Does any one has hands on experience with this protocol as I am trying to take the readings for standards on spectrophotometer at 220nm and I am getting everything in negative after using water as blank or reference. Any suggestions would be appreciated.
Natural organics interfere by spectral overlap. In some cases this can be managed by measuring at multiple wavelengths and doing a calculation. In others, you may need to remove some of the interfering organics. There is a good literature on this topic and methods in the APHA 'Standard Methods' compendium. The colorimetric methods based on reduction and diazotization are typically online and preceded by dialysis to decrease the concentration of organics since they can interfere with the reduction of nitrate to nitrite.
Hi Paul, thanks for your answer. In response I would like to mention that I made the wave spectrum from 200 to 230nm wavelength and got inconclusive results. Further, if we talk about interferrence of organics I can understand if it is in my sample but what about standards which were prepared in distilled water and nitrate salt. Moreover, I took the nitrate protocol from APHA standard methods book 20th edition.
For nitrate standards in water did you get an absorbance peak at the expected wavelength, and of the correct intensity and shape?
In response, I would like to mention that you should make the wave spectrum from 200 to 275 nm wavelength using uv -spectrophotometer with a quartize cell, to get the correct results. For nitrate standards use KNO3 salt.
@Paul.. I didnt get any peaks and all the standards were showing negative absorbance with respect to the reference which was distilled water.
@ Makia... I can try that, however according to the APHA standard method 220nm is the best wavelength where nitrate absorbs the most amount of uv light. Till now I have been using plastic disposable cells and rinsing them with 1N HCl. I can look for the quartize cell but I have my doubts that it will have that much effect. Yes, I have used potassium nitrate salt for making standards.
- Try using pure UV rated QUARTZ cuvette, not glass in the spectrophotometer.
- Make sure your samples are dissolved in the same solution as your reference.
- Run the Spectrophotometer's full test checks before using it to make sure the wavelength range is calibrated, the lamp energy is within specifications for use and no instrument problems are present.
- Run a check standard before analyzing samples.
- Always a good idea to review the instrument's specific User Manual before use and make sure you ask your professor for assistance too