Sunday, February 23, 2020

Principle and Procedure of Adsorption Chromatography

What is Adsorption Chromatography?
It is one of the oldest chromatography types and is most widely used in the organic laboratories in the form of column chromatography and thin-layer chromatography. TLC is used to analyze the compounds and to check reactions progress and column chromatography performs separation and purification of compounds. In adsorption chromatography, the stationary phase is a fine solid partitioned to maximize surface area. The analytes are interacting differently with the adsorbent since each has a different affinity. Hence, they separate different retention times.

Adsorption Chromatography Principle
Adsorption chromatography works on the principle that certain solid material, called adsorbent can hold compound onto their surface. The adsorbent beds will distinguish between the adsorbate based on their binding strength as eluent is continuously run over the stationary phase in the column; variations in the flow rate of the compound ultimately lead to separation of the analytes. The most widely used adsorbents in adsorption chromatography include silica, alumina, and charcoal. Based on their relative polarities the elution order of components from the adsorptive stationary phases can often be estimated. On polar adsorbents, compounds with the more polar functional groups are strongly retained and are therefore eluted earliest.

Types of Adsorption Chromatography
There are three main types of adsorption chromatography, such as column chromatography, thin-layer chromatography (TLC) and gas-solid chromatography.
Column chromatography is a commonly used technique of separating the compounds from the sample mixture. It used on small or large scans to separate and purify compounds. The mixture of samples moves through the stationary phase with the mobile phase and based on various degrees of adhesion it separates the compounds.
Thin-layer chromatography (TLC) is an analytical method of separation used for qualitative analysis and monitoring of the reaction as well as identifying unknown compounds.
Gas-solid chromatography (GC) is an analytical method for the separation of volatile compounds in which the mobile phase is gas and the stationary phase is suitable for solid support. The gas-solid chromatography, work on the principle of separation is adsorption and generally used for samples that had less solubility in the stationary phase.

Adsorption Chromatography Procedure
Before beginning an adsorption chromatography experiment, we must recognize the various components essential to perform the process
Stationary phase: The stationary the phase of adsorption chromatography is adsorbent.
Mobile Phase: In adsorption chromatography, either a liquid or a gas is used as a mobile phase.

Apparatus for TLC method
TLC jar: It helps to keep the atmosphere proper during isolation.
TLC plate: It is used as the stationary phase coated with a thin layer of silica, alumina, on glass plastic or aluminum.
Iodine chamber or UV chamber: It helps visualize the spots on the thin layer chromatography plate.
Capillary tube: It is used to apply the sample mixture.

Thin Layer Chromatography Diagram:
TLC Diagram

Experimental Procedure of Adsorption Chromatography
The following steps are involved in the TLC process.
  1. Prepare the developing container: Pour the solvent to a depth just below 0.5 cm inside the chamber to further saturate with the mobile phase or solvent vapor.
  2. Prepare the TLC plate: TLC plates are usually commercially available; just you need to cut them to the required size.
  3. Spot the TLC plate: Dip the micro-capillary into the sample solution, and subsequently touch its end gently on the plate at the suitable place.
  4. Develop the plate: In the chamber, place the prepared plate, cover it with the lid and leave untouched. Via capillary action, the solvent will raise the TLC plate.
  5. Visualize the spots: Remove the plate from the chamber, and if the plate has some colored spots, mark them. Most of the analytes are not colored and should be visualized by placing them in an iodine chamber or UV cabinet. Finally, calculate the Rf values for all the separated compounds.

Adsorption Chromatography Applications
  • It is used for the separation of amino acids.
  • In this technique, the analytes are purified from a sample mixtures according to certain physicochemical properties.
  • It is used to determine the concentration of a molecule.
  • It is used for the separation and identification of isomers.
  • The adsorption technique is useful for the identification of carbohydrates.
  • It is used for the separation and identification of various mixtures of volatile compounds.

The Advantages of Adsorption Chromatography are as Follows.
  • Adsorption Chromatography is an important tool for separating many molecules that cannot be distinguished from other methods.
  • Adsorption chromatography uses a broad range of mobile phases.
  • Very few types of equipment are used as opposed to other separation methods.
  • The molecules in the complex mixture can be easily separated.

The Disadvantages of Adsorption Chromatography are as Follows.
  • Automation is making it more complicated and expensive.
  • The major disadvantage of adsorption chromatography is that some solutes have longer retention times.
  • It can cause catalytic variations in the sample.
  • Results obtained by some methods of adsorption chromatography is difficult to reproduce.

Commonly asked questions on Adsorption chromatography are as follows.

What is adsorption chromatography?
Adsorption chromatography is a separation method, where the stationary phase is an adsorbent.

What is the basic principle of adsorption chromatography?
In chemistry, adsorption chromatography is a method of chromatography based on the adsorption principle.

What type of chromatography is adsorption chromatography?
Adsorption chromatography is a form of liquid chromatography in which components are retained at the support surface based on their adsorption and desorption.

What is the major advantage of adsorption chromatography?
The major advantage of adsorption chromatography is that it separates the mixture into individual compounds.

