REgeneration of HPLC column

[shantaram]

I am working with µbondapack ODS column 3.9*300mm (10µ). It is giving problems with peak resolution and pressure of column has also increased. Could you tell me the proper solvents for washing/regenerating this column to recover the efficiency.

[tom jupille]

If the problem is loss of bonded phase or dissolution of the underlying silica, then the column cannot be regenerated.

If the problem is caused by chemical contamination ("column killers") from the samples or the mobile phase, then you *may* be able to regenerate by flushing the column with something that is a good solvent for whatever type of contamination is present. This requires at least a guess as to the nature of the contamination, because a good solvent for fatty contaminants would be a bad solvent for salts or proteins (for example).

Aside from that, it's mostly common sense:
- do the regeneration in the "reverse flow" direction
- make sure any solvents you switch to/from are miscible and contain no insolubles (such as buffers).

In case of doubt, contact your local Waters rep.

[shantaram]

If the problem is loss of bonded phase or dissolution of the underlying silica, then the column cannot be regenerated.

If the problem is caused by chemical contamination ("column killers") from the samples or the mobile phase, then you *may* be able to regenerate by flushing the column with something that is a good solvent for whatever type of contamination is present. This requires at least a guess as to the nature of the contamination, because a good solvent for fatty contaminants would be a bad solvent for salts or proteins (for example).

Aside from that, it's mostly common sense:
- do the regeneration in the "reverse flow" direction
- make sure any solvents you switch to/from are miscible and contain no insolubles (such as buffers).

In case of doubt, contact your local Waters rep.

Thanks for the information.
I would like to add something that i was using mobile phase of pH 7.5, so there are chances of stationary phase dissolution. But plz tell me how i can escape from this situation in future.

[Consumer Products Guy]

I was using mobile phase of pH 7.5, so there are chances of stationary phase dissolution. But plz tell me how i can escape from this situation in future.

7.5 pH isn't too bad. Personally I would move into a more-modern column, such as a shorter, narrower-bore 3 or 5 micron column; you'd get improved results as well. That 300mm Bondapack 10 micron column technology is decades old, older than even my corduroy jacket !!!

Please don't tell me that you're assaying samples from the 1970s !!!

[tom jupille]

I would like to add something that i was using mobile phase of pH 7.5, so there are chances of stationary phase dissolution. But plz tell me how i can escape from this situation in future.

Two possible approaches:
1. Don't run at 7.5
2. Rework your methods for a more modern stationary phase.

As CPG pointed out, 7.5 is not terrible, but it *is* toward the upper end of recommended operating range. And Microbondapak was a good column in its day, but its day was 30 years ago.

Perhaps a more relevant question is "how old is the actual column?". Columns are, like tires on an automobile, a "consumable" item: they do wear out and must be replaced periodically.

[shantaram]

I would like to add something that i was using mobile phase of pH 7.5, so there are chances of stationary phase dissolution. But plz tell me how i can escape from this situation in future.

Two possible approaches:
1. Don't run at 7.5
2. Rework your methods for a more modern stationary phase.

As CPG pointed out, 7.5 is not terrible, but it *is* toward the upper end of recommended operating range. And Microbondapak was a good column in its day, but its day was 30 years ago.

Perhaps a more relevant question is "how old is the actual column?". Columns are, like tires on an automobile, a "consumable" item: they do wear out and must be replaced periodically.

Thanks a lot...

[danko]

Pressure increase should incite other thoughts than considerations of stationary phase dissolution!
Shantaram, what is the nature of your samples?

Best Regards

[shantaram]

[quote="danko"]Pressure increase should incite other thoughts than considerations of stationary phase dissolution!
Shantaram, what is the nature of your samples?

Best Regards[/quotes]
sample i am using for thw quantification is Leucovorin Ca(Calcium folinate)...
I am apologising for the delay in response...

[danko]

Without knowing too much about the chromatographic conditions applied to your method of analysis (hence not being completely certain of my hunch) I’d suspect some kind of precipitation of your analyte (or formulation compound/s) on the column and/or on the column frit.
It might be a good idea to examine the sample’s components’ solubility in the mobile phase – externally (i.e. outside of the HPLC system)

Best Regards