HPLC to UPLC transfer

[gooshi81]

I need to transfer an HPLC to UPLC method, does anybody have any idea if im suppose to get the exact same picture (HPLC vs. UPLC)? because i get RT ratio which is close to but not similar to the HPLC method.

[AA]

Unless you column chemistry is exactly the same, I would not expect the picture to look the same, similar, but not exactly the same. As well there other minor things that can change the chromatogram (column temperature differences, mis-calculated system volumes, incorrect detector settings, differences in injection mechanisms, and so on).

[Hfranz]

Gradient separations:
Following the gradient volume concept, you need to keep the ratio between the gradient volume and the colume volume constant. However, it is not possible to adapt the gradient delay volume exactly. You simply cannot adapt it precisely as only certain mixing and tubing options are available.
Let's pretend, your gradient delay volume relative to the separation column is bigger with the U-HPLC column. This might cause a longer isocratic hold step at the beginning of the run. Obviously, this might influence the retention of early peaks, i.e. they elute isocraticly/partly isocraticly. There are strategies to overcome this such as delayed injection, but this can hardy be considered a remedy. It is also not supported by all instument control softwares.

[gooshi81]

what is the UPLC closest chemistry to Hypersil BDS?

[Uwe Neue]

I agree that you can expect the same chromatogram only if the particle surface is the same in HPLC and UPLC.

If the column chemistry is the same, the scaling is rather straightforward. Assuming that you are dealing with a gradient:

1. Scale the gradient volume in proportion to the column volume. If you are running a gradient with multiple segments, the volume of each segment is scaled in proportion to the ratio of column volumes.

2. Change the flow rate with cross section and particle diameter. To go from a 4.6 mm 5 micron column to a 2.1 mm 1.7 micron column, you reduce the flow rate by a factor of 4.8 for the diameters and increase the flow rate by a factor of 3 for the particle size.

The delay volume of the UPLC system is rather small compared to many HPLC systems. Thus most of the time, you will need to add an isocratic section at the beginning of the gradient to simulate the gradient delay of the HPLC system. The volume of the delay is scaled in proportion to the column volume.

The UPLC system has some tools that help you with the calculations.

[gooshi81]

when i used the system calculation tool it gave me a shorter isocratic section at the begining of the gradient, why is that?

[unmgvar]

you can get a very close picture but a one to one transfer will never be possible. the closest you'll get depends on several factors:

1. column chemistry. if you moved between vendors from 5 micron to sub 2 micron, then the chemistry will be different for sure. even between different column types of the same vendora c-18 is not the same chemistry, maybe close but not the same.
2. your analitical system is most probably with an LPG pump. UPLC has a HPG pump. gradient formation is different
3. the dwell volumes are different and you must compensate either by bigger iscratic step or adding mixer size to the UPLC.

that is why moving a method from systems can vary from almost impossible to closely nice

[Uwe Neue]

The isocratic section at the beginning of the gradient is scaled using three factors:
1. the ratio in column volumes
2. the ratio of the flow rates
3. the difference in the gradient delay volumes between both systems

If you put the correct information into the calculator, it will do the entire exercise for you - automatically.

[Uwe Neue]

I just saw that you want to transfer from a Hypersil BDS column to a UPLC column. Since the column chemistry is different, you will get different results. In this case, I recommend not to scale the method, but to adopt the method using the optimize option. At the minimum, it is worth a try to get you to some suitable initial conditions, which can be fine-tuned in a second step.

Of course, if you are happy with what you got, then stick with it.