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Steps in Thin Layer Chromatography

In this steps in thin layer chromatography post we have briefly explained about principle, requirements, thin layer chromatography experiment procedure, Applications, uses and limitations of thin layer chromatography experiment.

Steps in Thin Layer Chromatography

Thin Layer Chromatography is an analytical tool commonly used in chemical laboratories to investigate the purity of organic compounds or to separate and analyse the constituents of complicated mixtures.

Thin layer chromatography experiment is a solid-liquid chromatography method, which means that the stationary phase is solid and the mobile phase is liquid. The stationary phase is made by covering a rectangular solid support with a very thin layer of polar adsorbent.

A glass, plastic, or metal plate, such as aluminium, can be used as a sturdy support. The most often utilised adsorbents are silica or alumina, which are deposited as uniformly thin layers on solid supports.

The adsorbent layers on analytical TLC plates are typically 250 µ m thick. Plates for preliminary separation might be as thick as 5 mm. To guarantee that the adsorbent adheres to the solid substrate, a binding agent such as calcium sulphate or gypsum is frequently included.

Principle

Thin layer chromatography experiment is done on a glass, plastic, or aluminium foil sheet that has been covered with a thin layer of adsorbent material, commonly silica gel, aluminium oxide (alumina), or cellulose. The stationary phase refers to the adsorbent layer.

A solvent or solvent combination (known as the mobile phase) is drawn up the plate via capillary action after the sample has been put to the plate. Separation is achieved because different analytes ascend the TLC plate at different rates.

It is thus based on the principles of adsorption chromatography, partition chromatography, or a mixture of the two, depending on the adsorbent, treatment, and solvents used. Components having a higher affinity for the stationary phase travel at a slower rate.

Once separated, the individual components are shown as spots at a specific level of movement on the plate, with components with a lower affinity for the stationary phase travelling faster. Detection techniques are used to determine their nature or character.

Requirements

Equipment

1. Chemical fume hood

2. Hot air oven

3. Weighing balance

Chemicals

1. Silica

2. Sodium acetate

Glassware

1. A pair of gloves

2. Development chamber

3. Filer paper

4. Glass capillaries

5. Glass microscopic slide

6. Glass pipette

7. Glass rods

8. Measuring cylinders

9. Pencil

10. Ruler

11. Tweezers

Thin Layer Chromatography Experiment

Plate Preparation

Thin layer chromatography experiment plates are readily accessible in the marketplace and are reasonably priced. The thin layer chromatography experiment plates, on the other hand, can be prepared by oneself. Plastic or glass is commonly used as the stationary phase support in commercially available thin layer chromatography experiment plates.

The plates are occasionally purchased as huge square sheets, which must be cut to the necessary size. If the plates have plastic support, they can easily be trimmed to the correct size using scissors.

Once the requisite dimensions are acquired, a line is drawn with a pencil parallel to the smaller side and 1 cm from one of the two tiny edges.

The to-be-analysed substance or mixture of compounds is dissolved in a suitable volatile solvent and spotted on the line. The plot spot is left to dry naturally.

Development

The eluant (a solvent or a mixture of solvents) is placed in a wide-mouth container known as the ‘development chamber.’

To produce a solvent saturated environment, a filter paper soaked in eluant is placed within the development chamber.

In the development chamber, put the dried thin layer chromatography experiment plate such that the solvent front is slightly below the sample spot (ensure that the plate does not touch the filter paper placed in the development chamber).

Due to capillary action, the solvent is taken up by the TLC adsorbent. The stationary phase (silica or alumina gel) is polar, but the eluant (mobile phase) is non-polar. The polarity of the molecules is used to divide them into the stationary and mobile phases.

The plate is removed using tweezers when the solvent front reaches within 1 cm of the top edge of the plate, and the solvent front is marked with a pencil. After that, the plate is air dried, and the spots are viewed.

Visualization

If the chemicals under investigation are coloured, they can be seen without any help. The majority of organic substances, on the other hand, are colourless. In most circumstances, ultraviolet light may be used to detect the spots.

Thin layer chromatography experiment plates containing fluorescent chemicals impregnated silica gel are commercially available. The compounds on the plates glow when exposed to UV light with a wavelength of 254 nm.

