Uses of chromatography

Different types of chromatography technique available according the requirement and the principle chromatography technique used.

Chromatography have vast area of the use in different industry.

Used in pharmaceutical industry:

Detection of impurity and unknown compound into the mixture during the process development and analytical method development  

Identify compound from the mixture or the dosage form (Tablets, Capsule)

To perform assay ad purity test of the product

To perform the organic volatile impurities into the mixture. Gas chromatography is used for the residual solvent test

For extraction purpose

Separation of the chiral compound from the mixture is the most widely used of the chromatography technique.

To purify the water for manufacturing process Chromatography is used. Cation anion exchange chromatography is used for Water purification system.

Check the quality of the drinking water air

 

Used in phytochemistry:

Phytochemistry is the origin of chromatography. chromatography was first developed by the Russian botanist Mikhail in 1903. In a column of calcium carbonate, a colorful separation of plant pigments was produced by him. Chromatography has since developed into an invaluable laboratory tool for the separation and identification of compounds.

Chromatography is used to obtain the pure compound from the plant. Column chromatography is used to isolation of the compound. TLC technique is used to identify the compound.

 

Used in food industry:

Now a day chromatography is one of the preferable techniques for testing of food in industry.

Chromatography technique also used in food industry in various way

To identify the additive into the food different types of the chromatography technique is used

One of the most common use of the chromatography is to find out the food spoilage

Detection of the nutrition of the food, Many types of amino acid, flavonoids, alkaloids, protein, organic acid, Vitamins, mineral etc.

Also used in liquor and alcohol testing industry to check the quality of the product

 

Used in polymer Industry:

Polymer Synthesis

To separate out the polymer based on the size

 

Forensic testing

HPLC in figure print and bioinformatics.

Gas chromatography is used to determination the alcohol and drugs level in someone body

 

In Biotechnology:

Separation and the purification of biological compound Separation of different compound of the blood.

Purification of the insulin is the example of using the chromatography

Plasma purification

Enzyme separation

Molecular biology research and the DNA

 

Environmental Industry

Effluent monitoring chromatography technique is used

In detection of small amount of the contaminants and the oil droplets pollutant different chromatography technique is used.

Most widely used in environmental research laboratory for the detection of the pollutant

 

In Vaccination:

Ebolla immunization.

 

There are different types of chromatography. According to the types of the chromatography there are different use of the chromatography.

Example:

Paper Chromatography

Paper chromatography is used for the identify the basic compound from the mixture the first use of the chromatography technique is separation of pigmentation from the plant extraction.

HPLC (High performance chromatography)

Ion exchange chromatography

Ion exchange chromatography are most widely used in purification of the water for the pharmaceutical industry. Ion exchange chromatography also used to monitor the fermentation process.

 

Size exclusion chromatography

As name suggested Size exclusion chromatography is used for the separation and purification of molecule depend on the size. To separate and purify the biological compound size exclusion chromatography is used.

Size exclusion chromatography is also used in separation of the molecular of the different size

 

Gas chromatography:

Gas chromatography is generally used for detection of volatile compound or the compound which can convert into their volatile form.

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Mobile Phase in Chromatography 

Thin Layer Experiment Procedure  

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Paper Chromatography  
Elution in Chromatography
Isocratic and Gradient Elution in Chromatography  

 

 

Gel Permeation Chromatography (Type of Size exclusion Chromatography)

Gel permeation chromatography is one of the high performance liquid chromatography. It is the one type of the Size exclusion chromatography.

There is two type of Size exclusion chromatography

Gel permeation chromatography

Gel filtration chromatography

The main difference between the Gel permeation chromatography and the gel filtration chromatography is the Mobile Phase

Gel permeation Chromatography:

Where the solid stationary phase and mobile phase is non-polar.

Gel permeation Chromatography Principle

The Basic principle of the Size Exclusion Chromatography is Smaller the particle of the analyte is trapped into the Column bed. The column bed act as sieve which trapped the smaller molecule of the analyte easily, Hence Smaller the molecule of the analyte, it retains for more period time in SEC column temporarily

Large molecules are not trapped in the column bed. Large molecule passes around the bed and excluded from the column bed. Larger the molecule quickly pass through the column

Large molecules are eluted first from the column and then small molecules are eluted

In other terms, we can say that large molecule has less retention time while the small molecule has more retention time.

 

Parts of HPLC (High Performance Liquid Chromatography)

HPLC is a technique for separation, identification and quantification of components in a mixture. It is especially suitable for compounds which are not easily volatilized, thermally unstable and have high molecular weights.

