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How to decrease run time for reversed phase LC

6 Nov 2014

There are several ways to accomplish this, although it boils down to roughly 4 possibilities.

  1. Use a shorter column length.

This may be appropriate if you have plenty of retention for your analyte(s), if you only have one or a few analytes AND if you do not have interference peaks in your chromatogram. It does, however, require purchasing a new column and shortening the length (L) does reduce the number of theoretical plates (N) in your column. The good news is that the end result might be better than you would expect. Shortening your column length by a factor of 2 does not reduce resolution (R) by a factor of 2, but only reduces it by a factor of 1.4, since R is proportional to the square root of N.

R for hpcl

N vs dp

  1. Optimize conditions for your existing column.

Look at the following and see what you might be able to tweak:

Flow rate- First of all, if you are running at less than the optimum flow rate, you should definitely increase it as long as your system can handle a little more pressure. Secondly, you may be able to go slightly above what is recommended as the optimum flow rate if you don’t need to have perfectly symmetric peaks and/or separation from other peaks. If you’re not sure what the optimal flow rate for your column should be, there are some listed in the blog post for Building up Pressure.

Mobile phase (s)- If you have not investigated this yet, try increasing the organic content of your mobile phase. You can also try using a different organic solvent. There are some cases when methanol will provide better selectivity, such as with biphenyl phases. Otherwise, acetonitrile may provide shorter retention times because it is more nonpolar. Solvent choice should be made on a case by case basis, though, as sometimes it may surprise you what works better. (Please keep in mind that this is only the case for reversed phase LC.)

Gradient- Try starting with higher organic and/or ramping up to more organic faster and see if that helps.

Temperature - Try increasing temperature. This usually speeds things up and often increases resolution also. Just make sure you don’t go beyond what is recommended for the column you have. Restek HPLC columns generally have a maximum temperature limit of 80°C. A good article to read on this topic is HPLC solutions #53: Temperature and Retention by John Dolan, published by Separation Science.

  1. Use a smaller particle size column

This will increase column efficiency and retain the efficiency even at higher flow rates. This is because efficiency is inversely proportional to the particle size, as shown in the two equations above. The following graph demonstrates the effect of linear velocity on H, the height equivalent of a theoretical plate. Keep in mind that a lower H value indicates greater efficiency, more plates (N) per unit length (L).

H vs dp and u

Since you will have more plates per unit length, you can either keep the same length and get increased separation or you can shorten the length accordingly to get the same separation with a shortened analysis time. The relationship between particle size and length is illustrated by the following equation.

L vs dp

Another option for smaller particles may be to increase the flow rate, since the column remains efficient at higher flow rates versus a comparable column with larger particle size, with all other dimensions remaining the same. For reference, please see the graph above of H vs. Linear Velocity, µ. Also keep in mind that any change in the inner diameter results in a change of linear velocity. A reduction in ID actually requires decreasing the flow rate to maintain the same linear velocity, so that must be considered as well. Generally, decreasing the ID is more useful in terms of saving solvent, not in shortening the analysis time.

Please note that decreasing particle size also results in a higher backpressure, so the instrument hardware must be considered when determining what particle size to use. The use of our 1.9 µm particle Pinnacle DB columns does require a UHPLC system (equipped for 1000 bar or higher max pressure).

  1. Use a superficially porous particle (SPP) column

In terms of Restek products, these are the Raptor columns. Otherwise known as core shell, particles in this column result in greater efficiency, similar to using a smaller particle, but without the backpressure that is normally associated with that particle size. A simple explanation of these benefits can be found at the blog post, What is SPP and when should I use a Raptor column?. Using SPP columns often produces a marked improvement in sharpness of peaks, resulting in greater overall sensitivity for the analysis. Often data can be produced that is UHPLC quality on an LC that is not technically a UHPLC system. If you need help determining whether to use a 2.7 µm of 5 µm column, please see the blog post Should I use a 2.7 or 5 µm Raptor column?.


I hope that you find this useful. Thank you for reading.