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Monday, August 31, 2020

Selection of the column for RP-HPLC

Column selection is the first and most important step for the development of an analytical method, whether it is gas chromatography or high-performance liquid chromatography. It is not possible to develop a rugged, robust, and reproducible method without high-performance and stable HPLC columns. Significantly, columns are reproducible and stable to avoid errors with irrelevant sample retention time during the development of the HPLC method.
The RP-HPLC column is selected based on the nature of the molecule and the information about the solute. Reversed-phase chromatography is the types of HPLC which have broad columns range such as dimethyl silane (C2), butylsilane (C4), octylsalane (C8), octadecylsalen (C18), cyanopropyl, BDS phenyl, and nitro, etc. A C18 or C8 column is specially made with less acidic, high-quality pure silica and is particularly suitable for the isolation of basic analytes. They are normally appropriate for all the samples and are strongly recommended by the chromatographers.
The following few points give guidance in the selection of column for RP-HPLC:
  • Almost all HPLC columns are made of steel. The major advantage of steel is that it is relatively inert to corrosion and pressure resistance.
  • It is significant for a better separation of solutes that there is no scoring, roughness, inside a chromatographic column.
  • Silica gel is the most popular stationary phase in high-performance liquid chromatography, which consists of Si atoms and oxygen atoms and it carries OH groups (silanol groups). It is available in different particle sizes and is used within a pH range of 1.00 to 8.00.
  • Polymer-based columns are also used in reversed-phase HPLC, providing higher pH stability than silica gel columns. It can be used for the isolation of strongly acidic or basic components.
  • Column packing materials of various types and sizes are available according to the nature of the compound and HPLC method.
1. Fully porous particles are the most used shapes in the HPLC column, which are available in 3, 5,     or 10 μm.
2. Coreshell particles have a solid core, and the outer shell is porous, resulting in improved efficiency in the separation. One major disadvantage of coreshell particles is that it has low load-ability compared to porous materials.
3. The monolithic phase column consists of a piece of porous material, like a silica gel. A disadvantage of monolithic phases is that the availability of selectivity is limited in contrast to fully porous materials.
  • The longer columns generally give a better separation because of the higher theoretical plate numbers. If the particle size decreases, then surface area available for coating increases. 5-μm particle size HPLC columns offer the best solution in terms of reproducibility efficiency, and reliability.
The C18 is the most common reversed-phase column may be used in several analyses. To decide which stationary phase or column is suitable, the C18 column is considered the first choice at the beginning of method development. Since it has longer carbon chains (typically 18 carbons) than other column and provides a wide surface area which offers an additional time of reaction between the bonded phases. The very-polar molecule does not interact with the C18 material and is eluted at the dead time, in such cases you can use C4, CN, diol, or phenyl phases. Conversely, if the molecules are very un-polar you can use silica, CN, or NH2 phase.
While doing the HPLC method development, there are some aspects to be considered: separation scheme, mobile phase, type of HPLC column, the diameter of the column (such as particle size, pore size, internal diameter, diameter, and carbon content), analytes of interest, pKa value, pH of the solvent system, detector, and injector, etc. Method development has different conditions in each case; each chromatographer should consider these factors when performing the HPLC analysis.

Commonly asked questions on the HPLC column are as follows.

How many types of HPLC columns are there?
There are four main types of HPLC columns-reversed phase HPLC column, normal phase HPLC column, the size-exclusion HPLC column, and the ion-exchange HPLC column.

Is c18 column polar or non-polar?
The C18 column is used in the reversed-phase chromatography; it’s packed with non-polar material as compared to the polar stationary phase. 

What is the difference between HPLC and RP-HPLC?
HPLC is the type of chromatography in which RP-HPLC is the most commonly used modes of HPLC. HPLC stands for high-performance liquid chromatography and RP-HPLC stand for reversed-phase high-performance liquid chromatography.

What is the difference between c8 and c18 HPLC columns?
Both C8 and C18 are the types of columns used in HPLC, the major difference between C8 and C18 is that C18 has 18 carbon atoms while C8 has only 8 carbon atoms.


