gradient LC question
Posted: Sat Aug 06, 2005 6:07 pm
by ashvin
Hi
Chromatographers
I have read that gradient runs are instrument specific.
is it true? WHY?
Ashvin
Posted: Mon Aug 08, 2005 9:17 am
by unmgvar
Hi Asvin,
I will not go into the mathematics and and heavy theory behind it, but the reproducability depends on mainly 2 parameters.
the first one is the pumps precision and the second one is the dwell volume of the pump and mixer. the difference between the manufacturers cause it that gradient methods for complex API's are hard to reproduce. sometimes even the different type of column oven is a factor.
the best for you to deal with this situation if you have different types of instruments in your lab is to use if you can a software like Dry lab or Chromsword. they will give you the best tips on how to resolve your problems.
Gradients
Posted: Fri Aug 12, 2005 2:19 pm
by chrisp
Hi Ashvin,
As stated by unmgvar, a number of things can have major effects on the chromatography obtained using gradient methods.
From my experience, the major variance from system to system (especially if systems are from different manufcaturers) is caused by the dwell volume of the system.
The dwell volume is in effect the total volume of liquid taken to fill the system from gradient proportioning valve, GPV (where the various solvents are mixed) to the detector itself.
When soem change in the gradient occurs, this change will not be seen in the chromatography until the changed mobile phase has reached the detector. If the volume of a HPLC instruments flow path is increased, then the change in mobile phase will take longer to reach the detector and hence the chromatography effect of the change will be seen later.
This can have major implications for the quality of the separation achieved, especially as gradient is usually used for more diffiuclt separations - you don't use gradient if isocratic works.
This leads to the use of a single manufacturer being advantageous to maintain the quality of separations with gradient elution, although this obviously can have implications if the method is to be transferred to other labs, etc. You must also take great care when replacing tubing on instruments, as significant increases in dwell volume can result.
Hope this helps a little
Chris