19th World Conference on Chromatography & Spectrometry March 20-21, 2019 New York, USA

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 analysed. 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 are 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 has numerous applications in both chemical and biological fields. In biochemical research, it is widely used in for the separation and identification of chemical compounds. This technique is employed to analyze complex mixtures of hydrocarbons in the petroleum industry.

Chromatography, as a separation method has a very high number of advantages over older techniques like crystallization, solvent extraction, and distillation. Without requiring an extensive fore knowledge of the identity, number, or relative amounts of the substances present, it has the capability of separating the components of a multicomponent chemical mixture.

Spectrometry is an Analytical technique which is mainly considered as the study of different types of interactions between the light and matter, reactions and measurements of radiation, intensity and different wavelengths.

Spectroscopy is an integral element of the scientific process within a variety of disciplines far from being a specialized and their unique field. It provides a theoretical backing to early quantum research in atomic structure and radiation, it also has a staggering number of other applied uses; Magnetic Resonance Imaging (MRI) and X-ray machines utilise a type of radio-frequency spectroscopy, which measures the unique makeup and physical properties of distant astral bodies through their wavelength and spectra, and it’s even used to test doping in many fields of sports!

Spectrometers are the chief instruments used in spectrometric analysis which are specialised pieces of equipment that measure wavelength and types of radiation.

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.

High Performance Liquid Chromatography is a highly improved form of column chromatography. This technique is makes it much faster as the solvent Instead of being allowed to drip through a column under gravity, it is forced through high pressures of up to 400 atmospheres.

 High Performance Liquid Chromatography allows us to use a very much smaller particle size for the column packing material which gives a much greater surface area for interactions between the Stationary phase and the molecules flowing through it. This allows a very good separation of the components of the mixture.

Mass Spectrometry:

Mass Spectrometry, also referred to as Mass Spec, is an analytical technique which is becoming increasingly important in the research of biosciences. For measuring the characteristics of individual molecules, a mass spectrometer converts them to ions as they can be able to be moved about and wield by their external electric and magnetic fields. Mass spectrometry is used in measuring the mass of different molecules within a sample accurately. By mass, even large biomolecules like proteins are identifiable, which means using mass spectroscopy the biologists can perform some very interesting experiments, probably add a new dimension to the research.

Mass Spectrometry is majorly useful in following techniques namely;

• Detecting the impurities in a sample.
• Study the protein content of some cell samples.
• Analyzing a purified protein.
• Identifying different molecules in a mixture.

The three major and important key stages to the spectrometer:

1. Ionization.
2. Acceleration 
3. Deflection.
4. Detection.

Gas chromatography (GC), also sometimes defined as Gas-Liquid Chromatography (GLC), is a separation technique in which Gas is used as the mobile phase. Gas chromatographic separation is typically "Packed" or "Capillary", which is always carried out in a column. Gas Chromatography is a technique which is very less expensive and easier in handling and often giving adequate performance. Capillary columns especially for complex mixtures generally give far superior resolution and although more expensive are becoming widely used one. Both columns are made from non-adsorbent and chemically inert materials. For Packed columns, stainless steel and glass are the usually used materials and for Capillary columns Quartz or Fused Silica are the generally used materials.

Gas chromatography is based on partition equilibrium of analyte between a solid or viscous liquid stationary phase and a mobile gas. The most often a silicone-based material is used for the preparation and Helium is most often used material for Mobile Phase. Inside a larger metal tube commonly known as a packed column, the stationary phase is adhered to the inside of a small-diameter glass or fused-silica or a solid matrix. The most common diameter inside a column is 0.53 – 0.18mm. This technique is widely used especially in Analytical Chemistry; nevertheless the high temperatures used in GC make it unsuitable for high molecular weight biopolymers or proteins, which are frequently encountered in biochemistry. It is highly handy for use in the Petrochemical, Environmental monitoring and Remediation, and Industrial Chemical fields. It is also used extensively in the extensive research of chemistry.

