Multidimensional wrote:
Some comments and questions to think about:
Q: Why are you injecting 150 ul into a 20 ul loop ? The accepted method of 3x loop volume for a full volume injection should be followed (in this example, 60ul total). Just odd to use 150ul for no reason ...
You describe "carryover", but I do not see any mention that you serviced whatever type of injection valve you are using. Valves require regular maintenance. Failure to maintain the value results in carryover. If you have not done so already, please properly service the valve (always a good idea to have the spare parts on-hand as this is a routine service).
Have you tried a true blank injection as part of you troubleshooting (a "Blank" injection involves injecting the mobile phase in the same volume as your sample)? The results are often very diagnostic.
From what you describe, either you are using the injector in an unusual way (plumbed wrong, wrong parts, wrong positions) OR far more likely the injector is worn out/broken.
Column fouling may also be contributing to the problems too.
"Wash" injections (you wrote: "dilutes the peaks recorded for the sample") , that is a follow up injection of mobile phase should never "dilute" any samples. I have no idea what you are referring to in this context. IN fact, for most types of analysis, a needle wash should not be needed unless the samples are rather 'sticky'.
You wrote: "The "binary" gradient is being used to mitigate the problems with frequent pump failure caused by air bubbles"; What gradient? It appears you are running isocratic with a RID (?). Pump cavitation is often caused by failure to maintain prime of the system. Common reasons for this include: a lack of continuous degassing of the mobile phase; sticking inlet or outlet valves; piston seal failure(s); clogged pickup frits; leaks or loose fittings. *If you are running a separate gradient step to wash the column down, what solvents are you using coupled to what time/flow program?
Thank you so much for your response. Regarding your questions:
1. The decision to use 150 uL was actually based on the 3x loop volume method. Unfortunately,
the lab I've joined had misplaced all the papers containing the specifications of the HPLC including the loop volume. So, when I was developing the method I had to
manually determine the loop volume by injecting liquid into the injector and measuring the subsequent volume of liquid drained/displaced from the injector.
This volume was approx. 50 uL, hence the decision to use 150 uL injection (3x 50uL). It wasn't until much later that I found out that the loop volume was only 20 uL.
2.
Yes, I did try a true blank for diagnosis. My approach was:
2.1 Inject a sample containing standards
2.2 Null injection which resulted in insignificant carryover, thus ruling out contamination in column.
2.3 True blank injection - where peaks for the standard compound from (2.1) were recorded with larger area than the sample/standard injection.
3. It's true that the sample injector hasn't been serviced in a very long time. I've requested the lab to schedule servicing soon. Since I'm a lowly Masters student, I'm not allowed to make any changes to the hardware.
4. We've only just installed a new column few weeks ago, so I don't think it's a major source of the issues.
5. Lactic acid is bit sticky/viscous.
6. Regarding the diluted peaks:
6.1 Since I usually analyze samples from different experimental conditions with high organic load (up to 25g/L), I observed carryover problems.
6.2 Following the null & true blank injections, I narrowed down the source of problem to the glass syringe and/or injector.
6.3 For the sample injectors (Rheodyne 7725i), I had to resort to "wash injections" using either true blank or distilled water to prevent compounds from one sample getting carried over to another.
6.4 Compared to older results for the same sample, the peak areas were significantly smaller for the sample prior to which I used wash injection. Since I use internal standards, I know that the peaks are in fact a lot smaller than they should be. But I don't know why.
7. I'm using the injector usage method I came across on this site and youtube.
7.1 Keep the injector in 'Load' position
7.2 Insert the syringe and inject the sample
7.3 Rotate the injector into the 'Inject' position to start the analysis but don't remove the syringe.
7.4 The injector and syringe stay in the same position as (7) until the end of the runtime.
If you don't mind I have a few stupid questions:
A. The peak areas will remain constant for all the sample injection volumes greater than the loop volume, is that correct? I.e In theory, a sample injection of 300 uL should give the same results as 150 uL & 20 uL (loop volume).
B. Is there any accepted method or protocol for cleaning/washing the rheodyne injectors after every (manual) sample injections?