retention time

[amaryl]

I thank you all for your help and advice.

I'm now checking the flow rate during the day. The pressure is very stable.
As soon as I finish, I'll have two different scenarios:
1. the pump can't work properly at that fixed flow rate
or
2. the column isn't the best column, a naughty C8.
In any case I think I'll have to review the method.

A further info is that now the Rts seem to be steady and my standard peak is now set at 10.7 mins, more or less. When the column was new, this Rt was 12.5 mins.
It seems that the column took two weeks to equilibrate...

I'll let you know the results of the flow check, as soon as it is over.

'Pump can't work properly'...so this implies the flow rate must be unstable. This decreasing RT suggests volume out is more than expected. Collect from the waste 10 ml in flask. Set your stop watch and flow rate at 1.0 ml/min. Volume should be 10 ml if pumps are working properly.

See the valves of the pump...does piston wobbles while moving in or out.
If pumps are working properly, flow rate should be 1.0 ml when set at 1.0 ml/min.

Even an excess of 100 microliter at flow rate of 1.0 ml/min will shift the RT.

How can you say its naughty. Is it some technical term or what I am thinking naughty

Check the LCGC article over retention time changes.

http://www.lcgcmag.com/lcgc/data/articl ... rticle.pdf


Why will column take two weeks to equilibrate. Its juzt that you are not able to set a proper chromatographic conditions.

Flow rate, pressure, Mobile phase composition, temperature...

Regards,

Amaryl.

[paolino]

I checked the flow. It is stable and what it flows is what I set the whole day long.
As I started analyses again on monday the RTs drifted again towards shortest time. New STD Rt is 10.5 mins
I think my column is spoiled.
FIY I'm analysing carotenes in a drug formulation.

[juddc]

I've seen similar RT behavior in isocratic runs and here are my two cents:

I don't necessarily think that your problem is linked to temperature because you'd only typically see a 1% change in RT for every degree C change in temperature. Your RT change seems to me to be a bit too great for that unless you have broad temperature swings in the lab on a daily basis. Also, pumps don't typically speed up in a repeatable manner on a daily basis. I'd bet this pump is dandy.

A question: How long do you equilibrate in the morning?

If quite a while (30-40 column volumes?), I'd not be surprised if you're loading up the head of the column with some material that is more hydrohphobic than your analyte during your run which then washes off when you're equilibrating the following day. You wouldn't necessarily see a change in backpressure because you're not blocking flow, just blocking your analyte's access to separation medium.

Try rinsing your column with a more hydrophobic mobile phase (Neat MeCN or 3:1 MeCN:IPA, maybe) every few runs and see what happens.

CJ

[juddc]

Don't toss the column before cleaning it and rechecking it. I'd bet without a cleaning step, the same thing will happen to any column you use to run the method. Heck, if you're determined to throw the column away, you can send it to me!

Good luck!

[amaryl]

A question: How long do you equilibrate in the morning?

If quite a while (30-40 column volumes?), I'd not be surprised if you're loading up the head of the column with some material that is more hydrohphobic than your analyte during your run which then washes off when you're equilibrating the following day. You wouldn't necessarily see a change in backpressure because you're not blocking flow, just blocking your analyte's access to separation medium.

Try rinsing your column with a more hydrophobic mobile phase (Neat MeCN or 3:1 MeCN:IPA, maybe) every few runs and see what happens.

CJ

From where this hydrophobic substance comes from...must be injecting solution...

If the injecting solution is containing some hydrophobic substance more hydrophobic than the analyte ...even if their is another equilbration of column following day, then won't it be still in to scene with next injection.

I could not get this sir, could you please elaborate on this.

Regards,

Amaryl.

[juddc]

If he has a relatively large quantity of hydrophobic material in his sample that is hanging up on the column, it may very well wash off the next day with a long equilibration in the morning, but it may NOT wash off between injections and therefore in some way shield his analyte from the C8 causing shorter retention. He says that if he continues running through the night, RT's keep getting shorter but if he stops, allowing the column to soak in MeOH overnight, then re-equilibrates, it's possible for performance to be restored IF the material isn't too tightly bound to the column. Being that he's assaying carotenoids, I'd have a look at the carrier / excipient mix as a culprit.

If he washes the hydrophobic substance off with a more hydrophobic mobile phase between injections, I'll bet that his chromatography will become much more reproducible.

I've had exactly this happen to me as recently as last month and several times over the last 15 years and washing the column well will solve the problem, IF that's the problem. I can't see him degrading the column with methanol and carotenoids. I don't think it's a pH issue as carotenoids are predominantly neutral, if I recall correctly, and he says his pump's OK.

I won't guarantee it, but I'd wager it's a good bet, given the information posted here.

So, the question is: Did he wash the column and what happened ???

[DR]

How close to a window is your LC? Does any sunlight hit your reservoirs?

