From broad to narrow peaks??

[esbena]

Hi.

We are setting up a method for measuring creatinine in serum/urine form mice.

For this I´m using a Zorbax SCX300 2.1mm x 50mm, 5u HPLC column with a 5 micron guard column in front.

My running solvent is : 5mM sodium acetate adjusted to pH 4.1 with acetic acid (glacial)

My creatinine standard is from Sigma Aldrich: creatinine anhydrous, ≥98%, 82.03 g/mol, pKa 4.83

And I´m make a two-fold dilution of my standard from 128 uM - 0.5uM

Acquity parameters:

- Flow: 0.35 ml/min
- Column temp 45C
- Autosampler temp 18C
- Inject vol: 3 ul
- Run time: 10 min
- UV: 225 nm

Back pressure was around 837 +/- 4 psi during entire run.

I get a average retention time of ~ 5.132 min, which is ok.

But my peak is broad.... Around 2 min.

I would like to get a smaller peak, but how?

Thanks

[tom jupille]

That's a very small column, so my first suspect would be extra-column volume.
How big is the guard column?
How long is the tubing between the injection valve and the column?
between the column and the detector?
What diameter is the tubing?

Overload is another possibility:
Does the peak width vary with the concentration of the standard?

[esbena]

Extra column volume?? What do you mean?

The peak width is somewhat the same in each run.

The guard column is 5 micron. Does the physical size of the guard column have a say? It is approx. half the size of the zorbax column.

Overload??? I only use 3 ul pr. run with a run time of 10 min...

[tom jupille]

Extra column volume?? What do you mean?

The volume of liquid in contact with the sample but not in the column. This typically comprises the sample loop, the transfer lines from injector to column, the detector cell volume, and any trapped liquid in connecting fittings. Because band broadening can occur not only in the column but also in this extra-column volume, it's important to keep it to a minimum. The typical recommendation is that the extra column volume should be less than about 15% of the column volume (in your case, the column volume is about 100-120 microliters).

If the guard column is packed with something other than the identical material you have in the analytical column, then the volume inside the guard column is part of the extra-column volume, so yes, the exact dimensions *can* matter.

The peak width is somewhat the same in each run.

What do you mean by "somewhat"? And for that matter, what is the size of the guard ("approx." half is not very informative). I'm trying to be helpful here, but you're not making it easy.

My reason for asking about the relationship between width and sample load was to see if mass-overload was a factor (if you are *not* overloading, peak width is independent of injected sample mass).

My reason for asking about the guard cartridge dimensions relates to the extra-column volume comments above. A common guard size is 5 x 0.46 mm, which works out to a volume of about 50 microliters.

[esbena]

Guard column:

5 micron
D 2.1mm
Length 12.5mm

[aceto_81]

0.35ml/min seems low for a 5µm column.
Why not use 1 ml/min? The increased speed will also narrow down your peak.
But that said, it looks like there are other issues too, like extra column volumes.
Be sure that there are no leaks.

Ace

[esbena]

There are no leaks.


I will try 1 ml/min flow this weekend.

But extra column volumes?? I don´t think. I only use 3 ul load each run

[esbena]

These samples I´m running now is only std. i.e. no serum/urine.

So should I try without the guard column, and see if this has an effect on the retention time, peak and so on??

?

[bisnettrj2]

What size is your injection loop? What length and internal diameter is your tubing to the column? What is your injection solvent? Does your system have a preheater in the column compartment (like an Agilent column compartment with the 3 and 6uL preheaters)?

[tom jupille]

Esbena, I don't want to sound critical, but an HPLC system is not like a microwave oven where you can walk up, put in your bowl of soup, and press the "warm" button. In order to use HPLC effectively, you really do have to understand how it works (particularly when problems arise). Get a copy of the book I recommended in your other thread (viewtopic.php?f=1&t=22935); it has a very good section on instrumentation that will help you understand what's going on and why we are asking these questions.

[esbena]

I found out the problem .

At 0.350 ml/min it looked like my creatinine std. was not eluted after 10 min run time, so that it actually eluted with the next load.

Then I tried 1 ml/min, which was much better.

Narrow peak and more stabile retention time