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Saturday, August 29, 2020

Advantages and disadvantages of buffer solution

Definition of the buffer: Buffer is a compound or mixtures of compounds that resist changes in pH by the addition of small amounts of acid or alkali by their presence in the solution.
A buffer solution is an aqueous solution that contains a weak base and its conjugate acid or a weak acid mixture and its conjugate base. It avoids the changes in pH or slight changes as applying small amounts of strong acid or base to it. Buffer solutions are widely used in a range of chemical applications as a way of maintaining pH at a nearly constant value. Several systems use buffering for pH regulation.
Advantages of buffer solution:
  • The major advantage of a buffer is that it is keeps the pH stable and increase the solubility of the substance or formulation.
  • Buffer solution plays a significant role in chromatography, as retention of ionizable molecules is extremely sensitive to the pH of the mobile phase in such cases the pH can be regulated by adding a buffer solution.
  • The buffer solution can remain stable for several weeks if it is stored in the fridge.
  • Different types of buffers such as acetate, ammonium, sodium, and phosphate, etc. are highly soluble in water.
  • The pKa of a buffer solution is the same as being body fluids; hence it is used in the dissolution testing to determine the extent and rate of solution formation from a dosage form.
  • It maintains the solubility of the therapeutic agent in the pharmaceutical product.
  • Buffer solution improves the stability of products where the active agent is pH-dependent.
  • It is non-toxic to cells and inhibits enzymatic reactions.
  • By using the buffer solution the onset of action of dosage form increases.
  • It is used to optimize biological activity.
  • Since the pH of the buffer solution is precise, standard buffers i.e. pH 07.00, 04.00, and 09.20 are used to calibrate the analytical instruments (pH meter).
Disadvantages of buffer solution:
  • The major disadvantage of the buffer solution is that it is an aqueous solution that can be contaminated with microorganisms and is not suitable for long-term storage, so you need to prepare a fresh buffer solution each time.
  • To adjust the pH of the solution, it is necessary to know the pKa value of the component. 
  • The drug dissolution may precipitate when the pH of the solution is not maintained properly.
  • Some types of the buffer are precipitated and tend to react.
  • Drug ingredients and some vitamins are dissolved only at a particular pH therefore, it is essential to maintain the correct pH of the solution.

Commonly asked questions about buffer solutions are as follows.

What are the types of buffer solutions?
There are two types of buffer solutions: acidic buffer solutions and alkaline buffers.

What is the range of pH scale?
The pH scale determines how acidic or basic a substance is in the range of 0.00 to 14.00 pH. A pH of 7.00 is neutral; a pH less than 7.00 is acidic and a pH greater than 7.00 is basic.

What are the applications of buffer solution in the pharmacy?
The buffer solutions are used in pharmaceutical industries for improving purity, increased stability, enhancing solubility, and optimizing the biological activity of the compounds or products.
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Wednesday, August 26, 2020

The separation mechanism of reversed-phase chromatography

Chromatography is a primary separation technique which is used in chemical analysis. It isolates the sample mixture by continuous partitioning between the mobile and stationary phases. The principle of chromatography involves the isolation obtained by dissolving compounds in a mobile phase and traveling over a stationary phase. Each compound of sample mixture has different retention time, as the compound with strongly interacting with the stationary phase appear to travel very slowly, while compounds that have less affinity tend to elute rapidly.
The high-performance liquid chromatography is one of the most powerful separation techniques since it can apply different types of separation techniques for different types of molecules. The reversed-phase chromatography is a type of HPLC that is currently the most broadly used method of isolation in HPLC since it is versatile, simple, separate the analytes of diverse polarity and molecular mass, and has more scope of the technique. 
The principle of reversed-phase chromatography depends on the interaction of the solutes with the hydrophobic group. It has analytical and preparative applications in the field of biochemical isolation and purification. It is typified by a mobile phase of polar properties and a stationary phase with non-polar properties. The reversed-phase chromatography does works in contrast to normal phase chromatography.
Separation mechanism of reversed-phase chromatography:
The separation mechanism in reversed-phase HPLC generally depends on the distribution of components between the mobile phase and the stationary phase. This process can also be interpreted as a type of component extraction between the stationary phase and the mobile phase. In the stationary phase, a non-polar substance dissolves preferentially, remains there preferentially, and is thus gradually eluted from the chromatographic column. Polar substances are easily eluted from the column, as they tend to remain in the solvent system. Subsequently, the retention time of the solutes varies as their distinct affinity for the stationary phase.
The stationary phase in reverse phase chromatography:
The stationary phase of reversed-phase HPLC is usually composed of hydrophobic alkyl chains that interact with the solutes. Octadecyl carbon chain (C18) bonded silica is the most popular column used in RP-HPLC as it has a longer carbon chain compared with the C4 and C8.
The mobile phase of reversed-phase chromatography:
The mobile phase used in reversed-phase HPLC has a certain amount of water or aqueous buffers and organic solvents are used to isolate as well as to obtain a symmetrical peak shape. The water-miscible solvents such as methanol, acetonitrile, and tetrahydrofuran are most commonly used organic solvents. The retention of ionizable compounds is very sensitive hence the pH of the mobile phase or buffer can play an important role.

