Chromatography Forum

I analyse aminoacids with the use of both OPA anf FMOC. I use an fluoriscensedetector.

Mobilphase are
A:Phosphate buffer with 0,8% THF
B:Mobil A(20%), methanol (50%), acetonitrille (30%).
This phase has a gradient from 100% A to 100% B and then back to 100% A.

Lateley (the last 6 month ) I get a ghostpeak starting when I have about 5%A and 95%B and this peak last until it go back to about 100% A (broad and unregular peak). From 22-25min into the analysis.

Something I have tested:
-New chemicals
-Several hundreds of blank run (still got the ghostpeak and it want get any smaller)
-Change mobilphase to natriumacetatebuffer
-New tubings in the HPLC system
-wash with 100mM NaOH through the system
-wash degasser after recomended methods from the supplier
-wash column
-new column

What HPLC system are you using?

What are the excitation/emission wavelengths? Does the ghost peak go away if you increase or decrease them both by 20nm?

Have you changed the solvent reserviors and filters?

Does the size of the peak increase 2x if you extend your gradient re-equilibration time 2x? Make 2 blank gradient runs with your standard program then make 2 runs with the equilibration time doubled.

What HPLC system are you using?

What are the excitation/emission wavelengths? Does the ghost peak go away if you increase or decrease them both by 20nm?

Have you changed the solvent reserviors and filters?

Does the size of the peak increase 2x if you extend your gradient re-equilibration time 2x? Make 2 blank gradeint runs with your standard programy then make 2 runs with the equilibration time doubled.

What HPLC system are you using?

What are the excitation/emission wavelengths? Does the ghost peak go away if you increase or decrease them both by 20nm?

Have you changed the solvent reserviors and filters?

Does the size of the peak increase 2x if you extend your gradient re-equilibration time 2x? Make 2 blank gradeint runs with your standard programy then make 2 runs with the equilibration time doubled.

What HPLC system are you using?

What are the excitation/emission wavelengths? Does the ghost peak go away if you increase or decrease them both by 20nm?

Have you changed the solvent reserviors and filters?

Does the size of the peak increase 2x if you extend your gradient re-equilibration time 2x? Make 2 blank gradeint runs with your standard programy then make 2 runs with the equilibration time doubled.

What HPLC system are you using?

What are the excitation/emission wavelengths? Does the ghost peak go away if you increase or decrease them both by 20nm?

Have you changed the solvent reserviors and filters?

Does the size of the peak increase 2x if you extend your gradient re-equilibration time 2x? Make 2 blank gradeint runs with your standard programy then make 2 runs with the equilibration time doubled.

What HPLC system are you using?

What are the excitation/emission wavelengths? Does the ghost peak go away if you increase or decrease them both by 20nm?

Have you changed the solvent reserviors and filters?

Does the size of the peak increase 2x if you extend your gradient re-equilibration time 2x? Make 2 blank gradeint runs with your standard programy then make 2 runs with the equilibration time doubled.

What HPLC system are you using?

What are the excitation/emission wavelengths? Does the ghost peak go away if you increase or decrease them both by 20nm?

Have you changed the solvent reserviors and filters?

Does the size of the peak increase 2x if you extend your gradient re-equilibration time 2x? Make 2 blank gradeint runs with your standard programy then make 2 runs with the equilibration time doubled.

It is an revers phase system.
All the solvents and filters are changed.
I have looked at the peak if I extend the re-equilibration time 2x, but there are now differense.
I use 260/315nm. I have not tried to change this because I feel I need to know why I get this ghostpeak. But I am pretty close to try something like that......

If the peak area does not increase when you double the re-equilibration time then your mobile phase A is clean but since you re-equil at 100%A you now nothing about mobile phase B.

Replace the column with a union, run a gradient from 100%A to 100%B over 10min with 10min isoratic holds at 100%A at the beginning and 100%B at the end. Does the detector signal go up linearly with the %B? Does it stabilize at 100%B?

What brand and model of HPLC system are you using?
What's your flow rate?
What are the dimensions and phase (C18) of your HPLC column?
What are you using as a needle wash solvent?

I have tryed the following gradient:
0-10min Mobile A (100%)
10-20min From mobile A (100%) to mobile B (100%)
20-30min Mobile B (100%)
30-40min From mobile B (100%) to mobile A (100%)
40-50min Mobile A (100%)

The following chromatogram occured:
0-17min Baseline
A very small increase in detector signal from 17-19min (70%B). A max signal occured at 21min (100% B). This signal holds around 100mv +-30mV til 28min. Then a very small decrease til 30min. From 30-32min it drops to the baseline again.
Hope you can read this chromatogram better than me

The HPLC is an Dionex Ultimate 3000.
The flow rate is 0,5ml/min.
The needle is washed with destilled water.
I use an Rested HPLC column with a length of 100mm, inner diameter 3,2mm, particle size 3µm and poresize 140Å.

Sine you ran this test with a column instead of a union its difficult to pinpoint the source of the signal. It could either mobile phase B or something adsorbed on the column.

Run the test with a union, not a column.

What is the bonded phase of your HPLC column, C18, C8, phenyl, etc?

What kind of union do you mean? Is it an back pressure union?

I am using a C18 column.

A standarde plumbing union that allows you connect the tubing that normally is connected to the column inlet to the tubing going to the detector.