Sample delivery issues with ALS Agilent 1329B

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

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After running through the step commands in LabAdvisor and using the "Draw" function, it appears as though the syringe is pulling very little if any liquid from the vial. I made sure to leave the cap loose so I do not believe this is a vacuum issue in the vial... Agilent recommends purchasing a new piston seal kit which I am going to do, but I really doubt that this is the only issue. Additionally, for what it's worth I figured I should mention that the fan above the tray in the autosampler compartment is no longer functioning and I am getting the error message "Heat exchanger defective"... Is there any possibility that there could be a greater electrical issue at hand that could also be effecting the ability of the metering device to function properly? I just think there is a larger issue rather than simply the piston seal in the metering device not allowing for proper suction in the syringe; and if that were the case wouldn't I maybe expect to see a leak in this area (currently no leak)?

Hello everyone, first post here so I apologize if I have posted in the wrong forum. I made sure to check and the LC Troubleshooting Bible before posting here but I very well may have overlooked something...

I am a novice to HPLC but have recently landed into a position in which I am responsible for operating an Agilent 1260 Infinity fitted with a G1329B autosampler. The previous operator has since left and I am doing my best to do a decent job. I recently had noticed some high backpressure and also observed some septa debris (after reverting back to non pre-slit caps) in the waste tube coming off the 6-port capillary (is that how I should call it? sorry haha not sure of the name). I went ahead and disassembled the syringe, needle seat, 6-port valve, and metering valve (all using the LabAdvisor software and using maintenance positions when applicable), cleaned all components to best of my abilities, and reassembled (unfortunately we dont have any replacement consumables on-site, and money is tight...). I did my best to take photos and reinstall everything in the order and position in which it was removed. Also, LabAdvisor indicates that it is a 100uL loop size and a seat capillary of 2.3uL (not sure if this is relevant but I figured I should include this).

My pressure is back to normal and I cannot find any leaks, but I have since injected a few (separately prepared) standards and have been getting consistently incorrect results. I really dont believe that the CRM has been compromised and rather that it is an issue with the instrument. I typically do 15 microliter injections and though that is somewhat difficult to visually observe, something in my gut tells me the injection volume is off. I had initially contacted Agilent for some support and their idea was to weigh a sample vial before and after injection (though I am slightly concerned about mass loss from piecing the septum in comparison to only 15uL leaving the vial...). I am deep-down hoping there is maybe some sort of air bubble or something hindering the metering valve's ability to inject the intended volume of sample; however, Agilent support does not lend me any hope toward this scenario...

In the LC Troubleshooting Bible I came across a recommendation to inject 10 shots of the same sample and check the standard deviation; but part of me is concerned that what if the injections are consistently off as I have observed this far... Are there any sort of additional diagnostic checks anyone can lend me to help me diagnose if it is in fact the injection volume being off? Has anyone else encountered a similar scenario? I tried to be very careful during all of my maintenance procedures to ensure that no metal parts were gouged or anything of the sort, but it is certainly a possibility since this issue has only occurred after my maintenance actions.

I look forward to any and all responses. Additionally, please let me know if there is any additional information that I can provide.

Thank you all for your time!

What company buys a 1260 HPLC system and can not afford to buy the most basic preventative maintenance parts and/or pay for professional service (or pay for someone to teach your group how to do the basic PM services)? What company can not "afford" to hire trained people to perform these basic services ? That sounds like very bad policy. I hope they do not run samples for clients!

Let us address a few of your concerns:

There is no "syringe" in an Agilent A/S. It uses a flow through high pressure HPLC pump, single piston, instead of a glass syringe. *It is actually built by using 1/2 of a std 100 ul pump head (some models use a 40 ul head, but the design is the same). It is good to still think of it as a "syringe" though, but in this case the 'plunger' has space all around it to allow liquid to flow through!

You stated that you made an attempt to take the sampler apart, clean it, then re-assemble using the old worn parts. First, mistake. Please order the routine maintenance parts for your A/S (a single piston seal for metering device, needle seat). Normally, no other parts are needed for routine maintenance of most of their samplers (unless of course they are damaged. Example, a bent or plugged needle. A/I pistons last a LONG TIME). I strongly recommend you hire a professional to do the basic services on all modules. This will insure that they are properly checked, inspected and re-assembled correctly. Money well spent!

The "fan". It sounds like you have a sample thermostatted module for your A/S. If you lift up the sample tray, then you will see the air channel which leads to the cooler/heater underneath it (Model G1330A or B). If the sample cooler is broken, then your sample temperature is no longer being controlled. This may or may not matter. It depends on what your method calls for and if sample temp must be controlled. If it needs it, have the G1330 repaired (or dispose of it and buy another one to replace it with. You can often find a working one on the web for very little money).

Here is a link to an article that might help you with the injector: "Troubleshooting HPLC Injectors (Manual and Automated) "; ... anual.html
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