Carryover problem

[digvijaysinh]

Dear All,

We run sequence as following way,

System blank(Only gradient run for Equilibration) (10ul injection)
Blank Solution (diluent) (10 ul injection)
SST mix (High concentration ref. sample as a std with impurities)(Main API peak has area @35000mAu.sec) (10ul injection)
Std(Low concentration Std) (Area @35mAu.sec) (10ul injection)
Std(Low concentration Std) (Area @35mAu.sec) (10ul injection from same vial as above)
Std(Low concentration Std) (Area @35mAu.sec) (10ul injection from same vial as above)

Std(Low concentration Std) (Area @35mAu.sec) (10ul injection from same vial as above)

Sample (Which having peaks like ref. std)
BKT Std (Low Concentration) (10ul injection from same vial as above)

BKT SST mix (High conc. Ref. sample) (10ul injection from same vial of SST mix)


Problem is that RSD of Low conc. Std is going beyond limit of 2%.

We have done following experiments & have following observations.
1) When injecting Diluent after SST mix, we got API peak of are in all Agilent HPLCs and we have only Agilent HPLCs.
2) When injected 4 injection of Diluent from same vial immediate after SST mix, API peak observed as in first diluent area of @1.5mAu@sec,in second run are of@13mAu.sec, in third run area of@10mAu.sec, in 4th run area of@7mAu.sec.
So question is why carry over is very less in very first blank & high in 2nd & 3rd balnk??
In most of the sequence area of Std is high for 2nd & 3rd Std and fails in rsd limit.

3) We have run two system blank(Only gradient run, no movement of any injector parts) immediately after SST mix, we observed no peak. That means no carry over from column. Then we immediately injected diluent solution,we observed peak. Then immediately injected 5 runs of Std, RSD found of 0.5%.Then again injected diluent, peak of area 1.5mAu.sec is observed.
4) Since peak of area 1.5mAu in diluent means carry over of approx. 0.05% (limit of system), but still affecting system suitability criteria.
5) We checked once by injecting from wash vial, we haven’t observed peak.
6) We have run sequence using different wash vials, one for SST mix & other for Std, but still , rsd for std has failed.

So, pl. suggest how carry over can be made 0.00% , so that this sequence can run successfully. Or if other than carry over problem, pl. suggest.

[Gerhard Kratz]

Please check your injection system. If possible implement an extra needle wash step. If you have a switching valve with sample loop please check the rotor seal. Good luck.