Chromatography Forum

We just purchased a second hand Agilent 1100. My manager and I have had previous HPLC experience at different companies. She worked with an Agilent 1100 previously and I previously worked with older Waters systems doing sugar analysis. We have a client who needs sugar analysis on their animal feeds on a weekly basis. They have been sending us samples for about two years now that I analyze using enzyme kits through Megazyme and a spectrophotometer. We bought the system to simplify this process and save us time and with our experience we figured we could get this system up and running as it was complete and had an RI detector. I used my prior experience to influence the purchase of a Biorad Aminex HPX-87C calcium column as this is what we used at my previous company and knew they worked well. I have attached a picture of my chromatograph and am trying to figure out the best steps forward for peak separation. The sample I am running is a standard I made containing a mix of the sugars that our client is wishing to analyze. When I ran pure samples of each individual sugar I had retention times of
Sucrose - 8.28
Maltose - 8.36
Lactose - 8.62
Glucose - 10.05
Galactose - 11.33
Fructose - 13.44

At my previous company, we only tested for glucose/fructose/maltose/trios/higher sugars and those peaks came off very distinct and easy to separate/integrate. However when I run this mix, I am only getting four distinct peaks and they are honestly quite ugly with lots of tailing. I am thinking the sucrose/maltose/lactose are all being lumped into one large peak and I absolutely need to find a way to get nicer looking/separate peaks in order to provide any sort of a reasonably accurate number to them on their samples. Does anyone have experience running a mix such as this? What worked for you to get those peaks separated out? Any help would be greatly appreciated!

System is set up as follows
pump speed: 0.6mL/min
detector temp: 45C
Column is a brand new Aminex HPX-87C
column temp: 80C (the maximum our system allows... we ran 85C at my previous company)
Mobile phase: heated DI H2O

Link to chromatograph (I don't know why it's saying it may contain mature content lol... it's definitely the chromatogram!)
https://imgur.com/a/XCyAnTO

Ooof, that resolution! First of all, could you let us in some more on your column dimensions, running buffers, etc. so we can get a better understanding of your system. This will include things like if you purchased it from a second-hand vendor or an individual and if you did any work to the system prior to running your samples, ie. running standards, flushing lines, etc.

In my opinion, the first thing you need to focus on is improving that resolution. Once we know more about your column and conditions, we will be able to improve your resolution and then we can move to the separation.

The retention of these compounds on your column looks good to me, but I am slightly worried that the peak at 5 minutes is your dead-time/void volume, in which your retention of these compounds would be pretty poor.

Let us know some more about your separation and hopefully we can help.

the 87C is 300x7.8mm, 9um particle size, pH range 5-9 and 8% cross linkage.

It has been nearly 10 years since I last used HPLCs but I was explaining to my manager that it looked pretty ugly to me and she agreed. So much tailing.

The system was a second hand unit. It actually came with a DAD detector but we got the RI detector from another third party seller.

https://www.proxibid.com/lotinformation ... -3rd-floor

Did you flush the reference cell of RID detector properly , with DI water ?
It may sometimes need half an hour to get a good baseline.

Also the lamp energy may need adjustment.

Did you flush the reference cell of RID detector properly , with DI water ?
It may sometimes need half an hour to get a good baseline.

Also the lamp energy may need adjustment.

We did flush and we did the RI calibration procedure. We had an issue one day where the system errored out and everything shut off leaving the hot column without water flowing through it for a brief period while it cooled down so hopefully that didn't do any damage to it.

When using RI detector, best to bypass the multi-channel gradient valve instead of just setting other channels to zero.

Agilent 1100 is a good unit.

I went ahead and did another flush of the cell as I don't believe we flushed it long enough in our initial flush. This greatly improved the resolution on the smaller peaks but I am still not getting good separation of the lactose and sucrose peaks. I did do a bit of digging and this column literature shows sucrose/maltose together as one peak and I may not be able to separate those two. Here is the updated pic of the resolution. I do not have the adapter to bypass the solvent switching portion of the pump at this time. I will have to purchase that.

https://imgur.com/a/Meu5i05

The Pb ( Lead ) form of this column seems more suitable for the seperation of lactose and sucrose .