Chromatography Forum

Hi all.

I could use some help here

I´m trying to measure creatinine by HPLC.

This is my setup:

Column:

Zorbax SCX 300, strong cation exchange, (Agilent), 50 mm x 2.1 mm, 5μ particle size with in-front five micron SCX guard column.

Solvent:

5mM sodium acetate
adjust to pH 4.1 with glacial acetic acid
degassed with 0.2micron filter.

creatinine anhydrous >98% (Sigma aldrich) disolved in solvent to give a 256 mM stock solution.

Temperature and flow: Column run at 50°C. at a flow of 1 ml/min. Backpressure at around 2200 - 2300 psi. Run time was 10 min. These parameters are set 45 – 60 min before run start to ensure a solid baseline (equilibration).

Detection: UV (deuterium source) at 225 nm (peptides).
Injection volume: 10 μl from autosampler. All samples are run in duplicates. Temperature of autosampler tray was 18 ± 0.5°C.

I keep getting tailing peaks during run...



Can anyone help with this??

This may be too simple a question, but are you just overloading the column?

I know there are lots of parameters that can affect peak shape. The most common one my lab is just too high a concentration. Do you have enough signal that a smaller loop would work? And does a smaller loop correct the peak shape?

The peak for all std samples are alike (256mM-1mM)...

And I have previously run the same setup with narrow peaks at 256 mM

Could it be the column packings? Or the assembly on the Acquity machine?

Have you tried changing the guard cartridge?

The peak for all std samples are alike (256mM-1mM)...

And I have previously run the same setup with narrow peaks at 256 mM

Could it be the column packings? Or the assembly on the Acquity machine?

If you have run the same column with excellent peak shapes before then it seems like a column issue. The simplest way to check the column integrity is to inject something unretained. If the unretained peak shows excessive tailing, then the column bed is not the same as before. I hope you are using the narrowest possible tubings and smallest possible connections. Believe it or not, a 2.1 mm column on an ordinary HPLC showed 6000 plates and with narrow tubings the plate count went to 14000!

A chemical reason for tailing is the fact that the mobile phase is too weak to elute your sample. At times 0.3 % acetic acid with 0.2% triethylamine as a buffer works wonders in improving the peak shapes. The pH is 4.1.

How about adding 0.08% TFA to the mobile phase? Would this have the same effect on peak width? Or should it be +.3% glacial acetic acid?

I will try a lower conc. of the standard (2mM-0.125mM) today with the same setup to rule out overloading

How about adding 0.08% TFA to the mobile phase? Would this have the same effect on peak width? Or should it be +.3% glacial acetic acid?

I will try a lower conc. of the standard (2mM-0.125mM) today with the same setup to rule out overloading

Both TFA and acetic acid combined with triethylamine improve the peak shapes. TFA elutes things much faster than acetic acid.

How about adding 0.08% TFA to the mobile phase? Would this have the same effect on peak width? Or should it be +.3% glacial acetic acid?

I will try a lower conc. of the standard (2mM-0.125mM) today with the same setup to rule out overloading

Both TFA and acetic acid combined with triethylamine improve the peak shapes. TFA elutes things much faster than acetic acid.

At what concentrations should I use?

I was thinking 0.08% TFA, 0.3% acetic acid and 14-20 mM TEA??

How about adding 0.08% TFA to the mobile phase? Would this have the same effect on peak width? Or should it be +.3% glacial acetic acid?

I will try a lower conc. of the standard (2mM-0.125mM) today with the same setup to rule out overloading

Both TFA and acetic acid combined with triethylamine improve the peak shapes. TFA elutes things much faster than acetic acid.

At what concentrations should I use?

I was thinking 0.08% TFA, 0.3% acetic acid and 14-20 mM TEA??

I had mentioned the concentrations in the previous posts: Try 0.3% Acetic acid + 0.2% TEA. This buffer has a pH of 4.

what is your sample solvent/matrix? If you are getting worse peaks shape with each injection, for me it looks like something is retain on column from solvent/matrix and it has impact on creatinine interaction with stationary phase.

I think I have solved the problem with the help from all of you.

Seems like the column was massively overloaded. And by adding 0.2% TEA to the solvent and by lowering concentration the creatinine peaks eluted at the same volume (sd+/- 0.06 ml).

However, the creatinine elutes very early on the chromatogram. Run time is 10 min and creatinine elutes at < 1min!!! Actually very close to my "injection noise" absorbance.

How may I change this?

I have read that change in flow rate has little effect on isocratic interaction, and the only things that may have influence on elution time is column temp and solvent "salt buffer" content.

Is this true and have any of you a good suggestion ? Thanks

[Flame alert: commercial hype follows]

Well, you could try our SCX material. The 200-Å pore version of PolySULFOETHYL A probably has 2x the cation-exchange capacity of that Zorbax material that you're using. Second change: Your flow rate is outrageously fast. With 2.1-mm i.d. columns, the usual flow rate is about 0.2 ml/min. You're running 5x faster than that, which accounts for the extraordinarily high backpressure that you're getting with a 50x2.1-mm column + guard cartridge. I assure you that you will see an increase in retention with a decrease in flow rate. You will also see an increase in peak fronting if you don't have connecting tubing that's short and of a narrow i.d. suitable for slow flow rates. It also helps to use a detector cell with a smaller volume at low flow rates.