0.45 or 0.20 µm membrane (sterile) filtration? Please also check blank nitrate values of filters and wash them nitrate free before use.

BTW, is it the nitrate concentration increasing or extinction (caused by turbidity) in spectroscopy?

thanks dear Thomas.

yes i checked with both .45 and .20 filters.the nitrate concentration increase i think its because of little turbidity .when i centrifuge the supernatanet again and again this error decrease.why filtration doesn't work well?my blank is redistiled water.

Yes Thomas Fischer suggestion is good one. if you getting algal cells in supernatant the size of the alga cell is below 2 micron or sometime 1.5 micron so first you centrifuge at maximum speed 10000 rpm or above than you collect the supernatant and filter through 0.2 micron filter followed by 0.45 micron filter. while filtration time keep the pressure in normal due to high pressure also filter will damage than you can get algal cells in the filtrate.

thanks Dr natarjan.

i also tried centrifuge at 10000 rpm  but stil some times increases.what should id do?

why this problem occurred?

I suggest to distinguish between nitrate concentration and extinction. Presumably, extinction may have two causes here: Nitrate and turbidity.

I am coming to this point, because nitrate has a specific (narrow band) spectral absorption (BTW, what method do you use for nitrate?), whereas turbidity usually has a broadband absorption. So you might cope with that problem using spectral background correction, or baseline subtraction. This will help you not only to specifically identify the nitrate peak, but also to determine the level of turbidity. Further, you can check for turbidity separately with the fluorescence cuvette housing of your spectrometer (chlorophyll?).

Please be also aware about spectral interferences of other compounds with nitrate. You didn't tell us too much about your experimental setup (matrix, cultivation solution, nitrate concentration level etc.) and your analytical methods.

You also might consider other methods for quality control. Ionic chromatography might not be the best suggestion with turbid solutions, anyway, your solutions already passed sterile filtration which normally will suffice for HPLC. So if you are ready to sacrifice an IC precolumn it might be worth a try. Another method would be ion sensitive electrodes. They, however, may interfere with other anions.

I suggest you to control the pressure of vacuum  filtering as

thanks Thomas

i solved the turbidity problem with centrifuging ar 12000 rpm. but still nitrate concentration increases .i think its related to interferences with other material. i growth algae in defined medium including NaNO3,K2hpo4,CaCl2,Mgso4. and i added 4 g/l NaHCO3 as carbon source(my culture is 50 liters). and i think bicarbonate is the problem. 

i used standard method(4500-B NO3) uv spectrophotometric method(220 nm and 275 nm).what should i do to remove interferences?

i can't use any other method in order to determine nitrate concentration.

I suspected that you were using the UV method. You can add HCl to your samples to remove carbonate interference, I am not exactly sure about the UV absorbance of the other anions, but you can check their influence by measuring your medium without nitrate after addition of HCl (you also can use this solution as blank when performing autozero).

Further, the method itself states that the correction at 275 nm varies with the type of organics. Is the reading at 275 nm more than 5% of the reading at 220 nm?

Hi, I am not sure which part of nitrate was analyzed in your research, the nitrate in meadium, or the nitrate in suspended particles. In your talks, I supported the method from Dr. Fischer, that is quite useful to HCl addition  in medium without nitrate for blank calibration. 

And other references may be helpful:

He S, Zhao J Q, Hu B. A Simple Method for Eliminating the Interference of CH3COONa in NO3--N Determination by UV Spectrophotometric Method[J]. Advanced Materials Research, 2014, 955-959:1172-1175.

Sandford R C, Exenberger A, Worsfold P J. Nitrogen cycling in natural waters using in situ, reagentless UV spectrophotometry with simultaneous determination of nitrate and nitrite.[J]. Environmental Science & Technology, 2007, 41(24):8420-5.

Kelly R T, Love N G. Ultraviolet spectrophotometric determination of nitrate: detecting nitrification rates and inhibition.[J]. Water Environment Research A Research Publication of the Water Environment Federation, 2007, 79(7):808-12.

Bastian R, Weberling R, Palilla F. Ultraviolet Spectrophotometric Determination of Nitrate. Application to Analysis of Alkaline Carbonates[J]. Analytical Chemistry, 2002, 29(12):470-4.

Thanks a lot Lu guangyuan for your help

yes i determine nitrate in medium.

as you and Dr Thomas said i used HCL and the absorbance decrease after acidification but it still is higher than the initial amount of nitrate.

thanks Dr Thomas for your help.

what do you mean using nitrate free medium as blank?

i only add bicarbonate to the deionized water and use it as blank or make the culture medium without algae and nitrate and then adding bicarbonate and using as blank ?it seems good suggestion doctor

no Dr the absorbance at 275 is less than 5% of absorbance at 220.

the acidification with HCl helped but didn't solved the problem.

for example my initial absorption at 220(with 1/100 dilution) was 0.360 .after culturing and adding bicarbonate it became 0.600 ! and after acidification with HCl it became 0.450 which still bigger than initial value

Hi Amir, 

I take it you're using a colorimetric assay? 

You have to be careful with the filtration of algal cultures for ntirate determination, if done under too much pressure/to much material applied to the filter, it could result in either cell lysis and leakage of nitrate, or cells simply crossing the filtrate. To guarantee good results I first centrifuge a small amount at a relatively low speed (>10,000g) then filter the supernatant. 

Also taking measurements at 750nm of these samples will indicate how much debris remains in your samples, could give you an idea of which preparation technique works best. 

I think Dr Thomas was suggesting you should use your standard media without adding nitrate as a blank.

I use an enzymatic method, as supplied by Superior enzymes, that we have scaled down to multiwell plates.  http://nitrate.com/store/index.php/nitrate

Hope that's helpful. 

Hi, Amir, as you said, 

You used the medium of bicarbonate adding into the deionized water without  algae as blank. If so, the blank of initial absorption at 220 nm could not be so high of 0.360. If the value is so high, that meaning the deionized water or bicarbonate was polluted with nitrate, or the spectroscopy instrument or color pools needs to check . You must make sure your procedure and equipment was all right, then take your determination.

Firstly, u should check deionized water, then If u add HCl into original medium of bicarbonate without algae, the blank value was also not less than 0.05, u need to change the bicarbonate. After that, you can do the culturing algae study. In common, u use filtration or centrifuge are both efficiency for removing turbidity of suspended solid particles or algae cells. To determine the nitrate in water is not so difficult.

In our previous study, the above two method was both ok, and the zinc-cadmium reducing and hydrochloride acid ethylene diamine colorimetric method under 540 nm for seawater was always right and efficiency.

If your culture have high Cl- concentration, the result of UV method could be backdrop. U need to add Ag+ to remove Cl-.

If the water have high turbidity, u can add Al(OH)3 drops to settle for sedimentation, then supernatant for determination.

If the water have other ionic impacts, you must use macroporous adsorption resin before determination.

Dear Lu

I do appreciate your help.

thanks a lot