High Sucrose Soybean Thin-Layer Chromatography (TLC)

High Sucrose Soybean

Thin-Layer Chromatography

(TLC)

High Sucrose Soybean

Seed Preparations

High Sucrose Soybean Seed Preparations

Step 1

• Measure out one cup of soybeans for each variety called for in the protocol.

• Place the soybeans in a beaker and completely cover with water.

• Let stand for 18-24 hours.

High Sucrose Soybean Seed Preparations

Step 2

• Drain the water off the soybeans using a kitchen strainer and rinse with cold tap water.

• Use the soybeans in the protocol per the directions.

High Sucrose Soybean

Thin-Layer Chromatography (TLC)

Activity

High Sucrose Soybean TLCSolution Preparation

Step 1

• Put the soybeans and an equal amount of distilled water in a blender and blend on high for one minute.

High Sucrose Soybean TLCSolution Preparation

Step 2

• After blending is complete, filter the soybean mixture through two #6 coffee filters placed in a kitchen strainer.

• Collect the filtrate in a clean beaker.

• Repeat for each variety of soybean required in protocol.

High Sucrose Soybean TLCSolution Preparation

Step 3

• Label three 1.5 ml tubes. One with “N” for normal, one with “S” for standards, and one with “H” for high sucrose.

High Sucrose Soybean TLCSolution Preparation

Step 4

• Each sample is then diluted to 1/5th its original concentration by placing one drop of filtrate from each variety into the appropriate 1.5 ml tube and adding four drops of distilled water.

High Sucrose Soybean TLCTLC Plate Preparation

Step 1

• Each group should prepare a TLC plate. Make sure to turn the plate white-side-up before starting.

• Use a ruler to make a very light pencil line across the plate 15 mm from the bottom.

High Sucrose Soybean TLCTLC Plate Preparation

Step 2

• Then make three very light marks 1 cm from each edge of the plate and one mark 1.5 cm for the first.

• Label very lightly with pencil the marks from left to right; “N”, “S”, and “H”.

High Sucrose Soybean TLCActivity Step 1

• Fill a 10 ul micro-pipette by placing it in the tube marked “N”. Capillary action will automatically fill the tube.

High Sucrose Soybean TLCActivity Step 2

• Place the end of the tube on the TLC plate at the “N” mark and let the liquid draw out until there is a ~2 mm wide stain.

• Repeat the procedure with tubes “S” and “H”.

• Let the spot dry completely.

High Sucrose Soybean TLCActivity Step 3

• After drying, place the plate in a jar containing 91% rubbing alcohol and cap the jar tightly. The amount of alcohol in the jar should not be higher than the 1.5 mm line on the plate.

High Sucrose Soybean TLCActivity Step 4

• Allow the alcohol solvent to rise to the top of the plate. This can take from 1 1/2-2hrs.

• Leave in the jar overnight.

High Sucrose Soybean TLCActivity Step 5

• Next day, remove the plate and dry it with a hair dryer set on low.

High Sucrose Soybean TLCActivity Step 6

• When the plate is completely dry, wear gloves to handle the plates.

• Place it in the dipping reagent and quickly remove it.

• Use the hair dryer set on high to dry and heat the plate.

High Sucrose Soybean TLCActivity Step 7

• After a few minutes of heating the plate, black spots will appear in the lanes. These represent the individual carbohydrates found in soybean.

• The instructor will help you analyze your results.

High Sucrose Soybean TLCTLC Plate Analysis

Sucrose

Raffinose

Stachyose