Negative Peak during run after injection

[skeetch79]

Let me start off by saying that I am a novice at HPLC - I am more a GC guy!

I am having an issue with the appearance of a negative peak after injection on my Agilent 1260 LC II system. The system is currently configured with a Phenomenex GuardColumn, 250mm x 4mm RP-C8 column and RID detector. Mobile phase is 100% Water, flow rate is 0.9ml/min, Column temp is 50C (both left and right) and Detector temp is 35C.

When I first got the LC I did not have this issue. My main analyte was Ethyl Alcohol. I ran Alcohol analysis for a month without issue. At some point, I had a need to analyze for Vanillin, so the 250x4mm column was swapped for a 125mmx4mm RP-C18 column and the mobile phase was changed from 100% Water to a changing gradient ratio of Acetonitrile and Acidified Water with Phosphoric Acid, pH <2.8 analyzed via DAD detector.

Between all of this switching the system was flushed with IPA and Water.

The need to analyze Alcohol arose again, so the original configuration was returned. This is when the negative peak problem arose. Longer story shortened a little, after multiple visits by the Agilent Tech, a swap of the degasser, replacement of the Needle, Needle Seat and Coil, multiple system flushes with the Agilent HPLC Flush solution, the problem still exists.

Strangely enough, when the system is in idle just running Mobile phase or when a no-injection blank is run the peak does not show up. The peak only shows after an injection. It even appears when injecting a vial with the mobile phase (100% Water).

I am at a loss. I can always explain further in more detail but I wanted to lay the ground work. I have attached a few screen shots of the Peak. Some screen shots show the negative peak while a column is installed, others show the negative peak with the column removed and a union installed connecting the column inlet capillary and outlet capillary together. Why this is even worse is that the negative peak elutes at the same place as my Alcohol peak.

https://drive.google.com/open?id=183AAbrKiNGJUsNIZcn2rjAcT_Bjb67jp

https://drive.google.com/open?id=1LCwDqIVLZa39Frary1xq2nJenadGXvRD

https://drive.google.com/open?id=1gD_IlAfNwTAGFhjcjn42MJkC1C75owyn

[Consumer Products Guy]

I'm still in shock seeing that you're using 250mm x 4.6mm columns !!! That is so 1990s !!!

By the way, when using refractive index, we always matched the detector temperature to the column temperature. And bypassed the Agilent multi-channel mixing valve with the little adapter.

[skeetch79]

I'm still in shock seeing that you're using 250mm x 4.6mm columns !!! That is so 1990s !!!

By the way, when using refractive index, we always matched the detector temperature to the column temperature. And bypassed the Agilent multi-channel mixing valve with the little adapter.

What can I say? I work for an international company based out of Germany. They developed the methods for company wide analysis around the globe. These established parameters ensure all generated data is comparable.

Can you please elaborate on what part the Agilent multi-channel mixing valve is and what little adapter you reference?

Thank you.

[Consumer Products Guy]

Can you please elaborate on what part the Agilent multi-channel mixing valve is and what little adapter you reference?

Your system cannot be UPLC because of the column sizes you detailed; Agilent 1260 is new since I've retired. Traditional Agilent quaternary pumps had a multi-channel mixing valve and binary pumps did not. Here's a photo of the adapter (on the right, looks like a fingertight nut but has female threads for low-pressure fitting. On the right. Agilent #0100-1847

https://www.agilent.com/store/en_US/Pro ... /0100-1847


Typical mixing valve for quaternary pump

What can I say? I work for an international company based out of Germany.

Sounds like little flexibility from your employer to change stuff (sounds like my employer).

[James_Ball]

Since this only happens when you have an injection and with or without a column, have you changed the rotor in the injection valve? If a contaminate gets into the valve or if the valve is worn and leaking a little, or plugged it can cause flow disturbances when it switches from flow through to inject and back. I am not certain but aren't RID very susceptible to disturbances in flow? It sounds like this is the only part the service engineer didn't replace.

[DoryFish]

This is a left-field idea, but wondered if you have tried any injection without a septa in the vial? I once spent a lot of time trying to find a contaminant that turned out to be associated with the septa. Can't recall if it was a new shipment or we changed our supplier, sorry.

[skeetch79]

Thank you for all of your responses. They all make sense.

I am starting to think it is air being introduced somewhere. A "no injection" blank does not result in a negative peak. Changing the flow rate moves the negative peak - i.e. slower flow, peak comes later. Also larger injection, larger peak. Smaller injection, smaller peak. I have also injected air from an empty vial and the negative peak gets larger. I am thinking it is somehow related to the injection valve, injection coil and/or metering valve. I have a 100ul coil on a system where at most I am injecting 20ul.

Is it possible something is not tightened properly and air is getting in? I have flushed this system with so much Agilent Flushing Solution (IPA/ACN/CH2Cl2/Cyclohexanes) it is silly at this point.

I see the negative peak when injecting the mobile phase from the autosampler.

[skeetch79]

....