The instrumentation of the HPLC system is quite complex there are so many critical parts in HPLC system. 

Following are the different parts of the HPLC system:

Solvent Reservoir

Tubing

Pumps

Injector system

Guard column

Analytical column

Detectors

Recorders & integrator

Waste collector

The liquid phase is pumped at a constant rate to the column packed with the stationary phase. Before entering the column the analysis sample is injected into the carrier stream. On reaching the column the sample components are selectively retained on the basis of physio-chemical interactions between the analyte molecules and the stationary phase. The mobile phase moving at a steady rate elutes the components based on the operating conditions. Detection techniques are employed for detection and quantification of the eluted components.

Details about the part of the HPLC system:

 

These are inert containers for mobile phase storage and transport. Generally transparent glass bottles are used so as to facilitate visual inspection of mobile phase level inside the container. Stainless steel particulate filters are provided inside for removal of particulate impurities in the mobile phase if any.

 Mobile phase serves to transport the sample to the system. Essential criteria of mobile phase are inertness to the sample components. Pure solvents or buffer combinations are commonly used. The mobile phase should be free of particulate impurities and degassed before use.

Following are the characteristic of the mobile phase:

-It should be inert to mobile phase.

-Made up of stainless steel or glass .

-Capacity:500ml or more than that.

-Degasing: to prevent formation of gas bubble in the detector .it is done by various methods: By distillation system, by vacuum pumping system, by heat solvent with stirring,

Tubing:

The nature of the tubing used to connect all parts of the system deserves some attention.

The inside diameter of the tubing prior to the injection device is not critical but the tubing should be inert have the ability to withstand pressure & be able to carry sufficient volume.

Pumps:

Variations in flow rates of the mobile phase effect elution time of sample components and result in errors. Pumps provide constant flow of mobile phase to the column under constant pressure

Ideal  requirement:

Produce very high pressure 5000-10000 psi.

Produce pulse free output.

Flow control and flow reproducible.

Flow rate of m.p. should be in the range of 0.1-10 ml/min.

All material of construction should be corrosion free like s.s., Teflon.

2 types of pumps available

    1. Constant flow rate.

     2. Constant pressure.

Injector System:

Injectors are used to provide constant volume injection of sample into the mobile phase stream. Inertness and reproducibility of injection are necessary to maintain high level of accuracy.

Ideal requirement:

Sample should be injected in a narrow plug.

Size of sample should be variable.

Should be reproducible.

System must

2 types of the injector system are there

Septum Injector and loop Injector 

Guard Column:

 Guard column has very small quantity of adsorbent and improves the life of the analytical Column. They do not have any contribution in the separation.

  It also acts as a pre filter to remove particulate matter, if any, and other material.

  Guard column has the same material as that of the analytical columns.

Analytical Columns:

A column is a stainless steel tube packed with stationary phase. It is a vital component and should be maintained properly as per supplier instructions for getting reproducibility separation efficiency run after run.

Analytical column is that important part of HPLC technique which decides the efficiency of separation.

 There are several stationary phases available depending upon the technique or mode of separation used.

Column material:

The columns are made up of either Stainless steel, glass, polyethylene and PEEK( poly ether ,ether ketone).

  Column length:  Varies from 5cm to 30cm

  Column diameter: Ranges from 2mm to 50mm

  Particle size      :  From 1μ to 20μ 

  Particle nature:  Spherical, uniform sized, porous materials are used.

Column Oven

Variation of temperature during the analytical run can result in changes of retention time of the separated eluting components. A column oven maintains constant column temperature using air circulation. This ensures a constant flow rate of the mobile phase through the column.

 

Detector

A detector gives specific response for the components separated by the column and also provides the required sensitivity. It has to be independent of any changes in mobile phase composition. Majority of the applications require UV-VIS detection though detectors based on other detection technique are also popular these days.

Ideal requirement:

High sensitivity.

Reproducible response &give response to all analyte.

Response should be linear over wide range of concentration.

Should not be sensitize to flow rate fluctuation or temp. Change or change in composition.

Capable of withstand at high pressure.

Data Acquisition & Control

Modern HPLC systems are computer based and software controls operational parameters such as mobile phase composition, temperature, flow rate, injection volume and sequence and also acquisition and treatment of output.

These are the main parts of a basic HPLC system more specialized equipment might also have solvent selection valves, vacuum degasser, auto samplers, column switchers, pre or post column derivatization and fraction collectors.

