The Principle
Gas chromatography (GC) is a technique used in analytical chemistry for separating and analysing gaseous compounds. Applications of GC are not limited to testing the purity of a given substance or separation of components in a mixture. An inert gas is preferably used as the mobile phase, e.g., helium or an unreactive gas like nitrogen. The stationary phase is a layer of liquid or polymer on solid support inside a column, which must be inert. The desired gaseous compounds interact with the coated stationary phase on the walls, and thus, each compound elutes at a different retention time. The column is located in an oven with a high temperature, so the gas can be controlled.
Applications
The Principle
HPLC (High Pressure Liquid Chromatography) is considered one of the most important separation techniques among all other techniques. It depends on the interaction of analytes with two phases, viz., stationary and mobile, based upon their affinity and thereby separation. Based upon the mechanism for separation, HPLC follows any of the following, but not limited to, mechanisms: adsorption, partition, ion exchange, ion pair, or size exclusion mechanism.
Applications
The HPLC technique is generally used for separation, identification, purification, and quantification of a compound from a mixture. The other major applications of HPLC include determination of percentage purity of APIs and separation of impurities; stability studies; tablet dissolution studies; pharmaceutical quality control; etc. The pharmaceutical and chemical industries benefit the most from HPLC. It is also useful in the food industry for quality control and to compare with standards given by the government.
The Principle
High Performance Thin Layer Chromatography (HPTLC) is a superior form of thin-layer chromatography (TLC). The principle involves the separation of samples by adsorption. The mobile phase moves by capillary action. Compounds move and get separated as per their affinities towards the stationary phase. Compounds with high affinity towards stationary phase travel slowly, while compounds with high affinity towards mobile phase and low affinity towards stationary phase travel fast, along with mobile phase. A number of improvements can be made in terms of automation in the loading of samples, the detection of spots and their quantification. These increase resolution and provide more accurate quantitative measurements. 2D chromatography with two different solvents increases spot capacity.
Applications
Major advantages of HPTLC include simplicity, low cost, multiple sample analysis at a time, high sample capacity, quick results, and the opportunity for multiple detection. It has numerous advantages over other chromatographic methods, including the presentation of results as an image. HPTLC is the most extensively applied method in the pharmaceutical industry, food and drug analysis, environmental analysis, clinical chemistry, forensic analysis, biochemistry, and cosmetic industries.
The Principle
A supercritical fluid has gaseous properties (able to penetrate) and liquid properties (able to dissolve materials). SFC is a hybrid of GC and LC. As the mobile phase is in liquid form below its critical temperature and above its critical pressure, the technique is liquid chromatography (LC). The mobile phase acts as a gas above its critical temperature and below its critical pressure, so the technique is called gas chromatography (GC). Use of carbon dioxide (CO2) or water in the form of a supercritical fluid provides a substitute for other solvents in the food industry and medical supplies. In SFC, the sample passes with a supercritical fluid through a separating column where the mixture is divided into unique bands. This happens based on the intensity of interaction between the analytes and the stationary phase. As these bands leave the column, their identities and quantities are determined by a detector.
Applications
SFC is useful and has a wide variety of applications, which include natural products, drugs, food, pesticides, herbicides, surfactants, polymers and polymer additives, fossil fuels, petroleum, explosives and propellants, etc.
The Principle
Atomic Absorption Spectroscopy (AAS) is a spectro-analytical method for the quantitative determination of chemical elements (metals) at very low concentrations of parts per million (ppm) or parts per billion (ppb) using the absorption of optical radiation (light) by free atoms in the gaseous state. AAS is based on the absorption of light by free metallic ions. At the high temperature, the sample is broken down into atoms, and it is the concentration of these atoms that is measured. The total amount of absorption depends upon the number of free ions and the degree to which the free ions absorb the radiation. In analytical chemistry, the technique is used to determine the concentration of a particular element in a sample to be analysed.
Applications
AAS can be used to determine different elements in solution, or directly in solid samples via electro thermal vaporization and is used in agricultural, chemical, environmental, pharmacological, biophysical, and archaeological and toxicological research. AAS is used to analyse soil and plants for necessary minerals required for growth. AAS has many uses in different areas of chemistry such as clinical analysis of metals in biological fluids and tissues such as whole blood, plasma, urine, saliva, brain tissue, liver, hair, muscle tissue, semen, in some pharmaceutical manufacturing processes, minute quantities of a catalyst that remain in the final drug product, and analysing water for its metal content.
