Key Difference – Paper vs Thin Layer vs Column Chromatography
Paper chromatography, thin layer chromatography, and column chromatography are three types of chromatographic techniques. The key difference between paper chromatography, thin layer chromatography and column chromatography are based on the type of stationary phase used in the chromatography technique. Paper chromatography uses a cellulose paper as its stationary phase, Thin Layer chromatography uses alumina or silica gel as its stationary phase, whereas Column chromatography uses a column packed with a suitable matrix material as its stationary phase.
In the process of separation and identification of biomolecules such as proteins and carbohydrates, chromatography is an important biophysical technique used. Chromatography separates compounds based on their solubility, size and charge. Based on the separation mechanism, chromatography uses mechanisms such as ion exchange, absorption, partition and size exclusion and there are three chromatographic techniques; namely, paper, thin layer, and column chromatography. Paper chromatography is based on the solid-liquid adsorption and solubility of the compound, and it uses a cellulose paper as the stationary phase. Thin layer chromatography is based on solid-liquid adsorption of molecules. It has a stationary phase that is typically made of alumina or silica gel and the mobile phase that is the solvent. Column chromatography uses a column packed with a matrix that is used to separate molecules mainly based on their size, affinity or its charge.
1. Overview of Chromatography and Key Difference
2. What is Paper Chromatography
3. What is Thin Layer Chromatography
4. What is Column Chromatography
5. Similarities Between Paper, Thin Layer and Column Chromatography
6. Side by Side Comparison – Paper vs Thin Layer vs Column Chromatography in Tabular Form
What is Paper Chromatography?
Paper chromatography is the simplest type of chromatography used, and it is not used for extensive research. It is mainly used in student laboratories to identify biomolecules such as amino acids and carbohydrates present in mixtures. Paper chromatography uses a stationary phase that is made using cellulose paper or Whatman filter paper and a mobile phase that is usually prepared using organic solvents such as n-butanol, etc. The stationary phase is saturated with water, making the stationary phase liquid. Thus, when the compounds are spotted and allowed to run in the presence of the mobile phase, depending on the solubility of the compounds, they are separated. Thus, upon the development of the chromatogram, staining can be done to determine the run the length of each compound. The retention factor can thereby be calculated.
Paper chromatography can be further classified as ascending paper chromatography and descending paper chromatography depending on the direction of the running solvent.
What is Thin Layer Chromatography?
Thin layer chromatography or TLC is a commonly used technique to identify different amino acids present in a mixture or for the identification of proteins. The technique of separation is based on the solid-liquid adsorption. During thin layer chromatography, a plate made of alumina or silica gel is used as the stationary phase. The solvent mixture varies upon the requirement and may use different combinations of organic compounds such as n-butanol, acetic acid, and water to prepare the solvent. The compounds to be separated are spotted on the plate and immersed in the solvent mixture. Once the solvent travels up based on the capillary action given by the plate, the compounds spotted on the plate also moves depending on their solubility in the solvent.
The detection of the spots after the chromatogram is run done by different staining procedures. Some use ninhydrin staining that is fairly a toxic method of staining. Modern thin layer chromatograms use fluorescence techniques to view the chromatogram after the run. Depending on the distances it has travelled, the retention time of each compound can be calculated. This can be used to identify the type of compound separated based on the mixture used. TLC is mainly used to identify amino acids in a protein mixture and also to separate different types of monosaccharides present in a mixture.
What is Column Chromatography?
Column chromatography is a broad term used to describe many types of chromatography techniques that use the column based method of separation. In column chromatography, a physical column is used with a packing material to separate the compounds. The separation may be based on different physical properties exhibited by the compounds. These properties can be the charge, size, 3D conformation and binding capacity, etc. Thus, the column packed with the matrix material acts as the stationary phase and the wash buffer applied to the column acts as the mobile phase.
If the molecules are separated based on the size, the packing material is packed in a manner that it leaves pores for the compounds to travel through. Thus, the larger molecules that cannot flow through the pores are eluted first, whereas the smaller molecules take much longer time to elute.
If the molecules are separated based on their charge, the stationary phase will contain either an anion or cation exchanger to which the compounds will get attracted based on their charge. Thus during the washing step, the non-bound compounds will be eluted. Upon adding the elution buffer, the bound charged compounds will be eluted. The detection of these eluents is mostly based on spectrophotometric techniques.
What are the Similarities Between Paper Thin Layer and Column Chromatography?
- All Paper Thin Layer and Column Chromatography three techniques are used for separation of biomolecules such as amino acids, proteins and carbohydrates.
- Paper Thin Layer and Column Chromatography techniques have a mobile phase and a stationary phase.
- Paper Thin Layer and Column Chromatography techniques use biophysical mechanisms for separation.
What is the Difference Between Paper Thin Layer and Column Chromatography?
Paper vs Thin Layer vs Column Chromatography
|Paper Chromatography||Paper chromatography is a chromatographic technique uses to separate compounds based on the liquid-liquid adsorption and solubility of the compound. It uses a cellulose paper as its stationary phase.|
|Thin Layer Chromatography||Thin layer chromatography is another chromatographic technique based on solid-liquid adsorption of molecules. It has a stationary phase made of alumina or silica gel and a solvent as the mobile phase, which is the solvent.|
|Column Chromatography||Column chromatography uses a column packed with a matrix that is used to separate molecules mainly based on their size, affinity or its charge.|
|Paper Chromatography||Paper made of nitrocellulose of Whatman is used as the stationary phase in paper chromatography.|
|Thin Layer Chromatography||Alumina or Silica gel is used as the stationary phase of the thin layer chromatography.|
|Column Chromatography||A column packed with suitable packing material is used as the stationary phase in the column chromatography.|
|Paper Chromatography||Running solvent is the mobile phase of the paper chromatography.|
|Thin Layer Chromatography||Running solvent is the mobile phase of the thin layer chromatography.|
|Column Chromatography||Wash buffer is the mobile phase of the column chromatography.|
|Mechanisms Used for Separation|
|Paper Chromatography||Paper chromatography is based on solid-liquid absorption.|
|Thin Layer Chromatography||Thin layer chromatography is based on solid-liquid absorption.|
|Column Chromatography||Column chromatography is based on size exclusion, charge and shape.|
|Paper Chromatography||Not required by paper chromatography.|
|Thin Layer Chromatography||Not required for thin layer chromatography.|
|Column Chromatography||Required in column chromatography.|
|Paper Chromatography||Staining and by determining the Retention factor.|
|Thin Layer Chromatography||Staining and by determining the Retention factor.|
|Column Chromatography||Spectrophotometric determination.|
Summary – Paper Thin Layer vs Column Chromatography
Paper chromatography, TLC and column chromatography are separation techniques used to separate biomolecules such as proteins, amino acids and carbohydrates (mainly monosaccharides). Paper chromatography uses a cellulose paper as the stationary phase, and the mechanism of separation is based on solid-liquid adsorption. TLC also uses solid-liquid adsorption mechanisms. The molecules are separated on the stationary phase, depending on their solubility in the mobile phase. Column chromatography uses physical properties such as size, shape, charge and the molecular weight of the compound to separate. The column packed with the matrix material acts as the stationary phase, whereas the wash buffer acts as the solvent phase. This is the difference between a paper thin layer and column chromatography.
1.Clark , Jim. “ THIN LAYER CHROMATOGRAPHY.” Thin Layer Chromatography, 2007. Available here
2.Coskun, Ozlem. “Separation Techniques: Chromatography.” Northern Clinics of Istanbul, Kare Publishing, 2016. Available here