Raman spectroscopy provides the information on molecular vibrations and crystal structures, which is one of the vibrational spectroscopic techniques used in Spectroscopy. To irradiate a sample, this technique uses a laser light source and generates an imperceptible amount of Raman scattered light, which is detected using a CCD camera as a Raman spectrum. In a Raman spectrum the characteristic fingerprinting pattern makes it possible to identify substances including polymorphs and evaluate local crystallinity, orientation and stress.

Some unique advantages of Raman Spectroscopy:

• Non-contact and non-destructive analysis
• High spatial resolution can be measured up to sub-micron scale
• Using a confocal optical system, In-depth analysis of transparent samples can be done
• Sample preparation is not needed
• Measuring of both organic and inorganic substances can be done.
• Measurement of samples in various states such as gas, liquid, solution, solid, crystal, emulsion can be done.
• Through a glass window samples in a chamber can be measured
• Typically, only 10 m.sec to 1 sec exposure to get a Raman spectrum
• Imaging analysis is possible by scanning the motorized stage or laser beam

Raman spectroscopy plays an effective role in departments of R&D, QA and QC in a variety of industrial and academic fields such as semiconductors, polymers, pharmaceuticals, batteries, life sciences and many other departments.

Liquid chromatography is an effective Analytical technique which is mainly used to separate a mixture of sample into its individual parts. There are several different types of chromatography that are classified based on the physical states, as this separations mainly occurs based on the interactions of the sample with the stationary and mobile phases because there are many stationary/mobile phase combinations that can be employed when separating a mixture of those phases. Liquid-solid Column Chromatography is the most popular Chromatography technique features a liquid mobile phase which slowly filters down through the solid stationary phase, bringing the separated components with it.

Based on each component's affinity for the mobile phase, components within a mixture are separated in a column. In a column, if the components are of different polarities and a mobile phase of a distinct polarity is passed through it, one component will migrate through the column faster than the other because same compound molecules will generally move in groups. Within the column, the compounds are separated into distinct bands. The corresponding bands can be seen if the components being separated are colored. Otherwise, the presence of the different bands can be detected by using some other instrumental analysis techniques such as UV-VIS spectroscopy as in high performance liquid chromatography (HPLC).

Liquid chromatography (LC) is a widely used method which is frequently coupled with mass spectrometry for sample ionization prior to analysis. In LC-MS, solubilized compounds (the mobile phase) are passed through a column packed with a stationary (solid) phase. Based on their weight and affinity for the mobile and stationary phases of the column, this effectively separates the compounds. Its anionization through loss of H+ ions also leads to fragmentation of the sample. The sample passes into the vacuum chamber of the mass spectrometer by following this step.

For larger and non-volatile molecules such as proteins and complex peptides, LC is the separation technique of choice. LC-MS offers broad sample coverage due to different column chemistries, such as reversed phase liquid chromatography, can be used when combined with MS. The ideal method for separating isomers, which have the same mass and will otherwise not be differentiated by a mass spectrometer, is LC. As a result, LC has largely replaced gel electrophoresis for molecular separation due to its superior resolving power and broad mass range. Finally, LC helps in reduce ion suppression, which occurs when two different molecules interact with one another and impede the process of complete ionization.

HPLC, which is commonly known as high performance liquid chromatography, has improved and largely replaced LC. HPLC was initially defined as High Pressure Liquid Chromatography which operates at a higher ranging of pressure from 50-350 bar. In contrast, LC majorly depends on gravity for the passage of the mobile phase through the column.

Gas Chromatography-Mass Spectrometry (GC/MS) is an Analytical instrumental technique, by which separation, identification and quantification of complex mixtures of chemicals can be done which comprises a gas chromatograph coupled to a mass spectrometer. This makes it eligible for the analysis of the hundreds of environmental materials relatively low molecular weight compounds. The compound must be sufficiently volatile and thermally stable in order to be analysed by GC/MS. Before GC/MS analysis, samples are usually analyzed as organic solutions consequently materials of interest (e.g. soils, sediments, tissues etc.) need to be extracted from solvent and the extract is subjected to various “wet chemical” techniques.

