Preparation Procedu res Preparation for TLC analysis i n cl udes prewashing the TLC plate, making fresh

4. Preparation Procedu res Preparation for TLC analysis i n cl udes prewashing the TLC plate, making fresh

eluent syste ms and detection reagents, and saturating the TLC chamber. The following preparation proced u res are started 24 hours prior to analysis:

1) Prepare cel l u l ose TLC plates The cel l u lose plate m u st be washed in methanol before analysis. This procedure takes approxi mately 4 hours . Place 3 0-60 m l of methanol i n a clean conventional TLC chamber. Allow the chamber to equilibrate with methanol for approximately 30 m i n utes . The cellu lose TLC plate is

i nserted vertically i nto the methanol, and the chamber is covered with the lid. Allow the methanol to rise to the top of the cellu lose TLC plate . Remove the plate from the chamber and d ry it in a fume hood. Sto re the cleaned cel l u lose TLC plate in a desiccator contai n i n g sil ica gel .

2 ) Prepare e l uent

Mix butanol, acetic aci d , and water i n an 80:20:20 volume ratio. Seal the sol ution in an amber bottle to mai ntain fresh ness before use. Prepare 60 m l o f t h e eluent fresh daily for a n analysis.

3) Prepare TLC chamber Presatu rate chamber with solvent system at least 4 hours before analysis. (N ote : It is useful to presatu rate the cham ber overn ight.) To do this, place

3 0-60 ml of the eluent inside a clean , d ry conventional TLC chamber. I nsert a saturation pad into the solvent syste m . Cover the cham ber with a l i d .

4) Prepare n i nhydrin detection reagent Weigh out 0. 1 58 g of ni n hydrin into a 250-ml amber bottle. Add 1 00 ml of ethano l . Mix thorough ly. The reagent can be stored in a refrigerator for 4-5 weeks.

5 . TLC Analysis Procedures

To an alyze protei n hydrolysates by TLC, the samples are spotted in i n d ivid ual lanes at the baseline of a prewas hed cellu lose plate. The plate is placed i n a satu­ rated conventional TLC chamber contai n i n g a saturation pad and the eluent (butanol : acetic acid : water, 80:20: 20) . The development of the plate is com plete

Identification of Proteins by Thin-Layer Chromatography

the visualization of the separation zones or spots. After 24 hours the plate can be documented .

The followi ng n i n e steps describe the proced ure for analysis:

1 ) Draw the baseline Using a ruler and pencil, li ghtly d raw a l i n e 1 cm from the bottom edge of the plate. Very l ightly m ark the lanes with short tick marks at intervals of 1 cm along this baseline, fo r a total of 1 9 lanes. In the u pper left corner, n u m ber the plate with a reference n u m ber, used to relate the TLC to i nformation in the research notes. Beside the n u m ber, place the date and the analyst's in itials. Place a mark 1 7 cm from the baseline as a reference to help determ i n e the completion of the deve lopment.

2) Apply the standard and reference solutions to the plate All solutions are applied followi ng the spotti ng proced ure noted in Protocol H . Apply 1 .0 � I o f t h e reference or standard solution t o a tick mark o n the origin of a lane using a capillary pi pette. The total vol u m e m ay be applied i n a series of smaller volumes to m i n i m ize the d i ameter of the spot. An air gun may be used to rapidly evaporate the carrier solvent between appl ica­ tio ns. Take care not to get the air gun too close to the pipette, as the

sample will evaporate.

3) Apply u n known sam ple solutions If pOSSible, apply each u n known sample i n two d i fferent vol umes. For exam ple, i n one lane apply 1 . 0 �I of the u n known sam ple, and in a seco nd lane apply 0.2 � I of the same solution . (The u n known sample may or may not

be very concentrated , and this proced ure m i n i m izes the possibil ity of over­ load i n g the plate .)

4) Develop the TLC plate Once the plate is spotted , either develop immediately or store i n a desiccator. To develop the plate , q u ickly i n sert the spotted cellu lose TLC plate i nto the

satu rated chamber, with the baseline o riented toward the bottom of the cham ber and the front faci ng away from the saturation pad . Replace the lid

of the cham ber. Do not leave the chamber open for any length of time, as the vapor phase equilibrium will be lost.

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