Tuesday, June 2, 2020

Mechanism of separation in paper chromatography

The chromatographic method works on the basis of four different separation mechanisms, such as partition, adsorption, size exclusion, and ion exchange.

What is paper chromatography?

Paper chromatography is one of the oldest and easiest separation techniques that have evolved along with other modern variants such as high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), gas chromatography (GC) and hyphenated techniques throughout the years. Paper chromatography is a planar chromatography in which the stationary phase is a cellulose filter paper, on which the compounds are isolated. The edge of the paper is immersed in a mobile phase or solvent, and it moves up the paper by capillary action so the compounds are isolated.

Mechanism of separation in paper chromatography:

The process consists of a sample apply as a spot close to a corner of a paper. The filter paper is initially saturated with some suitable solution to form the stationary liquid phase. An edge of the paper above the sample spot is then soaked in another solution, in that the analytes become soluble in different degrees. The solvent enters by capillary action in the stationary phase and, passing through the spot of the sample it carries different analytes of the sample.

The analytes travel with a solvent flowing in velocity that is dependent on their solubility in the stationary and mobile phase. The separation of analytes is achieved when their relative solubility varies between the two solvents. Both solvents are evaporated until the moving solvent reaches the farther edge of the stationary phase and the position of the separated analytes is recognized, generally, reagents are sprayed to appearance colored compounds with different substances. The isolated analytes show as spots on the path of the solvent and hence we can calculate the Rf value of the compound. If the mobile phase that flows in one direction and that cannot adequately separate all the molecules, then the paper can be turned 90° and the process can repeat with another mobile phase.

The principle of paper chromatography:

"partition" is the basic principle involved in paper chromatography, in which the solutes are partitioned or distributed among the liquid phases. In this method a special type of chromatography paper or a piece of filter paper used as a stationary-phase which is made with cellulose. The mixture of sample is applied to the paper and the edge of the paper is immersed in a mobile phase which is placed in the TLC chamber. The mobile phase or solvent runs on the paper under capillary action in paper pores. The capillary attraction of the solvent relies on the cohesive and adhesive forces allow the mobile phase to travel up the stationary phase because of created surface tension interaction from the forces.

The properties and structure of each molecule vary, so they have differences in the relative affinity for the stationary phase and the mobile phase. It is also in the form of adsorption chromatography between liquid and solid phases, in that the mobile phase is the liquid and a stationary phase is a solid surface. Though the most widely used paper chromatography operates on the partition chromatography theory.

Different types of paper chromatography:

There are 5 types of paper chromatography available, depending on how the chromatogram is developed.

Ascending chromatography:

As the name suggests, chromatograms are ascending in this type of paper chromatography. The chromatograms are developed due to the upward movement of the mobile phase on paper. The solvent is at the bottom of the TLC chamber. The paper tip below the sample spot immerses to the bottom and therefore the spots remain just above the mobile phase.

Descending chromatography:

In this form of paper chromatography, the paper is developed by the solvent travels downstream of the paper. The solvent is flowing from the top of the stationary phase and it moves by gravity.

Ascending- descending mode:

This is a hybrid type of paper chromatography, the mobile phase first flows-up over a stationary phase, which is folded onto the rod and starts traveling downwards after crossing the rod. This method allows longer-term development and gives higher resolution, particularly used for the complex sample mixtures.

Radial or horizontal mode:

The solvent travels from the center to the periphery of the circular filter paper. For the development of chromatograms, this is usually done in a covered petri dish instead of a TLC chamber. The wick at the center of the stationary phase is dipped in the solvent from which the mobile phase is drained on the stationary phase and moves the solutes radially as concentric rings to form the spot of the solute.

Two dimensional paper chromatography:

In this type of paper chromatography, stationary phase is first used with one mobile phase in one direction, and then another solvent is used in direction at right angles to the first, after the paper has been dried.


Commonly asked questions on chromatography are as follows.

What is the paper used in paper chromatography?
Paper is made of cellulose fiber, and cellulose is a polymer of simple sugar, glucose.

What are some examples of chromatography?
Column chromatography, thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), gas chromatography (GC), partition chromatography, ion-exchange chromatography, are some examples of chromatography.

What does paper chromatography separate?
Paper chromatography is used to isolate colored chemicals or compounds.

What factors affect paper chromatography?
Length of the paper, the composition of the mobile phase, quality of paper used, nature of solvents used, temperature, the thickness of the paper, the polarity of components, the pH of the solvent, and saturation of the solvent, etc. are the factors that affect paper chromatography.

