Preparation of reagents and TLC plate:

TLC plate :

1. 1. Prepare 20 ml of 20 mM sodium acetate solution.
   
   

2. Weigh 10 g of silica and add it to the 20 ml sodium acetate solution.


3. Mix thoroughly to obtain the slurry.


4. Wear the gloves and take 4 – 5 clean glass microscopic slides.


5. Place thin pieces of paper along the long sides of the slide as shown in figure 21.3, the thickness of paper determines the thickness of the adsorbent layer.




Figure 21.3: A diagram showing paper strips of equal dimensions placed along the long side of the glass slide


6. Pour the silica slurry on one edge of the slide and roll the glass spreader (glass rod) towards the other edge in a single stroke.


7. Air-dry the slides until they become white and smooth.


8. Bake the slides in a hot air oven at 120°C for 30 minutes.


9. Take out the coated slide and allow them to come to the room temperature.


10. The TLC plate is ready to use

Eluant : Butanol : Glacial acetic acid : Water (4 : 1 : 1)

• 1.  Pour 40 ml of 1-butanol, 10 ml of glacial acetic acid, and 10 ml of water in a 100 ml flask.

0.2% Ninhydrin solution :

1. 1.  Weigh 0.2 g of ninhydrin and dissolve it in 97 ml of 1-butanol.

2.  Add 3 ml of glacial acetic acid.

Procedure:

Spotting of samples on the TLC plate

1. 1.  Take a pencil and draw a light line, parallel to and 1 cm away from, one of the small edges. Be careful so that the silica layer does not get scraped off ( Note 3 ).

   2.  Prepare 1% (w/v) standard solutions of serine, phenylalanine, and lysine in water.

   3.  Using glass capillaries, load the serine standard solution, phenylalanine standard solution, lysine standard solution, and the given mixture of amino acids as four small spots on the spotting reference line (Figure 21.4).

2. 
   
3. Figure 21.4: A thin layer chromatographic plate with spotted samples

 

1. 4.  Load each of these solutions 5 times with intermittent drying using a hot-air blower or simply in the air.
   
   

5.  Air-dry the TLC plates for 10 minutes when the sample loading is complete; the plate can also be kept in the hot air oven at 70 ºC for 2-3 minutes for complete drying of the plate.

Development

1. 6.  Pour the eluant in the development chamber to get an eluant column of ~0.5 – 0.8 cm.
   
   
   7.  Take a piece of filter paper, dip it in the eluant and stick it inside the wall of the development chamber (Figure 21.5).
   

   8.  Close the development chamber and leave it aside for two hours to saturate the chamber with solvent vapors.
   

   9.  Place the dried TLC plate in the development chamber; ensure that the plate does not touch the filter paper kept inside the development chamber (Figure 21.5).

2. 
3. Figure 21.5: A thin layer chromatographic experiment set-up

 

10. Allow the eluant to rise till the eluant front reaches the desired height (~1 cm from the top edge of the plate).


11. Take out the plate and mark the solvent front using a pencil.


12.  Air-dry the plate for 20 minutes followed by 2 minutes drying in a hot air oven at 70 ºC.
 

Visualization and analysis

• 13.  Evenly spray the developed and dried TLC plate with the 0.2% ninhydrin solution (Notes 4 and 5 ).
  

  14.  Heat the plate at ~120 ºC for 3-5 minutes for drying the plate; purple-blue spots should appear under white light (Note 6 ).
  

  15.  Outline the spots using a pencil and measure the distance travelled by the spots relative to the spotting reference line.
  

  16.  Calculate the R_f values of the spots as shown in figure 21.2 and compare them with the standard spots.

Notes:

1. 1. It is tempting to score the glass multiple times to get a deeper cut but this usually makes it difficult for the glass to break along the scored line.
   
   

2. If the relative mobility of the components is very low, a run-over chromatography may be required to obtain separation of the components. In such cases the eluant reaches the top edge of the plate and starts dripping as an overflow. In such cases, it is not possible to determine the R_f values. However, R_s, the ratio of the distance travelled by the analyte to that travelled by a standard, can be determined.

1. 3.  Never use pen to mark the plate; the dyes present in the ink may appear in the chromatogram.
   
   

4.  The staining with ninhydrin should be done in a chemical fume hood and inhalation or contact with the skin should be avoided.


5.  Ninhydrin reacts with α-amino acids as shown in figure 21.6 to give purple-blue color except for proline that gives yellow-orange color.




Figure 21.6: Reaction of ninhydrin with an α-amino acid


2. 6. Proline is a secondary amine and gives yellow-orange color.