Iso-propanol gradients with U-HPLC

[Rob Burgess]

Hi all,

I'm wondering if anyone has tried this and got good results for a separation that was diffcult traditionally with either MeOH or AcN as the mobile phase.

Looking at the Snyder classification values i-propanol appears very similar to MeOH but is just might be different enough for certain RP applications.

I'm trying it at the moment and will model in DryLab and will feedback results soon.

U-HPLC - Acquity
Column: BEH, C18 (150 x 2.1 mm)
Mobile phase A = water + 0.1% formic
Mobile phase B = IPA + 0.1% formic
Flow rate: 0.18 mL/min
Column temp. 50°C

For drylab modelling two gradients:

5 to 100% B in 15 and 45 mins

PS. Does anyone know where I can obtain viscosity date for IPA-water mixtures?

[Bryan Evans]

According to Appendix II "Practical HPLC Method Development" 2nd Ed,
Snyder, Kirkland, Glajch:

Viscosity (cP)
ACN: 0.38
MeOH: 0.55
IPA: 2.40

There is a rather large increase in pressure when using IPA - you'll most likely have to
increase the column temperature substantially in order to keep the pressure down.

In contrast - below is a peptide separation done on Cadenza CD-C18
(150x2.0mm, 3um) using IPA - with column oven set at 40C:

http://www.silvertonesciences.com/files/TI057E.pdf

[Rob Burgess]

Hi Bryan,

Thanks for the information - I was after IPA-water mixtures specifically. I'm guessing such mixtures will be even higher than what you have quoted for standalaone solvent mixtures!

Cheers....

[HW Mueller]

I was not going to answer this as I don´t have the data for which you asked. But many ratios of IPA and aqu components never gave a backpressure above 200 bar with C-18 columns (4.6 x 250mm, 5µm).

[Uwe Neue]

IPA-water gradients have been used not infrequently in the biotech world for separation of peptides and proteins - even long before UPLC. Yes the pressure is higher, but if it gets you what you want, so what...

BTW, Rob: you can get a direct measure for the viscosity by running a slow gradient from water to IPA and measuring the backpressure. I have used this technique for measuring the viscosity of solvent mixtures. Works like a charm and is faster than trying to find unusual things in textbooks. You take the pressure in water as the viscosity of water under the circumstances (around 1 cP, depending on the temperature) and you are calibrated. The ratio of the measured pressure to the pressure in water is proportional to the viscosity of the mixture inside the column. If you want to be really picky, you can do step gradients...

[mbicking]

I have done the experiment that Ewe describes. The file is attached. It does indeed demonstrate the differences in pressure that result from using other modifiers.

The other interesting feature is the fact that water organic mixtures have higher viscosity that water alone. And the maximum viscosity appears at a different composition for each solvent.

[mbicking]

My apologies, correct spelling is Uwe.

[Bruce Hamilton]

The Rubber Handbook ( CRC Handbook of Chemistry and Physics ) has a section entitled "Concentrative Properties of Aqueous Solutions", which has quite detailed tables, including for density, RI, viscosity and conductivity at every few % difference in concentration.

Title isn't intuitive, but hiding amongst the salts are tables for various solvents, including Ethanol, Methanol, 2-Propanol mixtures with water . Good for a quick check, even though the effect of pressure isn't covered.

Please keep having fun,

Bruce Hamilton

[mbicking]

Years ago I also got the following plot via Waters. Same type of information (but missing IPA). Maybe the data are from the CRC Handbook.

[Rob Burgess]

Many thanks everyone for your informative replies - much appreciated as always.

Q/ As a general rule is iso-propanol a stronger or weaker solvent than AcN for RP-HPLC? Looking at my most recent gradient data it seems to suggest that it is stronger.

[zokitano]

Looking at my most recent gradient data it seems to suggest that it is stronger.

That's correct

Regards

[Uwe Neue]

Merlin,

the older data from Waters were measured at Waters, either in my lab or in one of my colleagues labs in CRD. Only much later did I become smart enough to use the gradient technique...

[mbicking]

Uwe:
Nice to hear there were some good people generating the plots. Even if they are old, they still do a nice job of explaining the effects. I still use this regularly.