Preparation Procedures Preparation for TLC analysis i n cl udes prewas h i ng the TLC plate and making fresh

4. Preparation Procedures Preparation for TLC analysis i n cl udes prewas h i ng the TLC plate and making fresh

eluent syste ms and detection reagents. The following preparation procedu res are started 24 hours prior to analysis:

1) Prepare si l ica TLC plates The s i l ica gel plate is washed with methanol, then heated before use. This proced ure takes about 2 hours . The plate can be washed i n a sandwich chamber or a conventional chamber. The proced u re described here uses a sandwich chamber.

Place 5 m l of methanol i n the trough of a TLC sandwich chamber. Position the silica gel plate so that the stationary phase faces the backing plate, and adj ust the spacing with the spaci ng guide. Attach the sil ica plate to the backi n g plate with two clips. Make s u re the clips are attached near the to p of the plate. I n sert the asse mbly vertically into the trough contai n i n g the methanol. Place the assembly inside a cylind rical TLC chamber and cover with the l i d . Allow the methanol to rise to the top of the silica gel plate . This process usually takes 20 m i n utes . Remove the plate from the chamber and

d ry it i n a fume hood. Place the plate in an oven at 1 00 °C for 1 hour to activate the sil ica gel . After cooling, store the clean , activated silica plate in a desiccator containing si lica gel . The plate i s ready for use after it cools (usually 30 m i n utes after it is removed from the oven).

Identification of Carbohydrates by Thin-Layer Chromatography

Backing plate Back of

TLC plate

Cylindrical chamber

2) Prepare eluent Prepare 60 m l of the elu ent fresh daily for an analysis. Mix aceton itrile and water in an 85 : 1 5 ratio. Seal the sol ution i n an amber bottle to mai n­

tain freshness.

3 ) Prepare cyl ind rical chamber

Presatu rate the cylind rical TLC chamber with 10 m l of the solvent system at least 30 min utes befo re analysis. Place the tro ugh of the sandwich cham ­

ber inside the cyl i n d rical chamber and cover it with a l i d .

Add 10 ml of eluent to cylindrical chamber

Cylindrical chamber

Sandwich chamber trough

4) Prepare aminohippuric acid detection reagent The detection reagent is a 0.3 % solution of aminohi ppuric acid with

3 % phthal ic acid in ethanol. Weigh out 0.24 g of aminohi ppuric acid and

2 .4 g of phthalic acid i nto a 250 ml amber bottle. Add 1 00 m l of etha- nol. Mix thoroughly until all solids are d issolved . This reagent can be stored for 2-3 weeks.

Striegel . Hill

5 . TLe Analysis Procedu res

To analyze carbohyd rates by TLC, the hyd rolysates of samples are spotted in i n d i ­ vidual lanes a t t h e baseline o f a prewashed, activated H PTLC silica gel plate. The plate is attached to the backing plate of a sandwich cham ber and i nserted

i nto a small trough contai n i n g the eluent (aceton itrile : water, 85: 1 5) . The development of the plate takes place inside a cyl i n d rical TLC chamber to red uce the effects of air cu rrents on the movement of the solvent front. This also

m i n i m izes the prematu re evaporation of the solvent syste m . The development of the plate is com plete when the eluent front travel s a d i stance of 8 cm from

the baseline. The plate is removed and d ried in a fume hood before spraying with the aminoh i ppuric acid reagent. Patte rns of fl uorescent spots are visible for each sample when the plate is vi ewed under u ltraviolet light. The plate m ay be

photographed fo r documentation immediately. The following nine steps describe the proced u re for analysis:

1 ) Draw the baseline

Using a ruler and penci l , lightly d raw a l i ne 1 cm from the bottom edge of the plate. Very l ightly m ark the lanes with short tick marks at interval s of 0.5 cm along this baseline, for a total of 1 9 lanes. In the upper left corner, n u m ber

the plate with a reference n u m ber, used to relate the TLC to information in the research notes. Beside the n u m ber, place the date and the operator's

i n itials . Place a mark 8 cm from the baseline as a reference to help determ ine the completion of the development. If this mark is scored heavily through the silica gel , it will be visible on both sides of the plate .

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