To the GC inlet the sample solution is injected where it is vaporized by the carrier gas (usually helium) and swept onto a chromatographic column. By virtue of their relative interaction with the coating of the column (stationary phase) and the carrier gas (mobile phase) the sample flows through the column and the compounds comprising the mixture of interest are separated. The compounds eluting from the column are converted to ions following part of the column passes through a heated transfer line and ends at the entrance to ion source.

For Ion production two potential methods exist. Electron ionisation (EI) is the most frequently used method is and chemical ionisation (CI) is the occasionally used alternative. A beam of electrons ionise the sample molecules resulting in the loss of one electron for Electron ionisation. A molecule with one electron missing is represented by M+ called as the molecular ion. In a mass spectrum when the resulting peak from this ion is seen, it gives the compound’s molecular weight. Usually fragments producing further smaller ions with characteristic relative abundances that provide a 'fingerprint' for that molecular structure due to the large amount of energy imparted to the molecular ion. This information may help to elucidate the structure of unknown components of mixtures and also used to identify compounds of interest.

Ion-Mobility Separation-Mass Spectrometry is an another term of Ion-Mobility Spectrometry-Mass Spectrometry (IMS-MS), is an analytical technique on a millisecond timescale separates gas phase ions using ion-mobility spectrometry and on a microsecond timescale uses mass spectrometry to identify components in a sample.

The phenomenon of ion mobility (IM) was first observed in the early 20th Century and harnessed later in ion mobility spectrometry (IMS) based on the movement/transport of charged particles under the influence of an electric field. This theory together with contributions from computational chemistry and gas-phase ion chemistry, there have been rapid advances in instrumental design, experimental methods through which the range of potential applications of contemporary IMS techniques have diversified. Having significant research/applied industrial potential and encompasses multi-/cross-disciplinary areas of science, Whilst IMS-Mass spectrometry (IMS-MS) have recently been recognized.

CE-MS, combining the high potency and backbone power of atomic number 58, with the high property and sensitivity inherent to MS, could be a terribly enticing analytical technique. However, CE-MS coupling, principally by means of ESI, wasn't simple to implement since a closed circuit is important not just for the action separation however additionally for associate degree economical ionization within the supply (with atomic number 58 and ESI currents within the vary of mA and atomic number 11, respectively). A solution for this problem is to ground the sprayer needle in order to distract all electrical energy from the CE to the ground and build an unbroken electrical field for ionization in the MS source. Though the sensitivity achieved with the utilization of a sheath flow is usually lower compared to sheath fewer interfaces, the hardiness of the previous system is usually higher and detection limits within the low femtomole vary are often achieved, particularly once the rate of flow of the sheath liquid is reduced to 500nL/min. The detection of the slim atomic number 58 peaks needs the utilization of a quick and sensitive spectrometer. IT and TOF systems square measure adequate detectors as a result of they acquire knowledge over an appropriate mass vary with rates of many spectra per second.

Thin-Layer Chromatography (TLC) is one of the major chromatographic techniques used to separate non-volatile mixtures. A sheet of glass, plastic, or aluminium foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide (alumina), or cellulose, are used in performing Thin-layer chromatography. The formed layer of adsorbent is known as the “Stationary Phase”.

Sample after applying on the plate, via capillary action a solvent or solvent mixture commonly known as the mobile phase is drawn up the plate as different group of analytes ascend the TLC plate at different rates, separation is achieved. The mobile phase when compared to stationary phase has different properties. For example, a much known polar substance with silica gel, non-polar mobile phases such as heptane is used. For Chemists, the mobile phase is a mixture to fine-tune the bulk properties of the mobile phase.

The spots are visualized after the experiment. Often we can do this simply by projecting ultraviolet light onto the sheet; phosphor is used to treat sheets, when compounds absorb the light impinging on a certain area dark spots appear on the sheet. To visualize spots, chemical processes can also be used; anisaldehyde, for example, with many compounds forms colored adducts, and most organic compounds will char by sulfuric acid, leaving a dark spot on the sheet.

