Title: Spectrophotometric determination of reactive phosphate in waters

Aim: The aim of this experiment is to analyze reactive phosphorus in south quay lake and
monash drain by spectrophotometer measurement.

Outline of method:

Calibration standards
of known
concentrations were
prepared

Concentration of
phosphate was then
determined by
plotting a calibration
graph.

Summary of results:
Blank data

1 drop of
phenolphthalein and
dropwise of sufficient
Naoh were added to
Monash drain and
South Quay samples
and 5 replicate blank
samples

Sulfuric acid and

combined reagent
were added to the
samples and blanks.

The absorbance of
all samples, samples
and blanks were
measure at 880nm
using a
spectrophotometer

The acidified
phosphomolybdenum
developed into a blue
solutions after 10
minutes.

Table 1. Absorbance, mean absorbance and standard deviation of the blank.

Blank no.
1
2
3
4
5

Abs 1
0.000
0.013
0.001
-0.001
0.010

Abs 2
0.008
0.009
0.010
0.014
0.000

Abs 3
0.006
0.008
0.006
0.013
0.001

Abs 4
0.004
0.004
-0.003
-0.010
0.009

Mean Abs

n-1

0.0050

0.0052

Calibration data
Table 2. The absorbance, mean and standard deviation of the phosphate
Phosphate
(gP/L)
0
50
100
150
200

Abs 1

0.045
0.070
0.116
0.138

Abs 2

0.040
0.077
0.121
0.143

Abs 3

Mean Abs

n-1

0.037
0.067
0.106
0.142

0.0050
0.0407
0.0713
0.1143
0.1410

0.0052
0.0040
0.0223
0.0076
0.0026

Calibration graph
0.16
0.14

f(x) = 0x + 0.01
R = 0.99

0.12
0.1
Absorbance at 880nm

0.08
0.06
0.04
0.02
0
0

50

100

150

200

250

Phosphate concentration (gP/L)

Figure 1. Graph of Absorbance at 880nm vs Concentration of phosphate (gP/L)

Sample data

Table 3. The absorbance at 880nm, concentration and mean concentration for the water samples.
Replicat
e
number

Abs 1

Abs 2

Abs 3

Conc 1

Conc 2

Conc 3

Sample
A1
Sample
A2
Sample
A3

0.030

0.035

0.033

35.2857

42.4286

39.5714

Sample
B1
Sample
B2
Sample
B3

QC

Mean
conc
(gP/L)

Mean
Conc
(gP/L)
dilution
corr.
78.1904

39.0952
0.029

0.028

0.024

33.8571

32.4286

26.7143

0.019

0.022

0.025

19.5714

23.8571

28.1429

62.0000
31.0000
47.7142

0.018

0.021

0.018

18.1429

22.4286

23.8571
Sample A mean
(gP/L)
n-1
18.1429 19.5715

0.014

0.017

0.013

12.4286

16.7143

11.0000

13.3810

26.7620

0.017

0.020

0.019

16.7143

21.0000

19.5714

19.0952

38.1905

Sample A mean
(gP/L)
n-1
Conc 3 Mean
Conc
(gP/L)

34.6984

Abs 1

Abs 2

Abs 3

Conc 1

Conc 2

0.077

0.080

0.073

102.428
6

n-1

5.0170

% RSD

106.714
3
4.9209

Sample A = South Quay Lake (upstream)

Sample B = Monash drain (downstream)

96.7143

101.952
4
% Relative Error

62.6349
15.2480
39.1430

6.8897
Mean
Conc
(gP/L)
dilution
corr.
1.9524

Sample calculations
Preparation of standards :
Using C1V1 = C2V2 where C1 is 50 gP/L, C2 is 1 gP/L and V2 is 250ml.
50 x V1 = 1 (250)
V1 = 5 ml of 50 gP/L
The same method was used to calculate the working stock at concentration of 100, 150 and 200
gP/L

