Chromatography Forum

Hey, I'm new to the board and LC and hope in time to provide advice and help from my experience on this forum and in lab.

Background:
I am using a Grace Alltech Alltima Amino column to seperate and quantify concentrations of fructose, dextrose, sucrose, maltose, maltotriose, and maltotetraose. My method of detection is via an old Shimadzu refractive index detector model RID-6A. Mobile phase is 70/30 Acetonitrile/Water @ 0.5 ml/min with an oven temp of 40 Celsius. I am in the calibration stage right now, having prepared 1000/2000/3000/4000/5000 PPM solutions for each individual sugar and solutions containing all sugars.

Problem:
I want to make sure I am properly stabilizing my RID detector.

Current Procedure:
Prior to running a sample I allow mobile phase to pass through the reference cell until it stabilizes at a value. I then adjust the balance of the detector so that the value is zeroed out. I then shut the reference solenoid, sealing the reference cell, and allowing mobile phase to pass through the sample cell. Theoretically, when only mobile phase is passing through the detector the detector should read "0.0" because the index of refraction shouldn't be changing, right? It spikes up to 0.013 and stabilizes there. Reopening the reference results in the balance going back to the previously zeroed state.

First Question:
The equipment was donated, I'm guessing its contaminated...but if its a consistent contamination then I can just adjust for that correct?

Second question:
There is an RIU Range that needs to be selected. The values I can choose ranges from 0.25 to 512 (X10^-6) (RIU). According to the (very undetailed) manual accompanying the detector, this range corresponds to the sensitivity of the detector. A smaller number appears to increase sensitivity. How do I know what value to choose? I ran one test @ 0.25 and one @ 512... the 0.25 test results in smoother peaks.

Attached I have a link to the picture of the RID detector I use. The line of buttons along the bottom of the faceplate are the values of the RIU range I can select.

http://www.spectralabsci.com/images/Shimadzu-RID-6A.jpg

I appreciate any help anyone can offer,

Mark

Hi Mark ,

Refractive Index detectors are a real headache , they are stabilising in a long time.
I may recommend you the following points :

The waste outlet tube must be 0.5mm ID ( not 0.25mm as usual ) , and must not be longer than approx. half a meter.
The cell is very sensitive against over pressure , and easily destroyed if the outlet is blocked by particles etc. or if you use a lower ID tubes causing much pressure.

Activate the cell temperature control switch at the back side of the detector.This will absolutely help to stabilize the detector.

Use a column oven ( if possible ) to control the temperature of the mobile phase in order to reduce the shift.
If you don't have a column oven , wrap the column with an insulator.

Clean the cell first with water , then acetone + acetonitrile mixture; both the reference and sample cells.

I think , the detector have a signal output for both for the integrator ( 1 volt , 10 volts ) and for the recorder ( 1 mV , 10 mV etc ).
If you use the integrator output , you get a proportional output voltage and attenuate it using attenuation function on the integrator ( or you can adjust the scale if you are using a software ).
If you use the recorder output , then you can adjust the output voltage by pressing the attenuation switches on the front of the detector.
Higher the value , lower the output voltage.

Finally , pass the mobile phase without connecting the column , both the reference and sample sides ( switch two or three times ) , then zero the detector.
After than connect the column and wait it to stabilize and zero the detector again.

I hope you find this information usefull.

balance of zeroing the signal needs to be done after both cells have been purged if i remember correctly