After the developing stage, the plate is irradiated with UV light. UV-absorbing analytes quench fluorescence and appear as dark patches against the brilliant green background.

The spots are pencilled in and the plate is removed from the UV light. If this procedure fails, the bands can be seen by exposing the plates to iodine vapours; iodine forms a dark-colored combination with a significant number of organic molecules.

Analysis

Thin layer chromatography experiment plates can be used to calculate what is called the retardation factor or the Rf value. Rf value of an analyte is the ratio of the distance traveled by the analyte to that travelled by the eluant front.

Thin Layer Chromatography

Steps in Thin Layer Chromatography

Applications

Purity of any sample: Thin layer chromatography experiment can be used to determine the purity of a sample. If any impurity is identified, the sample is directly compared to the standard or authentic sample; if any impurity is detected, it shows extra spots that may be easily detected.

Identification of compounds: thin layer chromatography experiment can be used to purify, isolate, and identify natural products such as volatile oil or essential oil, fixed oil, waxes, alkaloids, glycosides, steroids, and other chemicals.

Examination of reactions: thin layer chromatography experiment can be used to investigate the reaction mixture in order to determine whether it is complete or not. Other separation and purification procedures, such as distillation, molecular distillation, and so on, are also checked using this method.

Biochemical analysis: thin layer chromatography experiment is particularly effective in isolating or separating biochemical metabolites or constituents from body fluids such as blood plasma, serum, urine, and so on.

Chemistry: thin layer chromatography experiment is increasingly being utilised in chemistry for the separation and identification of molecules that are closely linked to one another. It’s also utilised in inorganic chemistry to identify cations and anions.

Pharmaceutical sector: In the pharmaceutical sector, thin layer chromatography experiment has been used to detect impurities in pharmacopoeial chemicals by a number of pharmacopoeias. Thin layer chromatography experiment has been used to test the quality of hypnotics, sedatives, anticonvulsant tranquillizers, antihistaminics, analgesics, local anaesthetics, and steroids. Separation of multicomponent pharmaceutical formulations is one of the most important applications of thin layer chromatography experiment.

Food and Cosmetics: Colours, preservatives, sweetening agents, and various cosmetic ingredients are separated and identified using the thin layer chromatography experiment in the food and cosmetic industries.

Advantages

1. Thin layer chromatography experiment necessitates less hardware. Separation of components is faster, resulting in time savings. The method is extremely sensitive and produces more precise findings.

2. Thin layer chromatography experiment has a lower detection limit for analytical samples that is one decimal lower than paper chromatography. Thin layer chromatography experiment also just requires extremely small amounts of a sample to analyse and produce findings. For labs with limited funds, this is a cost-effective solution.

3. To identify a sample in thin layer chromatography experiment, corrosive chemicals can be sprayed, however this is not possible in paper chromatography since cellulose is destroyed.

4. Because a large range of adsorbents are accessible, this approach is also used in adsorption, partition, and ion exchange chromatography.

5. Thin layer chromatography experiment breaks down the sample into its constituent parts. Scratching the plate’s granular layer will easily reveal these components. TLC also allows for the quantitative separation of spots.

6. Because the inorganic adsorbent backdrop does not glow, UV light may be used to see and identify the different components.

Limitations

1. The experiment’s results are difficult to duplicate.

2. Only applies to soluble combination components.

3. It’s qualitative research, not quantitative research.

4. This isn’t a straightforward procedure.

5. Because thin layer chromatography is an open system, humidity and temperature can have an impact on the results.

6. Because plate length is limited, the separation process is confined to a specific length.

7. It is unable to distinguish between enantiomers and some isomers.

8. The Rf values for the compounds of interest must be known ahead of time in order to identify individual compounds.

Further Readings

Reference

  1. https://www.milesscientific.com/applications-of-thin-layer-chromatography/
  2. https://microbenotes.com/thin-layer-chromatography/
  3. https://byjus.com/chemistry/thin-layer-chromatography-principle/
  4. https://open.umn.edu/opentextbooks/textbooks/biochemistry-free-for-all-ahern