What is the major difference between adsorption and partition chromatography?
The major difference between adsorption and partition chromatography is that the sample materials are physically interacting with the stationary and mobile phases used.

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Wednesday, February 19, 2020

Principle and Procedure of Affinity Chromatography

What is Affinity Chromatography?
Affinity chromatography is a form of liquid chromatography used to isolate and purify analytes in a specific way. It uses a reversible biological interaction called affinity that refers to the forced attraction used between the atoms in various degrees that cause them to remain in combination.
Affinity Chromatography Principle
Affinity chromatography is one of the most versatile and effective chromatographic methods for separating a complex mixture of a single or a group of components. It is based on very precise biological interactions amongst two components, such as enzyme-substrate interactions, antibodies, and antigens, or receptor and ligand. These typically reversible interactions are used for purification, in which molecules known as a molecule are placed on a solid matrix to form a stationary phase while components are in the mobile phase. Many of the widely used ligands are associated with matrices which are now commercially available and ready to use.
The stationary phase consists of a support medium to which the substratum is sympathetically bound, to reveal the reactive groups necessary to binding of the target molecule. As the crude sample mixture of molecules is traveled through the column, in the stationary phase, molecules with a binding site to the stationary phase bind while all other substances are eluted in the void volume of the chromatographic column. Once other molecules are eluted from the column, the bound target molecules can be overcome by including a competing ligand in the mobile phase, or by changing pH, ionic strength, or polarity state.
Affinity Chromatography Procedure
Before beginning an affinity chromatography experiment, we must recognize the various components essential to perform the process.
Matrix: It is passive support that can be directly or indirectly attached to a ligand.
Spacer arm: By eliminating any satirical hindrance effect it is used to enhance the binding between ligand and target molecule.
Ligand: It refers to a component that binds opposite to a particular target molecule.
Experimental Procedure of Affinity Chromatography
Column Preparation:
1.  Flush the column and stationary phase with the buffer solution to prepare the ligand for binding.
2.    Fill the column with solid supports such as agarose, safarose, and cellulose, etc. 
3.    Select the ligand according to the desired separation. 
4.    Attach the spacer arm between the ligand and the solid support.
Sample Loading: 
1.    Pour a sample mixture into a column and allow it to run at a controlled rate.
Elution of Ligand-Molecule:
2.  The desired component is recovered by adjusting favorable conditions for bound molecules to adhere.
Affinity Chromatography Applications
  • For all biological macromolecule separation and purification, affinity chromatography is used. 
  • It is used to purify antibodies, nucleic acids, and enzymes.
  • It is also used to decrease the amount of a substance in a complex mixture.
  • Affinity chromatography is used to notice what biological compounds bind to a given substance.
  • The affinity chromatography is used in clinical application.
  • Another application of affinity chromatography in analytical chemistry is a tool to study the kinetics of biological interaction.
The Advantages of Affinity Chromatography are as follows.
  • Target molecules can be obtained by affinity chromatography in a highly purified state. 
  • It can use to extract particular contaminants. 
  • Affinity chromatography has higher sensitivity than FID and FCD.
  • Affinity chromatography is used in vaccine production.
  • Affinity chromatography provides a high degree of purity.
  • It is used in the production of vaccines at pharmaceutical manufacturing to improve product quality.
  • It is doesn't depend on the temperature, buffer composition, pH, and ionic strength.
  • To improve the solubility of the affinity chromatography used.

The Disadvantages of Affinity Chromatography are as follows. 
  • It takes a lot of skill to do that.
  • This is a non-specific for adsorption compared to other chromatography techniques.
  • The ligands used in this chromatography are expensive.
  • Sometimes ligand leaks occur in affinity chromatography.
  • Metal-ion transfer and leakage lead to protein loss.
Commonly asked questions on affinity chromatography are as follows.

What is affinity chromatography?
Affinity chromatography is a technique of separating and analyzing biomolecules based on their structures or biological functions. It is a significant and useful separation method in biochemistry, biotechnology, environmental science, and pharmaceutical science.

What is the basic principle of affinity chromatography? 
The basic principle of affinity chromatography is that a bio-specific ligand is immobilized to a stationary phase or resin of the column matrix, such as agarose, or cellulose.

What type of chromatography is an affinity chromatography?
Affinity chromatography is a type of liquid chromatography used for the purification of specific biomolecules, including proteins.

What is the major advantage of affinity chromatography?
The major advantage of affinity chromatography is that it can achieve very high specificity and a high degree of purity.

What is the major difference between Affinity and Ion Exchange Chromatography?
The major difference between affinity and ion-exchange chromatography is that affinity chromatography is used to separate charged or uncharged molecules, while ion-exchange chromatography is used to separate charged molecules.

Monday, February 17, 2020

Principle and Procedure of Gas Chromatography

What is Gas Chromatography?
In analytical chemistry, gas chromatography (GC) is used to separate and determines the content of various analytes that can be vaporized without decomposition. The separation in gas chromatography is dependent on compound volatility. The more volatile compound is rapidly isolated, while the less volatile compound is slowly released. In this type of chromatography, the carrier gas is used as a mobile phase, usually, an inert gas such as nitrogen and helium is used, and as a stationary phase, a very thin layer of polymer or liquid on an inert solid support, inside a metal or glass tube is used, called as a column.