 

Related Post: 
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Stationary Phase in Chromatography  

Mobile Phase in Chromatography 

Thin Layer Experiment Procedure  

Thin Layer Chromatography: Basic, Application, and principle of  Thin Layer Chromatography 

Paper Chromatography  
Elution in Chromatography
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Difference between Ascending and Descending Chromatography

Descending chromatography and the ascending chromatography are the types of the planar chromatography. It depends on the direction of the mobile phase.

The main difference between the descending chromatography and ascending chromatography is the following:

When the mobile phase moves in the upward direction it is known as the Ascending chromatography. While descending chromatography move in downward the direction it is known as the Descending chromatography.

Ascending chromatography	Descending chromatography
The mobile phase moves in an upward direction.	The mobile phase moves to a downward direction.
The mobile phase move up by the capillary action	The mobile phase  move down by the gravitational force.
The solvent reservoir is at the top of the setup.	The solvent reservoir is the bottom of the setup.
Ascending chromatography has is a simple setup.	Descending chromatography has a complex setup.

 

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Chromatography Basic

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Size Exclusion Chromatography: Principle, Definition and types of chromatography

Size Exclusion Chromatography (SEC): Definition

Size Exclusion chromatography is one of HPLC technique which separates the component base on their size and in some case by the molecular weight and the shape of the molecules.

Size Exclusion Chromatography is generally used for the Separation of a large molecule or the macromolecule.

It also is known as the gel permeation chromatography and gel filtration chromatography depends on the Mobile phase used in Size exclusion Chromatography.

Gel filtration Chromatography:

When size-exclusion Chromatography is performed using the aqueous mobile phase it is known as the Gel Filtration Chromatography.

Gel Permeation Chromatography:

When size exclusion Chromatography is performed using the organic mobile phase it is known as the Gel Filtration Chromatography.

 

Size Exclusion Chromatography Principle

The Basic principle of the Size Exclusion Chromatography is Smaller the particle of the analyte is trapped into the Column bed. The column bed act as sieve which trapped the smaller molecule of the analyte easily, Hence Smaller the molecule of the analyte, it retains for more period time in SEC column temporarily

Large molecules are not trapped in the column bed. Large molecule passes around the bed and excluded from the column bed. Larger the molecule quickly pass through the column

Large molecules are eluted first from the column and then small molecules are eluted

In other terms, we can say that large molecule has less retention time while the small molecule has more retention time.

 

Component of Size Exclusion Chromatography:

Stationary Phase:

Chromatography column contain Stationary phase in the form of sphere or bead, It is also known as column bed. The beads of Stationary phase are semipermeable, porous cross-link polymer.

Smaller pore size for rapid desalting of protein or for protein purification. Intermediate pore size is used to separation of a relatively small protein. Large size is used for separation of biological molecular

E,g. Dextran (Sephadex),, Poly Acrylamide ( Sephacryl or BioGel P)., Agarose (Sepharose),

The stationary phase is chemically inert and stable; it does not react with the mobile phase or analyte to separate the component. ‘

 

Mobile Phase:

Aqueous and non-aqueous both are used as mobile phase.

The choice of the mobile phase is to depend on the type of separation to be achieved and material needs to be separated.

How it Works:

Polymer solution (Analyte) is dissolved on the mobile phase pass through the column. That solution passes through the column, The sample molecule passes through the column which contains the stationary phase in the form of a bead. Which also known as the column bed.

The sample molecule enters into the column, it moves along the column. The larger molecule excluded from the pores and travels through the column and eluted first while the small molecule enters into the pore and temporarily traps with the stationary phase (column Bed). Thus a molecule separated from each other. measured by one or more detectors when they elute from the column.

Buffer is used in Size exclusion chromatography. Separation should be perform within the broad PH. The buffer should not be cause the inactivation of or precipitation. It should maintain the stability of the analyte.

 

Advantages of Size Exclusion Chromatography

The sample is not decomposed in Size exclusion chromatography

Low quantity of the sample required for size exclusion Chromatography

 

Disadvantages

Sensitive equipment

Expensive

 Related Post: 

Chromatography: History, Definition and How it Works 

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Mobile Phase in Chromatography 

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Thin Layer Chromatography: Basic, Application, and principle of  Thin Layer Chromatography 

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Different between HPLC and GC

Different between HPLC and GC

HPLC: high performance Liquid chromatography

HPLC is one of the chromatography techniques where the stationary phase is liquid or solid and the mobile phase is liquid.

GC: Gas chromatography

GC is also one of the chromatography technique where the stationary phase is solid or liquid while the mobile phase is gas.

The main difference between the HPLC and GC is the mobile phase.

Example of mobile phase:

In HPLC chromatography liquid solvents are used as the mobile phase they may be the polar or the non polar solvent.

