Hi I'm Gerard Sharp. Welcome to the September 2011 issue of the monthly newsletter. This month the Tech Tip is on Factors affecting Resolution in HPLC.

Dr Gerard Sharp...

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Tech Tip - Factors Affecting Resolution in HPLC


There are essentially three factors in chromatography which can be changed to gain better resolution. These are:

  • Retention
  • Selectivity
  • Efficiency

Retention can be altered through a change in the mobile phase and the staionary phase. We can change selectivity by changing the nature of the column (stationary phase) and the solvent (mobile phase). Efficiency is a function of the column flow rate, particle size, column length and is measured by theoretical plates.

Defining Resolution

Firstly , we should define what we mean by resolution. Resolution is a function of the retention time difference of the two peaks and peak width. Resolution can be improved by either moving the peaks further apart or by decreasing the peak widths. 







Changing Retention

The easiest way to change retention of a component is to change the mobile phase strength. For reversed phase separations, this is a matter of decreasing the amount of organic modifier in the mobile phase. A decrease in the organic modifier (e.g. methanol) by 10% will have a 2-3 factor increase in the retention factor (k). The retention factor takes into account the time an unretained component (t0) takes to travel between the injector and the detector. It is a true measure of the retention of a component in the stationary phase. If the retention factor is less than 5, then changing the retention factor is the the first thing that should be tried as this will dramatically improve resolution. Ideally the retention factors for your peaks should sit between 1 and 5. A retention factor less than 1 means the compound is eluting too close to un retained components at the front of the chromatogram.


Changing Selectivity

Changing the mobile phase mix will also affect the selectivity (α) of the separation. The selectivity is a ratio of the two retention factors. This is not so predictable and mostly requires trial and error. As the mobile phase is changed the difference in the chemistry of the components in the mixture will result in different degrees of retention in the column and hence will aid separation.





Changing Efficiency

Efficiency is measured by theoretical plates (N) and the efficiency of the column can be found on test chromatograms provided with the new column. The higher the plate count, the better will be the separation. There are a few ways to increase the efficiency of the column and gain resolution.

Doubling the column length will double the plate count. Unfoutunately the increase in resolution is only 40%.

(see equation below).

Decreasing the particle size will increase resolution. This is the trend of column producers. 20 years ago the common particle size was 10µm. Now it is 3.5-5µm and sub 2µm particles becoming more common. The trade-off with smaller particles is the resistance in the column is higher and more back pressure is generated. Manufacturers of HPLC equipment have addressed this by pumps and other components capable of handling the higher pressures (1000 Bar and higher).

The sub 2µm particles columns can also be used to provide a higher plate count per meter but packed in a shorter length column. Shorter columns means shorter run times. For example, a 150mm x 5µm particle size column provides about the same plates as a 75mm x 3.5µm particles size. The 75mm will elute the peaks in half the time without a loss in resolution.

Resolution Equation

  • Resolution: Rs = (1/4)[(α-1)/α](N)½[k/(1+k)]
  • k = the average of two peaks

For those more mathematically suited, the relationship between resolution and the various factors is described above.



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