peak separation 2

[vanja13]

Here is the chromatogram.

Eluent is ACN: phosphate buffer 20 mM (pH 2,5) = 2 :1, Inertsil ODS 2 250 mm, 4,6 mm.
How can I get better separation of peak on 5,92 and the small one on 6,49 min? (replicate injections are quite different).
By reducing the amount of ACN?

[Bruce Hamilton]

Unfortunately, the chromatogram is very indistinct on my screen, more like looking through opaque glass, so I can't comment on the separation.

However, if the replicates are very variable, you have to sort out that problem first. Assuming that your retention times are constant, the areas of the small peak are presumably varying. If the large peak is also varying then you have even more problems to address.

The most obvious issue would be the integration, and you should have two sharp, well-defined peaks for the software to work with.

If not, then you probably should first decrease the sample injection volume, but still ensuring your smaller peak stays above the lower limit of quantitation.

Try to ensure your sample is dissolved in the mobile phase, and that you have nice peak shapes - eg play with column temperature etc to achieve the sharpest peak, perhaps try using a gradient, provided you have that option on your HPLC. Target about 8 - 12 mins retention of large peak for your column.

By increasing the organic component you should increase the retention, and may improve the separation ( depending on peak shape ) but you are already at 66% organic, so you have to be careful about issues such as solubility as well.

You may have to change to a different column chemistry, preferably one that moves the small peak either much further away, or in front of the large peak.

Good luck,

Bruce Hamilton

[tom jupille]

A few comments:

First of all, it looks like you saved the image as a 40 x 40 pixel bitmap. 400 x 400 is the usual recommendation.

Second, Bruce, I know you meant "decreasing" the organic solvent will increase retention.

Third, Vanja, you didn't give the flow rate, but assuming 1 mL/min, your k' values are fairly low (a 250 x 4.6mm id column has an internal volume of about 2.5 mL. You could decrease the % organic by 10% and triple your k' values. Other things being equal, that could increase your resolution by 15-20%. It will also make your second peak broader (and therefore shorter), so you may also have to increase the injection volume, as Bruce suggested.

[Bruce Hamilton]

Second, Bruce, I know you meant "decreasing" the organic solvent will increase retention.

My error. Thanks Tom. I'll try to check read my posts more carefully.

Please keep having fun.

Bruce Hamilton