The equation obtained from the graph is y = 0.0007x + 0.0053 where y is the absorbance value at
880nm and x is the phosphate concentration (gP/L)
Concentration for sample A1:y = 0.0007x + 0.0053
0.030 = 0.0007x + 0.0053
x = 0.030 0.0053
= 35. 2857 gP/L

Mean concentration for sample A :(Concentration A1 + Concentration A2 + Concentration A3) / 3

= (35.2857 + 42.4286 + 39.5714) / 3
= 39.0952 gP/L

All samples were diluted by a factor of 2. So the original concentration for sample A is:
Mean concentration sample A x 2
= 39.0952 x 2
= 78.1904 gP/L

Sample A mean:
(Original concentration A1 + Original concentration A2 + Original concentration A3) / 3
(78.1904 + 62.0000 + 47.7142) / 3
= 62.6349 gP/L
Sample A standard deviation:

( 15.5555 ) +(0.6349 ) +(14.9207)

2

31

= 15.2480
All the concentration, mean concentration, mean and standard deviation for sample B were
calculated using the same method.

% Relative standard deviation for QC:

(standard deviation/mean) x 100
= (5.0170/ 101.9524) x 100
= 4.9209 %

% Relative Error:
[(Actual Value Measured Value) / Actual Value] x 100
[(101.9524 100)/100] x 100
= 1.9524 %

From the graph, the sensitivity is 0.0007 g-1L

Molar mass of phosphorus is 30.97 g/mol
Sensitivity in mol-1L:
(7 x 10-10 L/g) x (30.97 g/mol)

= 2.1679 x 10-8 mol-L

Minimum detectable absorbance:
Sm = Sbl + ksbl
= 0.005 + 3(0.006)
Sm = 0.023

Limit of detection:
Cm = (Sbl + ksbl )/ m
= (0.023 0.005) / 0.0007
= 25.714 g/L

Summary Table
Table 4. Summary of the results.
Sensitivity
Minimum detectable absorbance
Limit of detection
Reproducibility

0.0007 g-1L
2.1679 x 10-8 mol-1L
0.023
25.714 g/L

n-1
% RSD

Accuracy

South
lake
15.2480
24.3443

Monash
drain
6.8897
19.8560

QC
5.0170
4.9209

% RE: 1.9524

Discussion and Conclusion

From the graph above, the R2 value is 0.9866 which indicates a strong correlation between the
concentration of phosphate and the absorbance at 880nm.
Accuracy is a measure of how close the result of the measurement comes to the "true", "actual",
or "accepted" value (Quevauviller, 2008). In this experiment, the accuracy of quality control is

101.9524 g/L while the actual value is 100 g/L, so the relative percentage error is 1.9524%.
The relative error of the measurement shows how large the error is in relation to the correct value
which compares the size of the error to the size of the object being measured. Since, the
percentage error is lower than 5%, the data is good because there is lesser error and the value is
almost close to the correct value. If the percentage error is more than 5%,
Precision is the reproducibility of results obtained in identical manner. The relative standard
deviation is widely used in analytical chemistry to express the precision and repeatability of an
assay (Robinson, Skelly Frame and Frame II, 2012). A lower percentage of RSD indicates a
lower variability in the data set. Equally, a higher percentage indicates the data set is more
varied. In this experiment monash drain has a lower percentage RSD which is 19.8560 %
compared to the South Quay lake which is 24.3443%. Hence, Monash drain has a lower
variability and is more precise compared to South Quay.
The sensitivity of this experiment is 0.0007 g-1L. This spectrophotometer has a minimum
detectable absorbance 0.023which means that the device can detect up to 0.023 for the blank.
Limit detection is used to detect the lowest concentration of the spectrophotometer which is
25.714 g/L. The higher concentration may be due to the reading value of the blank and the
standard solution. Colour of phosphomolybdenum blue which will fade in a long period of time
can also contribute to the error in the experiment as there is a prolog waiting time to take the
result for all the sample data. The % RSD for south quay, monash drain and quality control is
24.3443 %, 19.8560%, 4.9209% respectively.
Error in measurement may be represented by a tolerance interval (margin of error). The
spectrophotometer used in manufacturing often set tolerance intervals, or ranges in which
product measurements will be tolerated or accepted before they are considered flawed.
To avoid errors, make the measurement with an instrument that has the highest level of precision.
The smaller the unit, or fraction of a unit, on the spectrophotometer, the more precisely the
device can measure. The precision of a measuring instrument is determined by the smallest unit
to which it can measure (Kenkel, 2003). Besides that, randomizing samples or experiment could
also increase the accuracy by increasing the signal to noise ratio. Other than that, repeat the same
measure several times to get a good average value.
Hence it can be concluded that, the concentration of phosphate in South Quay (Sample A) is
62.6349 gP/L 15.2480 and the concentration of phosphate in Monash drain (Sample B) is
34.6984 gP/L 6.8897. This shows that monash drain is cleaner as there is lesser concentration
of phosphate present it water.

