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TLC Glossary

Learn the A to Z of Thin-Layer Chromatography

Use the TLC glossary to find the meanings of the most important terms used in thin-layer chromatography. You can browse alphabetically, search using keywords, or scroll down to view all TLC terms and definitions.

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Adsorption Chromatography

A chromatography technique in which sample components are separated based on their different degrees of adsorption to a stationary phase.


A substance of interest that is being analyzed, for example, by chromatographic separation.

Anticircular Development (Anticircular Elution)

A development method in which the sample and mobile phase are applied at the edge of a circle on the TLC plate, and both move toward the center. Opposite of circular development.

Ascending Development (Ascending Elution)

A development method in which the TLC plate is positioned vertically or slanted, and the mobile phase is introduced to its lower edge. The mobile phase travels up the plate due to capillary action.


Baseline (Start Line)

The line on which the sample is spotted on the TLC plate.

TLC Baseline (start line)


Additives used to hold the solid stationary phase to the inactive TLC plate.

Binding Selectivity

The characteristic affinity of a ligand to bind to a substrate in order to form a complex. The selectivity coefficient is the value of the equilibrium constant for the reaction of displacement of a ligand bound to a substrate by another ligand.

Bonded Phase

A stationary phase that is covalently bonded to the support particle.

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Chamber Saturation (Saturated Development)

The uniform distribution of the mobile phase vapor through the development chamber prior to chromatography.


The visual result of a separation, with different spots or patterns corresponding to different components of the separated mixture.

What is Chromatography?

A method of separation in which the components to be separated are distributed between two phases: a stationary phase and a mobile phase.

Circular Development (Circular Elution, Radial Development, Radial Elution)

A development method in which the sample and mobile phase are applied at a point in the center of the TLC plate and move outward in a circle. Opposite of anticircular development.

Column Chromatography

A separation technique in which the stationary phase is within a column. Column chromatography is typically used to purify individual compounds from mixtures.

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A device which allows parts of a developed chromatogram to be scanned with a beam of light of a specified wavelength in order to measure the absorption or fluorescence of UV or visible light, thus allowing quantitative analysis of the separated compounds.

Descending Development (Descending Elution)

A development method in which the mobile phase is introduced to the upper edge of the stationary phase, and the mobile phase travels downward due to gravity.

Distribution Constant (Distribution Coefficient, Partition Ratio)

Partition Chromatography

The distribution constant (Kc) is equal to the concentration of a component in the stationary phase divided by its concentration in the mobile phase.
Distribution Constant 

Kc: Distribution constant
Wi(S): Amount (mass) of component i in the stationary phase
Wi(M): Amount (mass) of component i in the mobile phase
VS: Volume of the stationary phase
VM: Volume of the mobile phase

The distribution constant (Kc) is also related to the retention volume (VR) of the sample component.
Retention Volume 

VR: Retention volume
VM: Volume of the mobile phase
VS: Volume of the stationary phase
Kc: Distribution Constant

Adsorption Chromatography

In adsorption chromatography, the concentration in the stationary phase is usually expressed per unit surface area.
Concentration in the stationary phase  

Kc: Distribution constant
Wi(S): Amount (mass) of component i in the stationary phase
Wi(M): Amount (mass) of component i in the mobile phase
AS: Surface area of the stationary phase
VM: Volume of the mobile phase

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Elute (Develop)

To separate substances by elution chromatography.

Elute (Power)

A measure of a solvent’s ability to "draw" an analyte off the adsorbent to which it is attached.

Elution (Development)

The process of extracting one material from another by washing with a solvent.

Elution Chamber (Development Chamber)

A container that is used to enclose the stationary and mobile phases in order to maintain a constant environment in the vapor phase.

Eluotropic Series

A list of different compounds in order of their eluting power for a specified adsorbent.


The process of balancing the saturation level of the TLC layer by the mobile-phase vapor prior to chromatography.

