments as well as practical applications in the field of low-field NMR spectroscopy/Relaxometry.

In Analytical science the important and attractive approach is visualization of single molecules, single cells, biological tissues and Nano materials. Also, analytical science is revolutionized by hybridization with other traditional analytical tools. Microscopy can be categorized into three different fields namely Optical microscopy, Electron microscopy, and Scanning probe microscopy. Recently, this field is rapidly progressing because of the rapid development of the computer and camera industries.

It is a qualitative and quantitative methods of analysis based on electrochemical phenomena occurring within a medium or at the phase boundary and related to changes in the structure, chemical composition, or concentration of the compound being analyzed. Electroanalytical methods containing the analyte measures the potential (volts) and/or current(amps) in an electrochemical cell . These methods can be categorized according to which aspects of the cell are controlled and measured. The four main categories are potentiometry which is used to measure the difference in electrode potentials, coulometry which is used to measure the transferred charge that is measured over time, amperometry is used to analyze the cell's current that is measured over time, and voltametry is used so as the cell's current is measured while actively altering the cell's potential. All the potentials and ampheres related to current can be estimated under the class of this electrochemical analysis.

Thermal analysis is a branch of materials science where the properties of materials are studied as they change with temperature. Several methods are commonly used – these are distinguished from one another by the property which is measured.The interaction of a material and heat are measured by Calorimetry and thermogravimetric analysis.It is used to control the temperature in a predetermined way - either by a continuous increase or decrease in temperature at a constant rate either by linear heating/cooling or by carrying out a series of determinations at different temperatures in a stepwise isothermal measurements. More advanced temperature profiles have been developed which use an oscillating which are usually sine or square wave of heating rate which is Modulated Temperature Thermal Analysis or modify the heating rate in response to changes in the system's properties by Sample Controlled Thermal Analysis.

"Hybrid" or "Hyphenated" techniques are produced by the Combinations of the above techniques. Today several examples are in popular use and new emerging hybrid techniques are under development. For example, gas chromatography-mass spectrometry, gas chromatography-infrared spectroscopy, liquid chromatography-mass spectrometry, liquid chromatography-NMR spectroscopy. Liquid chromatography-infrared spectroscopy and capillary electrophoresis-mass spectrometry. Hyphenated separation techniques refer to a combination of two (or more) techniques to detect and separate chemicals from solutions. Most often the other technique is some form of chromatography. Hyphenated techniques are widely used in chemistry and biochemistry. A slash is sometimes used instead of hyphen, especially if the name of one of the methods contains a hyphen itself.

Lab-on-a-Chip Devices for Particle and Cell Separation are used to either remove particles of interest, sort a mixed population of particles into subpopulations of like particles or concentrate (enrich) particles for downstream processing within microdevices. Methods for particle or cell separations exploit specific physical properties which include: fluorescence-based, magnetic-based, affinity-based, and cell density gradient-based separations depending on the properties of the particles of interest. Particle separation devices are often geared towards biomedical research and diagnostics particularly in blood cell separation and blood diagnostics.The numerical difference between observed value and true value is known as Error.The true value and observed value in chemical analysis can be related with each other by the equation E= O – T. Error of a measurement is an inverse measure of accurate measurement i.e. smaller the error greater the accuracy of the measurement.

Chromatography and Mass Spectrometry are commonly known to be the separation techniques which are playing a dominating role in the present scenario of the separations in the field of Analytical Chemistry which comes majorly under the department of Chemistry. As the generation is advancing the chemical analysis is also advancing according to its generations. As a result of this the seperation techniques became a major part in the chemistry which gets involved in most areas of chemistry and related departments. Some of the advances of chromatography and mass spectrometry are discussed based on their importance. 

• Handling Mass spectrometry for biomedical applications
• MALDI imaging mass spectrometry
• Biomarker discovery by Proteomics and mass spectrometry technologies
• Techniques of Electrospray ionization
• Overture in preparation of sample
• Approach in Instrumentation of Analytical Chemistry
• Advances in sample preparation and Interface design of MS
• Ionization and sampling developments
• Combination of Mass spectrometry with Microfluidics
• Advancements in different Spectrometric methods
• Mass spectrometry’s role in biomedical applications
• Mass Spectrometry in Physical and Biophysical processes
• The new progression in Triple Quadruapole GC-MS/MS
• Materializing the Tools and Applications
• HPLC and its applications
• Magnet design for NMR

A separation is a method which is used to achieve any phenomenon that converts a mixture of chemical substance into two or more distinct product mixtures, which may be referred to as mixture on which at least one of which is enriched in one or more of the mixture's constituents. Material mixtures separation processes are used to decrease the complexity of mixtures. Some of the representatives of this field are Chromatography, Electrophoresis and Field Flow Fractionation. Majorly between the constituents of a mixture, separations differ in chemical properties or physical properties such as size, shape, mass, density, or chemical affinity. They are classified particularly based on the particular differences they are using to achieve separation. Generally there is only physical movement and no substantial chemical modifications. If no single difference can be used to accomplish a desired separation, multiple operations will often be performed in combination to achieve the desired end.

Chromatography is an analytical technique commonly used for separating a mixture of chemical substances into its individual components, so that the individual components can be thoroughly analyzed. There are many types of chromatography e.g., liquid chromatography, gas chromatography, ion-exchange chromatography, affinity chromatography, but all of these employ the same basic principles.Chromatography may be either preparative or analytical technique. The main purpose of preparative chromatography is to separate the components of a mixture for later use which is a form of purification. Analytical chromatography is done normally with small amounts of samples which is majorly for establishing the presence or measuring the relative proportions of analytes in a mixture. The two are not mutually limited. Several types of chromatographic techniques are involved in the chromatography in which each type of chromatographic method deals with each type of particular separation techniques.

Chromatography is playing major roles in forensic science, ranging from toxicology to environmental analysis. Particularly, High-Performance Liquid Chromatography (HPLC) is a primary method of analysis in many laboratories. Maintaining a balance between practical solutions and the theoretical considerations involved in HPLC analysis, Forensic Applications of High Performance Liquid Chromatography uses real-life examples likely to be found within a forensic science laboratory to explain HPLC from a forensic perspective.