HPLC manual injector - inconsistent data (Feb/21/2009 )

Hey guys,

I just got myself into this HPLC business. Got it setup and all that. it's an old machine lying in the lab but recently some technician came and get it to back to work again.

However, recently when i tried it myself , i havent' been getting very promising result.

I am using a manual injector. What's bugging me is..

Everytime i injected, when the needle went into the hole, i always see liquid coming being "squeezed" out from the hole ( before i even press the syringe to inject liquid ).

Should this be a concern?

I haven't been seeing any peaks even though i m using standards which got me worry whether or not the sample is even reaching the column.

Thanks.

Okay

First thing...I hope its not gravity flow, just make sure that the injector is at an angle where the back flow cannot occur. In my HPLC, the injector can be moved.

Second...disconnect the sample loop and make sure its not blocked, you can put in in some solvent and sonicate to clear any blockage.

Third.....the sample loop keeps what its volume is and throws the rest through a tubing behind the injector, just inject lots and lots of solvent and clear that path, the extra should not come back but should go down that path.

This should help or let me know.

TC

also, make sure the injector is in the "load" position when putting sample into the loop. you move it to inject position when you want to apply what is in the loop to the column.

and, the sample loop should not be used dry or you will introduce bubbles into your column. you should fill the loop with mobile phase before applying sample and rinse with mobile phase between injections.

Alright i got this thing to work yesterday. I saw a peak from my standard. Solved it by continuously flushing the the injector loop with solvent. No more liquid leaking out when i put the needle inside. probably got something stuck in the loop .

Anyway ,

I got some problems with the pressure going down to zero now. It was working fine at 400 plus minus for a day or so. i let it go at 0.1ml/min overnight and it's down to zero . then when it's 1ml/min it got up to 400 plus for awhile but it went down to near zero after 10 minutes.

On another notes, i notice sometimes my peak retention time could double 3 min to 6 min. i am wondering what could be causing this problem .

My take...purge valve of one or both the pumps is not closed properly, so solvent leaks from there and u see low pressure.

I am not sure but this can lead to delay in retension time as the solvent is not really going to the column.........but can't say for sure.

TC

T C on Feb 23 2009, 11:54 PM said:

I got the problem sorted out. apparently it's the mixer that's giving much problem.
So in the end i made the solution with the appropriate ratio of solvents in one bottle manually and use 100% of that mixed solvent as my mobile phase.

Works like a charm !! and i probably should get someone to look at the system in the future.