What are the advantages of TLC?
TLC is a type of planar chromatography it has many advantages such as, it is very simple to use, it required less time to isolate the compounds, it requires less amount of sample, we can recover the sample by scratching the TLC plate, more sensitive than paper chromatography, and it can be upgraded to HPTLC.


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Partition chromatography mechanism of action

The chromatographic technique works based on four different separation mechanisms, such as partition, adsorption, size exclusion, and ion exchange.  

What is partition chromatography?

Partition chromatography is the basic method of distribution mechanism of liquid-liquid chromatography, specifically when the stationary phase and mobile phase both are liquids. Isolation by distribution depending on the relative solubility of the component in both phases. The basic principle of partition chromatography is used in several methods, such as high-performance liquid chromatography (HPLC), paper chromatography, high-performance thin-layer chromatography (HPTLC), and gas chromatography (GC). 

Separation mechanism of partition chromatography:

The stationary liquid phase of partition chromatography is coated on solid supports such as silica gel or cellulose powder. Assuming solid supports have not adsorption, the mobile and stationary phases move the compounds through the system at rates determined by the solubility of their components. Usually, the mobile and stationary phases do not need to be entirely immiscible; however, it is ideal to have a low degree of mutual solubility. Hydrophilic stationary phase solvents are commonly used in combination with hydrophobic mobile phases, or vice versa.

Principle of partition chromatography: 

Partition chromatography is the technique of separation in which the solute present in a sample mixture distributes more possibility in two liquid phases as the differences in partition coefficients. In this method, the analytes are isolated amongst two phases i.e. both the mobile phase and the stationary phase are in the identical phase. In this process of separation, on the stationary phase, the immiscible solid surface coated with the liquid surface is in the mobile phase. The liquid surface is immobilized by a stationary phase resulting in its being a stationary phase. The mobile phase travels over the stationary phase and the analytes are separated. Consequently, the analytes are preferably dispersed at any phase.  

Different types of partition chromatography:

Liquid-liquid chromatography: In this kind of partition chromatography both phases such as stationary phase and mobile phase are taken in liquid form. However, the liquid stationary phase is supported by coated by solid supports. Its sub-types are paper chromatography, column chromatography, and thin-layer chromatography 

Gas-liquid chromatography: In this form, gas is used as a mobile phase which is unreactive, and the stationary phase is a non-volatile liquid held on an inert solid support. 


Commonly asked questions on chromatography are as follows.

What are the types of partition chromatography?
Two types of partition chromatography are available, liquid-liquid chromatography and gas-liquid chromatography.

What is partition chromatography used for?
Partition chromatography is a process used for the isolation and identification of mixtures of closely related substances such as amino acids.

What are the two main types of chromatography?
Liquid chromatography (LC) and gas chromatography (GC) are the two major types of chromatography.

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

Why do we use a pencil in chromatography?
The starting line in paper chromatography is drawn using a pencil, as the pencil lead will not dissolve in the mobile phase, and does not affect the separation.


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Mechanism of size exclusion chromatography

The chromatographic method works on the basis of four different separation mechanisms, such as partition, adsorption, size exclusion, and ion exchange.
What is size exclusion chromatography?
Size exclusion chromatography is a type of chromatography also known as gel chromatography. It is a powerful separation technique based on the molecular size of the components, which is used to separate the components, especially the high-molecular-mass space. In this method, the porous material is used as a stationary phase and liquid is used as a mobile phase. Separation is achieved through the differential exclusion of the sample components as they pass through a bed of porous particles from the pores of the packing material. 

Separation mechanism of size exclusion chromatography:
The column bed of size exclusion has three practical components i.e. pore volume, void volume, and matrix volume.
The pore volume: The pore volume corresponds to the space of pore-lumen inside the particles.
The void volume: It refers to the excluded volume, that is, the space amongst the particles.
The matrix volume:  It refers to the solid component of the particles which fill the bed of the column.
The analytes larger than the size of the pore cannot penetrate the hole and are separated together as the first peak in the chromatogram. Analytes that can enter the pores will have an average residence time in their particles which depends on the size and shape of the analytes. Therefore, different analytics have dissimilar total travel time through chromatographic columns. Analytes have small in size and reside the longest on the column, which eluted together in the form the last peak in the chromatogram.

