Optimizing the Separation : HPLC

Optimizing the Separation

Once you have a separation you may want to optimize it.  You may wish to shallow out the gradient to improve the separation, or you may wish to shorten the run time.  Taking the illustration above one can see that all of the peptides are out by 40 minutes.  This does not mean that we can change this 80 min run into a 40 min run, but there is room for improvement.  The first step in the optimization is to determine the %B at which the last peak elutes.  If you look at the blue gradient line you might guess that the last peak elutes near 40%B but this would be incorrect.  All HPLC systems have a gradient delay.  The gradient delay is the time between when the software tells the pumps to start pumping at a certain mobile phase composition and the time it takes for that solvent composition to reach the column and have an effect.  A good guess for a gradient delay is 10 minutes.  This would mean that our guess for the final mobile phase composition for the 40 min peak would be approximately 30%B.  To observe the gradient delay time look at the illustration above and observe that the baseline returns to the starting conditions at 70 minutes and not at 60.1 minutes when our pumps have gone back to 2% B.  One must take care to avoid having the last peak elute on the "equilibration cliff", (at 70 min. in this example).  This can be avoided by ending the gradient at a %B that is slightly higher than that required by the last component.
Based on the separation shown at the top of this section one could rewrite the gradient to look like this:

This would make the gradient shallower and possibly give a better peak separation.  To shorten the run time one could rewrite the gradient to look like this:

This last change would cut 30 min. from the analysis time.  Shorter analysis times are always better for work efficiency.  With every minute you can cut from the HPLC method without sacrificing your chromatographic goals you will be rewarded with better work efficiency. With this change the last peak would most likely still elute at 40 minutes and the peptide separation would essentially remain the same as in the initial 60/60 analysis.
What is HPLC Equilibration?

The column must be equilibrated, re-equilibrated to the initial high aqueous solvent composition before another analysis can be performed.  Normally this re-equilibration is stuck onto the end of the gradient.  How much equilibration time is enough?  As a rule of thumb we give 20 minutes.  In reality it depends on the column length, flow rate and the hydrophobicity of your peptides.  Some chromatographers use 10 minutes as their standard equilibration time.  Equilibration is all about fitness of purpose.  You should determine the the equilibration time experimentally, the criteria will be, does my analyte really stick to the column and chromatograph appropriately and reproducibly with subsequent analyses. If you choose to do this part of the method development you will undoubtedly be rewarded with improved chromatography and better cycle time.

Should I Control Column Temperature?
Yes.  Scientists are control freaks.  If you can control a variable, control it!  Actually if you are performing automated analyses over a long period of time peak retention times can drift with changing ambient temperature.  It is common for many companies and institutions shut down the air conditioning at night to save money, which could result in shifting peak retention times due to dramatic changes in ambient temperature.  Many HPLCs provide the option to control column compartment temperature.  If your HPLC does not have this capability a heated column jacket can be purchased from many suppliers.  The most common running temperature is 40°C, this places the column compartment well above even the most extreme ambient temperature fluctuations.  In addition to maintaining constant temperature, temperature can be used to influence the chromatographic separation.  No chromatographic study is complete without a temperature study.  In our experience higher temperature is better, peaks will be sharper and elute earlier. It is not too uncommon to perform chromatography at 60°C and some daredevils even go to 80°C.  Remember though that higher temperature will lead to a shorter column lifetime and some columns may not be able to tolerate 60°C.  Consult the manufactures recommendations when experimenting with high temperature. After your runs are complete for the day it is advised that you turn off your column heater since high temperature leads to stationary phase deterioration.

Preparing for the First Run of the Day
One observation is that if you start up a reverse phase analysis from a dead stop with a column that has perhaps been sitting in high aqueous conditions for up to 10 hours the analysis will give irreproducible results.  Conventional wisdom has it, you want to first flush the column with the highest % organic of your method for at least 3 column volumes and then bring it back to the equilibrating condition.  This practice  may have the advantage of getting you to standard equilibration conditions faster and it will also clean your column.   A better alternative is to make the first run a blank run (or "preparation run") and then the next run can be your real analysis.  We prefer the second option because it should get you to the standard starting conditions more accurately.   However, often,  if we are in a hurry and the first option is quicker, well.....

After the Last Run of the Day
We store our columns in 50/50 methanol/water without any acid.  If you are using a salt, unlikely in LC/MS, wash your entire system, solvent bottles, HPLC, solvent lines, and column, into a non-salt containing solvent.   Salt may precipitate out and plug your HPLC or column or may cause corrosion.  Usually we flush with pure water first then leave the system in 50/50 methanol: water.  Some salts may precipitate out in high organic so an initial water wash is advised.  The 50/50 methanol:water solution helps to stop bacterial growth which can muck up your system.  Take care of your HPLC, it's the right thing to do!