Thin-layer chromatography

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Thin-layer chromatography
TLC black ink (cropped).jpg
Separation of black ink on a TLC plate
AcronymTLC
Classification Chromatography
Other techniques
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Thin-layer chromatography (TLC) is a chromatography technique that separates components in non-volatile mixtures. [1]

Contents

It is performed on a TLC plate made up of a non-reactive solid coated with a thin layer of adsorbent material. [2] This is called the stationary phase. [2] The sample is deposited on the plate, which is eluted with a solvent or solvent mixture known as the mobile phase (or eluent). [3] This solvent then moves up the plate via capillary action. [4] As with all chromatography, some compounds are more attracted to the mobile phase, while others are more attracted to the stationary phase. [5] Therefore, different compounds move up the TLC plate at different speeds and become separated. [6] To visualize colourless compounds, the plate is viewed under UV light or is stained. [7] Testing different stationary and mobile phases is often necessary to obtain well-defined and separated spots.[ citation needed ]

TLC is quick, simple, and gives high sensitivity for a relatively low cost. [5] It can monitor reaction progress, identify compounds in a mixture, determine purity, or purify small amounts of compound. [5]

Procedure

The process for TLC is similar to paper chromatography but provides faster runs, better separations, and the choice between different stationary phases. [5] Plates can be labelled before or after the chromatography process with a pencil or other implement that will not interfere with the process. [8]

There are four main stages to running a thin-layer chromatography plate: [3] [8]

Plate preparation: Using a capillary tube, a small amount of a concentrated solution of the sample is deposited near the bottom edge of a TLC plate. The solvent is allowed to completely evaporate before the next step. A vacuum chamber may be necessary for non-volatile solvents. To make sure there is sufficient compound to obtain a visible result, the spotting procedure can be repeated. Depending on the application, multiple different samples may be placed in a row the same distance from the bottom edge; each sample will move up the plate in its own "lane."

TLC of three amino acids and a sample (left) with an English translation (right) Amino acids TLC with English translation.png
TLC of three amino acids and a sample (left) with an English translation (right)

Development chamber preparation: The development solvent or solvent mixture is placed into a transparent container (separation/development chamber) to a depth of less than 1 centimetre. A strip of filter paper (aka "wick") is also placed along the container wall. This filter paper should touch the solvent and almost reach the top of the container. The container is covered with a lid and the solvent vapors are allowed to saturate the atmosphere of the container. Failure to do so results in poor separation and non-reproducible results.

Development: The TLC plate is placed in the container such that the sample spot(s) are not submerged into the mobile phase. The container is covered to prevent solvent evaporation. The solvent migrates up the plate by capillary action, meets the sample mixture, and carries it up the plate (elutes the sample). The plate is removed from the container before the solvent reaches the top of the plate; otherwise, the results will be misleading. The solvent front, the highest mark the solvent has travelled along the plate, is marked.

Visualization: The solvent evaporates from the plate. Visualization methods include UV light, staining, and many more.

Separation process and principle

The separation of compounds is due to the differences in their attraction to the stationary phase and because of differences in solubility in the solvent. [9] As a result, the compounds and the mobile phase compete for binding sites on the stationary phase. [9] Different compounds in the sample mixture travel at different rates due to the differences in their partition coefficients. [10] Different solvents, or different solvent mixtures, gives different separation. [5] The retardation factor (Rf), or retention factor, quantifies the results. It is the distance traveled by a given substance divided by the distance traveled by the mobile phase.[ citation needed ]

Development of a TLC plate. Spots that appear purple separate into red spots and blue spots. Tlc sequence.svg
Development of a TLC plate. Spots that appear purple separate into red spots and blue spots.

In normal-phase TLC, the stationary phase is polar. Silica gel is very common in normal-phase TLC. More polar compounds in a sample mixture interact more strongly with the polar stationary phase.[ citation needed ] As a result, more-polar compounds move less (resulting in smaller Rf) while less-polar compounds move higher up the plate (higher Rf). [10] A more-polar mobile phase also binds more strongly to the plate, competing more with the compound for binding sites; a more-polar mobile phase also dissolves polar compounds more. [10] As such, all compounds on the TLC plate move higher up the plate in polar solvent mixtures.[ citation needed ] "Strong" solvents move compounds higher up the plate, whereas "weak" solvents move them less. [11]