Example of polar solvent: Acetonitrile, Water , Dimethyl Sulfoxide 

Example of non polar solvent: Hexane , Chloroform 

In gas chromatography the mobile phase used is the inert gases following are the example of the mobile phase Helium, Nitrogen, Hydrogen, argon, and Air.

Compound to be analyzed

HPLC mainly used to analyze the less volatile compound. The sample should be in liquid form hence it should be soluble in the solvent. Gas chromatography only volatile compound is the analyze by the Gas chromatography. The chromatography sample shall be stable at high temperatures.

The temperature during the analysis

HPLC analysis is done at room temperature only. In GC analysis compound must be in vapor form to analyze. Gas chromatography requires a high temperature. For high temperature oven facility is available.

Column:

In HPLC column the liquid mobile phase is used and in gas chromatography gas is the mobile phase the liquid mobile phase creates more pressure than the gaseous mobile phase. Hence the column length of the gas chromatography is more than the HPLC column.

Generally, the length of the HPLC column is 5 to 50 cm long. The column of the HPLC is shorter wide and thick. Packed columns are used in HPLC.

 The gas chromatography column is long and narrow. Generally, the length of the column is 10 to 30 meters long. Two types of the column are used packed column and the capillary column.

Cost:

HPLC is costly than the Gas chromatography. The solvent of the high performance chromatography is costly than the gas. The second reason is HPLC requires the pressurize system to pump the liquid mobile phase. It is quite complex and the High performance liquid chromatography system requires more maintenance than the gas chromatography.

Method of detection of Sample:

In the detection of the HPLC UVs, RI, photodiode methods are used. While in gas chromatography FID (flame ionization detector), TCD (thermal conducting detector) different methods are. Generally, the method used in the detection of HPLC is nondestructive while the method of GC is destructive.

Now day’s mass the spectrometer is attached with the both high performance liquid (HPLC) and gas chromatography (GC) to achieve a better result.

Related Post: 
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Stationary Phase in Chromatography  

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Thin Layer Experiment Procedure  

Thin Layer Chromatography: Basic, Application, and principle of  Thin Layer Chromatography 

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Application of Affinity Chromatography

Affinity chromatography most widely used for the separation of the specified biomolecule from the mixture. There is a number of applications of affinity chromatography.

Following are the different application of the affinity chromatography:

·        Antibodies:- antigen is immobilized and used to isolate and purify the appropriate antibody. The antibodies in a serum sample specific for a particular antigenic determinant can be isolated by the use of affinity chromatography.

·        Isolation of insulin receptor of liver cell.

·        Enzymes:- Substrate inhibitors can be identified.

·        Purify and concentrate a molecule from a mixture into a buffering solution

·        Reduce the amount of a molecule in a mixture

·        Discern what biological compounds bind to a particular molecule, such as drugs

In Clinical analysis & biotechnology

·        Microscale Molecular Diagnosis.

·        HPAC Used to determine the presence of specific components of biomolecular families in mixtures, including biological fluids & cell & tissue extract.

·        Compared with standard techniques for clinical determination & quantitation of biomolecules, analytical HPAC has a specificity  & the speed of operation that allow rapid detection  & simultaneous binding activity characterization of a whole set of molecules that are related functionally

·        Use of boronate, lectin, protein A or protein G & immunoaffinity support for the direct quantification of solutes.

·        Affinity based chiral separation & used AC for the study of a drug or hormone interaction will binding proteins

·        In therapeutic extracorporeal plasmapheresis & large scale fractionation of human plasma with special emphasis on an industrial rather than analytical scale.

·        In the study of glycobiology, application of glycol affinity chromatography

·        The interaction between recommendation ricin A chain & several triazine dyes were investigated to improve the purification of r-ricin A from E.coli fermentation.

·        Interaction of R- &-S ibuprofen with human serum albumin is studied.

Applications in pharmaceutical analysis

·        Isolation & detection of hygromycin B in a biological fluid

·        Column consists of lysozymes & α- lactalbumin immobilized on an N-hydroxy succinimide, agarose support & evaluated for the purificator on hygromycin In biological fluid

·        Disopyramide by capillary affinity electrokinetic chromatography with human α1acid glycoprotein(AGP) as chiral selector is studied

·        The interaction between recommendation ricin A chain & several triazine dyes were investigated to improve the purification of r-ricin A from E.coli fermentation.

·        The work is also carried out to compare & evaluate specificity & binding capacity of commercial & in house affinity columns  used in the sample preparation for analysis of growth-promoting drugs.