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PART A: Operating Parameters and Calibration Solution Concentrations:
1.
TABLE # 1: Instumental Settings Information:
Instrument

Thermo scientific evolution 220 double beam

spectrophotometer

Data format

Absorbance

Start wavelength (nm)

700.00 nm

Stop wavelength (nm)

400.00 nm

Software

Insight:1.4

Application

Scan- Scan

Band width

1 nm

Integration time

0.100 sec

Data interval

1.00 nm

Baseline correction

100 %T

Scan speed

600.00 nm/min

Derivative order

None

Smooth level

None

Result Type

Peak Pick

Sample ID

Iron Fe2+ ,5.008 ppm, Scan 2

TABLE # 2: Maximum wavelength of Unknown Sample:

Wavelength(nm)

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Absorbance

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511.60

1.080

2. In order to calculate the actual concentration of Fe2+ in ppm ,first of all we need to find
out the mole of iron(ii)ammonium sulphate hexahydrate (Fe(NH4)2SO4 *6H2O) .
1.7583 grams of iron (ii) ammonium sulphate hexahydrate (Fe(NH4)2SO4 *6H2O) was
weighed and the size of the volumetric flask used for making the stock solution was
2000 ml.
The unknown sample containing iron in well water : FE #60
Hydroxyl amine = 100.1478 g/L
1,10-Phenonthraline = 400.07 g/4L
Molecular weight of Fe 2+ = 55.85 g/mole
Molecular weight of (Fe(NH4)2SO4 *6H2O) = 392.05 g
Actual Concentration in ppm of Fe2+ in the stock solution =
1.7538 g of salt
1 mole of salt
1 mole of Fe 55.85 g of Fe 1000 mg 1000 ml

2000 ml
392.05 g of salt 1 mole of salt 1mole of Fe
1g
1L
= 125.24 ppm of Fe 2+
Therefore,the concentration of Fe2+ stock solution was found to be 125.24 ppm

3. In order to calculate the actual concentration of

2+

Fe sub stock solution,

C1 = Concentration of the stock solution =125.2 ppm

V1 = Volume of stock solution transferred = 10.00 ml
C2 = Concentration of sub stock =?
V2 = Volume of volumetric flask used = 100.00 ml

C1V1 = C2 V2

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C2 =

C 1V 1
V2
125.2 ppm10.00 ml
100.00 ml

= 12.52 ppm
Therefore, the concentration of Fe2+ sub stock solution was found to be 12.52 ppm

4. The actual concentration in ppm of Fe2+ in external standard calibration solution based on
the volume of Fe2+ sub stock solution is:

C1 = concentration of the substock solution =12.52 ppm

V1 = volume of stock solution transferred = 1.00 ml
C2 = concentration of substock = ?
V2 = volume of volumetric flask used = 25.00 ml
C1V1 = C2 V2
C2 =

c1 v 1
v2
12.52 ppm1.00 ml
25.00 ml

= 0.5008 ppm

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125.2ppm

2000ml volumetric
fflask ffflask flask

5.

10
.0
0m

12.52ppm

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7.
5.00ml

m
00

111111111

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6.
TABLE # 3: External Standard Calibration Standards at 511.6 nm(max) :
Standard #

1
2
3
4
5

2+
Substock Fe volumes

2+
Calculated Fe

Absorbance at
511.6nm (Max)

pipette(ml)
1.00
3.00
5.00
7.00
10.0

concentration (ppm)
0.500
1.502
2.504
3.506
5.008

0.129
0.340
0.542
0.748
1.081

4.701
4.834
4.947

0.993
1.037
1.052

Unknown#
FE 60
FE 601
FE 602
FE 603

TABLE # 4: External Standard Calibration Standards at 570 nm(min):

Standard #

1
2
3
4
5

Substock
pipette(ml)
1.00
3.00
5.00
7.00
10.0

Unknown#
FE 60
FE 601
FE 602
FE 603

PATEL TVISHA

2+
fe

volumes

Calculated

2+
Fe

Absorbance at
570nm (Min)

concentration (ppm)
0.500
1.502
2.504
3.506
5.008

0.014
0.043
0.051
0.073
0.103

4.701
4.834
4.947

0.098
0.099
0.102

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PART B: Regression Method:
7.
TABLE # 5 : Regression equation and Correlation Coefficient obtained from Calibration
Graphs for 511 nm (max) and 570 nm (min)

Regression equation
(511.6 nm)
0.021 + (0.210)C

Correlation coefficient at 511.6 nm

Regression equation
(570 nm)
0.007 + (0.019)C

Correlation coefficient at 570 nm

1.000

0.982

8.
Calculating the unknown concentration of Fe2+ (ppm) for 511 nm wavelength :
Absorbance = 0.021 + (0.210)C
0.993 = 0.021 + (0.210)C
0.993 0.021 = (0.210)C
So, C =

0.972
0.210

= 4.628 ppm
TABLE # 6: Concentration of Fe2+ in unknown sample: (511.6 nm)
Serial #

Calculated
concentration in ppm

Absorbance at
511.6nm

1
2
3

4.628
4.838
4.909

0.993
1.037
1.052

PATEL TVISHA

Final
concentration in
ppm
57.85
60.47
61.36

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TABLE # 7: Concentration of Fe2+ in unknown sample: (570 nm)

Serial#

Calculated
concentration in ppm

Absorbance at 570
nm

1
2
3

4.789
4.842
5.000

0.098
0.099
0.102

Final
concentration in
ppm
59.86
60.52
62.50

9.
In order to calculate the final concentration of Fe2+ in original sample of well water, we
have to do the back calculation.(for 511.6 nm)
Final concentration of Fe2+ =

25 ml
4.628 ppm
2ml

= 57.85 ppm
10.
(i)

Performing the Q-test of any datum:

Qtest

Qexp

gap
= range

60.4757.85
61.3657.85

= 0.746

Qexp <Qtable

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0.746 < 0.970
It concludes that the value is being retained.
(ii)

Calculating the mean concentration at 511.6 nm :

Mean concentration of Fe2+ =

final concentration ppm

number of trials
57.85+ 60.47+61.36
3

= 59.89 ppm
(iii)

standard deviation=

Calculating the standard deviation

( x x )
n1

Where,
N=number of values
x=an individual value
x = mean or average of the value
(x- x )2 =sum of the square of the difference from the mean for all values.

