Chromatography Forum

I'm doing a RP-HPLC (C18, acetonitrile) gradient separation, and it just takes too long - 30 minutes per injection. The flow rate we've been using is 1ml/min. Do the higher rate machines (5 - 10 ml/min) really work? Do they explode, causing horrible bodily harm? The procedure also has a light reactor at the end - so we may not be able to use the fittings necessary for the higher rate.

We are playing with a more rapid gradient, but are there any other suggestions?


Best, Cytokid

Increase flow or temperature

You could try to see if a smaller column both in length and diameter is available.

What particle size / column dimension are you currently using?

We're using a 25 cm X 4.6mm Terra MS C18 column (Waters) w/ 5micron particle size. Temperature is a neat idea. Have you had good success with that? How high can/should we go?

Thanks,
-csutton

We recently published an article in LCGC online that describes how to do what you want to do. The theory is a bit tedious if you want to do it right, so the article gives a step-by-step description and tutorial. See http://www.nxtbook.com/nxtbooks/advanst ... tartpage=6

That's a LONG column.

First, what is the backpressure, and what are you willing to live with? Unless you are running methanol-water, your backpressure is possibly between 1000 and 1500 psi (70 and 100 bar). With this backpressure, you can run the system at 2 mL/min and half the run time...

You can also use a shorter column with smaller particles. Then you gain even more. If you go from a 25 cm 5 micron column to a 15 cm 3 micron column, you can go to 1.6 mL/min and cut your run time by a factor of 2.7 for the same performance.

We're using a 25 cm X 4.6mm Terra MS C18 column (Waters) w/ 5micron particle size. Temperature is a neat idea. Have you had good success with that? How high can/should we go?

Baby steps
Try to change +5 C (or -5 ) and flow 1 -> 1,3 or 1,5
If you have high pressure decrease flow (0,85)

You should try reducing the particle size to 3um. We have isocratic data comparing a 250x4.6mm (5um) column
to a Cadenza CD-C18 (150x4.6mm, 3um).

The result is the efficiency and back pressure are comparable (both values are slightly higher for the 3um column) -
but the run time is reduced by 40% (at the same flow rate):

http://www.imtakt.com/TecInfo/TI015E.pdf

Do they explode, causing horrible bodily harm?

You are funny. No they don't, not that I know of anyways.
You can start to play with parameters such as column type (do you REALLY need a C18?), Shorter column, smaller particle size, higher temperature, higher flow or a combination of all these factors to make a shorter method.

I note that you are using a light reactor at the end of the column - an increase in flow rate may decrease the yield.

In my experience all but the most complex separations can be done using a 10cm column. 25cm columns are a a hang over from the time when chromatography was done with 10u irregular particles.

A shorter column also gives greater sensitivity.

AdrianF, there are lots of people who only do "the most complex separations". They are not a hangover, nor do they have a hangover. For instance some years ago I helped someone with a C-14 labelling, we considered the synthesis and accompanying analysis a relatively simple thing. HPLC showed 4 main products. GC showed that the largest HPLC peak was composed of at least 5 substances. Even today I think this was a simple problem when compared to some of the forays into analysis of body fluids.
I can´t help but feel that the current fuzz about fast HPLC is an alibi for the lack of advance in the selectivity/resolution sector.
My suggestion out of the stalemate: Affinity chromatography with low affinity stationary phases.

My point was that a 25cm column is often used when a shorter one will do the job ie in analysing pharmaceutical products where there is one peak of interest. Maybe not a hang over but inertia!

In this case we need to know more about the problem to be able to give sensible advice eg how many other compounds in the mix respond to the light reactor?

for many of us who work in the regulated industry the so called Fast HPLC approach with smaller columns have its practical limitations. to get the most benefit, the instrument hardware often have to be modified to minimize dwell volume and extra column volume. we can't do much about the hardware configuration (especially for GMP instruments), so the benefits of shorter columns with smaller particles often get masked. I tried to start with a 4.6x50 mm sub 2 um column for a new related substances method. an 8 minute gradient can get the desired separation, but I had to wait for 5 minutes between injections to achieve reproducible results. when a method needs special care to perform, you worry about what would happen when it gets to the QC labs.

I tried to start with a 4.6x50 mm sub 2 um column for a new related substances method. an 8 minute gradient can get the desired separation, but I had to wait for 5 minutes between injections to achieve reproducible results. when a method needs special care to perform, you worry about what would happen when it gets to the QC labs.

How long would this equilibration time have been, if you used a longer column packed with larger particles?

I can´t help but feel that the current fuzz about fast HPLC is an alibi for the lack of advance in the selectivity/resolution sector.

I completely agree and would like to add some of my thoughts as well: Many places, the analysts set a series of samples (“Sample Setâ€