Negative baseline spike in HPLC

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Santosh@India
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Negative baseline spike in HPLC

Hello All,
I had a new Shimadzu LC2010 HPLC system recently installed. I encountered a problem of negative baseline spike at 2.7 min and a positive spike at 4.7 minutes, which is coming in all the injections, I changed a no. of mobile phaseas and the columns as well. The peak intensity is max at 200-230nm after which is goes on reducing, frequent flushing of the system also does not solve it. can any one please explaine that why these spikes are appearing and how to get rid of it?

Dr. Analytical
Dr. Analytical's picture
It would be easier to

It would be easier to diagnose if you could post an example of these dips.  Also, send information about your chromatography conditions.

Do the dips occur in all samples, including blanks?

Santosh@India
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Dr.

Dr.

I was injecting Caffeine standard to check the system performance,The chromatographic conditions are
Mobile Phase:MeOH:Water(50:50)
Column:-Luna C18, 250 X 4.6 mm, 5micron
Detector:-UV detector wavelength- 254nm and PDA 190nm to 800nm
Flow rate:-1mL/min
inj.vol:-10microL
Run time:-8 min
sample conc:-0.1% in water

These dips are occuring in all samples including blanks, but it is not appearing in baseline monitoring.The dips occuring even for different columns.
rgrds.

Dr. Analytical
Dr. Analytical's picture
The peak at 2.7 minutes is

The peak at 2.7 minutes is near the void time for this column.  Peaks at this location are commonly called "system" or "vacancy" peaks.  They are caused by a difference in composition between the injection solvent and the mobile phase.  The peaks can be positive or negative, and sometimes there may be mulitple peaks, and some can be at longer times (although the peak at 4.7 min. seems a little too long for this problem).  So, if your mobile phase is 50:50 MeOH:Water and your injection solvent is all water, you can expect to see system peak(s). 

To test this, inject a blank of mobile phase (collect what comes out the end of the column and inject).  You should see no peaks.  Try injecting just water, and then all methanol.  If these experiments do not explain the peaks, then you may have an impurity or contamination in one of your solvents, or in your glassware.

Please post the results of these studies so all can learn from them.

Santosh@India
Santosh@India's picture
Hi Dr.

Hi Dr.
Attached is the ppt.
rgrds.

Santosh@India
Santosh@India's picture
Check this one.

Check this one.