If the stationary phase is non-polar, like C18-functionalized silica plates, it is called reverse-phase TLC. In this case, non-polar compounds move less and polar compounds move more.[ citation needed ] The solvent mixture will also be much more polar than in normal-phase TLC. [11]

Solvent choice

An eluotropic series, which orders solvents by how much they move compounds, can help in selecting a mobile phase. [5] Solvents are also divided into solvent selectivity groups. [5] [12] Using solvents with different elution strengths or different selectivity groups can often give very different results. [5] [12] While single-solvent mobile phases can sometimes give good separation, some cases may require solvent mixtures. [13]

In normal-phase TLC, the most common solvent mixtures include ethyl acetate/hexanes (EtOAc/Hex) for less-polar compounds and methanol/dichloromethane (MeOH/DCM) for more polar compounds. [14] Different solvent mixtures and solvent ratios can help give better separation. [15] In reverse-phase TLC, solvent mixtures are typically water with a less-polar solvent: Typical choices are water with tetrahydrofuran (THF), acetonitrile (ACN), or methanol. [14]

Analysis

TLC plate visualised with UV-light Tlc plate (cropped).jpg
TLC plate visualised with UV-light

As the chemicals being separated may be colourless, several methods exist to visualise the spots:

Plate production

TLC plates are usually commercially available, with standard particle size ranges to improve reproducibility. [4] They are prepared by mixing the adsorbent, such as silica gel, with a small amount of inert binder like calcium sulfate (gypsum) and water. [18] This mixture is spread as a thick slurry on an unreactive carrier sheet, usually glass, thick aluminum foil, or plastic. The resultant plate is dried and activated by heating in an oven for thirty minutes at 110 °C. [18] The thickness of the absorbent layer is typically around 0.1–0.25 mm for analytical purposes and around 0.5–2.0 mm for preparative TLC. [19] Other adsorbent coatings include aluminium oxide (alumina), or cellulose. [18]

Applications

Reaction monitoring and characterization

TLC is a useful tool for reaction monitoring. [15] For this, the plate normally contains a spot of starting material, a spot from the reaction mixture, and a co-spot (or cross-spot) containing both. [4] [14] The analysis will show if the starting material disappeared and if any new products appeared. [14] This provides a quick and easy way to estimate how far a reaction has proceeded. In one study, TLC has been applied in the screening of organic reactions. [20] The researchers react an alcohol and a catalyst directly in the co-spot of a TLC plate before developing it. This provides quick and easy small-scale testing of different reagents.

TLC for reaction monitoring and choosing a purification solvent mixture (left)TLC from the resulting flash column chromatography (right) TLC Reaction monitoring and column chromatography.jpg
TLC for reaction monitoring and choosing a purification solvent mixture (left)TLC from the resulting flash column chromatography (right)

Compound characterization with TLC is also possible[ citation needed ] and is similar to reaction monitoring. However, rather than spotting with starting material and reaction mixture, it is with an unknown and a known compound. They may be the same compound if both spots have the same Rf and look the same under the chosen visualization method.[ citation needed ] However, co-elution complicates both reaction monitoring and characterization. This is because different compounds will move to the same spot on the plate. In such cases, different solvent mixtures may provide better separation. [21]

Purity and purification

TLC helps show the purity of a sample.[ citation needed ] A pure sample should only contain one spot by TLC. TLC is also useful for small-scale purification. [22] Because the separated compounds will be on different areas of the plate, a scientist can scrape off the stationary phase particles containing the desired compound and dissolve them into an appropriate solvent. [22] Once all the compound dissolves in the solvent, they filter out the silica particles, then evaporate the solvent to isolate the product. Big preparative TLC plates with thick silica gel coatings can separate more than 100 mg of material. [22]

For larger-scale purification and isolation, TLC is useful to quickly test solvent mixtures before running flash column chromatography on a large batch of impure material. [13] [23] A compound elutes from a column when the amount of solvent collected is equal to 1/Rf. [24] The eluent from flash column chromatography gets collected across several containers (for example, test tubes) called fractions. TLC helps show which fractions contain impurities and which contain pure compound.[ citation needed ]

Furthermore, two-dimensional TLC [4] can help check if a compound is stable on a particular stationary phase. This test requires two runs on a square-shaped TLC plate. The plate is rotated by 90º before the second run. If the target compound appears on the diagonal of the square, it is stable on the chosen stationary phase. Otherwise, it is decomposing on the plate. If this is the case, an alternative stationary phase may prevent this decomposition. [25]