Commonly asked questions on chromatography is as follows.

Why is reversed-phase chromatography useful?
The RP_HPLC is most widely used technique amongst the different types of HPLC chromatography since it has a hydrophobic stationary phase and can be used with the polar (hydrophilic), non-polar (hydrophobic), ionic and ionizable molecules to isolate their different molecules, which is depends on the procedure being used.

Which compound will elute first in a reversed-phase HPLC separation?
Polar compounds will elute first in a reversed-phase HPLC separation. The polar (aqueous) mobile phase is used in the reversed-phase HPLC As a consequence, in the polar solvent system hydrophobic solutes begin to adsorb into the hydrophobic stationary phase, and in the mobile phase, hydrophilic solutes pass through the column and are first eluted.

What is the difference between the normal phase and reversed-phase chromatography?
The major difference between reversed-phase chromatography and normal phase chromatography is that reverse phase chromatography has a non-polar stationary phase and a polar mobile phase while normal phase chromatography has a very polar stationary phase and a non-polar phase.

What is the major advantage of reversed-phase chromatography?
The major advantage of reversed-phase HPLC is that its hydrophobic stationary phase works well for the retention of most organic analytes. This allows the aqueous mobile phase to be used that includes water and the type of buffer solution such as phosphate, acetate, etc. RP-chromatography also has the advantage of being able to use pH according to the pKa of the molecule to improve the separation.

What are the major types of chromatography?
There are various types of chromatography has, such as high-performance liquid chromatography (HPLC), gas chromatography (GC), column chromatography, paper chromatography, thin-layer chromatography (TLC), and mass spectroscopy (LC-MS), etc.
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Tuesday, August 11, 2020

Reversed phase chromatography - an overview

learn about the principle of reversed-phase chromatography, and also learn stationary phase, mobile phase, and applications of reversed-phase liquid chromatography.

What is the reversed-phase HPLC?
Reversed-phase chromatography is a type of high-performance liquid chromatography, which is used to separate the solutes based on the hydrophobic interactions between the mobile phase and the stationary phase. RP-HPLC is the most widely used HPLC mode, and this mode is exactly the reverse of normal phase chromatography, in which the mobile phase is more polar than the stationary phase. In general, the stationary phase of reversed-phase chromatography is non-polar (silica-C18 column) and a moderately polar mobile aqueous phase.

The principle of chromatography:
· Chromatography is a separation technique in which molecules are contained within a mobile phase that travels through a stationary phase. 
· Normally, one phase is hydrophilic, and another is lipophilic. 
· The mixture of sample compounds interacts with these two phases differently. 
· As per the polarity of the molecules, they interact more or less time with the stationary phase and thus they eluted at different rates.

