HPLC 3000 Sample is not drawn up from the vial

[prk018]

Our lab has a HPLC Ultimate 3000 and I'm having an issue with our autosampler.

I've noticed that our sampler isn't injecting any samples from the sample vials. I visually checked that the needle is moving deep enough to be fully submerged in sample without touching the bottom of the vial, but when the syringe moves down, nothing from the vial is getting drawn out. I tried out different injection volumes, and with high injection volumes, a little bit of stuff does get drawn out, but nowhere near the actual volume that should be getting injected.

I already replaced the needle and the syringe to make sure there wasn't a clog in the pathway but sample is still not drawing up. It doesn't look like to me that the syringe is pulling in air and not liquid when it is supposed to be drawing up samples (although it's kinda hard to tell with clear liquids). I don't find any leaks in the pathway (e.g. from top of the syringe, injection valve, etc.) and I'm confused what else could be going on. Any suggestions?

(Also, unsure if this is related at all, but our syringe makes terrible squeaky noise when it moves up and down to rinse, inject, .... I took out the syringe and pushed the plunger a couple times myself, and it's not the syringe that's making that noise, so I'm assuming it's the hardware that moves the syringe.)

[bunnahabhain]

Try to put some oil on the spindle drive (sewing machine oil is well suitable), this should remove the squeaky noise.
As for the drawing up problem: Be sure that the wash liquid reservoir is filled with degassed liquid (we used methanol/water in the beginning, which caused trouble because it tends to produce bubbles. Isopropanol is much better).
Remove the syringe and flush it manually with isopropanol. Then fit it back into the valve block. Then start "prime syringe" with 25 cycles or so. If you see an air bubble which remains in the syringe during the first 5 cycles, remove the solvent line from the wash reservoir while the prime procedure is still running. When the syringe is pulled down, fill isopropanol into the open wash line port until the air bubble is gone (usually takes about 3-4 cycles). Then, during the upwards plunger movement, quickly reconnect and tighten the wash reservoir line.
Note: This procedure causes a lot of solvent dripping down, so be sure to use sufficient paper towels and gloves which are resistant to isopropanol. It needs some practicing to tighten the solvent line, so repeat it as necessary. Good luck!

[prk018]

Try to put some oil on the spindle drive (sewing machine oil is well suitable), this should remove the squeaky noise.
As for the drawing up problem: Be sure that the wash liquid reservoir is filled with degassed liquid (we used methanol/water in the beginning, which caused trouble because it tends to produce bubbles. Isopropanol is much better).
Remove the syringe and flush it manually with isopropanol. Then fit it back into the valve block. Then start "prime syringe" with 25 cycles or so. If you see an air bubble which remains in the syringe during the first 5 cycles, remove the solvent line from the wash reservoir while the prime procedure is still running. When the syringe is pulled down, fill isopropanol into the open wash line port until the air bubble is gone (usually takes about 3-4 cycles). Then, during the upwards plunger movement, quickly reconnect and tighten the wash reservoir line.
Note: This procedure causes a lot of solvent dripping down, so be sure to use sufficient paper towels and gloves which are resistant to isopropanol. It needs some practicing to tighten the solvent line, so repeat it as necessary. Good luck!

So I took your advice, switched out the wash reservoir, rinsed the syringe manually with IPA, and primed the syringe. I don't see any air bubbles during the prime cycles, and I'm wondering if air could have somehow gotten trapped somewhere in the flow path from the needle to the syringe. Is there any way I can pull air out manually using another syringe?

[bunnahabhain]

Other points you could try out:
- Is your sample highly viscous => Then try to reduce the DrawSpeed tp 5 µL/s or lower and see if things improve.
- Do you use septa with slit? If not, sample cannot be drawn correctly due to vaccum forming in the closed vial. Try to inject from an open vial to see if this helps.
- Your needle dips too deeply, hits the bottom of the vial and thus prevents the sample from entering the needle. Use ReagentLiquidHeight 2 mm or more.