Principle
Differential Scanning Calorimetry (DSC) is the most frequently used thermal analysis technique. It measures the temperatures and heat flows associated with transitions in sample and reference materials as a function of time and temperature in a controlled atmosphere. Both the sample and the reference are maintained at nearly the same temperature throughout the experiment. DSC identifies and characterises materials. A small amount of sample is heated, and if any transition occurs during this process, it will result in a slight difference in temperature between the sample and a reference sample; that is, differential scanning calorimetry measures the amount of energy (heat) absorbed or released by a sample as it is heated, cooled, or held at a constant temperature. Differential scanning calorimetry is fast, very sensitive and easy to use.
Applications
The major application of DSC is to check the compatibility of drugs with excipients. DSC analysis measures endothermic or exothermic reactions by a sample when it is heated, providing quantitative and qualitative data on heat absorption and heat evolution processes. DSC is widely used for examining polymeric materials to determine their thermal transitions. It is also used in the study of liquid crystals. DSC analysis is used to measure melting temperature, heat of fusion, latent heat of melting, glass transition temperature, crystalline phase transition temperature and energy, precipitation energy and temperature, denaturation temperatures, etc. It is also used to measure specific heat capacity, heat flow rate, thermal behaviour and its derivatization, such as heat transformation or any chemical change in the sample.
The Principle
Fourier Transform Infra-Red Spectroscopy (FTIR) is a technique used to obtain an infrared spectrum of absorption or emission of a solid or liquid. In FTIR analyses, infrared light from the light source passes through a Michelson interferometer along the optical path. The light beam split into two by the beam splitter is reflected from the moving mirror and fixed mirror before being recombined by the beam splitter. As the moving mirror makes reciprocating movements, the optical path difference to the fixed mirror changes in such a way that the phase difference changes with time. The light beams are recombined in the Michelson interferometer to produce interference light. The intensity of the interference light is recorded in an interferogram, with the optical path difference recorded along the horizontal axis.
Applications
FTIR spectra reveal the composition of solids and liquids. The most common use is in the identification of unknown materials. Apart from applications in pharmaceutical and chemical industries, it has applications in food sciences, forensic sciences, environmental sciences, chemical sciences, polymer and plastic industries, etc. It is used for structural elucidation in basic drug research; used in formulation development and validation; quality control processes for incoming and outgoing materials, etc. An FTIR microscope can be combined with a sample heating system, which allows changes in molecules to be measured for their thermophysical properties with image observation. The heating system uniformly heats or cools the measurement area in the IR microscope, assuring high-accuracy measurement. Temperature and measurement conditions are software controlled with simultaneous image scanning and IR measurement.
The Principle
High pressure homogenisation is a mechanical process that involves forcing fluid through a narrow homogenising nozzle at high pressure. The liquid is subjected to very high shear stress, which causes the formation of fine emulsion droplets. The smaller the nozzle aperture and the higher the pressure, the smaller the droplets that are produced, with the aim being to reduce particles and droplets from micron to nanometer sizes. In the piston-gap homogenizer, the macro-suspension coming from the sample container is forced to pass through a tiny gap; particle diminution is affected by shear force, cavitation, and impaction.
Applications
High pressure homogenizer applications require the most efficient fluid processing equipment for particle and droplet size reduction and cell disruption. Major applications of the homogenizer include high pressure pasteurization, particle size reduction, micro/nano emulsions, dispersions, and cell disruption. Pharmaceutical drug development and manufacturing, the milk industry, and the food and beverage processing industry are all major areas of application.
The Principle
The principle involved in lyophilisation, i.e. freeze drying, is a phenomenon called sublimation, where water passes directly from a solid state (ice) to the vapour state without passing through the liquid state. Water can sublimate at pressures and temperatures below the triple point. The material to be dried is first frozen and then subjected under a high vacuum to heat, so that the frozen liquid sublimes, leaving only solid, dried components of the original liquid. The concentration gradient of water vapour between the drying front and the condenser is the driving force for the removal of water during lyophilisation.