Different types of size exclusion chromatography:
There are two basic types of size exclusion chromatography are gel permeation chromatography (GPC) and gel filtration chromatography (GFC).
Gel permeation chromatography: In this type of size exclusion chromatography, uses a hydrophobic column packing material and a non-aqueous organic solvent uses to determine the molecular weight distribution of the molecule.
Gel filtration chromatography: In this type of size exclusion chromatography uses a hydrophilic packing material of columns and an aqueous mobile phase to separate and determines the molecular weight distribution of water-soluble molecules.

Principle of size exclusion chromatography: 
The selection of analytes based on their molecular size and shape uses the molecular sieve properties of a diversity of porous materials. Possibly the most widely used of these materials is a group of polymeric organic compounds that have a three-dimensional pores network that gives them gel properties. The basic principle of exclusion chromatography is quite easy. The gel particle of the column is in equilibrium with an effective solvent or mobile phase for separating the molecules. Large size components that are excluded from the pores can pass through the interstitial spaces, as smaller size components are distributed amongst the mobile phase inside and outside the molecular sieve and after that pass at a lower rate through the column.

Commonly asked questions on chromatography are as follows.

What is the size exclusion chromatography used for?
Size exclusion chromatography is mainly used for separating compounds according to their molecular size. It is also used for purification, molecular weight determination, desalting, and protein binding studies.

What is the stationary phase in size exclusion chromatography?
The stationary phase if size exclusion chromatography consists of a column which is commercially available with a group of various packing material pore sizes. The column of the SEC is packed with fine, porous beads, made of dextran polymer, agarose, or polyacrylamide.

Why is size exclusion chromatography important?
The size exclusion chromatography is an important technique in the fractionation of proteins, determining the fine structure of analytes, fractionation of water-soluble polymers, as well as the relative amount of certain analytes in foodstuffs.


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Separation mechanism of ion-exchange chromatography

The chromatographic method works on the basis of four different separation mechanisms, such as partition, adsorption, size exclusion, and ion exchange.
What is ion-exchange chromatography?
Ion exchange chromatography is a separation technique that allows ion exchangers to separate ions and polar molecules based on their affinity. It is the most powerful type of liquid chromatography since this method allows the analysis of a broad range of molecules. This method allows the isolation of related types of analytes which would be not possible to isolate by other techniques, as the charge carried by the interested molecule can be easily controlled by adjusting the pH of the buffer. 
Separation mechanism of ion-exchange chromatography:
Ion exchange is a separation mechanism depends on the relative ionic strength of the solutes. The stationary phase is a charged substance with the opposite charge, which would preferentially retain species. A positively charged stationary phase in anion exchange-HPLC is maintaining a negatively charged species. For a cation exchange, the opposite is true. In ion chromatography elution of analytes is influenced by the pH of the mobile phase, ionic-strength, and working temperature of the system. Ion exchange columns have distinct advantages over silica-based support; they can use the full pH range and high temperatures. Ion chromatography reduces the pre-treatment of the sample mixture and enhances the precision and accuracy of results.
Principle of ion-exchange chromatography:
The ion-exchange chromatography depends on the attraction amongst the oppositely charged stationary phase, which is called ion exchange. The ion exchange consists of an inert support medium attached covalently to the functional groups which can be positive or negative. The oppositely charged ions are bound to these covalently bound functional groups, which would be exchanged with charged ions in the sample mixture with a charge magnitude greater than ions bounded to the matrix. Consequently, if anion exchange chromatography is executed, the negatively charged compounds can more interact with the stationary phase and be exchanged for the charged ions, which are already bound to the matrix.
The procedure of ion-exchange chromatography:
  1. The separations in ion-exchange chromatography are generally performed in a column which is packed with an ion-exchanger and they are prepared of divinyl-benzene and styrene.
  2. The selection of exchanger relies on the particle to be isolated. The anionic exchanger is used to separate anions and to separate the cations the cationic exchanger is used.
  3. The first step in the IEC, the ion exchanger is filled in the column and then followed by buffer a sample is applied.
  4. Tris-buffer, acetate buffer, pyridine, phosphate buffer, and citrate buffer are broadly used.
  5. The analytes that have a more affinity towards the ion-exchanger, they will move down with buffer. In the next step in IEC, the tightly bound particles are separated using a compatible buffer.
  6. After the separation of these particles, they are analyzed spectroscopically.
  7. In the last step, the system has to re-equilibrate for the next purification in initial conditions.
Types of ion-exchange chromatography:
Two types of exchangers can be used in this method, namely, cationic and anionic exchangers.
Cationic exchangers: It has negatively charged groups and this would attract positively charged groups.
Anionic exchangers: It consists of positively charged groups and this would attract negatively charged molecules.

