Unstable Baseline (DAD) on Agilent Series 1100

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Hi all,

I am not sure if I am yelling into the ether, but I have been a lurker on these forums for a few months and they usually help a lot, so I thought I would give it a shot. If you couldn't tell from my username, I am a total newbie at HPLC (so please forgive me for my stupidity on certain things). I am also working on a slightly different software (Masshunter Data Acquisition to ChemStation for HPLC) than I have before. That being said, I am currently developing an assay that requires the determination of dwell volume/gradient delay volume/system delay volume (these have all been used interchangeably in the literature I have read, but correct me if I am wrong).
I attempted to do this by following a technique utilizing a mobile phase A of methanol and a mobile phase B of 0.1% acetone in meOH detailed on the HPLCtips blog (https://hplctips.blogspot.com/2017/02/d ... olume.html).

My mobile phases were as follows:
A: Water (HPLC-grade)
B: 0.1% acetone in water (also HPLC-grade)
I knew of similar tests run with 0.1 mM sodium nitrate in water as mobile phase B, so I thought this would be alright.

I removed the column and used a zero dead volume union. My gradient from 100% A to 100% B ran over the course of 10 minutes at a flow rate of 0.4 mL/min (very slow, I know, but we usually have issues with pressure** when we do have our column in and I wanted to keep the parameters similar enough) and a temperature of 40°C (again, just trying to keep parameters similar, though this may have been too warm for the pressurized acetone in retrospect). I flushed the system with 100% B for around half an hour, then 100% A for around half an hour. The baseline had stabilized at 0 with the water, and I ran my gradient.

I left for lunch and looked at my data when I returned. To my surprise, the signal looked more like a decreasing logarithmic function (it went WELL into the negatives). I ran the gradient again at ambient temperature to check, getting the same results.

I flushed with copious amounts of water at higher flow rates (1-2 mL/min) as I was worried a bubble had entered the system due to the acetone. Could this be the error? Should I remove the flow cell to sonicate it? Or is it a lost cause?

A couple of sources of error/factors which may be useful to the diagnosis of this issue:
Problem 1: Our lab did not have HPLC-grade acetone, but I was advised to use our anhydrous acetone (99.8%) in its place. It was not a new bottle, but it was septum-sealed and had to be removed using a cannula.
Problem 2: Adding even more to the gas being the issue, until very recently, the degasser was incorrectly installed (someone put the input lines into the output of the degasser and the output lines into the input). I reinstalled it correctly, but I am worried it may have done some damage (would this also explain the pressure issue?**).

**I am talking we are using a 2.1 mm x 50 mm column and it has a pressure ~ 100 bar with ACN alone

I am just at a total loss at the moment, so I would appreciate any help I can get greatly! Thank you so much.
What is the particle size of your column? I wouldn't think 100 bar is a particularly high backpressure (I often run up to 200 bar for a 3.5 um column).
A lot depends on your system. Many hplc pumps cannot supply an accurate gradient unless they are pumping against a substantial back-pressure. You removed the column and replaced it with a union, so if your pressure was only 100bar with ACN and a column, it's probably very low indeed with no column (even with water).

I suspect that you're not pumping the solvent mixes you think you are. You can add a low-dead-volume back pressure regulator in the path in place of the column and see if that improves the situation.
If your system pressure is high without any column in it, you need to get that sorted before figuring out the dwell volume as you're likely to change it a bit over the course of fixing that. Diagnose the pressure issue by breaking tubing connections and looking for a big pressure drop. Start at the detector and work your way back toward the pump to find the restriction.

0.1% Acetone absorbs strongly compared to water, so using water instead of MeOH - not a problem.
You don't want a column in there as it will inflate your "dwell volume".
I do suspect you got your A/B lines crossed.
I'd leave them and just change your percentages in the gradient program so that you're going from 100% B to 100% A and then rerun it after equilibrating at 100% B.
I'd also add a length of small bore peek tubing or a back pressure regulator to the UV detector waste port to provide a little back pressure (a couple hundred psi is enough to prevent our-gassing in a flow cell but not enough to break a flow cell, they're generally rated to at least 500 psi).

If you're figuring the dwell volume to a MSD, I suspect the back pressure regulator thing won't work (and is not needed).

The degasser should probably work in either direction (maybe not quite as well when backwards), but re-plumbing it may have resulted in some shedding of junk into the flow path.
Thanks,
DR
Image
wss wrote:
What is the particle size of your column? I wouldn't think 100 bar is a particularly high backpressure (I often run up to 200 bar for a 3.5 um column).

The particle size is 1.8 um (the column is a Zorbax RRHD Eclipse Plus C18 2.1 x 50 mm)! Sorry, did not specify the flow rate with the ACN: it is reaching ~100 bar at 0.2-0.25 mL/min with straight ACN.
New user: As you stated at the start of your post, you have absolutely no idea what you are doing. Your comments and questions makes this clear (sorry, not trying to be harsh, but this is a fact). To help you learn how to use the basic features of an HPLC system takes YEARS. A book or web forum can not provide the enormous amount of basic info you need to get started. Please contact someone at your place of business who has several years of industrial HPLC experience for assistance (or ask your employer to higher someone if they plan on using HPLC in any way for the work that they do. No one can learn this on their own in a few weeks or months). With a trained person present, these types of very basic questions could be solved and you could receive some on-the-job training too. You will be provided with an opportunity to learn, productively doing it this way.

A few comments to clear up many confusing questions an/or suggestions (in no particular order):
(1) You wanted to find a method for determining the gradient delay volume for YOUR specific HPLC system. You found a method to do so, then ignored it, using your own mobile phase choices, flow rate and conditions. This is a recipe for failure. Follow the instructions if you want any chance of success.
(2) When acetone is added as a tracer to a mobile phase (as it was in the example), it does not need to be "HPLC" grade. any technical grade would be fine, including some from the local paint store.
(3) Use a restrictor in-line (no column) when running the gradient delay volume test for best results. Sometimes a ZDU will work too, but in all cases, HPLC pumps need a certain amount of backpressure to operate well (this depends on brand, model, flow rate and mobile phase).
(4) For most types of vacuum degassers, it makes no difference which is the inlet/outlet line (maybe one or two exceptions out their, but the other 99% are fine).
(5) If you are really using a DAD with your method, then there is an enormous chance you have many other issues with your tests and methods too. Using a DAD requires specialized training in both the DAD software and hardware, it is best suited for use by more advanced level chromatographers (> 10 years). Incorrect DAD set up (really easy to set it up wrong) results in invalid data being collected and wrong conclusions being made.
(6) You wrote: "particle size is 1.8 um (the column is a Zorbax RRHD Eclipse Plus C18 2.1 x 50 mm)! Sorry, did not specify the flow rate with the ACN: it is reaching ~100 bar at 0.2-0.25 mL/min with straight ACN." - That backpressure sounds correct, not an issue.
(7) Before trying to calculate gradient delay volumes, learn a few basics of HPLC such as: Priming the pump; Calculating and Measuring Column Void Volume; Calculating Sampling Rate; Integration Basics; Measuring S/N of a std peak to judge baseline noise; Running TRUE blank samples... etc. These are types of basics we teach to students in the first year of learning HPLC and these are the concepts you will use all of the time running an analysis.
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