The closest I ever came to this sort of situation was when I had ~1% Et-O-Et/C6H14 eluent on silica column (normal phase analysis of biological sesqueterpenes). The reservoir was too close to a window (until I figured it out). The sun was heating up the mobile phase, driving off the Et-O-Et - caused all sorts of problems as the analyte was extremely sensitive to MP composition.

[juddc]

...unless I am mistaken, he wrote that his eluent is 100% methanol, therefore the MP composition is unlikely to be affected by evaporation.



CJ

PS -I like the pacing cat!

[syx]

Does methanol absorb moisture from air?

[paolino]

If he has a relatively large quantity of hydrophobic material in his sample that is hanging up on the column, it may very well wash off the next day with a long equilibration in the morning, but it may NOT wash off between injections and therefore in some way shield his analyte from the C8 causing shorter retention. He says that if he continues running through the night, RT's keep getting shorter but if he stops, allowing the column to soak in MeOH overnight, then re-equilibrates, it's possible for performance to be restored IF the material isn't too tightly bound to the column. Being that he's assaying carotenoids, I'd have a look at the carrier / excipient mix as a culprit.

If he washes the hydrophobic substance off with a more hydrophobic mobile phase between injections, I'll bet that his chromatography will become much more reproducible.

I've had exactly this happen to me as recently as last month and several times over the last 15 years and washing the column well will solve the problem, IF that's the problem. I can't see him degrading the column with methanol and carotenoids. I don't think it's a pH issue as carotenoids are predominantly neutral, if I recall correctly, and he says his pump's OK.

I won't guarantee it, but I'd wager it's a good bet, given the information posted here.

So, the question is: Did he wash the column and what happened ???

Just to sum up what happened to the column and what I checked afterwards:
- Rts are getting shorter although now they seem to be steady
- The pump is OK and the flow is regular.
- The column is quite new
- The eluent is HPLC grade and replaced every morning
- The temperature is controlled by thermostated oven
- There are neither leak, nor void nor bubbles in the system
- To avolid long equilibration times in the morning the column is flushed ccontinously

I now agree with Juddc as it seems that some excipients clogged the head of the column or spread over the silica surface, so that it became shorter. This is to be the most appropriate diagnosis, although it has some unanswered questions:
- if clogging was the cause, I'd see RTs getting shorter and shorter, while on the contrary they are now stable.
- I faced column clogging in the past but after thousands injections. It sounds very strange that after few injections of relatively pure material I found such a mess.

Anyway, washing the column should solve the problem.
Which is the best way?
I'd reverse the column before washing but some of you don't agree.
Moreover, what eluent to wash it? Methanol/acetonitrile? methanol/hexane?

[juddc]

If the RT's are now stable but shorter than what they were previously, you've apparently hit some sort of equilibrium. I'd guess that your column is as fully coated with whatever contaminant as it's going to get, but that's just a guess...

To figure out how to clean best, it would be good to know what your excipient mix is or at least what it's soluble in. Being that you're using a MeOH based MP and your samples are carotenes, I'd probably try IPA or EtOH (with a reduced flow rate and/or evlevated column temp to compensate for elevated viscosity), then move on to hexane. ACN can be a funny beast sometimes with regard to solubility and it's not miscible with hexane if I recall correctly.

I know I recommended a mix of IPA/ACN a while back, but have been thinking on this further!

Good luck and let us know what happens

[juddc]

If you are successful in cleaning the column up and getting it back to where it was originally, it might be relatively simple matter to develop a sample cleanup method rather that having a column rinse at the end of each run. Running your samples through reversed phase SPE *might* be sufficient to hang up whatever's messing up your column while allowing your analytes through. Careful solvent selection and analyte recovery experiments would be in order, of course. Your current solvent (MeOH) might be a place to start...

Again, just a thought...

[amaryl]

Check your column pamphlet too. Must be stating good procedures. Select the one as per your case.

LCGC article over column regeneration.

Regards,

Amaryl.

[paolino]

Hi, I'm back again with disappointing news.
I took you advice and wash the column WITHOUT REVERSING it, following the instruction of the column supplier.
I flushed the column @ 40° C with many and many volumes of methanol/ethanol, increasing the % of tha latter until 100% and then 10% dichloromethane in ethanol for a couple of hours.
I run the same procedure backwards until I flushed the column with 100% methanol overnight.
This morning I run a STD injection. The Rt is nearly the same bad Rt as before the washing, i.e. 10.5 mins. The pressure has the same value as when the colum was new.
This is going to drive me mad.
Any suggestions?

[JM]

Please check the water content of your mobile phase ( methanol) . The water content of methanol can very on storage . see the difference of moisture content of freshly opened bottle and old mobile phase . There may be a correlation between RT with the water content of methanol.

Good luck.

JM