I.

Introduction
Phosphates can play both a positive and negative role in the water.

Phosphates are not toxic to people or animals unless they are present in very
high levels.

Phosphates are present virtually in every detergent, including

household cleaners and laundry soap. If too much phosphate is present in the
water, the algae and weeds will grow rapidly, may choke the waterway, and use
up large amounts of precious oxygen. The result may be the death of many fish
and aquatic organisms. Phosphates can also be found in some foods. Digestive
problems could occur from extremely high levels of phosphate. Phosphates enter
waterways from human and animal waste, phosphorus rich bedrock, laundry,
cleaning, industrial effluents, and fertilizer runoff.
In this experiment, phosphate in water is analyzed. The principle of this
method involves the formation of molybdophosphoric acid, which is reduced to
the intensely colored complex, molybdenum blue.

Reagents

a. Ammonium molybdate reagent:

The solution is prepared by dissolving 25g of (NH 4)6Mo7O244H2O in 175 mL
purified water. Cautiously, 280 mL of concentrated H 2SO4 is added to 400 mL of
purified water. The acid solution is cooled, the molybdate solution is added, and
the entire mixture is diluted to 1 liter.
b. Stannous chloride reagent:
Dissolve 2.5 g of SnCl22H2O in 100 mL of glycerine. The mixture is gently
heated to hasten dissolution.
c. Stock 20.0 mg/L phosphate solution:
Dissolve 0.02866 g of KH2PO4 to1.0 liter of water.

d. Standard Phosphate solutions:

*1.0 mg/L standard: Place 5.00 mL of 20.0 mg/L phosphate solution

and dilute with enough water to give a final volume of 100 mL.
*2.0 mg/L standard: Place 10.00 mL of 20.0 mg/L phosphate solution
and dilute with enough water to give a final volume of 100 mL.
*3.0 mg/L standard: Place 15.00 mL of 20.0 mg/L phosphate solution
and dilute with enough water to give a final volume of 100 mL.
*4.0 mg/L standard: Place 20.00 mL of 20.0 mg/L phosphate solution
and dilute with enough water to give a final volume of 100 mL.
*5.0 mg/L standard: Place 25.00 mL of 20.0 mg/L phosphate solution
and dilute with enough water to give a final volume of 100 mL.
II.

Procedure
A. Determination of Absorbance from an unknown concentration solution
25 mL Water sample in an Erlenmeyer
flask

-Add 1.00 mL ammonium molybdate solution

-Swirl to mix
-Add 2 drops of stannous chloride
-Again, swirl to mix

-Let it stand for 5 10 minutes

Blue
-Set UV-Vis spectrophotometer with 650 nm
wavelength to zero absorbance using blank
solution
-Place blue sample in a cell
-Determine absorbance using the instrument
-Record result
-Perform three trials

Absorbance of the Unknown Solution

Determined

B. Determination of Absorbance from standard phosphate solutions:

25 mL of 1.0 mg/L phosphate solution
-place in an Erlenmeyer Flask

-Add 1.00 mL ammonium molybdate solution

-Swirl to mix
-Add 2 drops of stannous chloride
-Again, swirl to mix
-Let it stand for 5 10 minutes

Blue
-Set UV-Vis spectrophotometer with 650 nm
wavelength to zero absorbance using blank
solution
-Place blue sample in a cell
-Determine absorbance using the instrument
-Record result
-Repeat procedure using 2.0, 3.0. 4.0, 5.0 mg/L of
of

standard phosphate solution

-Make a plot of absorbance vs. concentration

Absorbance of the 5 different concentrations determined

C. Determination of Phosphate concentration from Unknown Solution

Absorbance of the Unknown Solution
-From the concentration and absorbance of 5
standards, determine slope and y-intercept
-From the absorbance of the unknown solution,
determine concentration using least-squares
method
-Compute again using the other 2 absorbances
-Compute the average concentration

