Tuesday, April 16, 2019

What is Instrumentation of HPLC

The separation of all chromatography techniques including high-performance liquid chromatography works under the identical basic principle. The molecules of the sample mixture are get separated due to the dissimilarity of their affinities for the stationary and mobile phase used in the chromatography system. High-performance liquid chromatography (HPLC) also has a stationary phase and mobile phase, HPLC is a highly advanced form of column liquid chromatography. In column chromatography, the solvent is passed with gravity through the stationary phase (e.g. Silica gel), but in the HPLC system, instead of under gravity the mobile phase and sample are run under the force at about 400-4000 PSI, which separates the molecules very rapidly. HPLC is a common technique for the separation of analytes in a complex mixture and which is used for the qualitative and quantitative study of molecules. Depending on the stationary phase system in the process, there are different types of HPLC, such as normal phase HPLC (NP-HPLC), reverse phase HPLC (RP-HPLC), size-exclusion HPLC, and ion-exchange HPLC.

The HPLC system consists of the following major components.
  • Solvent Reservoirs:
It fulfills the storage of HPLC solvents in sufficient quantity for uninterrupted operation of the system and it delivered to the pump by Teflon tube through the online degassing and filters. Generally, there are at least two reservoirs in a system, which are usually glass bottles. The reservoir and its attachment would be made of glass, Teflon, or stainless steel material so that the mobile phase is not contaminated. Generally, as a mobile phase, we are using HPLC grade methanol, acetonitrile, water, and buffer.
  • The pump of HPLC:
As the name, is required to be generated the pressure with reproducible and the constant flow of mobile phase through the system. The HPLC pump would be capable to take solvent with single or multiple reservoirs with pulse-free output at various flow rates to permit the controlled mixing of different solvents/mobile phases from multiple reservoirs. The reciprocating pump, syringe pump, and pneumatic pump are the types of HPLC pumps.
  • The Injector of HPLC:
The introduction of a sample in HPLC is a technique to inject the samples without interrupting the pressure and flow rate of the HPLC system. HPLC's work pressure is sufficiently high so that we cannot introduce a sample in the mobile phase by injecting the syringe, that's why we require an injector that gives reproducible results and without disturbing the system pressure and flow rate. Nowadays, modern injectors are autosamplers, that permit programmed injection of various volumes of sample, which takes from the vials in autosampler trays. Rheodyne injector, septum injector, and stop flow injector are the types of the HPLC injector.
  • The Column of HPLC:
The Column is the heart of the HPLC system, it actually generates a separation of molecules in the sample mixture. A column is situated after the injector where the mobile phase is in contact with the stationary phase, creating an interface with the enormous surface. The development of most chromatography in recent years has led to the design of many different approaches to increase this interfacial contact. Typically HPLC columns are available 30 to 250 mm long, 01 to 05 mm in diameter, and 03, 05 and 10 microns in the pore size. The column is filled with porous particles, which is made of polymer and bounded by a thin layer of silica and polystyrene (e.g. C8, C18 column)
  • The Detector of HPLC:
A detector is a tool used to detect the sample and column effluent, which is eluted from the column. The detector converts data into an electrical signal and is recorded by the system. The most common detector, UV/VIS is to be used in pharmaceutical analysis, It allows continuous monitoring of UV absorption over a particular wavelength or a range wavelength (using a PDA detector). The presence of components in the detector flow cell causes the change of absorbance.
Here are some types of HPLC detectors mentioned.
UV/VIS and PDA detector, Refractive index detector, Mass spectrometry (MS (MS), Fluorescence detector, Electrochemical detector, Conductivity detector, Light scattering detector, Infrared detector
  • Data Acquisition and Control System:
All parameters of the HPLC system are controlled through the computer-based system i.e. Run time, mobile phase composition, wavelength, flow rate, temperature, injection volume, sample sequence, etc. We can monitor the system continuously and check the pressure and other things using this system. Receives the data from the detector and monitors the system's performance and also allows integrating the obtained data.