The principle of reversed-phase HPLC: 
  • The principle of reversed-phase chromatography is based on the interaction of molecules with the hydrophobic group.
  • Reversed-phase liquid chromatography is an adsorption process that depends on a partitioning mechanism to effect isolation. The stationary phase In RP-HPLC is solid support applied with hydrophobic as well as hydrophilic groups.
  • This distribution is based on the medium's binding properties, hydrophobicity of the analytes, and composition of the mobile phase.
  • The polarity of a molecule that is similar to the column packing, their retention time is more as they are attracted to the particles more strongly. The polarity molecule that is similar to the mobile phase will preferably be attracted to it and move rapidly from the HPLC column.
The stationary phase of reversed-phase chromatography (RP-HPLC):
The column is the heart of HPLC chromatography, and the mobile phase can be used to control separation retention and selectivity. The reversed phase HPLC column of is filled with silica particles. The particles are usually characterized by particle size and pore size. Particle sizes vary typically from 3 to 50 microns, the 5 um is the most commonly used particle size. The stationary phase of RP-HPLC is typically composed of hydrophobic alkyl chains that interact with the components. C4, C8, and C18 are three chain lengths that are commonly used in the reversed-phase chromatography. 
Different types of columns are used in HPLC; however, C18 (Octyldecylsilane) is the most commonly used stationary phase of reversed-phase HPLC. It has 18 carbons that bonded to silica, which is more and a longer carbon chain than other columns used in RP-HPLC. As it has 18 carbon chains, it has a larger surface area which gives more time of interaction between the compounds and bonded phase. This is the major reason it covers a broad range of samples.
There are two types of reversed-phase chromatography column packing are used as follows.
  • Silica gel with chemically bonded alkyl-chains
  • Resin-based packing
The mobile phase of reversed-phase chromatography (RP-HPLC):
The role of the mobile phase in reversed-phase chromatography is to control the retention and selectivity of neutral and ionized compounds. In many RP-HPLC developed methods use a mixture of miscible organic solvent (methanol/acetonitrile) and water as the mobile phase. The reason for the organic solvents is to keep the polarity on adequate levels of the compound to dissolve in the solvent system and yet high enough to facilitate binding with the stationary phase of the preferred molecule. In some HPLC method developments, ion-pairing agents can be added to the mobile phase. When the compound to be separated is enters the column matrix it is allowed to dissociate from it by further decreasing the polarity by increasing the organic solvent concentration in the solvent system. This process of changing the concentration of organic solvent in the mobile phase to isolate a compound is called a gradient elution.
In some pharmaceutical analyses or research, several drugs are ionizable, i.e. acidic or basic. Therefore, the pH of the mobile phase must be controlled, as it can have a huge impact on the retention of components. Depending on the mobile phase pH, ionizable compounds that exist in ionized or non-ionized forms, and the ionized have significantly less retention than the non-ionized in reversed-phase liquid chromatography.
A buffer is a solution of a weak acid and its conjugate base, or a weak base and its conjugate acid. Significantly, the buffer has a pKa close to the preferred pH because the buffers best control the pH in their pKa. Different types of buffers such as phosphate buffer, acetate buffer, formate buffer, phosphoric acid, and its sodium or potassium salts are the commonly buffers for reversed-phase HPLC. The different solvent such as acetonitrile and methanol is the most commonly used organic solvents in RP-HPLC.

Applications of reversed-phase chromatography (RP-HPLC):
The reversed-phase chromatography is a significant analytical method used to separate the analytes, which is widely used in the analysis of pharmaceuticals, life-science research, forensics, chemical industries, biotechnology, food industries, and clinical diagnostics.

Advantages of reversed-phase HPLC:
  • It is the most commonly used technique of all the HPLC method as it isolates a wide variety of samples.
  • In RP-HPLC the water can use in the mobile phase with other solvents.
  • This separation method can be stable at all acid and basic pH values.
  • It is an inexpensive chromatographic technique.
  • It gives precise results even with the minimum amount of samples.
The commonly asked questions on HPLC are as follows.

How many types of injection are there in HPLC?
The three types of injectors are used in HPLC such as Rheodyne injector, septum injector, and stop flow injector.

What are the different types of detectors used in HPLC?
UV/VIS detector, photo-diode array (PDA), mass spectrometer, fluorescence, refractive index, electrochemical, electrical conductivity, and light scattering are the types of detector used in HPLC.

What are the types of HPLC chromatography?
There are four types of HPLC such as reversed-phase HPLC, normal phase HPLC, size-exclusion HPLC, and ion-exchange HPLC

How many types of pumps are there in HPLC?
There are three types of pumps reciprocating pump, syringe pump, and pneumatic pump that provides the required pressure and flow rates.

What is isocratic and gradient elution? 
There are two types of elution in HPLC i.e. isocratic and gradient. In isocratic elution, a constant composition of the mobile phase is pumped through the column during the whole analysis and in the gradient elution the composition of the mobile phase is steadily changed during the run.


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Monday, August 10, 2020

5 major applications of chromatography techniques

Chromatography is a widely used separation technique that has a number of important applications in the several sectors and industries such as pharmaceutical analysis, food industry, chemical industry, forensic science, and environmental analysis etc.

