Thin-Layer Chromatography (TLC): Principle, Method Development Steps, FAQs

Thin-layer chromatography (TLC) is a technique used to separate components/pharmaceuticals in a mobile phase.

It is one of the most widely used traditional chromatographic techniques in the pharmaceutical industry due to its reliability, simplicity, rapid results, and cost-effectiveness. In this article, I will share my expertise on TLC, method development steps, with case studies and FAQs. By the end of this article, your understanding of TLC will be strengthened, clearing up any doubts and enhancing your knowledge to a new level.

Thin-Layer Chromatography (TLC)

Thin-layer chromatography (TLC) is a technique used to separate components/pharmaceuticals in a mobile phase. It is carried out on a TLC plate, which consists of a non-reactive solid substrate coated with a thin layer of adsorbent material. A small amount of the sample is applied to the plate, which elutes with a solvent or solvent mixture known as the mobile phase (or eluent).

Thin-layer chromatography Principle

Thin-layer chromatography comes under complete chromatography. It contains the solid stationary phase (coated on the plate) and the liquid mobile phase. Separation is governed by the polarity principle. In other words, separation depends upon the polarity of each analyte, the stationary phase and the mobile phase. A spot of the sample mixture is made on the TLC plate using a syringe or capillary tube, and the plate is placed in the TLC chamber containing the mobile phase. The mobile phase is moved over the TLC plate. Each Analyte of the sample mixture interacts with the mobile phase as well as with the stationary phase. During interaction, the stationary phase wants to retain the analyte, whereas the mobile phase wants to carry the analyte with it. The more polar compound retains more on the stationary phase (on the TLC plate) and moves slowly on the TLC plate compared to the nonpolar or less polar compound. Due to the difference in polarity, each compound interacts with the stationary phase and the mobile phase different capacity and they separate. The relative mobility of the compound is described by the R_f or Retention factor value.

R_f value or Retention factor value

R_f value or Retention factor value is the ratio of the distance covered by the compound to the distance covered by the solvent (mobile phase).

Rf value is unique for each compound under specific conditions such as the stationary phase, temperature, thickness of the adsorbent layer, the amount of the spotted material and the mobile phase used

RF calculation

• a: Distance travelled by the compound
• b: distance travelled by the mobile phase

Components of The TLC

The following are the different components of the TLC:

1. Thin Layer Chromatography Plates: Commercial TLC plates are made of Aluminium and coated with a stationary phase like Silica gel or Aluminium oxide. A TLC plate can also be made using a glass plate or a plastic plate
2. Mobile phase: A solvent or a mixture of solvents with different polarities is used as a mobile phase. Generally, Hexane, heptane, chloroform, dichloromethane, ethyl acetate, ethanol, and isopropyl alcohol are used as solvents in the mobile phase. Acetic modifiers like formic acid, acetic acid and basic modifiers like ammonia, triethylamine amine and diethylamine are also used in the mobile phases based on the polarity of the analyte.
3. Modifiers: Acidic modifiers like formic acid, acetic acid and basic modifiers like ammonia, triethyl amine and diethylamine are also used in the mobile phases based on the polarity of the analyte.
4. TLC Chamber: The TLC chamber is made of glass, and it is used to develop the TLC plate. This maintains a stable environment during analyte spot development in the TLC process. It also prevents solvent evaporation and keeps the entire process dust-free.
5. Filter paper: It is used to equilibrate the TLC chamber with the mobile phase
6. Syringe: It is required to apply the sample/standard spot on the TLC
7. UV chamber: It is required to detect the analyte spot
8. Colour development reagent: Reagents like Iodine vapour, Ninhydrin solution, KMnO4 solution and alkaline tetrazolium blue solutions are used for colour development

Expert Tips: Thin-layer chromatography Method Development Steps

TLC separation is governed by polarity. The mobile phase is selected based on the polarity of each analyte in the sample mixture. For polar molecules polar mobile phase is used, and for non-polar molecules, non non-polar mobile phase is used. The following steps play an important role in the TLC method development:

The following steps play a vital role in the TLC method development:

1. How to select the TLC Mobile Phase?
2. How to Select a TLC plate?

1. How to select the Thin-layer chromatography Mobile Phase?

The mobile phase is designed based on the polarity of each analyte in the sample mixture. Use

• Polar mobile phase for polar molecules
• Nonpolar mobile phase for nonpolar molecules
• Modifiers in the mobile phase to control the polarity

2. How to Select a Thin-layer chromatography plate?

Silica and Alumina are commonly used as a stationary phase in TLC. Therefore, the scope to play with the stationary phase during TLC development is limited.