TLC is also an analytical method for the direct separation of enantiomers and the control of enantiomeric purity, e.g. active pharmaceutical ingredients (APIs) that are chiral. [26]

See also

Related Research Articles

In chemical analysis, chromatography is a laboratory technique for the separation of a mixture into its components. The mixture is dissolved in a fluid solvent called the mobile phase, which carries it through a system on which a material called the stationary phase is fixed. Because the different constituents of the mixture tend to have different affinities for the stationary phase and are retained for different lengths of time depending on their interactions with its surface sites, the constituents travel at different apparent velocities in the mobile fluid, causing them to separate. The separation is based on the differential partitioning between the mobile and the stationary phases. Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus affect the separation.

<span class="mw-page-title-main">High-performance liquid chromatography</span> Technique in analytical chemistry

High-performance liquid chromatography (HPLC), formerly referred to as high-pressure liquid chromatography, is a technique in analytical chemistry used to separate, identify, and quantify specific components in mixtures. The mixtures can originate from food, chemicals, pharmaceuticals, biological, environmental and agriculture, etc., which have been dissolved into liquid solutions.

<span class="mw-page-title-main">Gas chromatography</span> Type of chromatography

Gas chromatography (GC) is a common type of chromatography used in analytical chemistry for separating and analyzing compounds that can be vaporized without decomposition. Typical uses of GC include testing the purity of a particular substance, or separating the different components of a mixture. In preparative chromatography, GC can be used to prepare pure compounds from a mixture.

<span class="mw-page-title-main">Paper chromatography</span> Separation of coloured chemicals on paper

Paper chromatography is an analytical method used to separate coloured chemicals or substances. It is now primarily used as a teaching tool, having been replaced in the laboratory by other chromatography methods such as thin-layer chromatography (TLC).

<span class="mw-page-title-main">Column chromatography</span> Method to isolate a compound in a mixture

Column chromatography in chemistry is a chromatography method used to isolate a single chemical compound from a mixture. Chromatography is able to separate substances based on differential adsorption of compounds to the adsorbent; compounds move through the column at different rates, allowing them to be separated into fractions. The technique is widely applicable, as many different adsorbents can be used with a wide range of solvents. The technique can be used on scales from micrograms up to kilograms. The main advantage of column chromatography is the relatively low cost and disposability of the stationary phase used in the process. The latter prevents cross-contamination and stationary phase degradation due to recycling. Column chromatography can be done using gravity to move the solvent, or using compressed gas to push the solvent through the column.

<span class="mw-page-title-main">Solid-phase extraction</span> Process to separate compounds by properties

Solid-phase extraction (SPE) is a solid-liquid extractive technique, by which compounds that are dissolved or suspended in a liquid mixture are separated, isolated or purified, from other compounds in this mixture, according to their physical and chemical properties. Analytical laboratories use solid phase extraction to concentrate and purify samples for analysis. Solid phase extraction can be used to isolate analytes of interest from a wide variety of matrices, including urine, blood, water, beverages, soil, and animal tissue.

Chiral column chromatography is a variant of column chromatography that is employed for the separation of chiral compounds, i.e. enantiomers, in mixtures such as racemates or related compounds. The chiral stationary phase (CSP) is made of a support, usually silica based, on which a chiral reagent or a macromolecule with numerous chiral centers is bonded or immobilized.

Reversed-phase liquid chromatography (RP-LC) is a mode of liquid chromatography in which non-polar stationary phase and polar mobile phases are used for the separation of organic compounds. The vast majority of separations and analyses using high-performance liquid chromatography (HPLC) in recent years are done using the reversed phase mode. In the reversed phase mode, the sample components are retained in the system the more hydrophobic they are.

<span class="mw-page-title-main">Hydrophilic interaction chromatography</span> Type of chromatography

Hydrophilic interaction chromatography is a variant of normal phase liquid chromatography that partly overlaps with other chromatographic applications such as ion chromatography and reversed phase liquid chromatography. HILIC uses hydrophilic stationary phases with reversed-phase type eluents. The name was suggested by Andrew Alpert in his 1990 paper on the subject. He described the chromatographic mechanism for it as liquid-liquid partition chromatography where analytes elute in order of increasing polarity, a conclusion supported by a review and re-evaluation of published data.