Chromatography is essentially a method for the isolation of a sample mixture by continuous partition between two phases such as the mobile phase and stationary phase. The basic principle of chromatography consists of the separation of solutes obtained by dissolving the mixture of solutes in a mobile phase and traveling it over the stationary phase. Analytes that more strongly interactions they move slowly, while those analytes have a weak affinity they eluted rapidly.
 
Today different types of chromatography such as paper chromatography, column chromatography, high-performance liquid chromatography (HPLC), ion-exchange chromatography, thin-layer chromatography (TLC), gas chromatography (GC), liquid chromatography-mass spectroscopy (LC-MS) are being used for different purposes in across the world, let’s check some important uses of chromatography.

Applications of chromatography techniques

1. Applications of chromatography in the pharmaceutical analysis:

  • Different types of chromatography techniques are widely used in the pharma sector for qualitative and quantitative analysis of molecules.
  • As chromatography is easy, precise, rapid, and accurate. It can be adopted successfully and efficiently in bulk and pharmaceutical dosage form for routine quality control analysis of drugs.
  • Chromatography plays a significant role in the development of a new molecule, as it determines the structure, and monitors the synthesis or reaction process.
  • It is used to find out the amount of the drug from the pharmaceutical dosage forms.
  • Chromatography is used by pharmaceutical industries to prepare a huge amount of pure materials such as API and to also be analyzed purified compounds for trace contaminants.
  • It is used to separate the chiral compounds in pharmaceutical analysis.
  • Chromatography is a robust and efficient quantitative tool for the diagnosis and evaluation of diseases in drug and biomarker research.

2. Applications of chromatography in the food industry:

  • Chromatography is used in the processing of the food industry for quality control, by isolating proteins, preservatives, vitamins, and amino acids.
  • The presence of chemical additives, the nutritional value of processed foods such as junk food, fast food can be determined with the help of a variety of chromatographic techniques.
  • Many beverage industries use chromatography to confirm that every batch of their product is the same.
  • The chromatography is broadly used by food manufacturers in determining the contents of processed food and other foodstuffs.
  • It is used to determine food shelf-life, it is analyzed by which food spoils.

3. Applications of chromatography in the chemical industry:

  • The major application of chromatography in the chemical industry is that it is used to monitor the reaction and synthesis of a chemical and its products.
  • It is used in pesticide and oil industries to detect different contaminants present in the products.
  • It is used in the chemical industry for determining the pollutants in water, air, and chemicals.
  • The chemical industries are used chromatography to monitor the quality and purity of the samples.

4. Applications of chromatography in forensic science:

  • The chromatography is used in forensic science to collect the evidence and catch criminals.
  • Chromatography is used in blood tests that can determine the amount of alcohol, drugs, or toxic substances in the body after death.
  • Chromatography is also to determine if their poison in the body.
  • Gas chromatography is used to test samples of blood, urine, and clothes, to recognize criminals and to help them bring justice.
  • Since chromatography can reliably determine substances in the bloodstream, it is used for screening of athletes for doping or performance-enhancing drugs in sports.

5. Application of chromatography in the environmental analysis:

  • Chromatography is a widely used separation technique to determine the organic trace components in environmental samples.
  • Chromatography used for determining and identification environmentally ubiquitous pollutants.
  • Liquid chromatography with mass spectrometry (LC-MS) can be used to examine many different pesticide residues.
  • It is also used to determine the volatile and non-polar organic compounds (odors and non-odorous) in the air.
  • It is used to identify carcinogenic contaminants found in drinking or wastewater.
  • Some other applications of chromatography includes, applications in the life sciences, molecular biology, nanotechnology, petroleum industries, and diagnosis of disease, etc.

Commonly asked questions of chromatography are as follows.

What is the basic principle of chromatography?
Chromatography works on the principle that different molecules will have different solubility and adsorption amongst the two phases which they are to be isolated.

What are the four major types of chromatography?
The chromatography classifies based on four different separation mechanisms, such as adsorption chromatography partition chromatography, size exclusion chromatography, and ion-exchange chromatography.

What are the advantages of chromatography?
The advantages of chromatography include, it provides simple and rapid analysis with high resolution, it requires a very small amount of sample (Gram, PPM, and ng/ml), and it has a broad range of mobile phase and stationary phase to separate the components.

