Help!!!!

I am running an post column reaction IC gradient method and having problems with band broadening and tailing of the main peak. I have looked at the usual things but still this problem is there. Could anyone please help I am open to suggestions to try.

Thanks

Noname,
Would you consider listing the 'usual things'?

A lot of IC is optimizing to get good peak shape. Maybe you have a problem in the separation. Can another detector, perhaps not as sensitive as your PCR one, see the peaks at a higher concentration? That would isolate the PCR or the chromatography as the problem.

You can check if void-volume mixing causes the tailing by teeing in a pump between the detector and the column. If the problem stops, the reactor geometry sucks.

If the PCR method has slow kinetics, it might distort the peak shape. What is the effect of column flow, temperature, etc., on the peak shape?
Jim

Anonymous,
More information is needed in order to debug your problem. Can you tell us a bit more about your PCR plumbing scheme and relative flow rate of eluent and the post-column reagent? What sort of mixing tee are using? What detector are you using? Also, are you sure the tailing problem isn't caused by overloading the column? What column are using? How much are you injecting and what is your analyte concentration?

Jim/Chris

In answer to both Jim and Chris this is a small outline of my PCR system and what I have looked at:

Gradient pump (Delivering a mixture of two buffers one at pH 4.2 (A) other at pH 7.4 (B)both mainly salts) and reagent delivery pump (Containing ninhydrin) along with a mixing chamber (siutated after the pump heads and before the autosampler). System contains an autosampler, fast cation column and two reaction coils set at 130 degrees C, along with a UV detector set to 568nm. All dionex equipment.

Gradient is as follows 75% A 25% B for 3 minutes, 0% A 100% B from 3 minutes to 30 minutes then 30.1 to 50 minutes 75% A and 25% B (flow rate 1ml/min for both buffer and reagent pumps)

Injecting 20ul, analyte concentration about 0.25mg/ml.

Peaks all come off in approx first 25 -30 minutes. Peak shape is still OK but peak width has doubled.

I have looked at flow rate, column, tubing diameter and detector.

Hope this helps, still haven't got to bottom of problem so any help will be a bonus.

Anonymous,

Are your reaction coils knitted or packed? If they are packed reaction coils, check to see if there is a void formed at the top of the reaction coil. In either case, double check to make sure that the fittings haven't slipped, creating a void at one end. Often, unless the reaction coils are thoroughly rinsed after shutdown there can be post column reagent crystallization in the tubing of the reaction coil. At start up, this can result in damage to the reaction coil. It's possible this problem is caused by that sort of thing. Was there any specific event that correlated with onset of the current performance?

My bet is that the column has gone belly up. You won't see the ugliness of the peaks because the reaction coil is hiding it. Do you have another column around?

Noname
It sounds like your equipment should work fine. You use two reaction coils for ninhydrin? How long is the tubing in each coil? All post column reactors cause some peak broadening. Using two coils instead of one larger one of the same tubing length would add to the broadening. You speak of doubling the peak width with good peak shape. (the peak width doubles from what comparison?) That much sounds like everything is working just not optimal but maybe the best the system can do. The pH at 25 minutes is less than 5 still so your gradient is fairly subtle, shouldn’t cause problems. Probably with dwell and mixing the pH is probably still less than 5 at 30 minutes.
You can still test for reactor broadening. Speed up the ninhydrin flow. The peaks maybe shorter due to less reaction time, but narrower peaks will show it is where the broadening occurs.
Jim

If you are running aminoacids, you can use UV detector to find if a column or reactor are causing the problem. At 254 nm you can see the aromatic aminoacids (Trp, Phe). Just check the peak shape directly after column without PCR and after PCR (replace ninhidrin with water, do not heat the coils).

What about overloading, does the peak width change with concentration?