I have developed a RP-HPLC method to separate a mixture aryle compounds (mixture of iodonium and phsphonium aryl salts plus aryl phsphine) with the aid of a C18 Column, 100 Å, 3.5 μm, 2.1 mm X 30 mm. It was very effective (the Rs between the closest peaks is 2.6) using gradient elution in which two solvents were utilized to establish the mobile phase composition (acetonitrile/ammonium formate buffer) at flow rate 1 mL/min for either 5 or 5.5 minutes, plus 0.5 minutes or 1.0 minute as post time.
My question is: Can I separate the same mixture using a bigger column (for example column with the following features: C18 Column, 100 Å, 2.6 μm, 4.6 mm X 150 mm)?
Is it expected that the separation time will be significantly increased??
Thanks in advance to all responses
Saddam
In general yes! However, a bigger column may be more prone to turbulent flow (a bad thing in HPLC). Try to keep the internal dimensions and particle size the same. In any case you will have to experiment!
First question: Are you scaling up because you need to load more sample or to develop a more HPLC friendly method (easier to transfer and run on any system)?
- Normally, when we scale up we use the same or larger particle size. When we scale down, we use the same or smaller sized particle. This is due to the dramatic back-pressure changes which take place due to the packing material. What you are asking goes against this basic concept (but that is OK).
To change from a larger 3.5u particle packed in a tiny 2.1 x 30 mm column to a smaller, 2.6u particle in a much larger 4.6 x 150 mm column would provide for a very bad scaling result. To obtain similar results, you would have to maintain the same linear velocity. That means at 1.000 ml/min (your current flow rate) you would need to scale the flow rate up to between 4.5 and 5.0 (2.1 mm vs 4.6 mm goes up by the square). An aqueous mobile phase run at 4.5 to 5.0 ml/min (at 25C) would result in a column back-pressure of 600+ bars. The turbulence and heating plus pressure resulting from such a method all point to "do not try this at home". Now, simply change that particle back to the same 3.5u particle and you are getting closer to a similar method. You can calculate out the optimized flow rates, particle size, column length, ID, injection volumes (which will go up as your column gets bigger), gradient composition changes and even delay volume changes. If you do a keyword search on the web you can even find some free HPLC column scaling calculators too, but I always suggest you learn how to do the calculations yourself first (very easy math).
Note: I did not discuss how the change in dwell volume of your proposed method will effect the changes needed to arrive at an acceptable method. Only so many minutes in the day... time to turn off the computer for now.
I have included a few links to short articles for your leisure time reading below.
http://hplctips.blogspot.com/2011/08/pressure-drop-across-hplc-uhplc-column.html
http://hplctips.blogspot.com/2011/07/hplc-to-uhplc-conversion-notes.html
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