Phosphate concentration determined

III. Results and Discussion

Table 1 showing the absorbance of the blank solution and unknown
concentration solution

Blank Solution
T1

Absorbance
0.000
1.246

T2
T3

1.277
1.240

Table 2 showing the absorbance of the blank solution and 5 standard

phosphate solutions.
Absorbance
0.000
0.278
0.569
0.834
1.036
1.345

Blank Solution
1 ppm
2 ppm
3 ppm
4 ppm
5 ppm

From table 2, the slope, m=0.2601 and the y-intercept, b=0.0321

*Concentration (x) of T1
Given absorbance (y) = 1.246

y=mx +b
x=

x=

yb
m

1.2460.0321
0.2601

Concentration ( x )=T 1 =4.667 ppm

*Concentration (x) of T2
Given absorbance (y) = 1.277

y=mx +b

x=

x=

yb
m

1.2770.0321
0.2601

Concentration ( x )=T 2 =4.786 ppm

*Concentration (x) of T3
Given absorbance (y) = 1.240

y=mx +b
x=

x=

yb
m

1.2400.0321
0.2601

Concentration ( x )=T 2 =4.640 ppm

Average Absorbance=

Average Concentration=

1.246+1.277+1.240
=1.254
3

4.667+4.786+ 4.640
=4.698 ppm
3

Figure 1 showing Concentration (ppm) vs. Absorbance

1.6
1.4
f(x) = 0.26x + 0.02
R = 1

1.2
1
Absorbance

0.8
0.6
0.4
0.2
0
0

Concentration in ppm

Ammonium

heptamolybdate

is

often

referred

to

as

ammonium

paramolybdate or simply as ammonium molybdate, although ammonium molybdate

can also refer to ammonium orthomolybdate, (NH4)2MoO4, and several other
species. In the experiment, 1-ml of ammonium molybdate solution was added to
the sample and combines with phosphate ion under acid conditions to form
molybdophosphate complex. The reaction follows:

PO43- + 12 (NH4)2MoO4 + 24 H+

(NH4)3PO4 12MoO3 + 21 NH4+ + 12

H 2O
2 drops of stannous chloride was then added to the solution. The solution
turned blue for a couple of minutes after it was mixed by swirling. It produced a blue
color due to the formation of molybdenum blue and the depth of the blue color
indicates the amount of phosphate in water.The reaction follows:
(NH4)3PO4 12MoO3 + Sn2+

(molybdenum blue) + Sn2+

. The absorbance of the blue solution can be measured and the concentration
of phosphate in the solution can be calculated. The blue color solution wasnt
diluted to 100-fold since it wasnt very dark. Using the UV-vis spectrophotometer,
absorbance of the unknown concentration solution was determined. Three trials
were performed. As seen in table 1, variations in the result of absorbance are
observed. This can be accounted to personal errors on preparing the reagents
and/or not properly washing the cell. As computed, the average absorbance is
1.254. In this part, the concentration is still unknown.
In table 2, the absorbance of the five different standard phosphate solutions
was determined using the same method. Only one trial was conducted and we can
see that as the concentration (ppm) increases, the absorbance also increases, as
stated in Beer-Lambert law (the linear relationship between absorbance and
concentration of an absorber of electromagnetic radiation). The absorbance is
dependent upon the concentration of phosphate in the sample. Thus, manual errors
of pipetting the correct amount of water sample and reagent may affect the
absorbance of the blue complex which in turn may lead towards error in the
estimation of concentration of phosphate in the sample.
By plotting the values of absorbance and concentration, we can see
deviations of the points. As calculated the mean concentration of phosphate in the
sample is 4.698 ppm.

IV.

Conclusion
Using least-squares method, the concentration of phosphate in the water

sample was calculated to be 4.698 ppm. With careful manual techniques in

conducting the experiment, errors will be minimized which wil lead to better results.

V.