Applications
The major industries where lyophilisation is useful include, but are not limited to, pharmaceutical and biotechnological industries; the food industry; technological industries; and it is also useful to conserve special bacterial strains.
The Principle
The instrument uses the principle of dynamic light scattering to measure particle size. It is a measurement of the fluctuation in scattered light intensity with time. Fluctuation is mainly observed due to random Brownian movement of nanoparticles. The statistical behaviour of these fluctuations that occur in scattered intensity can be related to the diffusion of the particles. Larger particles diffuse more slowly than small particles, so one can readily relate particle size to the measured fluctuation in light scattering intensity. It also measures zeta potential. Nanoparticles or colloidal particles have a surface charge in suspension. When an electrical field is applied, the particles move due to the interaction between the charged particles and the applied field. The direction and velocity of motion are a function of particle charge, suspending medium, and electric field strength.
Applications
Particle size analysis is used to characterise the size distribution of particles in a given sample. Particle size analysis can be applied to solid materials, suspensions, emulsions, and even aerosols. It is also useful for the measurement of the zeta potential of nanoparticulate systems. Some industries and product types where particle sizing is used include pharmaceuticals, building materials, paints and coatings, food and beverages, aerosols, etc.
The Principle
The Spectrofluorometer works on the principle of fluorescence spectroscopy. This technique correlates fluorescent properties of some compounds in order to provide information regarding their concentration and chemical environment in a sample. The emission is observed either at a single wavelength or a scan is performed to record the intensity versus wavelength. The system has a high-quality optical system and also has high sensitivity.
Applications
The Spectrofluorometer is used to meet the demanding needs of laboratories in application areas like biochemistry, performing kinetics, stopped flow, titration, and anisotropy experiments. This technique has applications in the pharmaceutical industry, has environmental significance, geological applications, analytical chemistry, as well as biochemistry applications.
Principle
A total organic carbon (TOC) measurement is commonly used to determine the degree of organic contamination in water. TOC is an indirect measure of organic molecules present in water and measured as carbon. TOC is a highly sensitive, non-specific measurement of all organics present in a sample. Organic molecules are introduced into the water from the source, from purification, and from distribution system materials. TOC is measured for both process control purposes and to satisfy regulatory requirements. The high temperature combustion catalytic oxidation method achieves total combustion of samples by heating them in an oxygen-rich environment. The carbon dioxide generated by oxidation is detected using an infrared gas analyser (NDIR).
Applications
TOC is very important in detecting contaminants in drinking water, cooling water, water used in semiconductor manufacturing, and water for pharmaceutical use. TOC detection is an important measurement because of the effects it may have on the environment, human health, and manufacturing processes. It can be used to regulate the organic chemical discharge to the environment in a manufacturing plant. In addition, low TOC can confirm the absence of potentially harmful organic chemicals in water used to manufacture pharmaceutical products. TOC is also of interest in the field of potable water purification due to by-products of disinfection. Inorganic carbon poses little to no threat. The Total Organic Carbon analyser is used for environmental analysis, pharmaceutical and chemical industries, determination of carbon dioxide in food products, etc.
The Principle
A UV/VIS/NIR Spectrophotometer measures the reflection or absorbance characteristics of a sample. It measures in the range covering UV, visible, and near IR, from 200 to 3000 nm. Here, absorption is used to characterise materials. Radiation absorption may occur in a transmission or reflection mode. A spectrum is generated with a graph of absorption versus wavelength. The spectra is used to determine the spectral response of a colour sample in the visible region or the sample’s ultra-violet or near infra-red filtering characteristics or the difference in appearance between two pigments when viewed under different light sources.
Applications
A UV/VIS/NIR Spectrophotometer is useful for the measurement of the absorbance of liquids in the range of 200 to 2500 nm. In addition to measuring liquids, it is used to measure the transmittance and reflectance of solid samples. This technique gives basic information about the lmax of an unknown sample as well as a reflection spectra, which is useful to get preliminary information about the sample.
