Chromatography Forum

Hi
I currently experienced some distinct changes in elution time using our agilent 1200 system. So the percentage of B at which the peptide elute, has changed from 49% (7 months ago) to 62% (this week).

Method:
5-65% ACN (0.1% TFA)/Water (0.1% TFA) over 60 mins, flowrate 1ml/min

Channel B: Water
Channel C: ACN

So my initial thought was that it could be an issue with the multi-channel gradient valve, namely channel C, delivering less ACN then supposed, which could explain the increased retention time. However, swapping channels (B: ACN; C:Water), resulted in the exact same retention time!

Running the analysis on our 1260 system (with a new G1311B pump) using the same method, the analyte eluted at 53% B.

I have previously been told that using ACN can result in polymer formation damaging the gradient valves over time, and that flushing with isopropanol might help, however this haven't solved the issue in my case.

Any thoughts and/or advice is highly appreciated

\Erik

Did you used the same coloumn on both systems and is it the same from 7 month ago?

Is the pressure similar?

When the retention time did not change when changing the channels I think this problem is not caused by the pump.

Yes I used the same column on the 2 systems, as well as 7 months ago.

I'll have to take a look at the pressure, didn't think of it.

\Erik

Have you changed the frits at the inlet of the solvent lines?

If these become clogged they can restrict flow enough to alter the gradient, and if both are clogged you could theoretically have the problem follow the more viscous solvent, namely the water.

Did you used the same coloumn on both systems and is it the same from 7 month ago?

Is the pressure similar?

When the retention time did not change when changing the channels I think this problem is not caused by the pump.

Actually the pressure profile looks a bit odd, considering that increasing ACN concentration should result in a lower back pressure.

Run from April:


Run from last week:


Any idea what could cause this increase in pressure?

In advance thanks
Erik

Was the pressure the same on the other system with this coloumn?

Was the overall pressure higher than expected?

If I were you I would unscrew the head of the coloumn and remove carefully the fitting. There should be a "grey" seal. When there are some particles remove them. I use water and cotton swaps to do this. (My method runs with water.)

On my HPLC this is very often a problem that causes higher backpressure and odd baseline drifts.

Because of this problem i'm using special column heads with cartridges.

When this is your problem be aware that your retention time can change just by opening the column.

Please do not open the column before you know that you can open it without problems. (Special column, pressed material etc.)

The pressure profile indeed looks odd, an increase in pressure doesnt't fit to the increasing ACN concentration, as you already remarked. Even if there's some plugging on the column frit, this shouldn't happen. You didn't, by chance, use Methanol instead of acetonitrile?
I'd double check that your mobile phases are what they're supposed to be, that everything is on the right channel and that the gradient profile in the method is correct.

The pressure profile wasn't quite identical, for the faulty 1260 system the starting pressure was lower:



I'm not sure whether it's possible to remove the end of the column without damaging it, so instead I have flushed it with isopropanol in the opposite direction for 30 mins, hoping this will do the trick.

I'll get back once I know.

\Erik

The pressure profile indeed looks odd, an increase in pressure doesnt't fit to the increasing ACN concentration, as you already remarked. Even if there's some plugging on the column frit, this shouldn't happen. You didn't, by chance, use Methanol instead of acetonitrile?
I'd double check that your mobile phases are what they're supposed to be, that everything is on the right channel and that the gradient profile in the method is correct.

Yes, I never use methanol, only isopropanol for flushing. The thing is that when performed on our other system an identical pressure profile is seen, which makes me lean against asm's observations. If methanol was used instead of acetonitrile I would assume that an earlier elution would be observed?

\Erik

Have you changed the frits at the inlet of the solvent lines?

If these become clogged they can restrict flow enough to alter the gradient, and if both are clogged you could theoretically have the problem follow the more viscous solvent, namely the water.

Not yet, just ordered new ones.

\Erik

Well the reverse column flush didn't do it, same retention time same pressure profile. I think I'll order a new column and I guess I might have to consider using a guard column.

Have you tried to use the column the direction you flushed?

If there are particles on the front seal of the column there should be not that big problem when using it the other direction.

If it is for example a "normal" C18 column from Agilent there is no problem to open the head with two wrenches.

Even flushing them does not ensure that you'll get rid of particles.

Have you changed the temperature at the column oven? Do you "control" the oven or is it just "off"?

Have you run the HPLC without the column and is there any considerable backpressure?

I think you use an C18 column (lenght? ID?) with 5%ACN + 95% H2O at 1ml/min at the beginning? What is the pressure before the start?

Have you tried to use the column the direction you flushed? No, not yet.

If there are particles on the front seal of the column there should be not that big problem when using it the other direction.

If it is for example a "normal" C18 column from Agilent there is no problem to open the head with two wrenches.

Even flushing them does not ensure that you'll get rid of particles.

Have you changed the temperature at the column oven? Do you "control" the oven or is it just "off"? It's set to 25C.

Have you run the HPLC without the column and is there any considerable backpressure? Not yet.

I think you use an C18 column (lenght? ID?) with 5%ACN + 95% H2O at 1ml/min at the beginning? What is the pressure before the start? Yes it's a Phenomenex Kinetex C18-XB, 5um, 100Å, 4.6x250mm. The pressure at start of the method is 127 bar.

Thanks for taking the time.

\Erik

The pressure profile indeed looks odd, an increase in pressure doesnt't fit to the increasing ACN concentration, as you already remarked. Even if there's some plugging on the column frit, this shouldn't happen. You didn't, by chance, use Methanol instead of acetonitrile?
I'd double check that your mobile phases are what they're supposed to be, that everything is on the right channel and that the gradient profile in the method is correct.

Yes, I never use methanol, only isopropanol for flushing. The thing is that when performed on our other system an identical pressure profile is seen, which makes me lean against asm's observations. If methanol was used instead of acetonitrile I would assume that an earlier elution would be observed?

\Erik

No, methanol is a weaker eluent than acetonitrile in reversed-phase, so a shift to higher retention times should be observed, just as you did see.
Honestly, I still think there's something wrong with your mobile phases or gradient programme. That "odd" pressure profile cannot be explained by a clogged column frit. Even if the column is partially clogged, a gradient with rising ACN concentration should lead to lower backpressure over the course of the gradient. Did you properly flush all the propanol out of the system? A propanol gradient, similar to a methanol gradient, would lead to increasing pressure, just as you're seeing...

The pressure of 127 bar seems really normal to me when using 95% water on such a column. A clogged frit should cause a much higher pressure. So I think using the column the other way will affect nothing.

In this case i'm consistent with HPLCaddict.