Answers to Questions

1. What is Beer-Lambert Law?

Beer-Lambert law relates the absorption of light to the properties of the material
through which the light is traveling. The law states that there is a logarithmic
dependence between the transmission (or transmissivity), T, of light through a
substance and the product of the absorption coefficient of the substance, , and the
distance the light travels through the material (i.e., the path length), . The
absorption coefficient can, in turn, be written as a product of either a molar
absorptivity (extinction coefficient) of the absorber, , and the molar concentration c
of absorbing species in the material, or an absorption cross section, , and the
(number) density N' of absorbers.

2. What is complementary color? How is this important in colorimetric analysis?

Complementary colors lie across the diameter on the color wheel and combine to
form white light, so the color of a compound seen by the eye is the complement
of the color of light absorbed by a colored compound; thus it completes the color.
Colorimetric analysis is a method of determining the concentration of a chemical
element or chemical compound in a solution with the aid of a color reagent.

A color wheel, shown above, illustrates the approximate complementary

relationship between the wavelengths of light absorbed and the wavelengths
transmitted or reflected. For example, a blue substance, there would be a strong
absorbance of the complementary color of light, orange. For this case, the
absorption spectrum of a blue solution would have a maximum absorbance at a
wavelength corresponding to orange light.

3. What is the relationship between absorbance and transmittance?

Absorbance is the measure of the quantity of light that a sample neither
transmits nor reflects and is proportional to the concentration of a substance in a
solution. On the meter of the Spectronic 20, the absorbance is displayed on the
bottom scale, which is logarithmic. The transmittance of a sample is the ratio of the
intensity of the light that has passed through the sample to the intensity of the light
when it entered the sample ( T = I out / Iin ). The transmittance is displayed as a
percentage on the top scale of the meter of the Spectronic 20.To convert between
the absorbance and transmittance scales, use the equation: Absorbance = -log
(percent transmittance/100)

4.

Express the absorbances of standard phosphates solutions into percent

transmittance?

A = log (1/T) or A = 2 - log(%T)

10A = 1/T
T = 10-A
%T = 100(10-A)

Absorbance
0.000
0.278
0.569
0.834
1.036
1.345

Blank Solution
1 ppm
2 ppm
3 ppm
4 ppm
5 ppm

%Transmittance
100
52.7
27.0
14.7
9.20
4.52

5. Calculate the molar absorpitivity of the five standard solutions. Do the five
solutions have equal molar absorptivity?
A = ebc
A= absorbance
e = molar absorbtivity
b = cell path length in cm
c = concentration in moles/liter

e=

Absorbanc
e
0.278
0.569
0.834
1.036
1.345

Molar absorptivity
( Lcm-1mol-1 )
2.65 x 104
2.79 x 104
2.64 x 104
2.46 x 104
2.56 x 104

A
bc

Cell Path Length (cm)

1
1
1
1
1

Concentration
(M)
1.05 x 10-5
2.04 x 10-5
3.16 x 10-5
4.21 x 10-5
5.26 x 10-5

Molar absorptivitys of the standard solutions are not equal as seen in the table.
Though, the values are close to each other. This explains the deviations of points in
the linear graph.

6.

A solution containing 8.75 ppm KMnO4 has a transmittance of 0.743 in a 1.00

cm cell at 530 nm. Calculate the molar absorptivity of KMnO 4.

b=1.00 cm

c=

8.75 mg
KMn O4
L

x 1000 mg

mol
158.0339 g KMnO 4

5.54 x 10

mol
L

1
T

A=log
1
0.743

A=log
A=0.129

A=ebc

e=

A
bc

e=

0.129
( 1 ) (5.54 x 105 )

Molar absorptivity,
3

e=2.33 x 10 L mol cm

Mindanao University of Science and

Technology
C.M. Recto, Lapasan, Cagayan de Oro City

Experiment No. 1
Analysis of Phosphate in Water

Allen Mark T. Librado

Group 4
BS- Chemistry

S.Y. 2013- 2014