Questions:
1. Is there evidence from the sample measurements that the dissolved reactive phosphorus
concentrations are deemed to be above the Interim National Quality Standards (INWQS)
guideline values issued by Department of Environment (DOE) for a Class III water source?
From the website, the concentration of phosphate in class III is 0.1 mg/L which is 100 g/L
There is no evidence that the dissolved reactive phosphorus concentrations are deemed to be
above the INWQS because the mean concentrations for South Quay (62.6349 gP/L) and
Monash drain (34.6984 gP/L) are lower than the concentration of phosphate.

2. Estimate the improvement in sensitivity if a 4cm cell rather than a 1cm cell was used. Would
this change the detection limit?
The sensitivity of the sample will increase if a 4cm cell is used rather than 1 cm cell.
According to the Beer Lambert Law, the signal will increase if the path length used is
increased. This would change the detection limit. Higher sensitivity gives rise to a lower
detection limit as the intensity of the transmitted light decreases exponentially as the path
length of the sample increases (Koch, 2012).

3. How could you determine the accuracy of your experiment method?

Accuracy is a measure of how close the result of the measurement comes to the "true",
"actual", or "accepted" value. The accuracy of the experiment method can be determined by
using quality control of a known concentration which is used to give a theoretical value to be
used to compare with the sample result (Quevauviller, 2008). The % relative error is obtained
by dividing the data value over the quality control to compare the size of the error to the size
of the object being measured. The lesser the percentage relative error, the higher the accuracy
of the data obtained. To avoid errors and maximize the accuracy of results, make the
measurement with an instrument that has the highest level of precision. The smaller the unit,
or fraction of a unit, on the spectrophotometer, the more precisely the device can measure
(Kenkel, 2003).

4. Silicate also undergoes reactions with acidic molybdate to form silicomolybdic acid which
can also be reduced to molybdenum blue. Briefly describe an experiment that would enable
you to determine whether silicate is a chemical interference in the measurement of
phosphate.
Silicate can be tested through acid treatment and reaction with ammonium molybdate to give
a yellow coloured complex or silicomolybdate complex. Sodium sulphate is added as well to
reduce the complex. If the reaction turns blue, there may be possibility that silicate may be
present in the solution. To determine the absorption, absorption spectrum need to be tested
using spectrophotometer. The absorbance readings are then compared. High value of
absorbance reading indicates that silicate is a chemical interference in the measurement of
phosphate which leads to changes in intensity of the analyte signal in spectroscopy
(Henriksen, 2001).

References
Henriksen, A. (2001). Interference from silica in phosphate analysis. Analyst, 91(1081), pp.290291
.
Kenkel, J. (2003). Analytical chemistry for technicians. 1st ed. Boca Raton: Lewis Publishers.
Koch, W. (2012). Ten years of Analytical and Bioanalytical Chemistry. Analytical and bioanalytical
chemistry, 402(1), pp.186 -189.
Robinson, J., Skelly Frame, E. and Frame II, G. (2012). Undergraduate Instrumental Analysis, Sixth
Edition. 1st ed. Hoboken: Taylor and Francis.
Quevauviller, P. (2008). Quality Assurance in Environmental Monitoring. 1st ed. Wiley VCH.