Quantification of the results can be done based on the Retardation Factor (Rf) which states that the distance travelled by the substance divided by the total distance travelled by the mobile phase. In general, the stationary phase made up of a substance whose structure resembles will have low Rf, coming to the mobile phase, the one that has a similar structure will have high retardation factor. Depending on the exact condition of the mobile and stationary phase will change as Retardation factors are characteristic. For this reason, sample of a known compound to the sheet will be applied before running the experiment by chemists.

The study of the metabolism of living organisms in a wide range of conditions, including health and disease can be studied under the field of metabolomics. Muclear Magnetic Resonance (NMR) and mass spectrometry (MS) are the two main analytical techniques used for metabolomics which allow for the detection of many different metabolites. Precise quantitation and superior compound identification are the major strengths of NMR; however, its low sensitivity (metabolites must exceed 1µM) is a major weakness of the method.

In the range of femtomolar to attomolar MS can typically detect. MS can routinely analyze hundreds of compounds in a single sample and run, making it a very powerful and high-throughput process Coupled to either gas chromatography (GC), ion chromatography (IC) or liquid chromatography (LC). Metabolite identification has improved significantly with the advancement of high resolution accurate mass (HRAM) MS systems, as well as enhanced metabolite databases/libraries.To complement genomics and proteomics (multiomics) as core technologies in academic and industrial research labs Innovations, MS have also enabled metabolomics to emerge as its own field of study.

Study of all proteins in a biological system during specific biological events is known as Proteomics (e.g., cells, tissue, and organism). Because of the dynamic nature of protein expression, to study Genomics and proteomics together are considerably more difficult than genomics or even transcriptomics alone. Additionally, some form of posttranslational modification (PTM) will be undergone by the majority of proteins, further increasing proteomic complexity. Due in large part to technological developments in mass spectrometry the broad scope of proteomics is only beginning to be realized during the last 15 years.

To detect, identify and quantitate molecules based on their mass-to-charge (m/z) ratio, Mass spectrometry is a sensitive technique used. to trace heavy isotopes through biological systems, MS was first used in the biological sciences, originally to measure elemental atomic weights and the natural abundance of specific isotopes developed almost 100 years ago. In later years, to sequence oligonucleotides and peptides and analyze nucleotide structure MS was used. The study of protein structure by MS was enabled by the development of macromolecule ionization methods, including electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI).  To obtain protein mass "fingerprints" that could be matched to proteins and peptides in databases and helped and allowed scientists to trace out identity of unknown targets, by Ionization. Both in relative and absolute quantities new isotopic tagging methods led to the quantitation of target proteins. All these technological advancements have resulted in methods that successfully analyze samples in solid, liquid or gas states. The attomolar range (10-18) is the sensitivity of current mass spectrometers allows one to detect analytes of different concentrations.

Nuclear magnetic resonance (NMR) is a physical aspect in which magnetic field absorb and re-emit electromagnetic radiation in a nuclei. Depending on the strength of the magnetic field and the magnetic properties of the isotope of the atoms, this energy is at a specific resonance frequency which; the frequency is similar to television broadcasts (60–1000 MHz) VHF and UHF in practical applications. Observation of specific quantum mechanical magnetic properties of the atomic nucleus, NMR can be used. To study many other topics like molecular physics, crystals, and non-crystalline materials through nuclear magnetic resonance spectroscopy many scientific techniques exploit NMR phenomena. In advanced medical imaging techniques such as in magnetic resonance imaging (MRI) NMR is also routinely used.

Intrinsic magnetic moment and angular momentum can be found in odd number of protons and/or neutrons of isotopes. H and C are the most commonly studied nuclei, high-field NMR spectroscopy is used to study nuclei from isotopes of many other elements (e.g. H, Li, B, B, N, N, O, F, Na, Si, P, Cl, Cd, Xe, Pt) as well. Resonance frequency of a particular substance is directly proportional to the strength of the applied magnetic field is the key feature of NMR. In imaging techniques it is this feature that is exploited; in a non-uniform magnetic field if a sample is placed then the sample's nuclei’s resonance frequencies depend on where they are located in the field. Many efforts are made to develop increased field strength, often using superconductors, since the resolution of the imaging technique depends on the magnitude of magnetic field gradient. Using hyperpolarization, using multi-frequency techniques like two-dimensional, three-dimensional and higher-dimensional the effectiveness of NMR can also be improved.

