Monday, February 24, 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 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 materials, called adsorbents can hold compounds 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 polarity the elution order of components from the absorptivity are different.

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 is 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 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.

Procedure of Adsorption Chromatography:

Before beginning an adsorption chromatography experiment, we must recognize the various components essential to perform the process.
Stationary phase: The stationary 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:

Adsorption chromatography (TLC)
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
  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 a suitable place.
  4. Develop the plate: In the chamber, place the prepared plate, cover it with the lid and leave it 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 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 are difficult to reproduce.
Commonly asked questions on chromatography are as follows.

What is the basic principle of ion-exchange chromatography?
The exchange of ions is the basic principle of ion-exchange chromatography. Cationic and anionic exchangers are two forms of ion-exchange chromatography are used in this process.

What is column chromatography?
Column chromatography is a commonly used method of separating the compounds from complex mixtures. The chromatography, when performed in a column, is called column chromatography.

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.

What type of paper is used in paper chromatography?
Filter paper, chemically modified papers, acetylated papers, and pure cellulose papers of different grades like No.1, 2, 3, 4, 7, 17, 20, 40, 42, etc are used as stationary phases in paper chromatography.

What is the major advantage of HPLC?
One of the major advantages of high-performance liquid chromatography is that it has the ability to test a wide variety of samples.


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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 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. 
  • Affinity chromatography is used in clinical applications. 
  • 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 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 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.
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Monday, February 17, 2020

Principle and Procedure of Gas Chromatography

Learn the principle, types, applications of gas chromatography and how gas chromatography is works.

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 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 is 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.

Gas Chromatography Diagram:


Principle and Procedure of Gas Chromatography

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 their affinity with the mobile phase (gas) and the stationary phase are 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
  • 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 technique in pharmaceutical industries.
  • 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 is used in the analysis of flavor, fragrance, and food products.
  • GC is used to analyze the organic compounds in environmental samples.

Advantages of gas chromatography:

  • GC gives high sensitivity when 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.

Disadvantages of gas chromatography:

  • A major disadvantage of GC is that 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 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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Monday, February 10, 2020

Principle and Procedure of HPLC

Learn the principle of HPLC, types of HPLC, applications of HPLC and how high performance liquid chromatography works.

What is High-Performance Liquid Chromatography?

HPLC is an analytical technique in chemistry for the separation, identification, and quantification of the sample mixture. High-performance liquid chromatography is now one of analytical chemistry's most powerful tools. Since it's separate, identify and quantitate the analytes present in a sample mixture that may dissolve in a liquid.

High-Performance Liquid Chromatography (HPLC) Principle:

All chromatographic separation, such as thin-layer chromatography (TLC), column chromatography, HPTLC, and paper chromatography works under the same basic principle. HPLC is mainly a highly advanced form of column chromatography. The HPLC principle is based on the distribution of the component between a stationary phase (HPLC column) and a mobile phase (solvent). Depending on the chemical structure of the molecules they are retarded as passing the stationary phase. The intermolecular interactions among a sample's molecules and the packaging material determine their on-column period. Therefore, different components of a sample mixture are eluted at dissimilar retention times.


High-Performance Liquid Chromatography (HPLC) Diagram:


Principle and Procedure of HPLC

Different Types of HPLC:

Normal Phase HPLC: NP-HPLC separates the molecules according to polarity, in which the polar stationary phase and the non-polar mobile phase are used.
Reversed-Phase HPLC: The reverse phase chromatography works on the principle of hydrophobic interactions so the more nonpolar the analyte has, the longer it will be retained. In this the mobile phase is polar and the stationary phase is nonpolar in nature.
Size-Exclusion HPLC: Size Exclusion Chromatography (SEC) is a chromatographic process that separates molecules based solely on their size, in this technique molecules are separated by the column packing material on the basis of their exclusion from pores.
Ion-Exchange HPLC: It uses to separate the ions and polar molecules based on their affinity to the ion exchanger. Ion exchange chromatography is the most popular method for the purification of proteins and other charged molecules.

Experimental procedure of HPLC:

Before beginning an HPLC experiment, we must recognize the various components essential to perform the process.
HPLC Pump: The HPLC pump produces high pressure that gives a continuous and reproducible flow to the mobile phase throughout the HPLC system. E.g. Reciprocating pump, syringe pump, and pneumatic pump.
HPLC Mobile Phase: It is a solvent or contains a combination of water with organic solvents, an ideal amount of an aqueous solution with polar solvents, or mixtures of organic solvent.
HPLC Degasser: It is a tool for removing gas from a mobile phase used in HPLC.
HPLC Injector: A sample injector is a device used to inject samples solution into the HPLC system. E.g. Rheodyne injector, septum injector, and stop flow injector.
HPLC Column: The column is the key component of HPLC as it is responsible for separating the analytes of the sample mixture. Columns are now designed for use at high pressure in stainless steel tubes. Typically, silica gel is filled into the HPLC column known as the stationary phase.
HPLC Oven: It is a device used to control the temperature of a column.
HPLC Detector: A detector is a device used to detect compounds separated from a column. The detector transforms the effluent into an electrical signal and is recorded by the computerized system. E.g. UV/VIS detector, PDA detector, Mass detector (LCMS), Fluorescence detector, and Infrared detector, etc.