Commonly asked questions on chromatography are as follows.

What are the applications of ion-exchange chromatography?
Ion-exchange chromatography is the most commonly used in pharmaceutical, agricultural, biotechnology, environmental, and some other industry for the purification of different proteins, nucleic acids, polypeptides, antibiotics, and other charged bio-molecules.

What is the use of suppressor in ion chromatography?
The Suppressor is located between a detector and the column and serves to reduce the eluent’s background conductivity and increase the conductivity of the molecules.

What is the mobile phase in ion-exchange chromatography?
The mobile phase of ion-exchange chromatography is composed of an aqueous solution of an appropriate salt or a mixture of salts and some percentage of organic solvent.

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Mechanism behind adsorption chromatography

The chromatographic technique is working based on four different separation mechanisms, such as partition, adsorption, size exclusion, and ion exchange.
What is adsorption chromatography?
Adsorption chromatography is a kind of liquid chromatography in which the compounds are retained based on their adsorption and desorption at the stationary phase. Adsorption is the basic principle of adsorption chromatography. It is a separation method in which the isolation of a solute is depending on the interaction of adsorbate with the adsorbent. The sample mixtures of gas or liquid are separated when they move over the stationary phase and that adsorbs the multiple solutes at dissimilar rates.
Separation mechanism of adsorption chromatography:
The separation mechanism of adsorption chromatography depends on the difference in polarity between different molecules. If the molecule will more polar, it will adsorb more strongly by a polar stationary phase. Likewise, if the molecule will more non-polar, it will more strongly be adsorbed by the non-polar stationary phase. Throughout the process of surface adsorption chromatography, there is the competition between the sample components and the mobile phase for stationary phase adsorption sites. The low polarity components spend extra time in the mobile phase as compared with the highly polar components that are retained longer. Hence molecules of a sample mixture are eluted in order of increasing polarity. The mobile phase selection in adsorption chromatography is very significant. Mobile phase polarity must be selected to complement stationary phase preference. Normally, better separation of peaks is attained by low polar mobile phases and polar stationary phases. 
The stationary phase selection in adsorption chromatography is governed by the polarity of the analytes. If the molecules are too strongly adsorbed, they can be difficult to elute. Weakly polar samples on highly active absorbents should be separated, or there is little or no separation. In adsorption chromatography, there are three types of adsorbents are commonly used such as polar acidic, polar basic, and non-polar supports. The silica is the most commonly used support in adsorption chromatography since it is polar and acidic in nature. Porous silica gel and alumina these two are the most commonly used adsorbents in chromatography are. Alumina is a polar adsorbent and is used for the isolation of weakly or moderately polar solutes; the components that have greater polarity are retained more selectively by the stationary phase and are therefore the last to be separated from the column. Additionally, alumina is basic in nature and hence it retaining acidic molecules preferentially. For the above, the partition chromatography is occasionally used, in which an inert material is coated with a liquid phase. Hydrophobic interaction chromatography is a particular type of surface adsorption chromatography. The separating substance must be partially hydrophobic in nature. The separation is made easy by the difference in the interaction strength amongst these substances and the interchangeable matrix, replaced with hydrophobic groups. Such a process is widely used for preparative -scale isolation of proteins.
Principle of adsorption chromatography:
Adsorption chromatography works on the principle that adsorbent can hold the analytes on their surface. Alumina, silica, and charcoal are the most commonly used adsorbents in the analysis of adsorption chromatography. Adsorbent beds will differentiate between adsorbate based on their binding power as the analytes are run constantly on the stationary phase; dissimilarity in the flow rate of the will separates the analytes. Often the elution order of compounds from the adsorptive stationary phases can be determined based on their relative polarities. Compounds of the most polar functional groups are strongly retained on polar adsorbents, and are thus eluted more rapidly. 
Process of column adsorption chromatography:
Column adsorption chromatography it the type of chromatography in which columns packed with the solid stationary phase, a solvent or mobile phase moves through this column and different components are adsorbed into the solid. The components that absorb low will elute rapidly, components that are stronger absorb they will separate later, and the components are not adsorbed they simply run throughout the column.
1. First, a sample consists of analytes of different polarity is applied to the column of a polar adsorbent.
2. The mobile phase is gradually added by the sides of the glass column without disrupting the stationary phase. This mixture of solvents is added throughout the experiment as needed.
3. The column has a polar stationary phase, and therefore the compounds will interact with it. The polar compounds will interact while non-polar will not interact with the adsorbent.
4. Polar compounds will be retained in the column as it interacts with the stationary phase. Opposite of this, the non-polar compounds will elute rapidly.
5. If the non-polar compounds are separated, we may modify the mobile phase polarity to elute the remaining polar compounds in the column. 
6. Collect each constituent as they reach the end of the column.

