Irregularities and huge drifts

[alireza.shirkhany]

Hello,

I am having problem with sudden/changeable positive and negative drifts on my test.

Here is my gradient table:
time: 0 A:80% B:20%
time:20 A:32 B:68
time:21 A:32 B:68
time:21.01 A:80 B:20
time:25 A:80 B:20

Flow rate is 1mL/min.
Mobile A: Sodium Dihydrogen Phosphate (aq), pH=3.00
Mobile B: 70%(Acetonitrile):30%(Mobile A)
Auto-sampler temp:6 degree Celsius
DAD wavelenght: 210nm
Injection Vol.:20 micro-liter
Column: C18, 25 cm, 4.6 mm,5 microns
Run-time: 25min

I have been running this method for more than 100 times. Some of the chromatograms of blank and samples are chosen randomly between first 50 runs and are shown below.






Ok, as you see chromatograms of the blank are showing discrepancies as the absorbance at the first minutes of the run-time is getting low and is increasing toward the end of the run.

For other 50 runs, this effect is increased on both of the blanks and samples as below.





Finally, today I made a lot of tries.
First: I detached the column guard and changed the cartridge with a new one.
Second: I ran ACN (95%)/ Water (5%) into the system with and without the column attached to the column guard and the chromatogram below is obtained.


Then I injected the blank and the following chromatogram was obtained which is similar to previous chromatograms of blanks for the second-50 runs.


I don't know how can I get rid of this positive shift, increased absorbance and irregularities.

Please help me.

[cksoni]

Not able to see the images. if you can share the images may be helpful to understand the issue