Process for RP-HPLC:

  • Install column (C8 / C18) properly as required.
  • Prepare the mobile phase and fill it in the reservoir. (Solvent, Buffer, or combination of it)
  • Prepare samples in different concentrations, as needed.
  • Build a method, and fill parameters such as flow rate, the mobile phase composition, wavelength, oven temperature, and program time.
  • Create a sequence for samples and save it.
  • Purge the reservoirs.
  • Gradually increase system flow up to the required flow rate, and wait until the column is saturated and the baseline is corrected.
  • As you get a baseline, then inject the sample manually or by auto-sampler via injector (sample loop/syringe).
  • After analyzing the sample, study the retention time, tailing factor, capacity factor, and theoretical plates of each peak.
  • Repeat the process according to the number of samples.
  • Wash the column properly with HPLC-grade water, and methanol/ acetonitrile.

Applications of HPLC:

  • For pharmaceutical applications, the HPLC is used to monitor drug stability, tablet dissolution analysis of pharmaceutical dosages form, and quality control, etc.
  • For environmental applications, the HPLC is used for bio-monitoring of pollutants, and detection of phenolic compounds in drinking water, etc.
  • For forensics applications, the HPLC is used for the quantification of drugs in biological samples, determination of cocaine, steroids, and other abused drugs in blood, urine, etc.
  • For clinical applications, the HPLC is used for the analysis of urine, bilirubin, antibiotics, etc. in blood.
  • For food and beverage applications, the HPLC is used for the analysis of polycyclic compounds in vegetables, analysis of preservatives, sugar, and for measurement of quality of water and soft drinks.

Advantages of HPLC:

  • High-performance liquid chromatography provides a simple, automated, and highly accurate method of identifying certain chemical components in a sample.
  • The HPLC provides a quantitative and qualitative analysis that is simple and accurate.
  • It can be upgrading to mass spectroscopy.
  • Compared to other chromatographic techniques such as column chromatography, TLC, and paper chromatography, HPLC is fast, effective, and delivers high resolution.
  • The gradient elution is readily adaptable in HPLC.

Disadvantages of HPLC:

  • This requires a large number of expensive solvents, power supplies, and regular maintenance.
  • Need to be expertise, since it is more difficult for beginners.
  • The reliability of the HPLC separation process depends on the cleanliness of the mobile phase, sample, and proper system operation.
  • The contaminated column can affect the peak shapes.


Commonly asked questions on chromatography are as follows.

What is the basic principle of HPLC?
The HPLC's basic principle is to separate the molecules between the stationary phase and the mobile phase. Since molecules will have different partition coefficients, on that basis they will be separated.

What are the types of HPLC?
Normal phase HPLC, Reverse phase HPLC, Size-exclusion HPLC, and Ion-exchange HPLC, etc. are the types of HPLC based on the phase system (stationary) in the process.

What is the major advantage of HPLC?
One of the major advantages of high-performance liquid chromatography is that it has the ability to test a wide variety of samples.

What is the main difference between HPLC and HPTLC?
The main difference between HPLC and HPTLC is that HPLC separate a sample mixture using column, while HPTLC uses plate.

What kind of precautions to take during HPLC analysis?
  • Make sure the column is washed before and after analysis.
  • Solvents must be filtered through a 0.5 μm nylon filter membrane and degassed.
  • The sample must be particle-free, therefore filtered through a 0.2 μm nylon filter membrane.
  • Buffers like phosphate buffers, acetate buffers, etc. are very harmful to the HPLC system and columns they need to be washed properly.
  • Don’t overload the column.
  • Use appropriate flow rates to maintain system pressure.
  • Do not run HPLC systems at high back pressure.
  • Always use HPLC-grade solvents and water derived from reliable sources.
  • Use guard columns to protect against contamination and prolong column life.
  • Don't use a mobile phase or buffers with a highly acidic or basic pH.

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Wednesday, February 5, 2020

Principle and Procedure of Paper chromatography

Learn the principle, types, applications, and procedure of paper chromatography.

What is Paper Chromatography?