What are the factors affecting chromatography?
The mobile phase composition, pH of the mobile phase, stationary phase, system pressure, temperature, flow rate, the concentration of analytes, the particle size of adsorbent, and column dimensions are the factors that affect the separation, resolution, efficiency, Rf value, reproducibility, and retention time of chromatography.

What are the principle and types of column chromatography?
Column chromatography works on the principle of adsorption and it has five different types such as adsorption chromatography, ion-exchange chromatography, partition chromatography, and gel chromatography.

What are the applications of reversed-phase chromatography?
The reversed-phase chromatography has both analytical and preparative applications and commonly used in pharmaceuticals, food technology, medicine forensics, and biotechnology.
 
 


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Thursday, August 6, 2020

Chromatography principle, types, and applications

Chromatography is a physical separation process in which the different compounds are distributed between two phases. Learn the principle, types, and applications of chromatography.

What is chromatography?

Chromatography is one of the powerful and versatile methods that is used in all branches of science particularly in analytical chemistry. It is a separation technique that is obtained by distributing the analytes of a sample mixture between two phases, a stationary and a mobile phase. Each analyte has its properties, so they have a different affinity towards the stationary phase. 

The analytes of the sample mixture move at different speeds, causing them to separate from each other. It depends on the nature of the stationary phase and the mobile phase, which analytes travel more rapidly or more gradually. These different travel times of the separated analytes are called retention time.


The principle of chromatography (How does chromatography works?):

Chromatography is based on the principle of applying the mixture of the compounds to the surface or the stationary phase, they travel with the aid of a mobile phase and get separated. The factors that are involved in this process of isolation include molecular characteristics of a partition, affinity, and adsorption. 

Due to these variations, certain compounds remain in the stationary phase longer, and travel gradually throughout the chromatography process, whereas others are quickly eluted through the mobile phase.

Chromatography uses differences in the polarity of the molecules of the sample mixture. In this separation process, a liquid or solvent acts as a mobile phase and it runs through a layer of particles which is known as the stationary phase. A sample consisting of a mixture of compounds is introduced or injected into the mobile phase and passes through the stationary phase. 

Based on the relative affinity for mobile and stationary phases the compounds are isolated. The compounds having more affinity they are traveling slowly as compared with the compounds with a lower affinity. The results of isolated molecules are then used as qualitative and quantitative analysis by comparing them with standards.

Different types of chromatography:

The chromatographic separation can be executed with several different setups. Based on the molecular interaction, there is ion-exchange chromatography for ionic molecules, partition chromatography based on solubility, affinity chromatography with specific interactions, size exclusion chromatography based on molecular size, and adsorption chromatography for non-ionic polar molecules.

1. Planar chromatography:

The planar chromatography includes paper chromatography and thin-layer chromatography, which relies on the adhesion and cohesion forces of a solution that passes through the media. In paper chromatography, the cellulose paper acts as a stationary phase and in TLC a sheet which is coated with a thin layer of an adsorbent such as silica or alumina is used as a stationary phase. 

Analytes are separated according to their attraction to the adsorbent and appear after chromatographic separation as individual spots on the surface. Finally, the retention factor (Rf value) is used to identify an unknown substance.

The principle of paper chromatography and thin-layer chromatography (TLC) depends on the relative affinity of solutes towards both the phases. Paper chromatography has five different types such as ascending paper chromatography, descending paper chromatography, ascending-descending paper chromatography, circular paper chromatography, and two-dimensional paper chromatography, etc.

2. Column chromatography:

Column chromatography is a widely used technique for separating and purifying of different chemical compounds. It consists of a glass tube placed vertically, in which the mixture of samples along the mobile phase passes through a solid stationary phase and compounds gets separates according to their affinity. 

The column is packed with a solid adsorbent material which is called a stationary phase. Generally, silica gel or alumina used to pack the column using the dry method or wet method according to application.

Most commonly used column chromatography works on the principle of "partition" using the force of gravity. There are four different types of column chromatography are used in chemistry, such as adsorption chromatography, ion-exchange chromatography, partition chromatography, and gel chromatography, etc.

3.High-performance liquid chromatography (HPLC):

HPLC is the most popular and widely used analytical technique for the separation of the solutes. It is an advanced version of column chromatography. In HPLC, the sample is injected into the flow of the mobile phase, and then it is pumped under high pressure into an HPLC column. 