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Some Applications of Paper Chromatography

Paper chromatography is the kind of chromatography where it is at the stationary phase of cellulose paper, the solvent (Mobile phase) moves upward with the sample mixtures and as per the difference in affinity, they get separated. Following separate out the components as a result of their inter-migratory velocity, they are stained to make chromatogram visible or spray the solution (e.g. Ninhydrin solution) on them to make them visible. All types chromatography is working on a similar principle, for the various molecules in the solution, it is necessary that the molecule should have different properties such as the ability to dissolve in solvent or size and structure of the molecule. This is a simple and basic way to separate the components of the mixture, it is similar to the thin layer chromatography (TLC).
The Applications of Paper Chromatography:
  1. To detect these specific compounds, sugar, amino acids, lipids, and nucleic acids and other biomolecules can be easily identified by spraying with suitable reagents (e.g. Ninhydrin solution)
  2. To identify unknown organic and inorganic compounds the paper chromatography used in scientific studies.
  3. Paper chromatography is an effectual method to separate colored pigments from a mixture
  4. It can be used in a forensic laboratory.
  5. It is used to separate the complex mixture of components.
  6. Paper chromatography can use in qualitative analytical chemistry.
  7. Combining the unknown chromatogram with the chromatogram of known substances can help in providing clues to the crime and in drug abuse.
  8. It is applied to get pure compounds from the complex mixture.

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What is the Stationary Phase in Paper Chromatography

Paper chromatography is a kind of chromatography that works on a piece of special paper. This is a planner chromatography system in which cellulose filter-paper works as a stationary phase on which the compounds are separated. The basic principle involved in paper chromatography and thin-layer chromatography is partition chromatography in which the molecules are partitioned between liquid phases. Both TLC and paper chromatography is particularly applied for the separation of polar and non-polar compounds. Paper chromatography uses paper which acts as a stationary phase. The paper contains cellulose fibers, which are polymers -OH functional groups attached to polymer chains. These functional groups lead to retention and separation of surface absorbed analytes. Different molecules are balanced between the layer of the mobile phase and the water. There are various kinds of paper are were used, however, the Whatman filter papers number 1 and number 3 are found to be more suitable. An 18"x22" rectangular sheet of chromatography paper is usually available for analytical work. Apart from cellulose paper, to separate the different types of molecules different types of papers are also available which consists of silicone oil-impregnated papers, acetylated papers, silica, and alumina impregnated papers.


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What is Paper Chromatography and How Does it Work

Introduction to paper chromatography:
Paper chromatography is a technique of separation in analytical chemistry, which separates the mixture of analytes with the help of the mobile phase on a sheet of special paper that acts as a stationary phase. The process of paper chromatography is parallel to thin layer chromatography, so it is also used to teach the TLC process.
The principles of paper chromatography:
All chromatography techniques adhere to the same principle. According to other chromatography techniques, paper chromatography has a mobile phase and a stationary phase. The paper is used as a stationary phase and the solvent is used as a mobile phase. The mixtures of sample solution travel with the mobile phase through the stationary phase and get separated on the sheet of paper due to the difference in affinities of each component towards the stationary phase.
The stationary phase of paper chromatography:
Paper chromatography is parallel to TLC, the contrast is that the rather than utilizing a thin layer of silica on a sheet of metal, a special kind of paper that used as a stationary phase. This paper is composed of cellulose and contains polymers -OH functional groups. The other kinds of papers are also used in paper chromatography like silica and alumina impregnated papers, acetylated papers, and silicone oil-impregnated papers.
The process of paper chromatography:
In this chromatography, a special quality paper used as a stationary phase is called chromatography paper. The mobile phase is a solvent or mixture of solvents (e.g. Methanol, water, etc.). A sample solution of a complex mixture is spotted on a line approximately 2 cm above the paper, and after that suspended in the appropriate solvent in the chromatography chamber. The solvent rises up and flows over the spot by capillary action. Paper selectively holds different analytes in two phases as per to their different partition. The developed paper strip is called a chromatogram. The spot of different colored compounds appears on different heights from the initial position on the chromatogram. The spot of different colorless components can be seen either using a suitable spray reagent (e.g. Ninhydrin reagent) or under ultraviolet light (e.g. UV cabinet). The relative adsorption of separated components is expressed as a retention factor (Rf value).