Thin-layer chromatography Procedure

The following steps are involved in the TLC process;

• Make the point of spot application using a pencil. Keep in mind TLC spots should be evenly spaced and should not sink into the mobile phase.
• Apply the sample to the marked spots TLC plate using a syringe or a capillary
• Transfer the mobile phase to the TLC chamber
• Place the moistened (with mobile) filter paper along the inside wall of the TLC chamber. It maintains equal humidity and prevents the edge effect.
• Insert the prepared TLC plate into the chamber with the sample spot facing the mobile phase.
• Close the chamber and allow sufficient time (about 40 to 60 minutes) to separate each analyte spot during TLC development
• Remove the TLC plate from the chamber and allow it to dry
• Analysed the spot using UV light or by colour development (using KMnO4 or Iodine vapour)
• Calculate the R_f value

Applications

TLC is widely used for monitoring the reaction, identification test, purity test, related substances test, impurity profile test, compound purification, pesticide residue test, genotoxic impurity test and content test in the following industries:

• Research and development centre
• Pharmaceutical industries
• Pharmaceuticals formulation
• Cosmetic industries
• Pesticide industries
• Food industries
• Biochemical industries
• Polymer industries and
• Testing laboratory

Mobile phase optimisation

The composition of the solvent in the mobile phase is varied to get better separation between the spots.

• Increase the polar solvent (like ethanol, Isopropyl alcohol and Ethyl acetate) ratio to reduce/control the separation
• Decrease the polar solvent (like ethanol, Isopropyl alcohol and Ethyl acetate) ratio to increase the separation

Case study-1: Separation of N-(4-hydroxyphenyl)acetamide and 4-aminophenol by TLC

The following are the structure of Acetaminophen and 4 aminophenol:

Form the above structure it is clear that both compounds are polar but 4-aminophenol is more polar compared to N-(4-hydroxyphenyl)acetamide.

• Selection of TLC stationary phase: TLC plate containing Silica gel coating can be used
• Selection of mobile phase: Both 4-aminophenol is more polar compared to N-(4-hydroxyphenyl)acetamide are containing -NH and -OH functional groups. Hence polar mobile phase with basic modifier will be suitable choice. For example ; mixture of Hexane/Heptane (non polar) and Isopropyl alcohol/Ethanol (polar) with basic modifier like Triethyl amine/ammonia solution can be used
• Mobile phase optimisation: Increase the ratio of nonpolar solvent and reduce the modfier to get better separation between the spots
• Final TLC condition: In the following TLC mobile phase condition each spot of N-(4-hydroxyphenyl)acetamide and 4-aminophenol are well separated;
  • Final mobile phase: Hexane: Isopropyl alcohol:Triethylamine (90:10:0.05)

Case study 2: How to develop a TLC method for Tetrabutylammonium fluoride?

A bird’s eye view on method development approach:

• Molecular weight: C₁₆H₃₆FN
• Molecular weight: 261.46g/mol

Following is the structure of  Tetrabutylammonium fluoride

From the structure, it is clear that it is a quaternary ammonium salt. It does not have any chromophore and secondly, it is an ionic compound. Therefore it will neither analyzed on HPLC nor on GC.

The only possibility is to develop a qualitative TLC method. That is why widely qualitative TLC method is used for its content in Pharmaceutical substances.