The Principle
Many substances absorb light. However, some of them, after absorbing light of a particular wavelength and energy, emit light of a longer wavelength and less energy. Such substances are called “florescent substances.” The application of this phenomenon is the basis of the fluorescence microscope. Microbes are stained with a florescent dye and then illuminated with blue light. The dye absorbs blue light and emits green light. The specimen is previously stained with a fluorescent dye, such as acridine orange, acridine yellow, acriflavine, thioflavin S, thioflavin T, titan yellow G, etc. Certain portions of the specimen retain the dye, while others do not. The portions that retain the fluorescent dye absorb blue light and emit green light. The emitted green light goes upward and passes through the dichroic mirror. It reflects back blue light, if any, and allows only green light to pass through. Then, the light reaches a “barrier filter”. It allows green light to pass through to the eye and blocks out any residual blue light from the specimen, which might not have been completely reflected by the dichroic mirror. Thus, the eye perceives the stained portions of the specimen as glowing green objects against a jet-black background, whereas the unstained portions of the specimen remain invisible. Fluorescence microscopy requires a very powerful light source such as a xenon or mercury arch lamp.
Applications
Fluorescence microscopy is widely used in biology and medicine, as well as in other fields. Fluorescence techniques can be applied to all kinds of material, but generally, fluorescence microscopy is reserved for applications that require high sensitivity, i.e., to examine substances present in low concentrations. Fluorescence microscopy is also useful to detect particles below the resolution of a light microscope and in histochemistry to visualise substances that cannot be seen by conventional microscopy. Biological material is commonly stained in the same manner with a fluorescent stain and then observed under fluorescence microscopy.
The Principle
Flow cytometry means measuring the properties of cells when in motion. The basic principle of flow cytometry is the passage of cells in a single line in front of a laser so they can be detected, counted, and sorted. Cell components are fluorescently labelled and then excited by the laser to emit light at varying wavelengths. The fluorescence can then be measured to determine the amount and type of cells present in a sample. Up to thousands of particles per second can be analysed as they pass through the liquid stream. A beam of laser light is directed at a hydrodynamically focused stream of fluid that carries the cells.
Applications
The Attune NxT Flow Cytometer has multiple applications in the area of Immunophenotyping. It helps in designing a T-cell backbone panel and validating multicolour T-cell panels. It helps in multicolour immunophenotyping analysis of stained human whole blood using a no-lyse, no-wash protocol, with no compensation. It also helps in multiple parameter immunophenotyping of human lysed whole blood, which identifies multiple T-cell subsets and myeloid cells, multi-parameter immunophenotyping of human lysed whole blood identifies B cells, NK cells, multiple T-cell subsets and myeloid cells; multiparameter analysis of murine regulatory T-cells and dendritic cells and is used to detect murine regulatory T-cells. It also aids in the detection of platelets in whole blood, no-wash, no-lyse detection of leukocytes in human whole blood, and no-wash, no-lyse detection of phagocytic cells in human whole blood using the pHrodo BioParticles functional assay. It also has applications for rapid and accurate analysis of nuclear DNA content in plants and is used for the detection of fluorescent proteins. It is also used in the flow cytometry analysis of transcription factor expression during differentiation of hPSC-derived cardiomyocytes.
The Principle
The polymerase chain reaction (PCR) is a laboratory technique for DNA replication by selective amplification of target DNA. PCR uses the smallest sample of DNA to be cloned and amplifies it to millions of copies in just a few hours. PCR involves a primer-mediated enzymatic amplification of DNA. PCR is based on the ability of DNA polymerase to synthesise new strands of DNA complementary to the offered template strand. A primer is required because DNA polymerase can only add a nucleotide onto an already present 3′-OH group to add the first nucleotide. DNA polymerase then elongates its 3 ends by adding more nucleotides to generate an extended region of double-stranded DNA.
Applications
Major applications of PCR in various fields include medical applications like genetic testing, detection of infectious diseases causing genes, alteration of oncogenes, sensitive tissue typing, gene therapy; forensic applications like genetic fingerprinting in crime scenes, paternity testing, etc. Real-time PCR (or qPCR) has very wide applications in biological science, which include agricultural and food industries, gene expression analysis; the diagnosis of infectious diseases; and human genetic testing.