The isolation depends on the affinity of the solutes for particles of the analytical column and mobile phase. The mobile phase consists of a solvent/mixture of solvents or solvent with a buffer solution.

Different types of HPLC chromatography are used for various applications that are dependent on the stationary phase such as reversed-phase, normal phase, size-exclusion, and ion-exchange. For HPLC the different types of columns, different types of HPLC detectors (UV/PDA), different types of injectors (Rheodyne, septum/stop flow), and different types of HPLC pumps. (Reciprocating/syringe/pneumatic) are available.

4. Gas chromatography (GC):

In GC, an inert gas such as helium or nitrogen or argon is used as the mobile phase and is usually a solid or a liquid stationary phase composed of silicon polymers. The gas chromatography works on the mechanism of “partition”. The volatile mixture of sample is injected into the column with the stationary phase and is selectively adsorbed. The isolated molecules are recognized using a detector as they eluted from the column.

There are different types of columns (packed or capillary) and different types of detectors (flame ionization, thermal, and GC-MS) can be used for different applications of GC, usually, it depends on the type of sample used in GC. Results obtained from GC and HPLC have peak areas used as qualitative and quantitative analysis of the molecule. 

The shape of the peak needs to be symmetrical and it should not be beyond the limit of the tailing, fronting, and resolution of the peak.

5. Ion-exchange chromatography:

The ion-exchange chromatography is used to isolate ion and polar molecules, the isolation depends on the affinity toward the ion-exchanger. The basic principle involved is the exchange of ions, which can be used for almost any kind of charged molecule, such as amino acids, proteins, and nucleotides. Cationic and anionic exchangers are the types of ion-exchange chromatography.

Applications of chromatography:

Chromatography has many essential applications in several fields such as the pharmaceutical industry, food industry, chemical industry, environmental, beverage, and it's also used in forensics. Some of the important applications of chromatography are discussed below.
  • Chromatography is used to detect unknown components and purity of sample mixtures.
  • The HPLC, GC, and MS are used in forensic detection and crime scene investigation.
  • Chromatography is used in the separation of organic as well as inorganic components.
  • It is used for the determination of toxins, contaminants, nutrition, and food quality.
  • Chromatography used to monitor the drug synthesis reaction.
  • In the clinical diagnostics, it is used to determine the amount of substance found in blood and urine.
  • The applications of column chromatography include the separation of several classes of drugs such as plant extracts, aggregates, alkaloids glycosides, and amino acids.
  • Chromatography is used in the food manufacturing process for quality control.
  • The applications of TLC chromatography include the isolation and identification of preservatives, sweetening agents, dyes, and many other cosmetic products.
  • For testing the quality of water and air, it can be used.
  • Chromatography is used to isolate and analyze of vitamins, proteins, additives, and preservatives.
  • It is used to detect various contaminants in pesticides and oils.
  • The applications of paper chromatography consist to detect and separate color mixtures including pigments, it is used in analytical chemistry as a qualitative technique.

Advantages of chromatography:

  • The major advantage of chromatography is that a wide range of samples can be separated using different separation mechanisms.
  • It is a simple method for separating a complex sample mixture using the chromatography method.
  • It does not require a large amount of sample. To detect the sample is the use of a sample in just grams, ppm, and ng.
  • One person can perform or handle the experimental process of chromatography.
  • Chromatography results are rapid and precise.

The commonly asked questions on chromatography are as follows.

What is the major advantage of HPTLC over TLC?
The major advantage of HPTLC over TLC is that the HPTLC has more resolving power with faster development times and the consumption of solvent is less than TLC.

What are the factors that affecting resolution in HPLC chromatography?
It is an elution is a quantitative determination of how the two peaks can be distinguished in chromatographic separation. Three factors mainly affect the resolution i.e. separation, retention, and efficiency.

What is the principle of column chromatography?
Column chromatography is a type of chromatography in which the solutes to be isolated are distributed between a mobile phase and a stationary phase. In this method, different distributions depend on selective adsorption.

What type of chromatography is paper chromatography?
Paper chromatography is classified in planar chromatography in which processes are carried out on a specialized cellulose paper.

What is the importance of pKa value in HPLC method development?
The pKa is a way used to indicate the strength of an acid. It is a negative log of the acid dissociation constant or the value of. A low PKA value of a substance indicates a strong acid.


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