retention time delay

[yzhen77]

hallo,

I use RP-HPLC-FLD to detect aflatoxin with mobile phase H2O/MeOH/ACN (60/20/20). It's very stabil until yesterday, the total peaks delayed about 1 min. I suppose that the contamination from the samples can cause the peaks to be elutated earlier, it could be the case, because the day before yesterday a lot of samples ran, but can the contamination also cause peaks delay? By the way, I use the same batch mobil phase, and no system defect was observed. thanks a lot.

cheers,

Zhen

[Mattias]

Some suggestions:

Evaporation of solvent from the mobile phase?
New tubing installed?
Colder in the lab (if you run without thermostat)?

This assuming you are using the same bottle of mobile phase and same equipment as before.

[yzhen77]

hallo Mattias,

thank you for your replay. Actually I changed nothing in the system at all, the evaporation of the mobile phase should be excluded because the bottle is not open. The column is placed in the termostat oven.

Have tried the injection with Standard twice, didn't help, today I injected methanol (50%) and washed the column with the mobile phase, didn't seem to work. Try the injection with standard tomorrow again.

best regards,

Zhen

[AdrianF]

Flow rate? Try checking flow rate with a measuring cylinder or using a flow meter.
After many samples pressure could be higher and flow rate lower.

[Mark Tracy]

Are the retention times not as reproducible also? If so, you probably have a leak. Check the fittings, check-valves and piston seals. In rare cases the proportioning valve leaks and you get something else in your mobile phase.

[danko]

Adrian,
How can higher pressure cause lower flow rate? Unless leaks of course, but this is another story!

Best Regards

[Mattias]

I just recalled that we have seen season variations on our Alliance systems. Since that column oven has no preheating of the mobile phase, there is a temperature gradient over the column. That caused retention time variations even though the temperature was "fixed" in the thermostat. We got the shortest retention time in the afternoon, when the sun was shining on the instrument!

(I have no idea what instrument you use, but just in general)

[AdrianF]

Danko

Some years ago I used to put an electronic volumetric flow meter(Phasesep) at the end of each of my systems and found that higher pressure would lower the flow rate. With modern pumps in good condition this should not be the case.

[yzhen77]

hallo Adrian and Danko,

the ideas of the flow rate and temperatur of the mobile phase sound quite reasonable, specially I have noticed the enhance of the pressure after the samples. After the wash, the peaks eluated earlier but still not stable. thanks for your good ideas.

zhen

[yzhen77]

hallo Mark,

the retention time is stabil during a run, but not among the runs, although nothing was changed (I mean I did nothing). No leak was detected.

[unmgvar]

yzhen77,

i have a collegue that does your stuff for red peper powder.
he had a similar porblem maybe.
in is case he found out that is new FLD detector was more sensitive and he was sudenly seing stuff that was carried over from the previous injection.
the basic method was isocratic, to see that we were right we simply by made a longer run time and sure enough it was there instead of the next injection causing troubles.

[yzhen77]

the HPLC surprised me positively again. Now my peaks back almost to their positions (earlier) after I have run further serveral samples, dirrect after two aqueous (100% PBS) samples. Should it due to the shrinking of the surface area of C18 in aqueous? In which extent or could the sample solution influence the retention? I am not sure if it is the reason or just a accident.

zhen

[yzhen77]

what I can observe is that the room temperatur changed the retention time of my analytes dramaticlly.

[danko]

If the room temperature influences your retention times, a reliable indication of this would be the following observation: Higher temperature results in shorter retention time. I’ve seen an exception to this rule (in a HIC set up) but it belongs to the exotic observations.
Ones you confirm the temperature dependence, you should adopt a reliable column heating i.e. preheating.

Best Regards