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What is the Stationary Phase in Chromatography

Chromatography is a science to separate a mixture of components. To perform this, the sample passes through phases, these phases are known as the mobile phase (Liquid/Gas) and stationary phase (Solid). In this technique, the stationary phase remains constant, whereas the mobile phase flows through the system. Initially, a sample mixture is dissolved either in a mobile phase or suitable liquid (e.g. Methanol, Acetonitrile, and Water) that travels it in a stationary phase through a structure.
Chromatography depends on the principle where the molecules in the sample mixture are introduced into the solid, and while moving with the help of the mobile phase, the analytes are separated. In this separation process, efficient factor contains molecular characteristics associated with liquid-solid (partition), liquid-solid (adsorption), and affinity of the molecules. As of these dissimilarities, some analytes last longer in the stationary phase, and they are traveling gradually in the system, Whereas other molecules travel quickly and leave the chromatography system earlier.
In view of this approach, there are three components based on chromatography technology.
Mobile phase: The mobile phase is composed of liquid or gaseous components.
Stationary phase: This phase is made of a solid phase or, on the solid support surface, the liquid layer is adsorbed.
Molecules: Molecules to be separated, which is in the mixture sample.
Here are some types of chromatography and stationary phases which used in chromatography is mentioned.
  • Column chromatography: Silica gel, and alumina.
  • Paper chromatography: A special piece of filter paper or cellulose paper.
  • Thin-layer chromatography (TLC): Alumina, silica gel, or similar material is coated on metal, plastic film or a glass plate, as a thin layer.
  • Gas chromatography (GC): A microscopic layer of polymer on an inert solid support, inside the portion of metal or glass tubing.
  • High-pressure liquid chromatography (HPLC): Silica filled in the column.


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What are the Types of Chromatography

Chromatography is an analytical technique in chemistry that is based on the separation of a sample molecule in two phases, which is a mobile phase and a stationary phase. While a sample is passed with the mobile phase through a stationary phase the interactions happen between the stationary phase and the analytes or solutes, each molecule has different properties and based on the differences in these interactions they get separated. In many of its variations, chromatography is a crucial separation technique for the analysis or purification of one or several analytes in a mix that can include different types of complex components. Initially, it was developed in the form of column chromatography. But due to the limited efficiency and applications, it was modified and introduced advanced techniques of separation. Therefore, since advancement, we have many types of chromatography.
Various types of chromatography methods are available based on the points given below.
  1. It depends on the principle of isolation used.
  2. The physical states of the mobile and stationary phase.
  3. Based on the chemical nature (Polarity) of mobile and stationary phase used.
  4. As per the shape of the stationary phase.
  5. Chromatography based on the purpose of the testing.
  6. Depending on the chemical or physical nature of the stationary phase.
Different types of chromatography techniques:
  • Column chromatography
  • Paper chromatography
  • Thin-layer chromatography (TLC)
  • Gas chromatography
  • High-performance liquid chromatography (HPLC)
  • Affinity chromatography
  • Ion exchange chromatography
  • Size exclusion chromatography
  • Supercritical fluid chromatography
  • Gel filtration chromatography
There are various types of chromatography methods and are classified based on the separation mechanisms, the physical state of the mobile phase, and the shape of the bed.

