This protocol developed a method to estimate the yield of compounds on the TLC plate using the blue-LED illumination technique. The advantages of this method are that the blue-LED illumination technique is a safe, effective and cheap method, and allows the researcher to measure multiple samples at the same time. This method also can be applied to biochemistry and natural products chemistry.
To begin, dry the culture at 40 degrees Celsius for 24 hours. Transfer the dried culture into a 50 milliliter tube using sterilized tweezers and add 15 milliliters of ethyl acetate. Shake the mixture vigorously by vortexing for one minute and incubate the tube for one hour at 40 degrees Celsius with shaking at 200 RPM.
Then sonicate the mixture using a 40 kilohertz ultrasonic bath at 40 degrees Celsius for one hour. Next, centrifuge the mixture at 5, 000 G for one minute at room temperature and filter through an 11 micrometer filter paper. Extract the filtrate with an equal volume of sterile water in a separatory funnel.
After phase separation, collect the organic layer, evaporate it in a rotary evaporator, and dissolve the residue in two milliliters of ethyl acetate. Pack the column with normal phase silica gel as the stationary phase, and use n-hexane ethyl acetate and trifluoroacetic acid as the mobile phase. Load two milliliters of the extract onto the column and add the mobile phase solvent at a flow rate of one milliliter per minute to elute the extract.
After verifying the effluent by TLC to confirm the presence of lovastatin, evaporate in a rotary evaporator at 45 degrees Celsius until the solvent gets removed. Dissolve the residue in one milliliter of ethyl acetate and then mix with an equal volume of 1%trifluoroacetic acid. Centrifuge the mixture at 5, 000 G for one minute at room temperature, and collect the organic layer in a new glass tube.
Using a capillary pipette spot five microliters of samples and lovastatin standards onto the baseline of the TLC plate, leaving a border of one centimeter on the sides of the TLC plate. Then dry the TLC plate in a fume hood for five minutes at room temperature. Place the plate gently by forceps in a saturated glass chamber containing the mobile phase solvent.
Cover the chamber with a glass lid and allow the plate to develop fully. Remove the plate from the chamber when the solvents line reaches one centimeter from the top of the plate. Mark the solvent line with a pencil and dry the plate in the fume hood for 10 minutes at room temperature.
After drying, immediately soak the plate in 10%sulfuric acid solvent, and then dry in the fume hood for 10 minutes at room temperature. Next, place the plate on the heating panel until the brown spots appear. Transfer the plate to the blue-LED illuminator and scan using compatible freeware.
The results obtained from the bioassay method demonstrated that the R square between the dimensions of the inhibition zone and lovastatin standards was 0.99, and the sample yield was 0.56 milligrams as predicted by the regression model. The UV detection method shows that the R square between the lovastatin standards and the dimension of bands on the TLC plate was 0.97, and the sample yield predicted by the regression model was 0.53 milligrams. However, the band's edges were blurry and relatively low signal intensity bands were observed.
For the blue-LED illumination method, the R square between lovastatin standards and the band's dimension on the TLC plate was 0.98, and the sample yield was 0.54 milligrams as predicted by the regression model. The predicted yield using the blue-LED illuminator was closer to the bioassay method and clear bands were obtained. If the plate is overheated, the visualization of lovastatin may be difficult to observe.