{(57.8559.89 )2 + ( 60.4759.89 )2+ (61.3659.89 )2 }

31

{6.657 }
2

1.824

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(iv)

Calculating the confidence interval (95%) :

= x m

95

= 59.89

t XS
N

4.303 X 1.824
2

= 59.89 5.550
95

= 65.44 (by addition)

95

= 54.34 (by subtraction)

Table# 8: Summary table at 511.6 nm:

Mean concentration
in ppm
59.89

Standard deviation

1.824 %

True value at
95%confidence
interval
65.44 (by addition)
54.34 (by
subtraction)

Qexp

0.746

Table# 9: Summary table at 570 nm:

Mean concentration
in ppm
60.96

Standard deviation

1.373 %

True value at
95%confidence
interval
65.13 (by addition)
56.78 (by
subtraction)

Qexp

0.250

11.

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Based on the regression result at 511.6 nm, The mean value obtained is 59.89 ppm, if I compare
this value with the true value provided, my value nearly matches with SAMPLE D.

12. Relative error using 511.6 nm is:

Relative error =

value
100
|absolute
true value |

measured valuetrue value

|
| 100
Relative error =
true vale

|59.8964.60
|100
64.60

= 7.29 %

PART D: Qualitative Visible Spectrum:

13. Calculating the absorption coefficient at 511.6 nm ,using the following equation
a=

A
bc

Where,
A =absorption of a complex at 511.6 nm( maximum wavelength )
a =absorption coefficient
b =cuvette width(1cm)
c =concentration of the external used in mg/L

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a=

1.081
1 5.008
0.215 L/cm*mg

TABLE # 10: For Absorptivity co-efficient :

Serial #
1
2
3
4
5

Concentration in
ppm
0.500
1.502
2.504
3.505
5.008

Absorbance
(511.6 nm)
0.129
0.340
0.542
0.748
1.081

Absorptivity
coefficient L/cm*mg
0.025
0.067
0.108
0.149
0.215

14.
TABLE # 11: For Absorptivity Coefficient and the corresponding Slope:
Absorptivity coefficient
0.215

Slope value at 511.6 nm

0.210

Absorptivity coefficient
0.020

Slope value at 570nm

0.019

PART D: Discussion:
15.
I think the reason behind this is, as the wavelength increase, the absorbance of the
compound is being affected because it is known that the wavelength depends on the
presence of the light absorbing groups in the compound. Hence some compounds have
maximum absorption while some of them have minimum absorption depending upon the
capacity of the passage of the light through it. Even the solutions have different spectral
sensitivity at different wavelengths. Thus, affects the absorbance value.

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16.
a) As the value of the absorbance for both the wavelength is differs when compared.
Later, when external calibration plot is done between Absorbance vs. Concentration
we obtain different regression equation for both the wave length.
The reason behind this is that on one side we take 511.6 nm and on another side we
take 570nm which brings difference in regression equation because much of variation
is seen even wavelength. Also the linear regression shows the relation between the
concentration (x-variable) and the absorbance (y-variable) which is different for both
wavelengths observed.
b) According to the result obtained it can verified that 511.6 nm line shows the greatest
sensitivity toward the concentration of Fe+2 because absorbance value at 511.6 nm is
higher than absorbance value at 570nm.
17.
Upon examining the mean, standard deviation and true value for both the wavelengths,
it was observed that the mean concentration is 59.89 ppm (511.6 nm) and 60.96 nm
(570 nm), while standard deviation is 1.824 (511.6 nm) and 1.373 (570 nm), true
value at 95% confidence interval found is 65.44 (by addition) and 54.34 (by
subtraction) for 511.6 nm and 65.13 (by addition) and 56.78 nm (by subtraction)
for 570 nm.
But not much of the difference is seen between all the values obtained but the dilution
factor remains the same.

18.
In order to minimize the relative error one should recommend doing more accurate
dilution, in this experiment dilution is crucial because even a point difference can affect
the concentration of the solution.
Second recommendation to minimize the error is by accurate pipetting of each solution
and by proper calibration of instrument used in experiment.
19.
TABLE #12 : Absoptivity coefficient and Slope value
Wavelength(nm)

PATEL TVISHA

Absorptivity coefficient

12

Slope value

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510
570

0.210 L/cm* mg
0.020 L/cm* mg

0.210
0.019

As per lambert beers law,

So the absorptivity coefficient and slope value are similar.
Also, where the value of b=1, is ideal. So according to the equation both the criteria will become
same, so thats the reason, that the value of absorptivity coefficient and slope are similar.

20.
At 511.6 nm, Green colour is absorbed by Fe2+ 1,10- phenanthroline complex and
Yellowish green at 570 nm .Green colour is transmitted at 511.6 nm and Yellowish green
at 570nm by human eye.

REFERNCE:
Tyrer N., Rankin K., Chem 25415 Instrumental Analysis Laboratory Manual, Sheridan
College, Brampton, ON, fall 2013. Page no. 4.1-4.12

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