Commonly asked questions on chromatography are as follows.

What are the types of adsorption chromatography?
Adsorption chromatography has 3 types such as thin-layer chromatography (TLC), column chromatography, and gas-solid chromatography.

What is a partition and adsorption chromatography?
Partition and adsorption chromatography are both types of chromatography, in adsorption chromatography, separation is based on adsorption, in partition chromatography, and separation is based on partition.

Is paper chromatography partition or adsorption?
Paper chromatography uses a non-polar solvent for the separation of compounds, therefore it is a type of partition chromatography.

Which compound will elute first in column chromatography?
The non-polar compound will elute first in column chromatography. The order of elution of the compounds in column adsorption chromatography is non-polar- moderately Polar-most polar compound.

What is the use of adsorption chromatography?
Adsorption chromatography is used in many applications, normally, it is used to determine the concentration and purity of a solute, identifying of analytes and to separate a sample mixture into individual analytes.

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Factors affecting the resolution in HPLC chromatography

The choice of mobile phases, the number of sample components, the concentration of two different solvents used, sample solubility, detector efficiency (PDA or UV/VIS), and other factors may all affect resolution in HPLC separation.
 
High-performance liquid chromatography is a separation technique in chemistry, used for qualitative and quantitative analysis of components. In chromatographic separation, the resolution of elution is a quantitative measurement of how two peaks of analytes can be isolated.

Resolution is an important factor of each type of chromatographic technique, which is determined by three important parameters such as retention, efficiency, and selectivity. It is defined as the dissimilarity of RT between two peaks, divided by the width of the elution peaks.

To improve peak resolution of closely eluted molecules is an issue encountered by all chromatographers during the HPLC method development. The chemical differences between analytes can be used to get better separations. Chromatographers are used numerous parameters to develop the separation of closely related compounds.

Parameters that may be modified for the preferred separations consist of the stationary phase, mobile phase, flow rate, and temperature of the columns. Change the column particle size is another effective way used by many analyst. Small particle-sized columns generate high theoretical plate numbers by giving the Gaussian peak shape and are capable of resolving closely isolated peaks. Compounds that have a high molecular weight cannot separate on a small pore size column, therefore, they must resolve on a large pore size column.
 
The resolution equation states that three major parameters will influence the resolution in high-performance liquid chromatography.
1. Retention factor or capacity factor (k):
2. Separation factor (Selectivity):
3. Efficiency:
 
Selectivity is the factor that highest impact on the resolution as compared with other factors.
  • The particle size of the column
  • Column dimension
  • The activity of the adsorbent
  • Column overload
  • The temperature of the column
  • Degradation of HPLC column
  • Inadequate saturation of column
  • Mobile phase pH
  • The polarity of the solvents
  • Composition of the mobile phase
  • The viscosity of the mobile phase
  • The polarity of the sample
  • the pH of the sample
  • Structure of the solute
  • The flow rate of the system
  • Pressure on the system
The choice of a mobile phase (solvent system) and retention volume or volume time are also the most significant factors that can affect resolution in HPLC as more time for separation in the column causes the band spreading.


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What are the different types of columns used in HPLC
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Factors affecting column chromatography