In the following TLC condition it is quantified up to 0.05% in the pharmaceuticals

TLC condition

• TLC plate: Silica Gel 60 F254
• TLC mobile phase: mixture of Dichloromethane and Methanol in the rat io of 88:12.
• TLC chamber: Vertical chamber saturated with 25% ammonium hydroxide solution
• Diluent: Dichloromethane
• Sample concentration: about 150mg/ml in Dichloromethane
• Standard: Tetrabutylammonium fluoride 0.05% ( weigh about 7.5 mg of Tetrabutylammonium fluoride and dilute to 100 ml with diluent)
• Injection volume: 5µl
• Run: about 10 cm
• Detection: iodine vapour about 25 to 30 minutes

TLC Procedure

Apply 5µl of each standard and sample solution on the TLC plate and allow the spot to dry. Place the TLC plate in the equilibrated TLC chamber. Run the mobile phase up to 10 cm on the plate. Allow the plate to dry at the room temperature in the hood. Then put the TLC plate in the iodine chamber for 25 to 30 minutes. Compare the spot of Tetrabutylammonium fluoride in the sample with standard Tetrabutylammonium fluoride .

Thin-layer chromatography Troubleshooting

The following type of problem occurs during TLC analysis:

• Tailing in the analyte spot: If this problem occurs in the routine method then there is equilibration issue. Equilibrate the TLC system properly and then this tailing issue will be resolved
• Analyte spot is not moving from the point of application: If this type of problem occurs in the regular method then there is problem of solvent evaporation from the mobile phase. Prepare the fresh mobile phase, equilibrate the TLC system properly and then this issue will be resolved
• Analyte does not retain on the TLC plate and moves with mobile phase: If this type of problem occurs in the routine method then there is problem of mobile phase preparation. Prepare the fresh mobile phase, equilibrate the TLC system properly and then this issue will be resolved
• Distortion of the analyte’s spot: If this type of problem occurs in the routine method then there is a problem in the mobile phase and temperature of the lab. Prepare the fresh mobile phase, equilibrate the TLC system properly in temperature controlled environment and then this issue will be resolved

Advantages

The following are the TLC advantages:

• Selective technique with high separation power: Generally, normal phase TLC are used in the industries, and it is highly selective for qualitative analysis like identification test and purity test.
• Simple technique: It is a simple technique and does not need any special skill. Anyone can perform the TLC .
• Inexpensive technique: It is cost cost-effective analytical technique. That is the reason TLC is backbone of every industry
• Quick analysis: TLC is known for its fast results. One can get the result within 10 to 40 minutes. That is why it is widely used in the monitoring of the reaction.
• Versatile technique: It is a versatile technique and can be used for various types of compounds such as organic and inorganic compounds. For example content of Triethylamine and tetrabutyl ammonium bromide content in pharmaceuticals is performed by TLC
• High sensitivity: It is high sensitive instrument for qualitative analysis like identification and purity test
• Sample recovery: The Sample can easily be recovered after TLC analysis since volatile solvents are used in the TLC
• Visualisation: Analyte spots can easily be seen in the UV chamber or by color development
• Scale up potential: TLC technique can be used for scale-up after certain modification
• Regulatory acceptance: The TLC technique is acceptable by all regulatory agencies in the world

Disadvantages

The following are the disadvantage of the TLC technique

• Quantitative analysis: Less suitable for quantitative analysis
• Detection limit or sensitivity: The Sensitivity of the TLC is less than that of other chromatographic techniques, like HPLC/UPLC and GC
• Separation power: Limited stationary phases are available for the TLC, and hence, one can play only with the mobile phase during TLC development. That is the reason TLC has limited separation power.

New advancement in TLC

• TLC can also be performed in reverse phase chromatography mode (RPTLC). Now several new TLC plates containing C18 and C8 stationary phases are commercially available in the market.
• TLC is also used for Chiral purity using the chiral stationary phase.

RP-TLC stationary phase selection

TLC plates containing C18 and C8 stationary phases are used

RP-TLC stationary phase selection

Mixture of water and less polar organic solvents like tetrahydrofuran, methanol, ethanol, Isopropyl alcohol and acetonitrile are used.

