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Mass spectrometry is an analytical technique used to quantify material or identify unknown materials. the process involves conversion of sample into gaseous form which are then differentiated by their mass to charge ratio (m/ z). ions are separated on the availability and abundance in the mass spectrum. Mass spectrum shows the plot of ion abundance versus charge – to- mass ratio. Ion give the information of the structure and nature of the prime molecule of the sample.



It consists of 3 major components.




  1. Ion source –produce gaseous ions from the sample.


  2. Analyzer- region of spectrophotometer where ions are separated according to their mass to charge ratio.


  3. Detector system – for detecting the ions and producing the spectrum of the sample.



In addition, it should have other requirements like proper vacuum system, computer for controlling the whole process and for comparing and saving the data. Proper library for collection of all important data and results.



Following steps are being followed.




  • Sample is produced in an ionized form in gas phase.


  • Acceleration of the ions in an electric field, ions are emerged with the velocity proportional to mass to charge ratio.


  • Detect the ions coming from analyzer and measure their abundance with the detector which convert the signal into electric form.


  • Electric signal id transferred to computer for saving and for feedback.



 



Mass spectrometry is becoming vital field for analyzing biomolecules. Earlier electrophoresis and ultracentrifugation were used for similar information. But results were not as per demand because they are dependent on several ither factors also. Thus, only option left was calculation which was time consuming. Development of spectrometric technique not only saves times but help to analyze the structure of complex compounds.



Analysis of glycans/ lipids



Glycans are formed from several monosaccharides through glycosidic linkage which makes it complex to analyze. mass spectrometry is emerging technique for glycemic and glycobiology.



Lipids are made up of different class molecules. Mass spectrometric techniques help in analyzing such complex compounds. Most recent techniques are ESI (electrospray ionization) and triple quadrupole analyzers. Using MS, it is easy to determine structure molecular weight and branching of compounds.



Scope of mass spectroscopy and purification technique are electrophoresis, crystallization, purification, spectroscopy/ spectrometry in infrared.



Advantages




  • Very small sample weight can be detected.


  • It does not include emission or adsorption of light.


  • Simple spectra that in is unique an easily interpreted.


  • Sample purity is not important.



Chromatography, technique for separating the components, or samples of a mixture based on the relative amounts of each solute distributed between a moving fluid, called the mobile phase, and a thick stationary phase. The mobile phase may be either liquid or gas, while the stationary phase is either solid or liquid.



Kinetic molecular motion continuously exchanges solute molecules between the two phases. If, a particular solute, the distribution favors the moving fluid, the molecules will spend most of their time migrating with the stream and will be transported away from other species whose molecules are retained longer by the stationary phase. The ratio of the times spent in the moving and stationary regions is equal to the ratio of its concentrations in these regions, known as the partition coefficient.



Adsorption chromatography separation is based on differences between the adsorption affinities of sample components for the surface of active solid stationary phase. The components of mixture travel with different speed rate due to their non-covalent interaction with stationary phase. Partition chromatography separation is based on differences between solubilities of the sample component of stationary phase. In partition chromatography stationary phase is liquid and mobile phase is either liquid or gas.



In gas chromatography gas is the mobile phase. The two main division of gas chromatography is GAS –SOLID CHROMATOGARAPHY (GSC) and GAS –LIQUID CHROMATOGRAPHY (GLC). usually used for thermally stable compounds. mobile phase is called carrier gas, they are inert in nature generally used gases are helium, nitrogen, argon etc.  HPLC or high performance liquid chromatography is improved form of liquid chromatography. High pressure up to 400 atm is used. this is much faster method. both small particles and large surface area particles can easily be separated. A combination of high pressure and absorbent of small size results in high resolution power and less analysis time of sample.



DEVELOPING VACCINES



Chromatography is useful in adjudging which antibodies fight various diseases and viruses. Scientists used Chromatography in the fight against the Ebola virus, responsible for over 11,000 deaths, to develop the experimental immunization. The process was used to find out which antibodies are the most effective at neutralizing the virus.



 Food testing



The earlier methods of analysis were effective at determining the composition of raw samples but indecisive when analyzing processed meats, so a more precise method was searched. High performance liquid chromatography, combined with mass spectrometry (HPLC-MS) was successfully used.



 Beverage testing



Many drinks manufacturers use this technique to certify each bottle of their product is the same, so you can be confident on a consistent taste.



 Drug testing



As chromatography can specifically identify substances within the bloodstream, it is widely used in sport to test athletes for consuming or performance enhancing drugs.



Forensic testing



Chromatography is also used to help catch culprits. with programs like CSI, gas chromatography is used to analyze blood and cloth samples, helping to identify the offender.



Chromatographic technique will separate ionic species, inorganic or organic, and molecular species varying in size from the lightest and smallest, helium and hydrogen, to particulate matter such as single cells. It will separate several hundreds of components of unknown identity and concentrations, leaving the components unchanged. Amounts in the parts per billion range can be analyzed with some detectors. The solutes can range from polar to nonpolar. 



