Professional Documents
Culture Documents
In the experiment, the nitrate content of the water sample was determined through spectrophotometry at
220 nm and 275 nm wavelengths. The second value of wavelength was used to correct the read NO3- value from
the 220 nm because at the latter wavelength, the NO3- value given by the spectrophotometer might have
included dissolved organic matter.
The researchers checked if all apparatus needed for the experiment has been borrowed from the
laboratory custodian, Sir Francisco Pascua. The apparatus used for the experiment were rinsed with distilled
water then wiped with clean dry cloth; however, the pipettes used for drawing out reagents were hung upside
down to let the water drip out of its opening.
Preparation of Reagents
1N HCl solution
From the concentrated hydrochloric acid solution in the laboratory, a volume of the acid solution
was drawn out with a pipette then transferred into a 1000 mL volumetric flask containing distilled water. The
solution in the volumetric flask was diluted to the 1000 mL mark on the neck of the volumetric flask. The flask
was then capped and shook then labeled as 1N HCl. In shaking the volumetric flask, a member placed the
thumb of her right hand on top of the flasks cap and its bottom held by the other hand then the flask was tipped
back and forth until the solution has completely mixed. The volume of the concentrated solution needed to
make 1N HCl was calculated by:
V 1 C 1=V 2 C 2 (4.1)
where:
C1- 1N HCl
V1- unknown volume of HCl needed to make 1N HCl
C2- concentration of concentrated HCl solution
V2- volume of concentrated HCl
The 1N HCl solution was standardized against a standardized 1N NaOH solution to determine its
exact concentration and was determined by the formula:
V NaOH C NaOH =V HCl C HCl
(4.2)
where:
VNaOH sample volume of NaOH used in standardization process
CNaOH concentration of NaOH, 1N
VHCl volume of HCl used to titrate NaOH
CHCl unknown concentration of HCl
To determine the amount of NO3- in mg as well as its concentration, the researchers made use of
the equation:
10 microgram NO 3 1 mg NO 3
mg of NO 3=mL of stock nitrate solution x x
1mL solution 1000 microgram NO 3 (4.3)
mg mg NO 3
NO 3 concentration , =
L Total volume of sample (4.4)
The spectrophotometer was powered on and left to warm up. Next, the desired wavelength of 220 nm
was selected after which the cuvette containing the blank solution was placed in the sample compartment. The
sample compartment was then closed and the transmittance was adjusted to 100 %.
The intermediate nitrate solutions were first tested at 220 nm wavelength. A little amount of the first
sample, 3 mL NO3- solution, was used to rinse a separate cuvette. The cuvette was then filled with the sample to
approximately full and the cuvette was closed with its lid. The cuvette was then wiped with a clean dry paper
towel on its side before it was placed in the sample compartment. The compartment was closed and the
absorbance was read and recorded. The cuvette containing the NO3- sample was taken out and the volume it held
was disposed in a waste bottle. The cuvette was then rinsed with the next sample of 7 mL NO3- solution.
After rinsing the cuvette, it was filled to with the 7 mL NO3- solution and covered with its lid. The cuvette
was wiped again with a paper towel and placed in the sample compartment then the absorbance was recorded.
This procedure was repeated until the absorbances of all the NO3- samples were recorded.
The testing of the water sample was done after the recording the absorbance of the calibrating solution.
The cuvette containing the calibrating NO3- solution was taken out of the compartment and its content poured
into a waste bottle afterwhich it was rinsed twice with the water sample of the first trial. The cuvette was
covered and its side wiped with paper towel. The cuvette was placed in the sample compartment and the
absorbance of the water sample was recorded. The same cuvette was removed from the compartment and the
sample it held was poured into the waste bottle. Next, the cuvette was rinsed with the water sample for the
second trial and the same procedure was performed until its absorbance was read.
After acquiring the absorbances of the calibrating NO3- solution and the water sample, the
spectrophotometer was set to 275 nm wavelength and the instrument was again calibrated with the blank
solution to 100% transmittance. The same procedure was done to obtain the absorbances of both of the sample
at the said wavelength.
Clean up
The remaining calibrating solutions and the water sample were disposed off into the laboratory sink
together with the samples from the waste bottle. The apparatuses were washed with soap and water and the table
top of the laboratory tables were wiped with a dry rug. The water sample was returned to the laboratory fridge.
Computation
The researchers calculated for the corrected absorbance value of the calibrating NO3- solution and the
water sample by subtracting twice of the absorbance at 275 nm to that of the absorbance at 220 nm.
The concentration of the NO3- solution in the water sample was computed by first obtaining the equation
of the line created by the calibrating solution. The equation of the line was determined by plotting the computed
mg/L of the calibrating solution from Eq. 4.2 against the values of the computed corrected absorbance from Eq.
4.4. In the graphs, the concentration in mg/L was plotted in the abscissa region and the absorbance as the
ordinate.