Order of elution: Non-polar analyte retin more compared to polar analyte

Advantages of RP-TLC

• Humidity does affect the result since a mixture of aqueous and organic solvents is used
• Separation of analytes of different polarities, such as nonpolar, less polar and extremely high polar, can be possible
• RP-TLC results can be directly correlated with HPLC or UPLC
• A wide range of mobile phase selection availability and hence one can play a lot during mobile phase optimisation

Conclusion

Despite some limitations, TLC is widely used for various tests like mentoring the reaction, identifications, purity, related substances in the pharmaceutical industries due to its reliability of the result, simplicity and cost effectiveness. Now I hope this post has cleared all your doubts related to TLC and you can apply it more effectively in pharmaceutical development. For any queries or suggestion related to this article, write in the comment section or contact me using the contact form.

Related Topics:

• Relative Response Factor (RRF) in Pharmaceutical Analysis

FAQs

How is thin layer chromatography more superior than other chromatographic methods?

Due to quick result, costeffetiveness and simplicity it is more superior than other chromatography like HPLC and UPLC

How do you perform thin-layer chromatography?

Prepare the sample and a make sample-spot on the TLC plate. Develop the TLC in the TLC chamber and visualize the spot in UV chamber or by colour development

Can TLC be used for quantitative analysis?

Can be used but result will not be reproducible. TLC is mainly used for qualitative analysis

How long does it take to perform a TLC analysis?

TLC is a fast analysis band can be completed within an hour

Can TLC be used for analysing volatile compounds?

Not in all cases. But some of the volatile compounds, like triethylamine, is analyzed by TLC

Additional reading:

TLC Mobile Phase Chemistry: How to Understand

TLC can be operated in Normal phase chromatographic mode (NPC) and Reverse phase chromatographic mode (RPC). But it is widely used in NPC mode. The following solvents are widely used:

• Non-Polar Solvents (Hydrophobic)
  • Hexane (n-Hexane): Often used for non-polar compounds. It is commonly mixed with more polar solvents to adjust polarity.
  • Petroleum Ether: A mixture of hydrocarbons, often used for non-polar to moderately polar compounds.
  • Toluene: Slightly more polar than hexane and often used for moderately non-polar compounds.
  • Diethyl Ether: A low-polarity solvent, often used in mixtures with hexane or other solvents.
• Polar Solvents (Hydrophilic)
  • Ethyl Acetate: A relatively polar solvent used for moderately polar compounds, often in mixtures with non-polar solvents.
  • Acetone: Polar, good for separating compounds with moderate polarity.
  • Chloroform (CHCl₃): A moderately polar solvent, useful for separating a range of organic compounds.
  • Methanol (MeOH): Very polar, often used for highly polar compounds. Methanol is also commonly mixed with other solvents like chloroform, ethyl acetate, or water.
  • Water: Used in aqueous-based separations, often in combination with organic solvents.
• Mixed Solvent Systems
  • Hexane / Ethyl Acetate: A common mixture, where the ratio of hexane to ethyl acetate is adjusted based on the polarity of the compounds being separated. For example, a 9:1 mixture of hexane to ethyl acetate for non-polar compounds or a 1:1 mixture for more polar compounds.
  • Chloroform / Methanol: Often used for polar compounds, with varying ratios depending on the polarity of the analytes.
  • Hexane / Acetone: A mixture used for compounds with intermediate polarity, often in ratios like 7:3 or 9:1.
• Other Common Solvents or Mixtures
  • Dichloromethane (DCM): A moderately polar solvent, useful for a variety of organic compounds.
  • Hexane / Toluene: Used for non-polar to slightly polar compounds.
  • Acetonitrile (MeCN): A polar aprotic solvent, often used in gradient elution or in mixtures with other solvents.

Abbreviations

• TLC: Thin layer chromatography
• RPTLC: Reverse phase thin layer chromatography

Further reading:

• TLC
• https://www.sigmaaldrich.com/IN/en/applications/analytical-chemistry/thin-layer-chromatography
• https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/thin-layer-chromatography