Aqueous normal-phase chromatography (ANP) is a chromatographic technique that involves the mobile phase compositions and polarities between reversed-phase chromatography (RP) and normal-phase chromatography (NP), while the stationary phases are polar.

<span class="mw-page-title-main">Phosphomolybdic acid</span> Chemical compound

Phosphomolybdic acid is the heteropolymetalate with the formula H3[Mo12PO40]·12H2O. It is a yellow solid, although even slightly impure samples have a greenish coloration. It is also known as dodeca molybdophosphoric acid or PMA, is a yellow-green chemical compound that is freely soluble in water and polar organic solvents such as ethanol. It is used as a stain in histology and in organic synthesis.

In chromatography, the retardation factor (R) is the fraction of an analyte in the mobile phase of a chromatographic system. In planar chromatography in particular, the retardation factor RF is defined as the ratio of the distance traveled by the center of a spot to the distance traveled by the solvent front. Ideally, the values for RF are equivalent to the R values used in column chromatography.

<span class="mw-page-title-main">Elution</span> Extraction of a material by washing with a solvent

In analytical and organic chemistry, elution is the process of extracting one material from another by washing with a solvent: washing of loaded ion-exchange resins to remove captured ions, or eluting proteins or other biopolymers from a gel electrophoresis or chromatography column.

<span class="mw-page-title-main">Two-dimensional chromatography</span>

Two-dimensional chromatography is a type of chromatographic technique in which the injected sample is separated by passing through two different separation stages. Two different chromatographic columns are connected in sequence, and the effluent from the first system is transferred onto the second column. Typically the second column has a different separation mechanism, so that bands that are poorly resolved from the first column may be completely separated in the second column. Alternately, the two columns might run at different temperatures. During the second stage of separation the rate at which the separation occurs must be faster than the first stage, since there is still only a single detector. The plane surface is amenable to sequential development in two directions using two different solvents.

<span class="mw-page-title-main">High-performance thin-layer chromatography</span> Advanced technique to separate non-volatile substances

High-performance thin-layer chromatography (HPTLC) serves as an extension of thin-layer chromatography (TLC), offering robustness, simplicity, speed, and efficiency in the quantitative analysis of compounds. This TLC-based analytical technique enhances compound resolution for quantitative analysis. Some of these improvements involve employing higher-quality TLC plates with finer particle sizes in the stationary phase, leading to improved resolution. Additionally, the separation can be further refined through repeated plate development using a multiple development device. As a result, HPTLC provides superior resolution and lower Limit of Detection (LODs).

Partition chromatography theory and practice was introduced through the work and publications of Archer Martin and Richard Laurence Millington Synge during the 1940s. They would later receive the 1952 Nobel Prize in Chemistry "for their invention of partition chromatography".

<span class="mw-page-title-main">Countercurrent chromatography</span>

Countercurrent chromatography is a form of liquid–liquid chromatography that uses a liquid stationary phase that is held in place by inertia of the molecules composing the stationary phase accelerating toward the center of a centrifuge due to centripetal force and is used to separate, identify, and quantify the chemical components of a mixture. In its broadest sense, countercurrent chromatography encompasses a collection of related liquid chromatography techniques that employ two immiscible liquid phases without a solid support. The two liquid phases come in contact with each other as at least one phase is pumped through a column, a hollow tube or a series of chambers connected with channels, which contains both phases. The resulting dynamic mixing and settling action allows the components to be separated by their respective solubilities in the two phases. A wide variety of two-phase solvent systems consisting of at least two immiscible liquids may be employed to provide the proper selectivity for the desired separation.

Radial chromatography is a form of chromatography, a preparatory technique for separating chemical mixtures. It can also be referred to as centrifugal thin-layer chromatography. It is a common technique for isolating compounds and can be compared to column chromatography as a similar process. A common device used for this technique is a Chromatotron.