My system does have a Quanternary pump.

The fittings leading in to the pump from the Degasser look like the one on the left in your picture.

[skeetch79]

This is a left-field idea, but wondered if you have tried any injection without a septa in the vial? I once spent a lot of time trying to find a contaminant that turned out to be associated with the septa. Can't recall if it was a new shipment or we changed our supplier, sorry.

I have not tried this yet - I will give this a shot today and post the results.

[James_Ball]

Thank you for all of your responses. They all make sense.

I am starting to think it is air being introduced somewhere. A "no injection" blank does not result in a negative peak. Changing the flow rate moves the negative peak - i.e. slower flow, peak comes later. Also larger injection, larger peak. Smaller injection, smaller peak. I have also injected air from an empty vial and the negative peak gets larger. I am thinking it is somehow related to the injection valve, injection coil and/or metering valve. I have a 100ul coil on a system where at most I am injecting 20ul.

Is it possible something is not tightened properly and air is getting in? I have flushed this system with so much Agilent Flushing Solution (IPA/ACN/CH2Cl2/Cyclohexanes) it is silly at this point.

I see the negative peak when injecting the mobile phase from the autosampler.

If it is a normal Agilent autosampler, there is a small diameter plastic tube that comes from the 6 port valve on the autosampler as a drain for getting rid of the same amount of mobile phase as the sample volume you will draw up. This tube is supposed to fit into a slot in the side of the post that is right in front of the valve that also holds the drain tube from the pump above it. The opening of that small diameter tube has to be above the level of the needle seat( which is why it is in the slot) or it will form a siphon and draw air into the needle seat while the needle is in the vial drawing up sample. If the end of the tube is hanging down below the needle seat and valve or connected to a longer piece of tubing to go to a waste container, it will siphon even worse. I found that out a long time ago when I tried to connect that to a waste container below the bench, and every injection I did had air in it. Not sure this is what is happening but letting you know about it just in case.

[skeetch79]

Thank you for all of your responses. They all make sense.

I am starting to think it is air being introduced somewhere. A "no injection" blank does not result in a negative peak. Changing the flow rate moves the negative peak - i.e. slower flow, peak comes later. Also larger injection, larger peak. Smaller injection, smaller peak. I have also injected air from an empty vial and the negative peak gets larger. I am thinking it is somehow related to the injection valve, injection coil and/or metering valve. I have a 100ul coil on a system where at most I am injecting 20ul.

Is it possible something is not tightened properly and air is getting in? I have flushed this system with so much Agilent Flushing Solution (IPA/ACN/CH2Cl2/Cyclohexanes) it is silly at this point.

I see the negative peak when injecting the mobile phase from the autosampler.

If it is a normal Agilent autosampler, there is a small diameter plastic tube that comes from the 6 port valve on the autosampler as a drain for getting rid of the same amount of mobile phase as the sample volume you will draw up. This tube is supposed to fit into a slot in the side of the post that is right in front of the valve that also holds the drain tube from the pump above it. The opening of that small diameter tube has to be above the level of the needle seat( which is why it is in the slot) or it will form a siphon and draw air into the needle seat while the needle is in the vial drawing up sample. If the end of the tube is hanging down below the needle seat and valve or connected to a longer piece of tubing to go to a waste container, it will siphon even worse. I found that out a long time ago when I tried to connect that to a waste container below the bench, and every injection I did had air in it. Not sure this is what is happening but letting you know about it just in case.

Hi James,
Please see the attached pictures of my AutoSampler Valve. Please advise which port the small plastic tube comes out of. The only small plastic tube I have is to drain the Injector area after a needle wash.

https://drive.google.com/open?id=1iUzuvAQOrj1dUzzuLcl1NU32boCZl0CW

https://drive.google.com/open?id=1iXmfGObE8kHMgU4CJPzYIta-uCJYtsX3

[skeetch79]

So after much troubleshooting the problem appears to be arising from the Degasser. Upon sampling the Mobile Phase (Milli-Q Water) from the Reservoir Bottle atop the instrument, before the Degasser, after the Degasser and multiple other points in the system and a final sample of the mobile phase exiting the RID and injecting these samples on our Q.C.'s LC instrument - the negative peak appears right after the Degasser. The LC Chromatogram of the Water from the Reservoir Bottle and just before the Degasser does not show any peaks. Sampling right after the Degasser and before the Quaternary pump the negative peak appears. I am thrilled to find the trouble component, but I am still at a loss as to how the Degasser is causing this issue. It is a contaminant which is not persistent in the system - it appears within samples after the Degasser but then is not present in the sample of the Milli-Q Water from the Reservoir bottle.

Is the Degasser introducing air? Are one of the other mobile phases somehow contaminating the mobile phase in use? A Mystery!

EDIT (UPDATE): The problem was also traced back to the Multichannel Gradient Mixing Valve. IT appears one of the channels was not fully closing and was leading to contamination. Replacing both the Degasser and MCGV resolved the issue.