Chromatography Forum

Hello, Guys

I'm developing a method to analyse sorbitol in grape juices.
I tried water as mobile phase. The baseline is good, the standards are good. But I had some coelunt with sorbitol.
So, I tried change the mobile phase. I tried use 10% of acetonitrile and 90% of water. The separation is better. But the baseline drifts up and down. The noise is so big and the analyte peak is so small that the results aren't accurate.

I use a refractive index deterctor (RI).

The baseline drifts just when I use acetonitrile:water (10:90) as mobile phase.
I mixed the water and the acetonitrile before use it. I'm using just one channel pump for the mobile phase.
The acetonitrile barnd is Merck, gradient grade for liquid chromatography.

The column is an Aminex HPX 87C, from Bio Rad.
Flow: 0,6 mL/min.
Oven temperature: 80°C.

What can I do?

Thank you!

We assayed for sorbitol in soap and liquid soap, but used GC after derivatization.

When we used refractive index detector on our Agilent HPLC systems, we bypassed the multichannel gradient valve (Agilent supplies an adapter to go directly from the degasser to the pump's active inlet valve). So we also did pre-mix the mobile phase.

RI detectors are very sensitive to composition (obviously!), temperature, and flow rate. You have eliminated composition by pre-mixing your mobile phase, so the remaining problems are temperature and flow.

You referred to "drift" and "noise". If the problem is drift (a relatively slow monotonic change), that would point the finger at temperature. Make sure that your temperature control system is working properly and the temperature is not oscillating (it doesn't take much of a change to show up a few tenths of a degree will do it). If you are using an air-bath type oven, you might consider adding some thermal mass (e.g., a block of aluminum zip-tied to the column).

If it's truly noise, is it random or rhythmic? if it's rhythmic, look to see if it is synchronized with the pump stroke. If it is, clean your check valve(s) and if that doesn't help, consider adding a pulse dampener to the flow path.

Do you have a backpressure regulator device after your RI?
This helped us in the past to get better baselines (but we don't use RI a lot).

Maybe add some piece of 0.1mm ID tubing at the RI outlet, but ensure to not exceed the maximum pressure of your RI cell (keep in mind other operating conditions as lower temp, flowrates and other solvents)
An online calculator from Waters can be found here:
https://www.waters.com/webassets/other/ ... itters.htm) (seems to use the max solvent viscosities at 20°C)

Additionaly I've found in this product brochure, that ACN/Water forms an azeotrop which has a boiling point of 76°C (which is below your operating temp).
https://www.ineos.com/globalassets/ineo ... ochure.pdf

This, without enough backpressure, may lead to bubble formation within your RI-cell. (I don't know how to handle this in the reference cell?)

'Baseline Stabilty' is a very common concern when running any analysis with a RID. Attention to the details of temperature settings (esp not setting the different thermostats to completely different temperatures), insulating all of the lines, keeping the system away from drafts/windows/vents helps a lot. The column used should be in a column oven that can control the temperature (keep it stable). Don't overlook the pump and degassing too! ALL mobile phases used should be continuously degassed to stabilize (Equil) the amount of gas dissolved in the solutions over time (RI effects) and no cavitation or other problems with the HPLC pump must be present (issues with mixing valves, clogged frits, stuck valves).

Here is a link to a FREE article which addresses your concern. Maybe it will help you troubleshoot the issue.

"HPLC Baseline Stabilization Tips for Refractive Index Detectors (RI or RID)"; https://hplctips.blogspot.com/2018/02/h ... s-for.html

You probably have an in-line degasser, but it might be worthwhile to sonicate MP under vacuum for 20 seconds, then continuously sparge with Nitrogen. Given how touch RI detectors are.

"Ice9" wrote: "then continuously sparge with Nitrogen".

Warning: HPLC training is critical to avoiding problems with HPLC methods. Please, NEVER ever sparge your mobile phase solutions with Nitrogen gas. Nitrogen is soluble in the solution so you will be ADDING gas to the mobile phase (something that chromatograpphers do not wish to do). If you choose to sparge with gas, then please use Helium which is NOT soluble in the mobile phase and will displace other gases for better performance (and better UV transparency).

Here is a link to an article which may help educate chromatographers about why proper HPLC training is critical and the importance of degasser sparging gas choice.

"HPLC Solution Degassing, Sparging With the Wrong Gas (Gas Choice Matters)"; https://hplctips.blogspot.com/2013/05/h ... g-gas.html

Hello

Thank you so much.

How can I add this pulse dampener to the flow path?

Is it a setting or I need some extra equipament...

I have never worked with it...

Thank you!

RI detectors are very sensitive to composition (obviously!), temperature, and flow rate. You have eliminated composition by pre-mixing your mobile phase, so the remaining problems are temperature and flow.

You referred to "drift" and "noise". If the problem is drift (a relatively slow monotonic change), that would point the finger at temperature. Make sure that your temperature control system is working properly and the temperature is not oscillating (it doesn't take much of a change to show up a few tenths of a degree will do it). If you are using an air-bath type oven, you might consider adding some thermal mass (e.g., a block of aluminum zip-tied to the column).

If it's truly noise, is it random or rhythmic? if it's rhythmic, look to see if it is synchronized with the pump stroke. If it is, clean your check valve(s) and if that doesn't help, consider adding a pulse dampener to the flow path.

It's a piece of hardware that essentially acts as a shock absorber. Here's a description of how it works:
http://hplc.chem.shu.edu/NEW/HPLC_Book/ ... _damp.html

On many HPLC systems, a pulse damper is built-in (the Agilent 1100 and 1200 series pumps, for example). If your system does not have on built-in, or if it's inadequate for your purpose, or if it's failing, you can buy them (for example:
https://www.hplc-asi.com/standard-hplc-pulse-dampers/ ). They would usually be installed in the flow path immediately downstream from the pump.