Paper chromatography is one of the kinds of processes that operate on a piece of special paper. It is a method of planar chromatography in which a cellulose filter paper works as a stationary phase in which the compounds are separated. Paper chromatography is the most effective technique of separation, particularly in the analysis of chemical compounds and lipid samples. Several types of chromatography, such as liquid chromatography, ion-exchange chromatography, gas chromatography, thin-layer chromatography (TLC), column chromatography, and affinity chromatography have the same basic principles.

Paper Chromatography Principle:

In paper chromatography, the adsorption chromatography or partition chromatography is involved in which components are divided or distributed between liquid phases. In this, a special chromatography paper is used in which water is trapped which is the stationary phase. A sample solution is spotted on the base of the page. In the appropriate solvent, the paper strip is suspended and the mobile phase rises through capillary action to the top of the paper. The analytes are selectively retained on the stationary phase (cellulose paper). The spots of different analytes move to different heights along the mobile phase and each component in the sample mixture separates based on varying degrees of adhesion.

Experimental Procedure of Paper Chromatography:

Choose an appropriate type of development: It’s decided based on the solvent, mixture, paper complexity, etc. As they are easy to execute, radial type or ascending paper chromatography is usually employed. It's also simple to handle, the chromatogram obtained is quicker.
Selecting an appropriate filter paper: Based on sample quality and pores size, the filter paper is selected.
Sample preparation: Sample preparation involves dissolving the sample in an appropriate solvent that is used in the process of mobile phase making.
Sample spot on paper: The sample mixture should be spotted in a proper position on the paper using a capillary tube.
Development of chromatogram: The development of chromatograms is spotted by immersing the stationary phase (Paper) into the mobile phase. Because of the paper's capillary action, the mobile process passes over the paper sample.
Paper drying and compound detection: Upon the development of the chromatogram, the paper is dried by an air dryer. Detecting solution also is sprayed on developed paper and dried to recognize the spots of the sample chromatogram. Paper with varying bands of analytes is observed under UV-light and further Rf values are calculated.

Paper Chromatography Applications: 

  • Paper chromatography is used for separating polar and non-polar compounds.
  • It is used for the detection and separation of color mixtures including pigments as a qualitative analytical chemistry technique.
  • Paper chromatography is used for identifying sugars, nucleic acids, lipids, amino acids, and other biomolecules.
  • It is used in DNA/RNA sequencing.
  • It is used to test pharmaceutical purity.
  • It is used for the detection of pollutants in food and beverages.
  • For investigations and criminal trials, paper chromatography is used in forensic research.
  • This is used for ripening and fermentation study.
  • It is used for the analysis of cosmetics.
  • Paper chromatography is used for component separation and purification.
  • Paper chromatography can be applied to monitor chemical synthesis reactions.

Different types of paper chromatography:

Ascending Paper Chromatography: As the name suggests the developing solvent or mobile phase travels in an upward direction by the capillary action.
Descending Paper Chromatography: In such paper chromatography, due to the gravitational pull the direction of the solvent flow is downward
Ascending-Descending Paper Chromatography: In this type of paper chromatography the solvent first moves up on the paper folded over a rod and after crossing the rod it continues its path downwards.
Horizontal or Circular Paper Chromatography: In this method, the sample mixture is employed in the center of a round paper placed on a flat surface.
Two Dimensional Paper Chromatography: With the aid of two-dimensional paper chromatography, substances that have the same Rf values can be solved.

Advantages of Paper Chromatography:

  • Compounds are separated within a short time.
  • Analysis of the paper chromatography required small sample quantities.
  • It is an inexpensive technique compared to other chromatographic methods.
  • It is possible to identify both organic and inorganic compounds.
  • Paper chromatography is easy to use and set up.

Disadvantages of Paper Chromatography:

  • The use of paper chromatography techniques cannot separate volatile substances.
  • Paper chromatography cannot separate large sample volumes.
  • The paper chromatography has less accuracy compared to TLC, HPTLC, and HPLC.
  • It is unable to separate the complex mixture.
  • It cannot save results for long periods.
The questions commonly asked about paper chromatography are as follows.

What is the basic principle of paper chromatography?
The principle involved is partition chromatography in which the compounds are distributed or partitioned into liquid phases.

What is paper chromatography?
Paper chromatography is a method of separating and identifying mixtures, especially pigments.

What is the major advantage of paper chromatography?
The main advantages of paper chromatography are simplicity, rapid separation, low cost and have good sensitivity.

What type of paper is used in paper chromatography?
Filter paper, chemically modified papers, acetylated papers, and pure cellulose papers of different grades like No.1, 2, 3, 4, 7, 17, 20, 40, 42, etc are used as stationary phases in paper chromatography.

What is the major difference between thin layer chromatography and paper chromatography?
The major difference between TLC and paper chromatography is that the stationary phase of the paper chromatography is cellulose paper while TLC uses a thin layer of silica or alumina supported on a flat surface.

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