Thermoresponsive polymers can be used as stationary phase in liquid chromatography. Here, the polarity of the stationary phase can be varied by temperature changes, altering the power of separation without changing the column or solvent composition. Thermally related benefits of gas chromatography can now be applied to classes of compounds that are restricted to liquid chromatography due to their thermolability. In place of solvent gradient elution, thermoresponsive polymers allow the use of temperature gradients under purely aqueous isocratic conditions. The versatility of the system is controlled not only through changing temperature, but through the addition of modifying moieties that allow for a choice of enhanced hydrophobic interaction, or by introducing the prospect of electrostatic interaction. These developments have already introduced major improvements to the fields of hydrophobic interaction chromatography, size exclusion chromatography, ion exchange chromatography, and affinity chromatography separations as well as pseudo-solid phase extractions.

<span class="mw-page-title-main">Chiral thin-layer chromatography</span>

Chiral thin-layer chromatography is a variant of liquid chromatography that is employed for the separation of enantiomers. It is necessary to use either

References

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  2. 1 2 Zhang, Meizhen; Yu, Qian; Guo, Jiaqi; Wu, Bo; Kong, Xianming (2022-11-28). "Review of Thin-Layer Chromatography Tandem with Surface-Enhanced Raman Spectroscopy for Detection of Analytes in Mixture Samples". Biosensors. 12 (11): 937. doi: 10.3390/bios12110937 . ISSN   2079-6374. PMC   9687685 . PMID   36354446.
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  4. 1 2 3 4 5 6 7 Stahl, Egon (1969), Stahl, Egon (ed.), "Apparatus and General Techniquesss in TLC", Thin-Layer Chromatography: A Laboratory Handbook, Berlin, Heidelberg: Springer, pp. 52–86, doi:10.1007/978-3-642-88488-7_3, ISBN   978-3-642-88488-7 , retrieved 2023-03-29
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  10. 1 2 3 Thin Layer Chromatography (TLC): Principle with animation
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  12. 1 2 Johnson, Andrew R.; Vitha, Mark F. (2011-01-28). "Chromatographic selectivity triangles". Journal of Chromatography A. EDITORS' CHOICE V. 1218 (4): 556–586. doi:10.1016/j.chroma.2010.09.046. ISSN   0021-9673. PMID   21067756.
  13. 1 2 Snyder, Lloyd R.; Kirkland, Joseph J.; Dolan, John W. (2009-11-11). Introduction to Modern Liquid Chromatography. Hoboken, NJ, USA: John Wiley & Sons, Inc. doi:10.1002/9780470508183.app1. ISBN   978-0-470-50818-3.
  14. 1 2 3 4 "SiliaPlate - TLC Practical Guide". SiliCycle. March 28, 2023.
  15. 1 2 Dickson, Hamilton; Kittredge, Kevin W.; Sarquis, Arlyne M. (1 July 2004). "Thin-Layer Chromatography: The "Eyes" of the Organic Chemist". Journal of Chemical Education. 81 (7): 1023. Bibcode:2004JChEd..81.1023D. doi:10.1021/ed081p1023. ISSN   0021-9584.
  16. ↑ Thin Layer Chromatography stains http://www.reachdevices.com/TLC_stains.html
  17. ↑ Jork, H., Funk, W., Fischer, W., Wimmer, H. (1994): Thin-Layer Chromatography: Reagents and Detection Methods, Volume 1b, VCH, Weinheim
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  19. ↑ Tables showing the thickness value of commercial regular and preparative Thin Layer Chromatography plates
  20. ↑ TLC plates as a convenient platform for solvent-free reactions Jonathan M. Stoddard, Lien Nguyen, Hector Mata-Chavez and Kelly Nguyen Chem. Commun., 2007, 1240–1241, doi : 10.1039/b616311d
  21. ↑ Bickler, Bob (22 November 2022). "Why is Solvent Evaluation by TLC Important for Good Flash Chromatography Results?". Biotage. Retrieved 1 April 2022.
  22. 1 2 3 Wing, R. E.; Bemiller, J. N. (1972-01-01), Whistler, Roy L.; BeMiller, James N. (eds.), "[8] - Preparative Thin-Layer Chromatography", General Carbohydrate Method, Academic Press, pp. 60–64, doi:10.1016/b978-0-12-746206-6.50015-1, ISBN   978-0-12-746206-6 , retrieved 2023-04-01
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  24. ↑ Fair, Justin D.; Kormos, Chad M. (2008-11-21). "Flash column chromatograms estimated from thin-layer chromatography data". Journal of Chromatography A. 1211 (1): 49–54. doi:10.1016/j.chroma.2008.09.085. ISSN   0021-9673. PMID   18849041.
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Bibliography

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