How to transfer UPLC method to HPLC method
Answers
-
If you have a method devlopment program like DryLab, this kind of thing is relatively straightforward.
Have you the option perhaps of developing two methods, one UPLC (much quicker obviously) and the second a HPLC method? We're pondering whether we should do just that at the moment. For early phase work we'd look to exploit UPLC and at the point of widescale late development switchover look to develop a second HPLC method. then the choice is down to our end transfer partners as to what method they can/should use. Obviously a higher "critical mass" of UPLC's within labs would help address this, but until capital budgets improve this is how we are going to play it I guess.
0 -
This is possible but there are numerous things to take into account. I will try and use a gradient example to illustrate the process. I used the Waters prep calculator to help me with these calculations.
UPLC method
Column - ACQUITY BEH 2.1X50 mm
Flow - 0.60 mL/min
Gradient 5-95% over 3 minutes
Injection volume 3 uL
Things you must deal with
Select a column
Select a flow rate
Scale injection volume
Scale and modify the gradient table
Deal with differences in system volume
Adjust re-equil time
Things that we don't have to deal with (keep these all the same)
Column Temp
Solvent A composition
Solvent B composition
Wavelength
Step 1 - Column Chemistry-XBridge is virtually identical to BEH so this choice is quite simple, if you have chosen to use to use one of the other UPLC chemistries you would need to refer to the Waters column selectivity chart to pick an suitable column
Step 2 - Choose a particle size for you HPLC column, usually 3.5 um or 5 um (I will use 5 um for this example)
Step 3- Choose a column length - first thing to do is calculate L/Dp for the UPLC column (50 mm/ 1.7 um = 29412). An equivalent column length for the 3.5 um would be (100 mm/3.5 um = 28571) or for the 5 um (150mm/5um= 30000). You must try and maintain L/Dp as best you can. For purposes of this exercise I will go with the 5 um so I will need a 150 mm column.
Step 4-Choose a column i.d. This is a personal preference, so I will randomly choose a good old 4.6 mm i.d. column.
So, I now have a column
Step 5-Select a flow rate. Typically, UPLC flow rates are not scalable so you have to choose a flow rate suitable to you column. To illustrate the point a scaled for rate of 0.60 mL/min on a 2.1 mm column would be equal to a flow rate of about 2.9 mL/min on the 4.6 mm column. You could do this but you may not have enough backpressure range on your HPLC system and its not likely in an area of highest efficiency for that column so I would choose a typical flow rate for a 4.6 mm column of 1.4 mL/min.
Step 6 - scale the injection volume - 3 uL injection volume on a 2.1 mm column scales to a 43.2 uL injection on a 4.6 mm column.
Step 7 Gradient transfer
You need to have the same number of steps and ensure that you have about the same number of volumes of solvent passing through the column.
So my gradient of 95/5 to 5/95 over 3 minutes scales to about 18 minutes using the 1.4 mL/minute flow rate.
Step 8 System volume adjustment - You may have to add an isocratic hold to the method to account for the increased system volume especially if there are early eluting peaks.
Step 9- Try the new method and adjust as required. Remember that column re-equilibration time has significantly increased compared to the UPLC method.
After all of that you also must remember that you will lose resolution compared to the UPLC method and there may be reductions in sensitivity as well.
0 -
Grace:
This is a great question - does the customer have an ACQUITY UPLC system yet?
You have received answers from the eCommunity as it is very possible to do, with the strategies that have been summarized. The one thing that is missing is the end user, can we have them involved in the dialogue directly? Also it would be great to know which Shimadzu systems represent the bulk of his systems. Other users with the same system can comment even more specifically.
Hope this helps!
Liz
0 -
Hello all,
Thanks for your prompt and excellent reply.
It is best if we can support them a calculator to transmit UPLC method to HPLC method by using Waters columns.
Best regards!
Grace
0 -
Hello, there:
Several parameters need to be considered and scaled properly for UPLC-HPLC method transfer.
Please search a poster on Waters website titled "Considerations for Stationary Phase Selectivity when Transferring Methods Between HPLC and UPLC Technology" in 2009 HPLC conference.
Hope this helps.
0