What are the Different Types of Chromatography

Chromatography is an analytical technique in chemistry and it is used to separate the compounds of analytes into their constituents. All types of chromatography techniques work on the same principle. All of these contain a mobile phase consists of gas or liquid and a stationary phase consist of solid or liquid. The sample mixture travels through the stationary phase with the mobile phase, due to the difference in the affinities of components, they are separated.
The various types of chromatography are mentioned here.
Adsorption Chromatography:
To separate the components the adsorption chromatography is the oldest kind of chromatography, it is based on the principle of adsorption in which the separation depends on the interaction of the adsorbate with the adsorbent. In this type of chromatography, the liquid or gaseous phase (Mobile phase) is used on the surface of the solid stationary phase. The equilibration amongst the mobile phase and the stationary phase is for the separation of different solutes.
There are three main types of adsorption chromatography are thin-layer chromatography (TLC), column chromatography, and gas-solid chromatography.
Partition Chromatography:
Partition chromatography is a kind of chromatography used for the separation of components. In this type of method, the analytes present in the sample mixture distribute more probability in two liquid phases due to differences in the partition coefficient. It relies on the retention factor (K) and distribution coefficient (Kd) of the molecules using the liquid for the stationary phases. The liquid-liquid chromatography and bonded-phase liquid chromatography are types of partition chromatography.
Ion Exchange Chromatography:
Ion chromatography is an effective method for the separation of charged particles; it involves the separation of ionizable components on the basis of their total charge.  Ion exchange chromatography is often executed as column chromatography. This method allows the separation of equivalent kind of analytes which will be complex to isolate by other techniques, as the charge made by the interested molecule can be easily manipulated by altering the buffer pH. Ion exchange chromatography has two types anion-exchange and cation-exchange chromatography.
Molecular Exclusion Chromatography:
It is also recognized as gel filtration or gel permeation chromatography. In this chromatography method lack an alluring interaction amongst stationary phase and molecules. The liquid/gas phase travels via a porous gel that separates the analytes as per its size. Generally, the pores are small and remove large molecules, however, it allows small analytes to enter the gel so that they flow in large quantities. This is the cause that large molecules move through a column at an accelerated rate compared to smaller ones. Two basic types of size exclusion chromatography are a gel permeation chromatography (GPC) and the second is gel filtration chromatography (GFC).
Affinity Chromatography:
It is a method in which the distinction of absorption relies on the particular relationship between the substances and the desired component in the ligand. Affinity chromatography is the mainly discriminating type of chromatography. It employs a specific interaction amongst a type of solute analyte and another analyte, which is immobilized at the stationary phase.




How Does Chromatography Work

Chromatography is used to separate the mixture of analytes into their components. There are all types of chromatography methods work on a similar principle. All of them have a mobile phase (Gas/Liquid) and a stationary phase (Solid/Liquid).  The mobile phase runs through the stationary phase and moves the analytes of the mixture together. Various components travel at different rates and get separated, because of differences in the affinities of different components for the stationary and mobile phase.
As soon as the liquid begins to travel past the solid, some of its molecules are sucked towards the surface of the stationary phase and temporarily attach there before being into the liquid they originated from. This swap of molecules amongst the solid and liquid surfaces is a type of adhesive effect called adsorption. Keep in mind that the liquid is a mixture of different solvents/liquids. Every analyte passes through the adsorption in little dissimilar ways and uses less or more time in the liquid or solid phase. Compared to the liquid phase in a solid phase liquid can remain longer, therefore it will travel gradually on the solid; Another one can remain more time in liquid and less time in solid so it will be a bit more rapidly. In order to work chromatography efficiently, we require components of the mobile phase in order to separate as much as possible, since they move beyond the stationary phase.
There are different types of chromatography, here are some mentioned: thin-layer chromatography (TLC), gel permeation chromatography, high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), high-performance thin-layer chromatography (HPTLC), ion-exchange chromatography, paper chromatography, affinity chromatography, column chromatography,  and gas chromatography, etc.



What is Chromatography and How Does it Work

Chromatography is one of the well-known technique for the separation of components for qualitative and quantitative analysis on the basis of the relative quantity of each solute distributed among a stationary and mobile phase. The mobile phase can be either liquid or gas, whereas the stationary phase is either a solid or a liquid.
The mixture of different components enters a process of chromatography and various analytes are passed through the system at dissimilar rates. These disparity rates of migration provide a separation when the mixture moves on the adsorptive materials. These differential rates of analytes provide different when the mixture of sample runs on the adsorptive material. Frequent actions of sorption or desorption occurring during sample movement on the stationary bed determine the rates.
•    Having more adsorption in the stationary phase, the molecule or component will slowly move through the column.
•    Having higher solubility in the mobile phase, then the molecule or component will move faster through the column.
So, the dissimilarity between the mentioned factors determines the differential rates, on which various components can travel through a column. The solubility and adsorption of a component will be modified by selecting the suitable mobile phase stationary phase. All chromatographic separation works under the identical basic principle; each component interacts with other chemical species in an attribute manner. Due to the difference in affinities to different analytes for the stationary and mobile phase used chromatography separates a sample.
The principle of implementing chromatography that is used as a method of quantitative analysis in addition to its separation is to have an acceptable separation within an appropriate time period. There are different types of chromatography method are developed, some of them include thin-layer chromatography (TLC), gas chromatography, paper chromatography, column chromatography, ion-exchange chromatography, high-performance thin-layer chromatography (HPTLC), high-performance liquid chromatography (HPLC), affinity chromatography, and gel permeation chromatography.

