retention time

[paolino]

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

I'm puzzled.
I guess that if some water was present in the eluent mixture, the Rt would be greater, wouldn't it? As it is more polar than the methanol for such a column and such analytes, the eluition should be slowed down.
Anyway I don't think water uptake in methanol should be taken into account. In fact I use fresh HPLC grade methanol from a new bottle every day: if water was a problem I'd see Rts vary during the day and the day after the Rt should become normal.
This seems to be a long-term 'desease' of the column: clogging? collapse? whothehellknows?
I feel very far from the conclusion, unless I change the column.

[juddc]

OK...this might seem a bit off the wall - desperate even - but since going more hydrophobic didn't help, maybe flushing with a more hydrophilic MP could help. I don't really expect it to help, but as a last ditch effort prior to replacing the column, try 50/50 MeOH/Water.

I think your column might just be too full of goop to save... sorry .

Next round, buy a spare column in addition to your replacement, work up a sample cleanup and install a pre-column if you have not already. I still think it's a sample related column problem.

[JMB]

Paolino,

If RT changes this much when the flow rate and temp. are both constant, it has to be due to a change in any or all of (1) mobile phase composition, (2) column state, (3) composition of sample diluent.

(1) as previously suggested, methanol is quite hygroscopic and you may have a varying water content.

(2) water will partially deactivate the silica (upon which the C8 is bonded) and if the carotenoids are being retained by both a reversed phase (C8) and normal phase (SiO2) mechanism, then the retention may well be changed.

(3) any chance the diluent composition changes in a vial with a pierced septum ??

If the water content cannot be trusted, then you might consider adding say 1-2% to the methanol in order to swamp any gradual water absorption from the atmosphere.

JMB

[paolino]

Paolino,

If RT changes this much when the flow rate and temp. are both constant, it has to be due to a change in any or all of (1) mobile phase composition, (2) column state, (3) composition of sample diluent.

(1) as previously suggested, methanol is quite hygroscopic and you may have a varying water content.

(2) water will partially deactivate the silica (upon which the C8 is bonded) and if the carotenoids are being retained by both a reversed phase (C8) and normal phase (SiO2) mechanism, then the retention may well be changed.

(3) any chance the diluent composition changes in a vial with a pierced septum ??

If the water content cannot be trusted, then you might consider adding say 1-2% to the methanol in order to swamp any gradual water absorption from the atmosphere.

JMB

1. OK.
2. This method was tuned to 100% methanol after starting with methanol/ water mixture as eluent but Rts were very long. I tried also more hydrophobic eluents such as methanol/ethanol (absolute) or methanol/hexane or isopropanol but none of them was satisfactory in terms of resolution for both carotenoids and excipients.
So, now I'm facing a choice:
- add water and suffer longer analyses;
- add water and ethanol for instance in order to swamp moisture absorption but not to lenghten Rts;
- avoid moisture uptake (molecular sieves?);
- review the method.
What will be the most practical choice?
I do not have much time to dedicate to long lasting review as the HPLC station is working continuously, even if the method is changed.

3. vials are OK, no chance of pierced septa.

Any help is very very welcome.

[paolino]

Hi everybody,
the latest news from the column:
1. water in methanol seems not to be the cause for the shortening of Rts. I've checked H2O content and this is neglectable.
2. I washed the column overnight with a gradient of metanol/water from 100% to 50% MeOH. RTs keep on being shorter.
3. The STD Rt is now stable (at 10.5) mins as well as the other peaks
4. I have seen only a slight increase in pressure - say 2% - from the earliest injections up to now.
5. column performances are only slightly affected by this problem and peak are well-resolved anyway.
So, I guess I'll keep my column as it is now.
Thank you all.
Bye.
P.