Agilent HPLC system - Chromatogram assessment

[Deppizzymo]

welcome.

And add a bit of backpressure to the RI-cell.
This helped me in the past and is mentioned and explained by John Dolan in the article posted by TylerSmith in your other thread about the equlibration time

Can you bypass a column and simply connect the autosampler directly to the RI detector and inject water? I thought maybe if I did that I could eliminate one thing at a time that might be causing my noise. Maybe that was one of your suggestions with this

"- replace the column by a piece of tubing (e.g. 1m x 0.1 mm, to have some backpressure on the pump so they work properly), run and equilibrate with 100% MeOH (or ACN, or water); then make multiple injections from of e.g. caffeine in the same solvent and compare the peak areas."

[TylerSmith123]

Hi Depizzymo,

This is exactly what that comment is saying. Typically, instead of just adding new line in completely, the column is substituted with a union or a small back-pressure generator for the pumps. However, as Hollow pointed out, a back-pressure restrictor can be found following many RI detectors that act as insulators for RI detectors themselves. These two things are different. Your comment describes the unions (aka, bypassing the column), while Hollow is describing a back-pressure restrictor (installed after the RI detector). I hope this helps somewhat.
I hope you update us more on your issue-- the more specific you are in your details/experiments, the better-able we will be to help. Do not take much of this advice for granted, it may be a $5000 instrument, but getting accurate and repeatable results is priceless.

[Deppizzymo]

Hi Depizzymo,

This is exactly what that comment is saying. Typically, instead of just adding new line in completely, the column is substituted with a union or a small back-pressure generator for the pumps. However, as Hollow pointed out, a back-pressure restrictor can be found following many RI detectors that act as insulators for RI detectors themselves. These two things are different. Your comment describes the unions (aka, bypassing the column), while Hollow is describing a back-pressure restrictor (installed after the RI detector). I hope this helps somewhat.
I hope you update us more on your issue-- the more specific you are in your details/experiments, the better-able we will be to help. Do not take much of this advice for granted, it may be a $5000 instrument, but getting accurate and repeatable results is priceless.

Today I connected my column heater directly to my RI detector and it essentially shows no more baseline noise when running a blank. It went from a fluctuation of ~500 nRIU up and down up and down to a much more stable looking ~100nRIU fluctuation. It seems that the baseline noise is column related in some fashion. Can the back pressure increase from the column cause noise like this? Or could it just be the column itself? I am running about 75 bar with the column in line and ~5-10 without.

[Hollow]

Hi Depizzymo

(almost) as TylerSmitth said, I mentioned both options.

1: to add some backpressure on the RI cell will help.
If your RI limit is about 5 bar, then you should be ok with adding 50mm x 0.127 mm tubing (normally red-coded PEEK) to the detector's outlet. This should be ok for flows up to 2.0 ml/min of methanol/water mixtures. But double check with your RI specifications and change to your needs. If you normally run higher flow rates, then probably use a wider tubing but longer (pressure scales linear in length but with 1/diameter^4)

2: similar to what you did, but just replace the normal column with a piece of tubing. E.g. prepare a 1000 x 0.1 mm (black-coded) PEEK with two unions on both sides and use it like a "dummy column". The goal of this is to generate some more backpressure on the check valves and pump seals, so they work properly. With only a union, the backpressure may be too low and some PQ-test may fail because of this. The 1 m x 0.1 mm tubing will generate about 40 bar with methanol @1 ml/min.

Back to your issue:
it seems the baseline ripple is pressure related. (I hardly can imagine why the column itself should give this cyclic ripple, so I attribute it to general "pressure").
So it may be good to repeat the test with the "dummy column" in place to see if the noise increases again. You may also test different pure solvents or flow rates to vary the pressure.

This pressure correlated baseline ripple brought another topic to mind, +/- specific for Agilent systems.
Check the solvent compressibility settings in your method.
There should be/is a "calibration tubing" from Agilent to determine the proper value for your mobile phase. For pure solvents, I guess there should be some default values available/implemented in the software. Check if these are correct or reset to default.

Search the forum for "agilent compressibilty" and you will find other threads with similar issues.
There is also a blog post about the compressibility setting on Agilent systems: https://hplctips.blogspot.com/2011/10/h ... ility.html

"uncle google" also finds an entry on the Agilent Knowledge portal: https://community.agilent.com/knowledge ... penlab-cds


In one of the previous threads, it was also mentioned that a replacement of an old and dirty outlet frit at the pump (somewhere at the purge valve) increased the situation a lot and even decreased the back pressure significantly.
You may also check/replace that if the general state of your 2nd-hand LC is unknown.

Once the pressure ripple is improved and ok, remember to also check the autosamplers injection accuracy/repeatability. ...this was how your original question started...