Chromatography Forum

Hi, everyone,

Recently I do NPLC-RID for a compound. Mobile phase: Hexane: IPA: CH3COOH = 950:50:1. Flow rate: 1.0ml/min. Column temperature: 30C. RID: 40C. The baseline is very unstable. Give me some suggestion! Thanks!

guoyang,

To avoid or decrease such an effect you have to match the detector and column temperature (either both 30 C or both 40C) and to insulate the tubing between the column and the detector (I use a piece of insulating foam for that).

bhuvfe

2 things - wil I agree match the temp I prefer having the RI temp lower than the column. Also you look to have a little pump noise - I would chack that there is no air a chack valve or pump head.

I've always been told that the RID temperature should be higher than the column temperature, which is contrary to what the two previous posters have stated. Perhaps someone else will post their experience to clarify this. I set the RID temperature to be 10C higher than the column temperature.

Insulating the lines, as Bhuvfe mentioned, is also a common practice. Some other basic things to look at: make sure your reference cell is properly and adequately purged; make certain the air temperature around your instrument is constant (I recently had an issue where the HPLC with the RID was directly underneath the air conditioning vent).

I hope this helps.

What kind of fluctutations are you seeing and what model detector is it? With the Agilent RID I used to see about 10 to 100 or so nanoRIU fluctutations in the baseline peak to peak. It helps to have a bit of backpressure which can be maintained by putting the waste tube into the liquid portion of the wasted container and put the waste container up on top of the HPLC.

Whether you heat or cool depends on the instrument. I think my Agilent didn't have cool but only heat so if you put the RI cooler than the column you had no control at all. For that reason I agree with last poster. Safest to set the RI 10 degrees higher than the column.

Sure, there's an overall rise. But I think the bigger issue is the cyclic "humps".

Personally, I'd match up the temperatures of the column compartment and the RID, and insulate the transfer line. And with RID, I tend to pre-mix the mobile phase and bypass with a fitting any low-pressure mixing valve. Are you using a vacuum degasser?

Thank you very much for your suggestion!
It is very useful.
I use aglient 1100 (quaternary pump) with vacuum degasser.
I aslo asked service engineer and he gave me two suggestion: (1)make sure the detection cell and feference cell is clear;
(2) bypass proportioning valve and use adapter to be connected on the Active Inlet Valve and connect the solvent tube directly to the active inlet valve (instead of connecting to the proportioning valve).
In addition, keeping ambient temperature is constant is very important.
But I don't understand why baseline is so regular and like a sine curve or cosine curve?

guoyang we are talking nRIU
generally like in UV we look at m(units)
so anyway you should be way lower then the actual peak scales you are going to look at.
what is the peak height of the compound in your application. look at that first to see if it matters. you are taking of a change of 0.2mRIU
why Agilent gives such a weird scaling i have no idea but it certainly helps to confuse things.

how long after you start working do you start looking at the signal?
if only a few minutes after igniting the RI and column oven then wait further. especially old RI need several hours of equilibration.

have you looked at signal pressure while you looked at that RI signal?

and if you bypass the proportioning valve and it actually helps then your pump is faulty.

I suspect that one of your degassers has gone south on you based on the period of the fluctuation. Try the other two channels.

The first thing I would do to get an answer to the question
"But I don't understand why baseline is so regular and like a sine curve or cosine curve?"
is to change the flow rate and see what the results are.

The reason I use a lower temperature is due to the fact that most columns I use for RI separations (sugar and organic acids) are heated higher than the maximum temp allowed for the RI detector. It often is easier to have the detector set lower and therefore the temperature fluctuations are less. This has been true for every manufacturer of LC equipment I have used (Waters, Shimazdu, Thermo, Agilent).

When I have been able to use I higher temperature, I often found out you have to insulate the tubing between the detector and the column since a fluctuation in the amount of heat needed causes the baseline to rise or fall.

The reason I use a lower temperature is due to the fact that most columns I use for RI separations (sugar and organic acids) are heated higher than the maximum temp allowed for the RI detector. It often is easier to have the detector set lower and therefore the temperature fluctuations are less. This has been true for every manufacturer of LC equipment I have used (Waters, Shimazdu, Thermo, Agilent).

When I have been able to use I higher temperature, I often found out you have to insulate the tubing between the detector and the column since a fluctuation in the amount of heat needed causes the baseline to rise or fall.

That is classic pump noise or pulse dampener noise. If an air bubble is trapped in the system somewhere, you will see this type of noise (although the noise is usually has a faster periodicity to it).

I think this unstable baseline maybe due to low column pressure (40bar in above chromatographic condition). Recently I use other column with higher coulmn pressure, the baseline is better.

I think this unstable baseline maybe due to low column pressure (40bar in above chromatographic condition). Recently I use other column with higher coulmn pressure, the baseline is better.

Try pushing just IPA through the column with the problem. I have a feeling air is trapped in the column or was ran dry. Since IPA has a higher viscosity than hexane, you should be able to push the air out of the column with it.