Gas Chromatography Principle
Gas chromatography follows on the principle of the partitioning of volatile compounds with the mobile phase (gaseous) and stationary phase (liquid or solid). The separation speed of molecules through the column are based on the affinity for the stationary phase; the molecules which are partitioned in the gas first come out, whereas the other eluted later.
The sample solution of complex mixtures injected into the instrument enters a stream of gas that travels through the column and in which the different components are separated. The detector (usually FID, GC-MS) determines the number of analytes exiting the column. For analysis of a sample with an unknown concentration, a standard sample with a known concentration is injected into the sample injector. To calculate the concentration, the peak area and retention time of the standard sample compare with the test sample.
In this type of chromatography, gas is always used as a mobile phase and the stationary phase is either solid or liquid. When the stationary phase is solid, it is called gas-solid chromatography and if it is liquid, it is called gas-liquid chromatography. GC is similar to HPLC and TLC in principle but has some differences.

Gas Chromatography Diagram

Experimental Procedure of Gas Chromatography:

Before beginning a Gas Chromatography experiment, we must recognize the various components essential to perform the process.
It is composed of four major components.
1. Carrier Gas: Since, carrier gas (hydrogen, helium) is used as a mobile phase in GC it plays an important role in isolation.
2. Injector: The port is intended to inject samples into the GC by manual or autosampler.
3. Oven: The temperature of the column in GC is controlled by an oven to manage the separation and retention time of the analytes.
4. GC column: A column, in which the molecules, depending on its affinity with the mobile phase (gas) and stationary phase is separated into individual analytes.
5. Detector: The detector used to determine the composition and concentration of a sample.

Procedure of Gas Chromatography:
  • Maintain inlet and outlet pressure of gases with the help of a regulator mounted on the control panel.
  • Install the required column (Packed/Capillary) and it should not leak.
  • Create and download the method and sequence from software and before injection ignites the flame. The parameters such as injector temperature, detector temperature, oven temperature, flow/pressure of the gas, and sequence of the sample/vial, Etc.
  • Prepare samples as needed.
  • Download the method of the software and ignite the flame before injection.
  • Saturate GC column with mobile phase until baseline.
  • Inject the sample manually by syringe or Autosampler by filling the vial at least halfway.
  • Depending on their affinity to the stationary phase, the components in the sample mixture are isolated from the column.
  • At different times the separated analytes reach the detector and are recorded by the computerized system.
  • From the chromatogram, the retention time (RT), peak area, column efficiency, tailing factor, peak height, and the number of theoretical plates can be calculated.
Gas Chromatography Applications
  • Gas chromatography is commonly used as a routine analytical tool in the quantitative analysis of drugs as well as pharmaceuticals.
  • It is used for both medicinal and forensic applications for the quantification of drugs and their metabolites in blood and urine.
  • GC is used in pesticide and volatile analysis.
  • Gas chromatography techniques are used in many fields of forensic science.
  • GC used in the analysis of flavor, fragrance and food products.
  • GC is used to analyze the organic compounds in environmental samples.
  • Gas chromatography is used to determine organic volatile impurities.
The Advantages of Gas Chromatography are as follows.
  • GC gives high sensitivity when it used with a thermal detector.
  • GC has an additional column length compared to high-performance liquid chromatography, so it is possible to separate complex mixtures of analytes with high resolution.
  • The analysis of the samples is faster than HPLC.
  • Gas chromatography has a high-resolution power than other methods such as HPLC, TLC, and column chromatography, etc.
  • A small amount of sample is required for separation.
  • This method provides comparatively high accuracy and precision.
The Disadvantages of Gas Chromatography are as follows.
  • A major disadvantage of GC is that the only volatile samples can be analyzed. 
  • Except for mass spectroscopy, most GC detectors are destructive. 
  • It is unable to recover isolated samples.
  • In HPLC and TLC selectivity is best compared to GC, since a mobile process can be easily replaced changed.
Commonly asked questions on gas chromatography are as follows.

What is the Basic Principle of GC?
GC separation is based on different distributions of the component molecules which are separated between the stationary phase (column) and the mobile phase (gas).

What are the Types of Gas Chromatography?
Based on the stationary phase used, there are two types of gas chromatography, such as gas-liquid chromatography (GLC) and gas-solid chromatography (GSC).

What is the Major Difference between GC and HPLC?
The main difference between GC and HPLC is the mobile phase used. In GC the sample mixture compounds are separated using gas as a mobile phase and in HPLC the liquid is used as a mobile phase.

What is the Major Advantage of Gas Chromatography?
The major advantage of GC is a rapid analysis with high efficiency.

How many types of GC detectors are there?
Flame ionization (FID), Electron capture (ECD), Thermal conductivity (TCD) Flame photometric (FPD), Nitrogen phosphorous (NPD) and Mass spectrometer (MS) are detectors that are used in gas chromatography.

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