Separations are fast varying from analysis times of a few minutes to several hours. The earlier world would have considered a time of several hours to separate multicomponent mixtures to be miraculously fast. Now several hours is reduced and there is much emphasis on increasing speed.



One of the major challenges in the medicine today is developing new therapies that improve human health. To help address these challenges the utilization of analytical technologies has been employed; to perform more experiments in a shorter time with increased data quality. In the last years various analytical strategies have been established to enhance separation speed and efficiency in liquid chromatography applications. Liquid chromatography is an important tool for monitoring drugs and their metabolites. Additionally, liquid chromatography has played an important role in pharmacokinetics and metabolism studies at these drug development stages. Current trends in fast liquid chromatographic separations involve technologies, fused-core columns, high-temperature liquid chromatography (HTLC) and ultra-high performance liquid chromatography (UHPLC). The high specificity in combination with high sensitivity makes it an attractive method to traditional methodology used for daily applications. 



Aspects of environmental quality can also be evaluating and monitored using chromatography. The concentration of pollutants in water, soil, and air are each determined firstly by chromatography in tandem with other characterization techniques such as mass spectrometry. This process has proven boon in city planning and assessing the impact of industry on locations and the planet in general



HPLC can be used in both qualitative and quantitative applications that are for both sample quantification and identification. Routine phase HPLC is rarely used now, almost all HPLC separation can be performed in reverse phase. Reverse phase HPLC (RPLC) is ineffective in for 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, polymer chemistry, environmental chemistry and green chemistry.



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, either in their active pharmaceutical ingredient or during the process of their discovery, development, and manufacturing.



Chromatography and spectroscopy are techniques, i.e., their types of information are unique and are specific. HPLC is a technique for separation, quantification, and identification of components in a sample mixture. It is specifically suitable for compounds which are not easily volatilized, thermally unstable and have high molecular weights. 



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



Fingerprinting is a quality 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 and displays integrated quality information. Comprehensiveness and fuzziness are the two important traits of a fingerprint. The similarity of fingerprints is established through these traits. Fingerprint analysis focuses on accurate identification, and not on precise calculation. The comparison of fingerprints declares similarity and the fingerprints compared do not need to be the same. When it is impossible to find out all the complex components of a medicine fingerprints can be used to check the authenticity of the intrinsic quality of the medicine.



HPLC is a popular method of analysis for natural products because of its high accuracy 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) has been utilized for the qualitative and quantitative determination of various types of Phyto-constituents like alkaloids, glycosides, tannins, tri-terpenes, etc. HPLC methods are used for analyzing of drug in biological fluids and pharmaceutical dosage forms. HPLC determination with spectroscopic detection is useful for daily quality control of drugs in pharmaceutical dosage forms and studies.



A chromatographic detector can create both identity and concentration elution of components in the mobile phase stream. Various range of detectors are available to meet different sample conditions. some detectors are UV-VIS, Photo diode array, fluorescence, and mass spectroscopic detectors. Some bulk Property detectors include refractive index electrochemical and light scattering detectors.




  • Can separate complex mixture with great precision.


  • Less material consumption


  • Fast analyzing technique


  • Many samples can run at a time in one run.


  • Well established instrument with control system



Ionization refers to the generation of gas phase ions appropriate for resolution in a mass analyzer or mass filter in mass spectrometry. The ion source is where ionization takes place. There are a variety of ion sources accessible, each with its own set of benefits and drawbacks for certain applications.  For example, electron ionization (EI) causes a high degree of fragmentation, resulting in highly detailed mass spectra that, when properly analyzed, can provide valuable information for structural elucidation/characterization and aid in the identification of unknown compounds by comparing them to mass spectral libraries obtained under identical operating conditions.



Inductively coupled plasma (ICP) sources are commonly employed for cation analysis on a variety of sample types. To atomize introduced sample molecules and further strip the outer electrons from those atoms, a plasma that is electrically neutral overall but has had a significant fraction of its atoms ionized by high temperature is utilized in this source. Because argon atoms have a higher first ionization energy than any other element except, He, F, and Ne, but a lower second ionization energy than all other elements except the most electropositive metals, plasma is frequently created from argon gas.



 Photoionization can be employed in investigations aimed at understanding chemical kinetics mechanisms and isomeric product branching using mass spectrometry. In such cases, a high-energy photon, such as an X-ray or an ultraviolet photon, is used to dissolve stable gaseous molecules in a carrier gas such as He or Ar. A tunable photon energy can be used in conjunction with the charge ratio m/z to obtain a photoionization efficiency curve that can be used to fingerprint molecular and ionic species when a synchrotron light source is used.



Environmental and healthcare applications are both possible applications for ambient ionization. Ions develop in an ion source outside the mass spectrometer in these approaches. Because the samples do not require any prior separation or preparation, sampling becomes simple. DESI, SESI, LAESI, and Desorption atmospheric-pressure chemical ionization (DAPCI) are some examples of ambient ionization processes