Hi,

What temperature are you running at? 

I'm a frequent user of the 1000A version in 2.1mm ID and normally I run this at 70 degrees and 0.4 mL/min. The pressure at starting conditions is also around 70 bar. So I guess you could easily increase flow to, for example, 0.4.

Do you also use MS detection? That could eventually become a limiting factor with higher flow rates.

Hope this helps, good luck! 

There is usually a sheet with a test chromatogram included with the column. I tend to run columns at the flow rate indicated for their test sample, as I assume that is close to the van Deempter minimum (best resolution). Different solvents and compounds may have the best resolution at a slightly different flow rate but those flow rates are close to what the manufacturer uses, in my experience. If the QC sheet was lost, look at the example chromatograms for your column on the manufacturer's web site.

Note: The QC test sheet is a very poor indicator of quality or of an example in how to use the column. The column vendor optimizes those test conditions to get the column to pass as easy a test as possible in the shortest time as possible (insures good peak shape). For in-house QC, always develop your own test method using an appropriate sample/std. This will provide you with a useful benchmark to compare when the column's performance is questioned. The vendor's criteria and goals are often very different than your own...

Quick comments about Flow Rate: The efficiency of an HPLC column varies with flow rate (just like it does with particle size, ID, temperature and length). A higher flow rate does not always result in better performance. Their are many parameters effected by such changes. Flow rate optimization requires optimization of the maximum number of theoretical plates (optimization, not maximization). Generically, we start with flow rate values that provide easy linear velocities [v = L / t0 ]. Examples: 4.6 mm ID ~ 1.000 ml/min; 2.1 mm ID ~ 200 ul/min; 1.0 mm ID ~ 50 ul/min. These nice round values will get you close to the optimum values (though they are all a bit high).

Also, we do not develop HPLC methods by trying to max out the pressure specs of a specific column. Moderate pressure is good (keeps everything primed). Very high pressures often result in shorter column and instrument life-times. We optimize the method to the sample within the limits of our instrumentation and conditions.  If you are at or near the optimized linear flow rate, increasing the flow rate alone will not improve your separation. Instead, you want to improve your method to retain and elute the sample peaks in less total time by changing the mobile phase conditions to do so. Once you have done so, then you can experiment with temperature and higher flow rates (i.e. 50% increase in flow rate) and compare the results. BTW: Column washes should always be done separately, not part of the analysis method. This saves times, removes interference, improves integration and allows you to use a stronger mobile phase solution to wash down, then re-equil the column before use.

Thank you all for the helpful suggestions! I think I will definitely tweak my method before I do anything with the flow rate.

Additionally, any advice on how to thoroughly clean these type of columns between runs? I have colleagues who do injections of 10-50% formic acid, but I've just been running 95% ACN until I get a baseline absorbance reading through the instrument. I'd appreciate any thoughts on this, thanks!

Kanishk: For column cleaning, always consult the column manufacturer first for their best advice (this is true for all columns).

The PLRP-S column is a gel column made up of polystyrene divinylbenzene beads. Never use or wash these columns using pure water. Some organic must be used at all times. A wash solution containing a high concentration of MeOH (instead of ACN) should be used along with the strong ACN mixture you are currently using (95/5 ACN/H20) should do a good job of general cleaning (it is what I use and I alternate between them). Additionally, I would not use high concentrations of Formic acid at all. 1% formic acid in either: ACN/Water or MeOH/Water mix should be fine.

Thanks, Bill!

Will try an ACN gradient with 1 % formic acid (that's what Agilent recommends as well).

I disagree about the comment on the QC test. The manufacturer does want a fast and easy test, but they also don't want to ship a batch of poor columns either. The manufacturer of the column looks at retention time, plate count, and peak shape. They also want the peaks to look as good as possible to the end user. I also note that, in example separations, they run the columns close to, if not at, the flow rate in their QC sheet. Their flow rate is a good starting point, and the method for your compound and resolution can be optimized from that point.

I do agree with using your own QC, with compounds similar to what you purify. The column manufacturer has to choose something to test their columns, and it is usually nothing like anything I purify. If a purification is to be run many times, I agree it may be fruitful to optimize the flow rate. Many purifications are run only a few times, and the time spent on optimization, provided there is sufficient resolution, may be more profitably applied to other work.