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Flow Constant (Velocity Constant)

A measure of the migration rate of the solvent front. It can be used to determine development times for different separation distances, given that the mobile and stationary phases, chamber type and temperature remain constant.

Fluorescence Quenching

With TLC layers that contain a fluorescence indicator, UV-active substances in the sample extinguish (quench) the fluorescence and appear as dark spots on a bright background.

Freundlich Equation (Freundlich Adsorption Isotherm)

An empirical relationship between the concentration of a solute on the surface of an adsorbent and its concentration in the liquid with which it is in contact.

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Gaussian Distribution (Normal Distribution)

A continuous distribution that is symmetrical around its mean. The mean, median, and mode of a Gaussian distribution are equal.

Gradient Layer

A TLC layer that possesses a gradual transition in some property.

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hRF Value

The hRF value is equal to the retardation factor (RF value) multiplied by 100. To calculate hRF values, the distance Zs is measured from the start line to the midpoint of the substance zone.
TLC hRF value 

Horizontal Development (Horizontal Elution)

A development method in which the TLC plate is positioned horizontally, and the mobile phase moves along the plate due to capillary action.

What is HPTLC?

A high-performance version of thin-layer chromatography (TLC), which allows automation of different steps, increased resolution, and more accurate quantitative measurements.

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Modification of the separation properties of the TLC layer by using appropriate additives.

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Liquid Chromatography (LC)

A separation technique in which the mobile phase is a liquid. Liquid chromatography can be performed in a column or on a plate.

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What is Mass Spectrometry (MS)?

A technique used for qualitative and quantitative analysis of chemicals in a sample by measuring the mass-to-charge ratio and extent of gas-phase ions.

Matrix-Assisted Laser Desorption/Ionization (MALDI)

A soft ionization technique used in mass spectrometry to analyze biomolecules and large organic molecules that tend to be fragile and fragment with conventional ionization methods.

Migration Distance

The run distance, run height, or developing distance, calculated as the ratio of the distance moved by the solute to that moved by the solvent (mobile phase).
 Merck:/Freestyle/LE-Lab-Essentials/Chromatography/LE-Graphic Migration Distance-05102016.jpg

Mobile Phase (Solvent System)

The phase that moves along the stationary phase (TLC layer) and separates the sample.
The mobile phase may be a liquid (liquid chromatography, capillary electro-chromatography), a gas (gas chromatography), or a supercritical fluid (supercritical-fluid chromatography).

Mobile-Phase Distance

The distance traveled by the mobile phase along the stationary phase from the starting (application) line to the mobile phase front. 

Mobile-Phase Front (Solvent Front)

The leading edge of the mobile phase as it travels along the stationary phase.
Except in radial development, the mobile phase front is a straight line parallel to the mobile phase surface.

Mobile Phase Viscosity (η)

The viscosity of the mobile phase at the temperature of the stationary phase.

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Normal-Phase Chromatography

A development method used in liquid chromatography in which the stationary phase is more polar than the mobile phase.
Compare with: Reversed-phase chromatography

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Particle Diameter (dP)

The average diameter of solid particles.

Partition Chromatography

A separation method that is based on differences between the solubilities of the sample components in the stationary phase (gas chromatography), or on differences between the solubilities of the components in the mobile and stationary phases (liquid chromatography).

Planar Chromatography (Open-bed Chromatography)

A separation technique in which the stationary phase is a plane.
The plane can be a paper (paper chromatography), or a layer of solid particles spread on a support, such as a glass plate (thin-layer chromatography).

Polytetrafluoroethylene (PTFE)

A high-molecular-weight compound of carbon and fluorine (a fluorocarbon solid), which is a synthetic fluoropolymer of tetrafluoroethylene.
PTFE is hydrophobic, inert to most chemicals, and has one of the lowest coefficients of friction against any solid.

Preparative-Layer Chromatography (PLC)

A modified version of TLC in which preparative layer (PLC) plates with thicker layers are used to separate and purify greater quantities of samples – from milligrams to grams.

