HPLC manual injector problem?!

[hanming86]

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 being "squeezed" out from the hole ( before i even pressed 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.

My suspect is something is going weird with the loop.

IS there anything you could suggest to help me troubleshoot this problem that would help a lot on this end.

The system is Rheodyne 7725 . Manual injector.

Thanks.

[Kostas Petritis]

As you say that you are new on this:

Make sure that you are injecting with a HPLC and not GC syringe (the needle is different).

Make sure that your loop is not plugged

Make sure that you understand the load and inject positions of your valve

Try using an HPLC needle of a higher volume that will make a tighter connection when you inject (i.e. if you are using 10 uL Hamilton syringe, switch to a 25-50 uL as the needle OD is larger).

[HW Mueller]

There are two things that come to mind when liquid comes out at inserting the needle: a.The rotor is not pressing on the stator, sufficiently, or it is shot (doesnt seal). b.The waste outlets are too high so that waste runs back into the valve.
Regarding no peaks: Did you try to inject without the column?
If the fault really lies with the Rheodyne one can add to the advice given by Kostas that one needs to whatch for drainage of the loop during filling via hanging tubes attached to the waste outlet combined with a bad needle seat (seal), or a wrong injection sequence (taking out the syringe before switching to inject).

[hanming86]

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 .

[Kostas Petritis]

Maybe it is time to give us some more details on column dimensions, mobile phase, pressure units (are you referring to bars or psi)?

Propably your pump is not designed to operate well at 0.1 ml/min and looses it's prime. Try to purge (without the column) and try again...

[tom jupille]

Your retention time shifts and the pressure changes are both consistent with flow rate problems (essentially the pump losing prime).

If you're new to HPLC, there are a number of free tutorials available on the web. Several of them are linked in the FAQ section (click on the FAQ link near the upper left of the screen here); drill down to "additional information".