Scaling Up for Prep HPLC

[kaha37]

Hello Everyone
I'm new to the forum and am looking for some help and advice regarding prep hplc. After 10 years doing analytical HPLC I have recently started a job where I need to run prep HPLC. Not having ever done any prep HPLC before I'm pretty much trying to muddle through the prep method development and scale up using bits of information from books and catalogues.

The way the chemists in the department have been running the system previously is to get an okay analytical method (preferably isocratic) and then just multiply everything by 3 (?!) apart from the flow rate which they always run at 5ml/min for prep and then inject 1ml of their sample solution (I don't think they prepare them quantitatively - just a bit in some solvent) - does this make any sense at all? Having read bits and pieces in books it sounds a bit dubious to me.

Can someone with some experience in this area point me in the right direction please - is it always advisable to use the scale up calculations and determine the maximum sample load on the analytical column as described in the literature or will I have just as much success with the rough and ready approach being used by the chemists?
Apologies if these are stupid questions.
Thanks in advance for your help.
Karen

[abn70]

For a fresh beginners like you. You can develop a method using an analytical column and then try to scale up using the scale up procedure. The sample loading on a prep column depends up on length and ID. Use the scale up procdure for sample load. Similarly, this holds good for establishing the column flow rate.

[Bruce Hamilton]

There are some wonderful free resources awailable on the web, mainly at instrument and column suppliers like Waters, Agilent, Varian etc. Just search their literature databases for information on preparative HPLC.

Agilent also has a free booklet, " Principles in Preparative HPLC - a primer " - March 2004 Pub 5989-0652EN ( might be available on their website as a pdf file - have a look ). I'm sure other suppliers have similar generic guides.

5 ml/min is probably considered "semi-prep" by many, so you might find that the rough and ready methods are just fine. For larger preparative systems ( 20+ ml/min ) scaleup issues are more important, as you want to minimise solvent use and maximise sample loading - so there will be less evaporation / freeze drying to perform.

If they are your clients, and the current procedures work, don't change anything unless you are certain you will provide improved service that they want. People may only want the prep to knock out specific junk, as it hurts the next step or can not be removed later. They may not want optimum methods, just simple solvent systems that are easily removed.

Many if the same rules for analytical apply to preparative, with perhaps the only major difference being that rapid elution is usually more desirable, provided acceptable resolution is achieved.

Scaling to column is useful if you have lots of columns, but often choice is limited anyway, so you scale the analytical to the column, and choose the column size according to sample size, depending on how many runs you want to do - refer above booklet and websites for details.

Provided their formula works, I'd just keep using it - they'll tell you when the separation is unacceptable, then investigate further. I'd also investigate if you have to do multiple runs on samples, but a bigger column usually is the fastest solution if separation is already optimal.

I hope this helps,

Bruce Hamilton

[Uwe Neue]

The first things in prep are very straightforward. You first want to optimize your load on an analytical column for which you either have a prep column or you can buy a prep column. Do not change the packing going from the analytical column to the prep column!!! Once you have optimized the load on the analytical column, you can calculate the amount and the volume that you can load on the prep column by simply using the ratio of the column volumes.
Other things, such as the flow rate and setup for the gradient, are also very straightforward. You need to know the capability of your prep pump both in terms of flow rate and pressure capability. If the separation is isocratic, this is all you need to know. From the ratios of the column volumes, the flow rates, and the retention time under analytical conditions, you can estimate the retention time under preparative conditions.
If the separation is a gradient separation, you can scale the gradient by scaling the gradient volume with the column volumes. A few small adjustments may be needed, such as taking account of the gradient delay volume of your instrument, but this is also very straighforward. A formula for this is in my book on "HPLC Columns". But in most cases, and if you are not too picky, the simple ratioing of the column volumes will do fine.

As you can see, the gradient volume needs to be scaled by the column volume. Therefore there is a significant advantage if you optimize the analytical separation before the scaling while keeping the scaling in mind. If you just want to prep one particular peak in a chromatogram, you should opimize the separation on the analytical column around this peak.

Thus there is only one calculation that accounts for everything in prep scaling: the ratio of the column volumes, and this is very simple.

A few other tricks can be used for advanced use. You may not always be free in the solvents used to dissolve the sample, and the best solvents are often not compatible with your preparative chromatography. In such a case, you need to consider a set-up with the capability of at-column dilution....
However, all the last stuff is nothing to worry about yet for a beginner.