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4.2 Column Chromatography

4.2.1 PREPARATION OF THE COLUMN (PDF version)

4.2.2 ELUTION OF THE COLUMN                                 


                           
When product is close running with another spot collect 20 fractions instead of 10.

Advantages of gradient elution method versus standard method:

  1. It works very well at separating products  - very rare that products come out mixed.
  2. Uses less silica
  3. Uses less solvent
  4. Is quicker to carry out: to prepare & elute the column should take no more than 1hr for the average column. The whole process including combining, evaporating fractions and clean up should take overall no more than 2 hours.

 

4.2.3 Detailed Procedure                                 

You will need the the following prepared solutions for TLC and running the column:
2.5L of each of the following combinations:- hex, 1%EA/hex, 2.5%EA/hex, 5%EA/hex, 10%EA/hex, 25%EA/hex, 50%EA/hex, 100%EA, CH2Cl2, 1%MeOH

Steps:

1. Find TLC solvent system

2. Determine column solvents

3. Select column size

4. Prepare the column

5. Add the loading solvent

6. Add the crude product mixture to the column

7. Add a small ammount of the column setup solvent

8/11. Fill column with setup solvent and other solvents with gradient order

12. TLC all the tubes



1. Find TLC solvent system
: where the desired product has a Rf ~0.3 using the set mixture of combinations (see table).

Use EtOAc/hexane mixtures. For very polar compounds use MeOH/CH2Cl2 mixtures. A good place to start is 25% EtOAc/hex then adjust according to the relative polarity.

NOTE: ALWAYS KEEP A SAMPLE OF THE CRUDE MIXTURE.


2. Determine column solvents: the column set up solvent will be the least polar of the solvents used: hexane or CH2Cl2.

Use the solvent table to calculate the required solvents as follows: identify the TLC solvent on the table and then elute with the previous 3 solvent combinations on the table. The product should elute from the column when you change to the 3rd solvent, i.e it will elute before you arrive at the calculated TLC solvent. you should not need to use this solvent combination.

For example: TLC in 25%EtOAc/hexane


3. Select column size
: based on the table (refers to mass of expected product – excess reagent doesn´t count).


4. Prepare the column:
add a thin layer of sand of aproximately 1 cm.

Then fill the column with silica (always in a gently motion) to a depth of approx 8cm – for easy to separate mixtures this can be reduced to 6cm or increased to 10cm for difficult to separate mixtures – it is generally best to use to LESS rather than more silica! For very apolar products more silica can be used and for very polar  compounds less should be used.

 

You should try to get a plane line of sand and silica, so they must be gently inserted in the column and then with your hand tap repeteadly in the column to get it as plane as possible.
Confirm if you have a stable column in the 8 cm line of silica.


5. Add the loading solvent:
this will be hexane (or CH2Cl2 for polar compounds) and compact the silica.

 

Once again, do not forget to tap gently in the column to straighten the silica layer.

Locate the erlenmeyer under the column and ready to start.

In order to compact the silica, the bellows are attached and then start slowly pumping to pass the hexane through the layers, compacting the column.

ALWAYS leave some solvent in the column (about 2 cm).
And then using a pipette or glass rod to slowly insert the solvent, in order not to disturb the silica layer.


6. Add the crude product mixture to the column: REMEMBER!! ALWAYS KEEP A SMALL SAMPLE OF THE CRUDE MIXTURE FOR LATER TLC ANALYSIS.
Dissolve the crude mixture in a polar solvent such as ether or CH2Cl2

Measure silica (approx 8 times the volume or 4 times the mass ( e.g 1g crude: absorb onto 4g/8ml of silica).

Add it to absorb the crude mixture.

Perform rotoevaporation.
If after evaporation it does not form a fine powder add a slight bit more silica reabsorb and add to the packed column (in this procedure, we didn't encounter that problem).
This is how the crude mixture should look after the rotovap, like a dried powder.
Add to the packed column the fine powder.
Wash the walls of the column.
If the column set up solvent is CH2Cl2 the crude product can be dissolved in CH2Cl2 and added via pipette. Do not use this method for non polar EtOAc/hex mixtures!!! or the column will fail!!!!!
Use the pear and pass it again.
Pass it all through the column, and then it should look like presented in the picture.


7. Add a small ammount of the column setup solvent
Do this in order to wash the walls of the column and wet the silica/product – push all solvent into the silica – protect this layer of silica by ADDING SAND – approximately 1cm.

 


8. Fill column with setup solvent (in this procedure hexane)
until the top of the column and elute all of this solvent into an erlenmeyer flask.
 
9. Change the solvent to the first in the gradient
and collect 10 tubes (or the equivalent volume in an erlenmeyer flask – without stopping the column and making TLCs of the collected fractions – use the table to calculate the required fraction size.
For close running spots you can increase the number of fractions collected for each gradient to 20. 
 
10. Change the solvent to the second in the gradient
and collect 10 tubes – if the product has an impurity you can TLC the fractions at this point to see if if it is all removed – if not continue with this eluent until it is all off.

11. Change the solvent to the third in the gradient and collect 10 tubes.

12. TLC all the tubes - TLC every second tube on one single plate. Spot a sample of the crude mixture as a reference. The product should appear between tubes 20-30.
Example of TLC eluted in 25% EtOAc/hexane
 
13.
If the product is just starting to elute maintain the polarity of the 3rd solvent until it is all off the column or increase to the solvent system of the TLC if there are no impurities directly below it.
 
14.
If further elution is necessary continue and then re-TLC every fraction in the area of interest – in the above example fractions 22 to 30.

 

Next - 4.3 Distillation