Question

while doing FPLC my dye(MHI-148) got stuck in column? . so how can i clean the column efficient? and why some particle stuck in column?

Answer 1

While doing FPLC, the dye (MHI-148) got stuck in column.

The 2nd part of the question, i.e-" Why some particles get suck in column ?" is answered first.

This may be due to the size of particulate matter, that is being passed in or along with the mobile phase in the flow through. If the size of the dye or any particle is larger than the size of the pores, clogging is bound to happen. These pores are designed to let the mobile phase pass through them smoothly and not the particles.

Well, it can be easily prevented by having a close look at what is there in the sample.

1. Its recommended that one should know the size of particles in the sample. Its always a good idea to pre-filter the buffers and samples before use by passing them through syrige filters. The choice of size of syringe filter to be used should depend upon the pore size of LC column.

2. If the particle size is very large, one can also centrifuge the sample and use the supernatant for LC column. This way the pellet of particular matter will be prevented from entering the LC column.

3. Also, degassed buffers should be used to avoid formation of air bubbles in the column.

Now, the 1st part of the question- "How to clean the column efficiently?"

Well, column cleaning varies with the kind of gel filtration medium used.

In general, these are the steps of routine cleaning and its recommended to clean the columns after 10- 20 seperations.

1. Wash with 0.5M NaOH. The volume used should be equivalent to 1 column volume.

2. Wash with distilled water. Here also, the volume used should be equivalent to 1 column volume.

3. Equilibirate it with buffer until the baseline and pH are stable. Here, the volume used should be equivalent to 2 column volumes.

Moreover, inorder to remove the contamination, that is visible as a dark band in the column (eg. in this case where dye is stuck). following steps are generally recommended-

1. Reverse the flow and wash the column. All the washing steps discussed below should be performed at room temperature at a low flow rate of 25cm/h.

2. In order to remove hydrophobic proteins or lipoproteins, the columns should be washed with 1M NaOH and then distilled water. For both, the volume used should be equivalent to 4 column volumes.

3. Well, to get rid of lipids or highly hydrophobic proteins, its required that columns are washed with 30% isopropanol. The volume used should be equivalent to half of the column volume. Further, the column is washed with distilled water. The volume used should be equivalent to 2 column volumes.

4. After washing is done, the coulms are equilibrated with buffer before starting the experiment. The columns are equilibrated until stable baseline and pH are achieved. The volume of buffer used can be equivalent to 4-5 column volumes.