Problem of area count of a tricky molecule

[Echis]

Dear Friends,

I am facing below mentioned peculiar problem with a particular product.

The area count of the standard (10 µg/ml) when injected on a brand new column (Inertsil ODS-3, 250 x 4.6 mm, 5 µ) is around 60 (Agilent 1100 series HPLC), after one day when a fresh standard is loaded its area count comes to ~40, in around three days the area count comes to ~15. The problem of solution stability can be ruled out as when 2 days old standard solution is injected on a new column the area count comes ~60.

Mobile Phase: Buffer : ACN (99:1)

Buffer: Milli-Q water + H3PO4 (0.6ml/Lt H2O) adjusted to pH 3.0 with NaOH soln

Wavelength is 210 nm. Even after washing with pure ACN there is no improvement in the area counts

can any boarder suggest the possible cause?

[Hemal]

As mentioned your molecule is tricky, is your standard used remains stable under storage conditions?

[Echis]

Yeah, it is quite stable. If a standard is injected after 48 hrs of preparation on a new column then also the area count of ~60 is obtained. I think that answers the stability issue.

Rgds

[HW Mueller]

The second new column also gave lower areas on subsequent injections??
No flow, retention time, peak shape changes? Are you shure that your columns are equilibrated? Do your compounds ionize?
Dewetting seems to be ruled out if ACN does not rejuvenate the column (anyway, that should influence area only if it causes a flow change?).

[Mark Tracy]

If you substance is a good complexing agent, buildup of metals and/or corrosion on the column could cause this, along with serious changes in peak shape.

[Hemal]

• Echis: The problem of solution stability can be ruled out as when 2 days old standard solution is injected on a new column the area count comes ~60.
  
  Hemal:As mentioned your molecule is tricky, is your standard used remains stable under storage conditions?
  
  Echis: Yeah, it is quite stable. If a standard is injected after 48 hrs of preparation on a new column then also the area count of ~60 is obtained. I think that answers the stability issue.

Since you are getting the same response on the solution prepared 2 days before & low response for solution prepared freshly, possibility of column problems won't be there if retention time is not changed & system suitability passes.

I will insist to check the standard stability and/or standard preparation correlation (n=2 to 3). Standard in solution may be stable.

[Echis]

I think u misunderstood me..... iwanted to say that even if 2 day old standard is injected on a new column then also it gives area same as that prepared freshly and injected on a new column (~60). Whereas Even if freshly prepared standard is injected on a 2 analysis old standard the peak area is reduced (~15-20).
Rgds

[Echis]

Yes, the second and third column also behaved in similar fashion
Rgds