Separation by Chromatography

Chromatographic Separation of Sodium Fluorescein and Methylene Blue

In this two-part lab the students will:
• Learn the techniques associated with thin layer and column chromatography.
• Determine the effects of the stationary phase in separating a mixture by chromatography.
• Determine the effects of the mobile phase in separating a mixture by chromatography.
• Conduct a separation using column chromatography.

In the first week, the students will determine the best solvent system for separating a methylene blue/sodium fluorescein mixture by thin layer chromatography using silica gel and alumina as the stationary phase. In the second week, the students will separate a methylene blue/sodium fluorescein mixture by column chromatography with silica gel as the stationary phase.

Part I: Thin Layer Chromatography (TLC)
1. Obtain 2 silica-gel-coated TLC sheets and 1 alumina coated TLC sheet. Measure up 1 cm from the bottom of the TLC strip. Taking care not to damage the white, powdery stationary phase, use a #2 pencil to draw a line across the width of the strip. Carefully mark two evenly spaced spots along the line to delineate where to spot the dye solution.
2. The TLC strip can now be spotted. Use the capillary tube to apply two dots of the dye onto the TLC strip as in the figure 1 below.

3. Transfer enough solvent into a 200 mL beaker to achieve a level of about 0.5 cm deep. Cover the developing chamber with a glass lid or with a watch glass if a beaker is used. For best results, the developing chamber’s atmosphere must be saturated with the solvent. This can be accomplished by lining the developing chamber with filter paper. Place a trimmed sheet of filter paper in the jar.

Perform the following 3 TLC experiments:

Stationary phase
Mobile phase
TLC 1
Silica gel
95% ethanol
TLC 2
Silica gel
1:1 water : acetic acid
TLC 3 alumina 95% ethanol

4. Place the TLC strip into the developing chamber with the spotted end at the bottom. Cover the developing chamber as shown in Figure 2.

5. The solvent will be slowly absorbed by the TLC strip. When it is about a centimeter from the top, remove the strip from the chamber and immediately mark the solvent front with a pencil.

6. Calculate the Retention Factor, Rf, for both the sodium fluorescein and the methylene blue as shown in figure 3. The spot should be measured at the middle of the spot.

Part 2: Column Chromatography

Each group will conduct a column chromatography experiment to separate the dye mixture using silica gel as the stationary phase.

1. Insert a small plug of cotton into the neck of a Pasteur pipette. The cotton plug must be packed well but not too tightly into the tip using a long piece of wire. The cotton plug is then covered with a thin layer of sand (about 0.3 g).

2. Silica gel (about 1 g) is added to the column and it is packed by gently tapping the outside wall of the column with your finger or a pencil. The eluting solvent (95% ethanol) is then added to the column and allowed to flow through to “wet” the column. If needed, add more solvent.

3. Allow the solvent to flow through the column until only about 0.5 cm remains above the stationary phase.

4. Carefully, add about 0.5 mL of the methylene blue/sodium fluorescein mixture to the top of the column.
5. Allow the mixture to flow just below stationary phase (silica gel). Carefully add a thin layer of sand and fill the column with eluting solvent.

6. Allow the solvent to elute through the column. When the solvent gets close to the top sand layer, use a dropper pipette to add more solvent. Never allow the column to become dry.

7. When the first component of the mixture appears ready to elute from the column, place an empty 50-mL Erlenmeyer flask under the stopcock to collect the eluent. Once the first component has been eluted, remove the flask and label it.

8. Add 50% glacial acetic acid to the top of the column. Collect the second dye component in another Erlenmeyer flask.