Chromatography Forum

dblux_

The peak is present , so as u suggest "contaminated switching valve" but what should i do , do i need to flush again ?,,, thanks

thohry

i inject a series and the size is changed ! what shall i do next ? ,,, thanks


HW Mueller& Tom

if is it t0 noise how can i get rid of it , because i used to seperate one compound but my supervisor suggest to not study this compound because it was eluted before this big peak ! and this peak come between and the shape of the peak become very very big in the chromatogram with analyte of ( 0.01 ppm ) .I feel the proplem is coming from injection port there is something hide inside

Unless one does charge exclusion chromatography, or some other speciality, one does not bother with peaks at "to" or peaks ahead of it. That is why normally one adjusts retention times of analytes to be considerably after "to".

Unless one does charge exclusion chromatography, or some other speciality, one does not bother with peaks at "to" or peaks ahead of it. That is why normally one adjusts retention times of analytes to be considerably after "to".

HW Mueller

My dear as i understand from u that is no proplem to have this big unknown peak at 5 min and this what my supervisor say because he said all the peaks that are before the analyte we can ignore !! .Because i am separating two compounds all are eluted after 8min but do you thing will my sepaqration will be accepted in things like conference .Beacuse i really tired to solve this problem ( one year pass ) but with no success and also it is funny to say in the paper the reason of why this peak appear is unknown do u thing so by the way the troubleshooting book suggest that a hardware proplem but the system pressure is ideal

"Have you tried to inject a clean solvent several time and changing the injection volume : 1; 5; 10 microlitter and see if the ghost peak changes in size ?
Strange really.

"i inject a series and the size is changed ! what shall i do next ? ,,, thanks.

You are probably seeing a solvent peak. The fact that the peak gets bigger when you inject more solvent leads me to believe this is the case, and you should therefore take HW Mueller's advice and ignore it.

Hope you find the answer to your situation.

Just a note - I have seen carryover with Shimadzu autosamplers before. If your analyte is a base, add some acid to the wash and purge it really really well.

You still have not told us your flow rate!

The "dead time" (t0) of a column is the dead volume (Vm) divided by the flow rate. The dead volume of a 4.6 mm id column is approximately 0.01 times the length in millimeters, so your 250 x 4.6 mm column has a dead volume of about 2.5 ml (give or take 15%). At a flow rate of 1 mL/min, your column will have a dead time of about 2.5 minutes.

When discussing separation chemistry, it is best to refer to retention in k' units, where k' is the ratio of retention time to dead time, minus 1:
k' = (tR/t0) - 1 (k' is independent of column dimensions or flow rate).

The US FDA suggests that any peaks being quantitated should have k' values greater than 2, and most chromatography textbooks recommend never, ever trying to quantitate a peak with k' less than 1. A big part of the reason is that injection of a sample upsets the equilibrium of the chromatographic system. This "chaotic" state passes through the column and shows up as baseline noise around t0.

So, if those peaks are not of interest to you, they can safely be ignored; they are artifacts of the chromatographic process.

tom

Thanks indeed u r genious my t0 is exactly at what you said ( 2.5 !) and unlnown peak double this time always ( 5 min) .

bisnettrj2
thanks , i made him blind from my horribe questions ,,, sorry tom