The interaction of infrared radiation with matter is involved by Infrared spectroscopy (IR spectroscopy or vibrational spectroscopy). Mostly based on absorption spectroscopy, it covers a range of techniques. It can be used to identify and study chemicals as with all spectroscopic techniques. The state of the Samples may be either solid, liquid, or gas. Infrared spectrometer (or spectrophotometer) is an instrument used to produce an infrared spectrum by the method or technique of infrared spectroscopy. On the vertical axis vs frequency or wavelength on the horizontal axis of an IR spectrum can be visualized in a graph of infrared light absorbance (or transmittance).  Reciprocal centimetres (sometimes called wave numbers) are the typical units of frequency used in IR spectra, with the symbol cm−1. Micrometres are the Units commonly given in IR wavelength, symbol μm, which are related to wave numbers in a reciprocal way. Fourier transform infrared (FTIR) spectrometer is a common used laboratory instrument. Two-dimensional IR is also possible as discussed below.

Considering the relation to the visible spectrum, usually the three regions that are classified infrared portion of the electromagnetic spectrum are; the near- infrared, mid- infrared and far- infrared radiations. Excite overtone or harmonic vibrations are don by the higher-energy near-IR, approximately 14000–4000 cm−1 (0.8–2.5 μm wavelength). To study the fundamental vibrations and associated rotational-vibrational structure, approximately 4000–400 cm−1 (2.5–25 μm) may be used by the mid-infrared spectroscopy. Lying adjacent to the microwave region the far-infrared spectroscopy, approximately 400–10 cm−1 (25–1000 μm, has low energy and may be used for rotational spectroscopy. Based on the relative molecular or electromagnetic properties the names and classifications of these sub regions are conventions, and are only loosely arranged.

For assuring the safety of water supplies detailed chemical analysis of water is a prerequisite. To assure water quality and to assess environmental impacts, limitations in our ability helps to identify contaminants in water. The compound range that is amenable to specific identification is due to the recent improvements in analytical techniques having expanded the "analytical window". Much has been learned about the occurrence, fate, and transport of organic chemicals in the environment using these improved tools. "Analytical frontiers" are represented by the boundaries of the analytical window and are continuously expanded. By the contaminant concentration and chemical properties, such as molecular weight, polarity, chemical lability, and structural complexity these boundaries are defined loosely.

In typical natural water samples, compared to the total organic carbon, the mass of compounds that is within the analytical window is small. In conjunction with derivatization, the specifically identified compounds accounted for less than 12 percentage of the organic carbon in different groundwater samples are analysed using coupled Gas Chromatography/Mass Spectroscopy (GC/MS). This fraction typically remains uncharacterized or only in aggregate form for 80 percentage of the total organic carbon. Because of a lack of reference spectra and/or reference compounds, majority of the contaminants that are detected, most remain unidentified and/or unquantified even though good mass spectra are obtained. To propose structures using spectral determinations, the information that can be deduced from the mass spectra may be sufficient. However, because of a lack of reference spectra, verification and quantification of the proposed structures are often impossible.

Forensic science is defined as the use of science to answer questions that are majorly related to the field of interest to a legal system. In the current scenario a common person is far more familiar with forensic science majorly due to the promotion through many popular televisions series, books and films where the mysterious cases are analysed and the way the clues usually discovered by the scientists through laboratory techniques.

In solving many criminal cases, Forensic analytical techniques play a major role for detection of the culprit. Finger printing, analysis of DNA, recognition of voice, analysis of hand writing, ballistics, autopsy etc. is forensic methods to detect a reason for crime or death. Most important areas of materials science, physical science and life science are used in forensic analytical techniques. Some hidden metals and chemicals are traced by the techniques such as chromatography will be used as forensic analytical technique. The age of an unknown human body will be even estimated through some forensic analytical techniques. In some forensic analytical techniques, data will be analysed using digital forensics.