Column chromatography is one of the most effective methods for isolating and purifying of solids and liquid compounds. It is a solid-liquid method in which the mobile phase is a liquid and the stationary phase is a solid. The basic principle of column chromatography is adsorption. The sample mixture is placed over the column and dissolved in the solvent or mobile phase and travels through the stationary phase. Based on their relative affinities of analytes are separated into a different fraction. The separation process or mobile phase is optimized using thin-layer chromatography (TLC) before performing column chromatography. There are 4 types of column chromatography are available such as partition chromatography, adsorption chromatography, ion-exchange chromatography, and gel chromatography.
The separation, resolution, retention time, and efficiency of column chromatography is dependent on different factors, some of the key factors include.
The polarity of the mobile phase: The polarity of the mobile phase is related to the resolution, Rf value, and separation of analytes, the drastic variations in the polarity of the solvent can affect the result of column chromatography.
Nature of the solvent: The flow rate of the solvent can be affected by its viscosity. Thus, the solvent has a low viscosity that is efficient compared to a more viscous solvent.
Quality of solvents: High-quality solvents can give much better reproducibility than low-quality solvents.
Solvents selection: The solvents or mobile phase used must be compatible with the matrix; in this case, the TLC is a very effective way to optimize the mobile phase.
Flow rate: Changes in the flow rate between the processes can affect column efficiency; hence the components need to be isolated with a suitable flow rate.
Packing of the column: Column packing is an essential aspect of resolution, proper packing of columns is very crucial to obtain better performance. 
Column dimension: Chromatographers use different dimensions of a column, it depends on the process and usage. Length, height, and width of columns are important factors for the separation of molecules, hence appropriate column dimensions need to be selected.
The particle size of the adsorbent: The movement of the adsorbent is dependable on their surface area, for enhancing the surface area of adsorbent, the particle size can be decreased, and therefore the activity of the adsorbent increases.
Pressure: The increase or decrease of pressure between the separation can affect the results.
The temperature: Temperature is also an important factor in chromatographic separation because variations in temperature can affect the separation process and in some cases, it degrades the sample.
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Factors affecting thin layer chromatography

Thin layer chromatography is commonly considered as an easy, quick, and cheap technique for the separation, and tentative detection of a broad range of components. The several steps involved in thin layer chromatography. First, the sample spotted on a coated plate. Then the TLC plate is placed in a covered TLC chamber containing a small amount of a mobile phase or a solvent. As the mobile phase moves to the plate by capillary action, analytes are traveling at different rates because of their interaction with the stationary phase and mobile phase. After the development of chromatograms by achieving different spots or bands, the plate is removed and the compounds are visualized. Once the compounds are visuals the Rf value can be determined. The resolution of TLC is better than paper chromatography and equal to or less than high-performance liquid chromatography (HPLC).
The various factors that affecting the resolution, separation and Rf value in TLC are as follows.
Mobile phase composition: The suitable combination of solvents in a mobile phase is necessary to separate the components this will mainly affect the separation and resolution.
Selection and quality of solvent:  As the mobile phase carries the compound up the TLC plate, the solvent selection will more impact on the separation in thin layer chromatography. Each time of analysis the high-quality solvents are needed to be used as low-quality solvents affect isolation
The polarity of the solvent or mobile phase: The solvent polarity is related to the Rf value, resolution, and separation of molecules, the difference in polarity of solvent maybe affects the result of TLC.
Solvent saturation: RF values in thin layer chromatography mainly rely on the solvent saturation of the atmosphere in the TLC chamber.
The concentration of the sample: The proper concentration of the sample has been required since a very concentrated and diluted sample cannot be properly separated.
Spotting of the TLC plate: Sampling techniques may also affect the retention factor, with the more volume of the sample resulting in a larger and a diffuse band of compounds, making it unclear to determine the distance correctly.
TLC Plate: Mostly silica or alumina is used to prepare the plate. The TLC plate can affect the retention factor, resolution, and separation of molecules since the uniformity and thickness of the absorbent layer can vary from plate to plate.
Temperature: The slight changes in the temperature of the system can influence the result, hence control of the working temperature may be needed.
Sampling technique: Every chromatographer has a different technique to apply the sample; these variations can cause to change the retention factor.

Commonly asked questions on TLC chromatography are as follows.
Why iodine is used in TLC?
The TLC plate is saturated with iodine vapor when the eluted TLC plate is put in the iodine chamber and separated spots become light brown since iodine has a high vapor pressure.
What are the advantages of thin-layer chromatography?
TLC is rapid, with multiple simultaneous samples separated. It requires a very small sample amount, it can be in micro-litre. TLC has higher Sensitivity compared to paper chromatography. Separated molecules can be seen by UV light. Silica gives better selectivity.
What are the disadvantages of TLC?
Compared to HPLC, GC, and column chromatography, TCL has a short stationary phase. Just qualitative analysis is possible. It is possible to separate only soluble components. Automation (HPTLC) makes it more expensive. The results can be affected by humidity and temperature, as it is an open system
What are the uses of thin-layer chromatography?
TLC is used to separate and determine the number of solutes present in the sample mixture. It can be used for identifying the analytes, determining their purity. The progress of a reaction can be monitored by TLC. It is broadly used in the isolation of compounds of pharmaceutical formulations.
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