5 Key Factors Affecting Fermentation Process

Title (Aim):

To separate and identify the components of a mixture of amino acids using Thin Layer Chromatography (TLC).

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Theory:

Thin Layer Chromatography (TLC) is a simple and widely used technique for separating non-volatile mixtures. It is important in the identification and purification of compounds. TLC is faster and more sensitive than paper chromatography and can separate a wide variety of biological and chemical substances. In TLC, the stationary phase is a thin layer of adsorbent material (usually silica gel or alumina) coated onto a glass, plastic, or aluminum plate. The mobile phase is a suitable solvent or solvent mixture that moves up the plate by capillary action, carrying different compounds to different heights based on their polarity and solubility.

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Principle:

TLC operates on the principle of adsorption chromatography, where the separation is based on the differential adsorption of compounds to the stationary phase and their solubility in the mobile phase. Components with stronger interactions (higher adsorption) with the stationary phase travel slower, while those with weaker interactions move faster. After the run, the separated compounds can be visualized using detection reagents (e.g., ninhydrin for amino acids). Each component's Rf value (Retention factor) can be calculated and compared with standard values for identification.

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Requirements:

• TLC plates (pre-coated with silica gel)

• Amino acid mixture (e.g., glycine, alanine, leucine)

• Standard amino acids (optional)

• Capillary tubes or micropipette

• Chromatography chamber or glass beaker with lid

• Solvent system (e.g., n-butanol:acetic acid:water in 4:1:5 ratio)

• Pencil and ruler

• Ninhydrin solution (0.1% in acetone or ethanol)

• Spray bottle or cotton swab

• Hot air oven or hair dryer

• Forceps and gloves

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Procedure:

1. Take a TLC plate and using a pencil, draw a horizontal baseline approximately 1 cm from the bottom edge.

2. Mark 3–4 points along the baseline to spot the samples (mixture and standards).

3. Use a capillary tube or micropipette to apply a small amount of each amino acid solution at the marked points. Let each spot dry completely and reapply 2–3 times for better visibility.

4. Prepare the TLC chamber by pouring the solvent system to a depth of 0.5–1 cm and close the lid to allow saturation of vapors.

5. Once the spots are dry, carefully place the TLC plate vertically in the chamber, ensuring the solvent does not touch the spots.

6. Allow the solvent to travel up the plate undisturbed until it is about 3/4th of the plate’s height.

7. Remove the TLC plate and immediately mark the solvent front with a pencil. Let the plate dry completely.

8. Spray the plate with ninhydrin solution in a fume hood or apply it gently with a cotton swab.

9. Dry the plate in a hot air oven (or hair dryer) at 60–70°C for 5–10 minutes to visualize the spots.

10. Measure the distance travelled by each spot and the solvent front from the baseline.

11. Calculate the Rf value using the formula:
    $R_f = \frac{\text{Distance travelled by the compound}}{\text{Distance travelled by the solvent front}}$

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Observation:

Spot No.	Sample Name	Distance travelled by spot (cm)	Distance travelled by solvent front (cm)	Rf value
1	Amino acid A	X₁	Y	X₁/Y
2	Amino acid B	X₂	Y	X₂/Y
3	Amino acid mixture	X₃, X₄, ...	Y	X₃/Y, X₄/Y

Note: Compare observed Rf values with literature values to identify unknown components.

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Result:

The TLC plate showed the separation of different amino acids present in the mixture. The Rf values matched with those of standard amino acids, confirming their presence in the sample.

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Conclusion:

Thin Layer Chromatography effectively separated and identified the components of an amino acid mixture based on their adsorption and solubility. This practical demonstrates the utility of TLC in biochemical analysis and compound identification.

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