hplc

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cdf246
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hplc

 I am trying to separate quercetin dihydrate using HPLC using a mobile phase of methanol and phosphoric acid. However, I cannot seem to separate the compound and I have tried different detection wavelengths at 280nm and 360nm but does not seem to work. Has anyone got any suggestions please?

Dr. Analytical
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Please give us more

Please give us more information about what your separation conditions.

Column type (including dimensions and particle size), flow rate, injection volume, injected concentration, and injection solvent.

You say you are trying to separate quercetin, but separate from what?  Is there another peak in the chromatogram?  Are you getting no retention?  What are the differences at 280 and 360 nm?  Why did you try those wavelengths?

cdf246
cdf246's picture
 The column is a C18, 5cmx4

 The column is a C18, 5cmx4.6mm, 5um particle size. Flow rate is 1ml/min, injection volume is 20ul, concentration is 100ug/ml and the injection solvent is methanol and phosphoric acid. The compound is quercetin dihydrate standard. I have tried increasing the compound concentration, injection volume and changing the solvent composition but the chromatogram does not show a peak at all. The reason I tried these wavelengths is because these are the wavelengths for this compound. 

Dr. Analytical
Dr. Analytical's picture
Are you getting any signal at

Are you getting any signal at all?  Is there at least a little noise in the baseline?  Is there drift?  You might also see an irregular response (both positive and negative peaks) at around 0.3 - 0.5 minutes.  Are you able to inject any other samples and get a response?

Have you verified the flow rate out of the end of the column?

If you want to check your column, prepare a 100 ug/mL solution of toluene (or benzene, or any other small aromatic) in methanol and make an injection.  Monitor at 220 nm.  You should see a large peak at about 0.3 - 0.5 min.  If you don't see a peak, then there is something wrong with your system (detector, injector, etc.).

Once you have these answers and/or results, please write back.

cdf246
cdf246's picture
 When I inject other

 When I inject other standards I get a response using the same column, but it is just this compound that does not give a response. When I test the compound (quercetin) at 280nm I get a large negative peak at about 40 minutes, and at 360 nm there is just noise on the baseline with no positive or negative peaks.

Dr. Analytical
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What detector do you have? 

What detector do you have?  Is it a diode array (and from Agilent)?  If so, what are the settings in the detector setup screen. 

The negative peak is usually just a settings problem with the diode array, but I will need to see what they are first.  Also, do you have an absorbance spectrum for this compound?   The spectra may be in the data file, if you are using a diode array.

cdf246
cdf246's picture
 It is a diode array detector

 It is a diode array detector (not  Agilent). The absorbance spectrum for this compound is 350-370nm. But I am getting responses with the other compounds I am testing, so could it be the detector?

Dr. Analytical
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What model is the diode array

What model is the diode array?  Does it have a sample wavelength setting and a "reference" wavelength setting?

cdf246
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The diode array has a sample

The diode array has a sample wavelength setting and reference wavelength setting. The reference wavelength setting is 360 nm.

Dr. Analytical
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In these diode array systems,

In these diode array systems, the software usually measures the absorbance at the "sample" wavelength and subtracts the absorbance at the "reference" wavelength.  This approach tends to reduce system noise.

In your case, the compound you want to measure absorbs at 360, so when you measure at other wavelengths, the software is subtracting a large absorbance at 360.  That is why you get a negative peak.

Try setting the reference to a longer wavelength (say 450 nm), where the compound does not absorb.  You may have to turn on the visible lamp, if the instrument has one.