Chromatography Forum

Hello all,
I would like to get some general suggestions on how to speed up my HPLC analysis. My current method includes:
Column XTerra RP18, 3.0x150mm, 3.5 u
Mobile phase water, ACN (gradient)
flow rate 0.5ml/min
Detection PDA
The run time is 30min. I have been asked to reduce my run time to maximum 10min using a shorter column and increasing the flow rate. Any suggestions on how to speed up my method without compromising resolution?

without compromising resolution?

Three-word answer: "smaller particle size". Do a search here on the forum for "UPLC" and you will find more than you wanted to know (both pro and con!).

How far you can go, depends on some details of the analysis. If you do not have any tightly eluting peak pairs, you can buy a 5 cm column, run the gradient at the same flow rate in 10 minutes, and you are done (and your boss is happy). If you want a bit more sophistication, use a 5 cm x 4.6 mm column instead of the 3 mm column, and run at 1.2 mL/min. Your response will get a little bit better, but the likelyhood that you run into post-column bandspreading problems (and thus a much inferior separation) is diminished.

However, if you do this, the peak width will be wider on the shorter column (in theory by about 70%, but it may be less in reality, if you use the larger i.d. column). Consequently, this is not a good idea, if you have some very closely coeluting pairs in your chromatogram.

If this were indeed the case, you should still use this short column, but now you will need to reoptimize the separation by playing with the usual method development tools: flow rate, temperature, solvent etc.

You can potentially also do the same analysis on the existing column with a steeper gradient, i.e. a 10-minute gradient. However, your peaks will flip around (or at least change their resolution patterns), and you need to redevelop the separation.

If your separation allows it, the fastest path to happiness is to buy a short column.

What is the nature of your analytes? How many compounds ou have in your mixture?

In addition to all the suggestions given, you
can try using a 3 micron column at a shorter length
(the 3 micron can be used on your existing HPLC).

Just another tool you have available to you. Here's an example
comparing a 5 micron column to a 3 micron column:

http://www.silvertonesciences.com/modul ... TI058E.pdf

Bryan,

He is already using a small particle size column (i.e. 3.5 um)...

Have you tried monolythic columns?

Check www.phenomenex.com/Phen/Products/onyx/index.htm

Maybe be this is a solution for you!

Have you tried monolythic columns?

Check www.phenomenex.com/Phen/Products/onyx/index.htm

Maybe be this is a solution for you!

yep, we have have had some good success with these. 100x4.6mm C8 has worked a treat for some proteins in plasma.

you can also try 50x2mm or 50x4.6mm columns in a 2u particle size.
we also have found MercuryMS columns to work well to shorten run times, 20x4.0mm in 2u particle sizes. most manufactures have the 2u particle size now.

but as already stated, whenever you go to shorter columns, separation sometimes becomes an issue.

Thanks to everybody for your suggestions!
I will try some of the different options that each of you suggested.
About the nature of my analytes, SIELC_Tech, we have various types of compounds. I am trying to develop a general method for purity analysis that could be applied to most of our compounds, of course after adjusting some parameters for each one.
Thanks again, and if you think of something else, please let me know!

This kind of separation problem (?) to me seems ideally suited to modelling software. Half hour modelling your fast separation can equate to days if not weeks of endless real chromatographic runs.

PS. DryLab software seems pretty user friendly and is quite accurate from my experience of using it.

Dear Rob:

Have you tried Osiris software?

You might get info and download a demo version at www.datalys.net

Rafael,

We've made friends now, haven't we?
One question. Have you read my post on Ibandronate?
I'm waiting for a conductity detector to arrive.. meanwhile I need to optimize the method that Ferris suggested.
There is this problem with detection at 195 nm. My boss doesn't want to accept it...
Can you give me any suggestions? In case, reply in the topic page devoted to Ibandronate as the subject is different here.
Thanks a lot... and sorry for today...
you know.. pressure at work...