High Pressure Issues

[s]

Hi,

I have a TOSOH G4000SW gel filtration column. I had labelled some dextrans with Fluorescein isothiocyanate (FITC), a flourescent dye, and ran through column. I am having pressure issues when cleaning column in reverse direction with 20% methanol and am wondering if it is b/c the any residual dye (that was in my sample, not conjugated with dextran) did not come off column. I just can't get a flow going without pressure going crazy high. Should I be using a different solvent to clean FITC? There are no pressure issues due to system, already checked. This is just an idea, I really have no idea why I can't get flow through.

See http://www.sigmaaldrich.com/catalog/sea ... IGMA/F7250
for FITC structure.

S

[s]

I wanted to give everyone more information...

I have a new inlet column frit, installed 3/9/06
I have a new guard column, put in flow path 3/9/06
I also have a new frit in precolumn filter, put in flow path 3/9/06

Still, with flow rate at 0.01ml/min (not a typo), with column in reverse direciton, I got P to exceed 200psi, which is limit for my column when running 20% methanol. I also attempted to run water thru in reverse direction and got same P issues.

I have since, installed column in downflow direciton (i.e., regular flow direction), passed water through. So far, I have had no pressure issues at 0.01ml/min, 0.03ml/min, and 0.1ml/min. I DON'T UNDERSTAND WHY. CAN SOMEONE EXPLAIN TO ME WHY WATER CAN GET THROUGH...I KNOW IT IS SMALLER THAN CH30H but how can difference be so pronounced and how come it wouldn't go thru in reverse direction.

Even though water is going thru in downflow direction, this doesn't help me. I need to make sure column is clean, which would mean putting it back in reverse flow and passing methanol which it doesn't want to.

I would be ever so grateful if anyone has any explanation or ... well,
while writing this message, the pressure exceeded with water in downflow direciton at 0.1ml/min. Ok, so new question, why did it work for an hour w/ no issues and now, it won't flow thru at 0.1ml/min?

Note: I had posted yesterday about bubble problem but have since popped. I did see unevenness in resin on inlet side, should I just pull out and replace? I don't see how void (i am still hesitant to say there is a void) could cause P issues.


Extremely Frustrated.
Sue

[tom jupille]

Mixtures of water and methanol can have substantially higher viscosity than either water or methanol alone (I don't have the viscosity tables handy to give you specific numbers) so it's not unusual to see a higher back pressure.

That said, unless you're using narrow-bore columns, you should not see a pressure problem at those flow rates. Per the other thread, it's quite possible that you have precipitated material plugging up your packing bed.

[s]

do you have any suggestions for how to get precipitate out? What solutions to use? Obviously, methanol is not working. Is there someting that will slip in there and dissolve precipitate? Is water the only solution?

Also, do you think that residual FITC dye could have precipitated on the column resin? I had accidentally used a very high concentration of sample...15mg/ml 1K FITC-dextran on column, could that have caused problems?

I pretty much agree too, something has precipitated in my column. Is there any soln?

Sue

[tom jupille]

I've never worked with it, so I can't be of much help, sorry. On the other hand, even though it's an expensive column, it may be more cost-effective to simply replace it. You can then try fixing the old one at your leisure.

[s]

That's true, i can replace it but if i don't like that i don't know what i did wrong. if you have suggestions for how to maintain silica column, that would be great. do you think fitc dye had any effects? Thanks for your help though.

Sue

[HW Mueller]

Actually, one does checks into solvents which may clean a column before opening it. Your bubble/void could have been created during opening.... It doesn´t take too much special skill to kill a column by opening it.
On the solvents, one uses something that dissolves potential precipitates. You need to be careful to see to it that consecutive solvents are miscible and are allowed by the manufacturer (who usually gives good cleaning proceedures). Recently, I helped someone who was at the end with their nerves, because they tried to wash water out of their C-18 column with methanol, but the pump stopped soon after the methanol had hit the column, thereafter a few seconds on each restarting of the pump. Since this was done per phone I was stumped, went to that lab and saw that the pump pressure setting was way too low (a pressure recommendation was confused by the operator with the max allowable pressure). After slightly raising the setting, the pump happily did its job. Maybe your low flow rates are also based on a confusion?
There is no rule prohibiting you from washing in the forward direction (discussed extensively, before). If you get water through there you should be able to pass any other common HPLC solvent (with the restriction mentioned above). The only exception that I have seen to this is that organics MeOH, ACN) precipitated proteins on the frit.

[s]

Hi,

Thanks for the input. I will check pump settings.

How does one make sure not to kill column upon disconnection?

I recall perfectly that I was rinsing out column with water in downflow direction and no pressure problems.

It was only after I disconnected column, installed in reverse direction, and started methanol wash, did I have Pressure problems.

So, it is quite possible that I created bubble during disconneciton? How could I have avoided this?

Sue

[s]

Hi,

Found something interesting on both ends of column...looks like wx? It is rather deep. I have accepted that I need to by a new column. No wonder I can't get flow to go through. Anyone have a clue what it is and/or what is caused by? I have a TOSOH G4000SW column.

S

[HW Mueller]

Wax? How did the stuff look like that you injected? Honey?
One can only prevent, with certainty, an air bubble caused by opening a collumn by not opening it.

[s]

I worked with fitc labelled dextrans and nonlabelled dextrans with pbs as eluent.

s