Preparation Procedu res P reparation for TLC analysis i ncludes prewashing the TLC plate and making fresh

4. Preparation Procedu res P reparation for TLC analysis i ncludes prewashing the TLC plate and making fresh

eluent systems and detection reage nts. The fol lowing preparation procedu res are started 24 hours prior to analysis:

1) Prepare si l ica TLe plates The silica gel plate is washed with methanol, then heated before use. This proced u re takes about 2 hours . The plate can be washed in a sandwich

chamber or a conventional chamber. The proced ure described here u ses a sandwich chamber.

Place 5 m l of methanol i n the trough of a TLC sandwich chamber. Position the sil ica gel plate so that the stationary phase faces the backing plate and adj ust the spaci ng using the spacing guide. Attach the silica plate to the backi n g plate with two clips. Make sure that the clips are attached near the top of the plate. I n sert the assembly vertical ly i nto the trough contai n i n g the methan ol. Place all parts i n side a cylindrical TLC chamber and cove r with the l i d . Al low 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 cham ber and d ry it in a fume hood . Place the plate in an oven at 1 00 °C fo r 1 hour to activate the silica gel . Cool and store the clean , activated silica plate i n a desiccator contai n i n g s i l ica gel . The plate is ready for use after it cools (usually

Identification of Waxes by Thin-Layer Chromatography

+- Clip � Backing plate

Back of TLC plate

Cylindrical chamber

Trough

2) Prepare el uent Prepare 60 m l of the eluent fresh daily for an analysis. Mix petroleum ether, ethyl ether, and acetic acid i n a 90: 1 0 : 1 ratio . Seal the solution in an amber bottle to maintain freshness.

3) Prepare cyl ind rical chamber Presatu rate the cylind rical TLC chamber with 1 0 m l of the solvent system at least 30 m i n utes before analysis. Place the trough of the sandwich chamber

i nside the cylind rical cham ber. Cover the cyl i n d rical chamber with a l i d .

Add 10 ml of eluent to cylindrical chamber

Cylindrical chamber

Sandwich chamber trough

4) Prepare anisal dehyde detection reagent Measu re 0 . 5 ml of anisaldehyde into a 250-ml amber bottle . Add 1 0 ml of concentrated acetic aci d , 84.5 ml of methanol, and 5 ml of concentrated

su lfuric acid . Mix thoroughly. The reagent can be stored fo r 1 -2 weeks. Reminder: H ighly corrosive concentrated acetic acid and su lfuric acid are used in this detection reagent. They should be hand led with extremecare i n

Striegel . H i l l

5. TLC Analysis Procedures

To an alyze waxes by TLC, the chloroform extracts of sam ples are spotted in i n d i ­ vidual lanes a t t h e baseline o f a prewashed, activated H PTLC si lica g e l plate . The

plate is attached to the backing plate of a sandwich chamber and i nserted into a small tro ugh contai ning the eluent (petroleum ether : ethyl eth er : acetic acid ,

90: 1 0 : 1 ) . The development o f the plate takes place inside a cylind rical TLC chamber to m i n i m ize the effects of air cu rrents on the movement of the solvent fro nt. This also m i n i m izes the premature evaporation of the solvent syste m . The

development of the plate is com plete when the eluent front reaches a d i stance of

8 cm from the basel i n e . The plate is removed and d ried i n a fume hood before spraying with the an isaldehyde reagent. Patte rns of fl uorescent spots are visi ble for each sample when the plate is viewed under ultraviolet light. The plate may be

photographed fo r documentation i m med iately. 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 line 1 cm from the bottom edge of the plate. Very lightly mark 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, u sed 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 com pletion of the development. If this mark is scored heavi ly through the silica gel , it will be visible on both sides of the plate.

Top #, date, initials

s 8 em u

Baseline

Bottom

2) Apply the reference solutions to the plate All solutions are applied fo llowing the spotti ng proced u res noted in Protocol

H. Approxi mately 1 0 min utes before analysis, gently heat the reference solu­ tions to 40 °C on a hot plate. Apply 0.5 f,l l of each reference solution on a tick mark at the origin of a lane, using a capil lary pi pette. The total volume may

be applied i n a series of smaller volumes to m i n i m ize the diameter of the spot. An air gun may be u sed to rapidly evaporate the carrier solvent between appl ications. Take care not to get the air gun too close to the

pipette, as the sample will evaporate.

Identification of Waxes by Thin-Layer Chromatography 111

lane apply 1 . 0 JJ I of the u n known sam ple, and i n a second lane apply 0.2 JJ I of the same solution. (The u n known sample may or may not be very concen­ trated , and this proced ure m i n i m izes the possibil ity of overload ing the plate . )

4 ) Develop t h e TLC plate Once the plate is spotted, develop i m mediately or store i n a desiccator. Open the lid of the cylind rical cham ber slightly, and q u ickly place 5 m l of eluent in the trough of a TLC sandwich chamber, located i n side the cylind rical chamber. Position the silica gel plate so the stationary phase faces the backi n g plate and adj u st spaci ng between the edge of the plate and the rim of the back ing plate with the spaci ng guide. Attach the sil ica plate to the backing plate with two clips. Make sure that the clips are attached

near the top of the plate , next to the spacers . Quickly open the cyl i n d rical cham ber and set the bottom edge of this assembly i nto the trough con­ tai ning the eluent. 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.

Backing plate Back of

TLC plate

Cylindrical chamber

Trough

5) Completion of development Develop the plate for a d istance of 8 cm. Development usually takes about

1 5 m i n utes.

6) D ry the plate Remove the plate from the chamber and let it d ry for about 30 m i n utes at

room tem perature in the fume hood .

7) Prepare to spray plate Sprayi ng of an ani saldehyde reagent should always be performed u nder a well -ventilated fume hood or some other device to ensure effective removal of the reagent cloud and solvent vapors, which are corrosive and toxic. Protective glasses, laboratory gloves, and a resp i rator should always be

worn d u ring spraying. Set the plate on a clean, d ry spray stand i n side

Striegel . H i l l

8) Spray plate w ith anisaldehyde Hold the reagent sprayer 8-1 0 cm from the su rface of the TLC plate and spray the plate slowly back and forth, then u p and dow n , u ntil the plate is evenly covered. This is usually when the thin layer of sil ica gel is wetted with

j u st enough detection reagent to begin to turn transparent.

" . ' ..... � ..

Start

: ..... . . :'

Stop

9) Heat plate

D ry the plate for 1 5-20 m i n utes i n the fume hood , then place it i n a preheated oven at 1 00 °C for 1 0 m i n utes.