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Reference Standard

A standardized substance that acts as a measurement benchmark for similar substances.
Reference standards are used by pharmaceutical and related industries to help ensure the identity, strength, quality, and purity of products.

Relative Retardation (Rrel)

The ratio of the retardation factor of the sample component to the retardation factor of the reference standard.
Relative retardation is also equal to the ratio of the distances traveled by the component and the reference substance, since the solvent front is the same for both.

Rrel = RF(i) / RF(st) = bi / bst

Rrel: relative retardation
RF(i): retardation factor of the component
RF(i): retardation factor of the reference standard
bi: distance traveled by the spot of component
bst: distance traveled by the reference substance

Retardation Factor (RFValue)

The ratio of the distance traveled by the center of a spot of component to the distance traveled by the mobile phase.
The retardation factor is unique for every compound (when the same stationary and mobile phases are used). Hence, it can be used to prove the identity of compounds by comparison with reference standards.

RF = b / a

RF: retardation factor
b: distance traveled by the spot of component
a: distance traveled by the mobile phase

The retardation factor is always smaller than one, and usually given to two decimal places. For simplicity, the hRF value may be used instead, which is equal to the RF value multiplied by 100.

The RM value is a logarithmic function of the RF value:

Merck:/Freestyle/LE-Lab-Essentials/Chromatography/LE-Retardation Factor-05102016.jpg

What is Reversed-Phase Chromatography?

A development method used in liquid chromatography in which the mobile phase is significantly more polar than the stationary phase.
Compare with: Normal-phase chromatography

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A mixture consisting of a number of components, which is to be analyzed by chromatographic separation.

Sample Components (Analytes, Eluites)

The chemically pure components of the sample.
Sample components can be retained permanently by the stationary phase, partially retained (develop at different times), or unretained (not delayed).

Sandwich Chamber

A development chamber whose walls are close enough to the TLC plate to allow faster equilibration.


The variable strengths of the interactions between the sample components and the stationary phase (ΔhRF).

Separation Efficiency

The degree of resolution of the sample components in the TLC system.


The sample components in partition chromatography.

Solute Distance

The distance traveled by the solute from the starting (application) point or line to the center of the solute spot.
For non-circular solute spots, an imaginary circle is used whose diameter is the smallest axis of the spot.


Any substance capable of solubilizing another substance, especially the liquid mobile phase.

Solvent System

See mobile phase


An almost circular zone containing the sample component before or after TLC development.

Spot Diameter

The width of the sample component spot before or after chromatography.

Spotting Device

A syringe or micropipette used to deliver a fixed volume of sample as a spot or streak at the starting point or line of the TLC layer.

Starting Point (Starting Line)

The point or line on a TLC layer where the substance to be separated is applied.

Stationary Phase

A TLC plate coated with a layer of sorbent material.
TLC layers can be prepared in the lab, but they are normally purchased as pre-coated plates. There are various options available, including silica, aluminum oxide, cellulose, and modified or mixed-layer plates. The choice should be based on the properties of the sample and the application goals.

Stepwise Development (Stepwise Elution)

A method of development in which the composition of the mobile phase is changed in steps during a single chromatographic run.

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What is Thin-Layer Chromatography (TLC)

A technique used to separate components in a mixture based on their different polarities.

Two-Dimensional Chromatography (Two-Dimensional Development)

A TLC method in which the sample is first developed in one direction, and then again in a direction at right angles to the first. A different mobile phase is used for each elution.

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Ultraviolet (UV) Light

Electromagnetic radiation with a wavelength of 10 nm to 400 nm, which is shorter than that of visible light but longer than X-rays.

Unsaturated Development (Unsaturated Elution)

Thin-layer chromatography performed in a development chamber without chamber saturation.

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Visualization Chamber

A device in which the TLC plate is viewed under controlled-wavelength light, usually after spraying with chemical reagents to allow the detection of the separated components as visible spots under specified conditions.

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Zone (or Band)

A region on the TLC layer where one or more sample components are located.

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