Chromatography Forum

ear all
I am using Shimadzu LC 10 ATVP HPLC system with PDA detection
Mobile phase: MeOH:Buffer(pH 4.3 KH2PO4) in 50:50ratio isocratic

Problem: i am getting a peak at RT 5.6 with area ranging from 6000-9000
I have run the same on HPLC prominence series but there i do not find any peak.
this peak is coming with everything even with water, methanol, IPA.
i have clean up the system and but still facing the problem for almost 1 month.
injector port is also replaced, also all four pumps run indivisually gives same peak.

kindly suggest me cure

other conditions are mentioned below.
reply asap
thanks
rohit

Make: Shimadzu

Model: LC-10 AT/vp/

Pump: LC-10 AT/vp/

Detector: SPDM 10 A/vp /DAD

Flow Rate: 0.5ml/min

Sample volume:10ug

Injector: SIL 10 A autoinjector

Software: CLASS /vp/

Column: Luna 5 C(18) 150 X 4.6mm 5u

Pressure: 10MPa / min

Column Temperature: 30 C

Injector Temperature: 10 C

Final pH of pH of Mobile Phase: 5.3
1 MeOH: Buffer pH 4.3(KH2PO4) 50:50(Mobile phase)

Injector rinse: 50% MeOH RT 5.6
2
MeOH: Buffer pH 4.3(KH2PO4)

Injector rinse: mobile phase RT 5.6
3 MeOH: H2O 50:50

Injector rinse: 50% MeOH RT 5.6
4 Pump A,B,C,D
100% Mobile Phase RT 5.3-5.5
5 IPA through whole system thenMobile phase

RT 5.6
6 Mobile phase collected fro RT 5-8min and subjected to MS
282.5 mol wt peak
7 Millipore Water from different source
Peak exist
8 Buffer from different batch
Peak exist
9 Different columns (C18)
Peak exist
10 Cleaning with DMSO
Peak exist
11 Replacement of injector port
Peak exist
12 Using gradient method
Peak exist
13 Change of flow rate
RT changes
14 Using Shimadzu prominence series
No peak
15 MeCN as mobile phase but no separation
No peak

I do not how but like to guess it is due to contamination. Can you call your vendor to see if they can help to diagnose? good luck

Did you try to replace the loop?

I'd try replacing the high pressure seal & doing several high % organic injections w/ short run times between them (no column needed) - sounds like you have something in the injector.

If that doesn't do it, you may want to replace the rotor & stator portions of your 6 port valve.