help required for analysis

[rrm1987]

i was analyzing toxin in two different instruments.The specifications were:
1)column:kinetex2.6 micron,100 mmX 4.6 mm;C 18.

i observed tailing in the chromatogram in one instrument and in other instrument the peak was perfect at 276 nm.I realized the problem is with detector.what could be the reason and how to overcome this?

[Petrus Hemstrom]

Most likely the flow cells have different volumes in the detectors. The tailing one is either too large causing dilution or too small causing turbulence (I have only seen that with prep flow rates though). It could also be a tubing problem.

[HPLCaddict]

First of all what makes you think this is a detector problem? Did you swap detectors between both machines?
If you certainly can rule out chemistry problems (i.e. column and mobile phase) I'd have a look at the fittings first. If there's a dead volume somewhere it might lead to tailing.

[rrm1987]

dead volume means? how to overcome this?

[rrm1987]

not the same detector.each instrument has different detector.

[BFR_83]

Other advices:
- Decrease injection volume in the system you are having problems with peak tailing.
- Change solvent composition of the HPLC samples (use weak solvents for reverse phase to dissolve the samples, such as water or a mixture of warter and methanol, if possible).

[HPLCaddict]

If the connection fittings are not perfect, there might be small "pockets" (aka dead volume) that act as a mixing chamber which leads to broadened and eventually tailing peaks.
One important thing one might overlook: What about detector parameters in the methods on both machines? have a look at the "response time"/"rise time"/whateveritscalledonyoursystem. If it's to high, it might lead to tailing peaks although chromatography itself is perfect.

[rrm1987]

can you tell me how to change this Rise time/retention time in the HPLC system?i am using Schimadzu LC-20A prominence binary gradient system.

[HPLCaddict]

can you tell me how to change this Rise time/retention time in the HPLC system?i am using Schimadzu LC-20A prominence binary gradient system.

Unfortunately I'm not familiar with this system. In the chromatography data software you should find this parameter in the method setup.

[rrm1987]

i could not find any parameter "rise/response time" in the new method settings option.can you elobrate on how to do set raise/response time and what value should we give?

[HPLCaddict]

Which software are you using?

[rrm1987]

Schimadzu's LC lab solution

[HPLCaddict]

Sorry, I don't know that software and couldn't find a manual online. Perhaps someone else might help??
In the method setup under the detector parameters look for something like "response time", "rise time", "time constant" or similar. Unfortunately the different manufacturers use different names. The parameter in question is in seconds, usually in the range 0.1 - 2.0 sec.

[rrm1987]

thank you.I could find time constant in that settings.What is the default number for that?

[HPLCaddict]

There is no default. You have to adjust the setting according to your chromatography .
Generally, a lower setting will give you a noisier baseline but probably better peak shapes and reduced peak widths.
In your case, I'd try the lowest setting possible, just to see if peak shape improves (less tailing). If yes, you might increase the setting to higher values in order to find a good compromise between peak shape and baseline noise. If peak shape does not improve with the lowest setting, well, then you have to look somewhere else for the cause...