Thursday, April 11, 2019

Difference between IR and NMR Spectroscopy

The interaction of light with substance to identify the substance properties called spectroscopy. Chemical analysis can be done both qualitatively and quantitatively by many analytical methods, but a large area of analysis is done using spectroscopy, in which IR and NMR Spectroscopy are the most useful. Infrared spectroscopy (IR) is a potent tool to identify various functional groups, whereas to determine the structure of organic compound the Nuclear magnetic resonance (NMR) spectroscopy is one of the valuable techniques.
Both are the forms of absorption spectroscopy, however, they didn’t work the same. IR spectroscopy means infrared spectroscopy and NMR spectroscopy means nuclear magnetic resonance spectroscopy. The basic difference between IR and NMR spectroscopy is that the Infrared spectroscopy determines the frequency of vibrations between atoms in a molecule, and it used to identify the functional groups present in the matter. NMR calculates the nuclear magnetic resonance, where the frequency of resonance is influenced by electrons protecting the nucleus and it used to determine the structure of organic compounds.


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Difference between Spectroscopy and Chromatography

The key difference between spectroscopy and chromatography is that spectroscopy is the analysis of interaction among analyte and electromagnetic radiation and chromatography is the isolation of mixture into individual compounds with the help of the mobile phase and stationary phase.
Spectroscopy is an interaction of electromagnetic radiation with the component may absorb or emission sample passed from one energy state to another. The photon interacts with the sample while an electromagnetic radiation beam goes through a sample, they can be reflected, absorbed, and refracted. The radiation can affect the chemical bonds and electrons in a sample. In several cases, the absorbed radiation leads to low energy photon emission. Spectroscopy sees how the phenomenon of radiation affects the sample solution. Absorbed and emitted spectra apply to obtain about the component information since the interaction relies on the wavelength, so there are numerous kinds of spectroscopy, here are some examples of spectroscopy mentioned.
Ultraviolet and Visible Spectroscopy, Infrared spectroscopy, Fourier Transform Spectroscopy, Mass Spectrometry, Raman Spectroscopy, Attenuated Total Reflectance Spectroscopy, Laser Spectroscopy, Atomic Absorption Spectroscopy, Electron Spectroscopy, Gamma-ray Spectroscopy, X-ray Spectroscopy.


Chromatography is one of the most significant methods in analytical chemistry to isolate the sample mixture between the stationary and mobile phases depending on their different distribution. The different molecules or components travel through the stationary phase with the mobile phase at different rates in the sample, they are separated into a series of bands. There are four most important types of chromatography are available i.e. Liquid Chromatography, Gas Chromatography, Thin-layer Chromatography, Paper, and Column Chromatography.


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Tuesday, April 2, 2019

Advantages of Nanotechnology

Nanotechnology contains structures and properties that manipulate on the nanoscale. Nanotechnology incorporates computing, medicine, science, engineering, and robotics on this scale, which is called nanoscale. Nanotechnology gives the ability of novel and faster types of computers, life-saving medical devices and treatments, and more capable power sources. To calculate the advantages and disadvantages of nanotechnology, let's first learn about the benefits that this technique brings. 
  • Manufacturing Advantages
Nanotechnology is already providing new substances that can revolutionize several areas of manufacturing such as nanoparticles and nanotubes. Nanotechnology can actually change many electronic applications, products, and processes. For example, nano transistor, OLED, a plasma display, nano diode, quantum computer and many more. 
  • Energy Advantages of Nanotechnology
Nanotechnology can transform the ways we get energy and use it. It is probable that nanotechnology can make solar energy economical by decreasing the rate of construction of solar panels and associated apparatus. Nanotechnology will also open new ways to produce and store energy. 
  • Medical Advantages of Nanotechnology
Nanotechnology is significant in drug development and discovery in the field of medicine. It has the probable to bring the most important progress in pharmaceuticals and its material such as nanotubes, aerogels and other packing material need to be lightweight, durable, and strong. 
  • Environmental Advantages of Nanotechnology
It can expect a significant contribution to the protection of the environment and climate of decreasing greenhouse gases and hazardous wastes, energy, and water using nanotechnological products, processes, and applications.