This modified procedure allows for recycling of the reverse phase thin layer chromatographic
plate. The results obtained using recycled plates are identical to results obtained using new plates as long as the absorbent surface is not disturbed. The number of times a plate may be recycled is limited by any disruption in the adsorbent surface due to handling of the plates, the actual spotting process and the residue left by the solvent front. Each time a plate has been recycled, it must be removed sooner from the developing chamber so that the residue from the previous solvent fronts do not overlap. With normal care, 80% of the plates can be recycled for a total of 3 to 4 cycles before the normal wear and tear of the experiment damages the plate surface.
Examine the plate, make sure there are no scratches or gaps in the adsorbent surface. Clean the plates by carefully soaking them individually in a bath of dichloromethane for 20 minutes. This dissolves the polystyrene on the surface without affecting the adsorbent. The pencil line and a line of residue from the solvent front does not dissolve. In order to recycle a reverse phase TLC plate, pencil marks indicating baselines, spots and solvent fronts much be avoided and the plate is developed a distance of 5 mm shorter than the previous development. This is so the residue from the previous solvent does not interfere with the current solvent front. This can be accomplished by substituting the following procedure for determining the molecular weight by thin layer chromatography.
- Prepare 100 mL of 71:29 stock solution of dichloromethane/methanol by pipetting 71 mL
of dichloromethane in a clean, dried stoppered bottle and adding 29 mL of methanol. Mix
the solution thoroughly and keep stoppered when not in use. This stock solution is
enough for two separate slide developments, as 50 mL is needed for each.
- Prepare a capillary spotter for each standard or sample that will be examined. Using
capillary tubing, draw out the tubing into a fine narrow spotter using a bunsen
- Place the plate on a piece of wide ruled paper. This allows for easy visualization
of lanes on the TLC plate. If care is taken during the spotting procedure, 10 lanes can
easily be run on a 10 cm plate without risk of overlap. Place a plastic ruler
perpendicular to the base of the plate, parallel to the wide rule lines on the paper.
This enables the student to place the spot 1 cm
from the bottom of the plate. By gently sliding the ruler across the place, multiple
spots may be introduced with the ruler serving as both a guide for the 1 cm baseline and
as a guide for the individual lanes. (See Fig.2). The 10 lanes allow several
permutations of standards and samples on the same plate. Typically, 5-7 standards are
needed for the calibration curve, leaving 3-5 lanes for samples to be analyzed. The
standards and samples should have molecular weights between 2,000 and 100,000 and be
prepared by dissolving 5 mg of polystyrene in approximately 1 mL of dichloromethane.
- Place a standard sample spot 1 cm from the bottom of the plate in the center of a
lane. Care should be taken to avoid overloading the plate at any one site. Check the
standards and sample spots with the UV lamp. If the spots can be visualized under the UV
lamp, then enough sample has been placed on the plate at that site.
- Place 50 mL of the stock solution in a 1000 mL beaker. This amount of solvent should
be sufficient to cover the bottom of the 10 cm x 10 cm reverse phase TLC plate without
being above the spots. Place this beaker or an equivalent developing chamber in a
location free of drafts.
- Place the TLC plate in the developing chamber gently. Lean the plate against the
side of the beaker so that the plate only touches the beaker at the top and the bottom.
Do not cover.
- Do not disturb the plate while it is developing. Remove the plate when the solvent
comes within 5 mm of the previous solvent front and gently measure the distance the
current solvent front has traveled with a ruler.
- Allow the plate to dry and examine it with the UV lamp. Place a plastic ruler
perpendicular to the base of the plate, parallel to the wide rule lines on the paper and
record the distance from the baseline to the bottom, the midpoint and the top of each
spot. (See Fig. 1 and 2).
- Determine the Rf of each of the recorded points for each spot by dividing
the distance from the baseline by the distance from the baseline that the solvent
- Plot log MW of each standard versus Rf for the bottom and the midpoint of
each corresponding spot. Obtain 2 calibration curves. The results of each should
correlate best with a second power polynomial fit. If access to a curve fitting program
is limited, reasonable results are obtained for a linear fit for a Log MW versus
Rf bottom and Rf midpoint.
- Using the calibration curves produced in Step 10, determine the molecular weights of
the bottom and midpoint for each of the unknown polystyrene samples.
- Under the conditions discussed above, the molecular weight derived from the bottom of
a spot correlates with the weight average molecular weight (Mw) for the sample. The
molecular weight derived from the midpoint of the spot correlates with the number average
molecular weight (Mn) for the sample.
- Calculate the polydispersity index for each sample by dividing Mw by Mn.
Once all vital information has been recorded, the plate may be soaked for 20 minutes in a
dichloromethane bath. This removes the polystyrene from the plate. Remove the plate from the
bath and allow it to dry. If the surface of the adsorbent has not been disturbed, the plate has been successfully recycled.