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SCHOOL OF LIFE
SCIENCES
ANALYTICAL
TECHNIQUES
FRS2007M
LAB REPORT
SHANKAR VAHESAN
13452128

Significance of AAS, Fluorescence and UV/VIS Spectroscopy in Testing
for Lead Poisoning
Introduction
To begin with, the atomic absorption spectroscopy is a widespread technique used
to determine elements in various samples by using the free, vaporised atoms
generated in the atomiser
...
The beam of light from the hollow-cathode lamp excites
the ground state atoms in the flame enabling absorption to be measured related to
concentration of the gas-phase atoms (Slavin and Slavin, 1978)
...
A
calibration curve of known concentrations can be produced in order to determine
other absorbance
...
A detector accurately measures the light and a
monochromator is required to isolate the specific wavelength of light to be measured
(Beaty, 1978)
...
In this case, the blood would have to be centrifuged in order to
separate the lead from the dissolved glucose, mineral ions, proteins and hormones
in the plasma and then the sample has to be digested with nitric acid to eliminate the
organic matter at a high temperature to prevent interferences during data analysis
...

Applications of fluorescence spectroscopy include the analysis of structural changes
and the determination of whether a tryptophan is in an aqueous environment or
within the protein (www2
...
ac
...
Moreover, when a fluorophore such
as Eosin or Fluorescein is excited into a higher state, the fluorescence signals
information about the environment and structure of the fluorophore (Gell, 2006)
...


Furthermore, Ultraviolet-Visible spectroscopy uses light in the visible range to
measure transitions from the ground to the excited state in which is absorbed
(Perkampus, 1992)
...
One of the beams passes through the cuvette containing the transparent
solution of the compound studied, whereas the reference beam passes through the
blank
...
The UV/VIS
spectrophotometer can be used to calculate the concentration of an analyte using its
response which is similar to using calibration curves
...
In addition, this technique can be used to measure the
reflectance of light
...
Nevertheless, it can also be used clinically to
calculate absorbance of eosin which is linked to the concentration of eosin in Mallory
bodies
...

 To compare and identify the fluorescence and absorptivity properties of eosin
and fluorescein
...


Materials and Methods
The lab manual was followed for the use of the Perkine Elmer AAS 800 in order to
carry out the Lead analysis and for the use of the Shimadzu UV-1650p and the
Eclipse Fluorescence Spectrophotometer to carry out the Eosin and Fluorescein
analysis (Goddard, 2014)
...


Results
Practical 1 - Analysis of Lead Using Atomic Absorption Spectroscopy
Equation used to calculate volume of lead stock solution required (Richards, 2012):
C1V1 = C2V2
 C1 = concentration of stock concentration
 V1 = volume to be removed from concentrated stock solution
 C2 = final concentration of diluted solution
 V2 = final volume of diluted solution
Calculated example:
 C1 = 10 V1= 50/1000 C2=100 V2= X/1000
 10 x 50/1000 = 100 x X/1000
 X = (10 x 50) / 100 = 5mL

Calibration

Total Volume (mL)

Volume of Lead Stock Solution

Standard (mg L-1)

(100mg L-1) required (mL)

0

50

0

1

50

0
...
Calculated volumes of Lead stock solution at 100mg/L required to prepare
five calibration standards and analyse absorbance using AAS
...
0061

0
...
0055

1

0
...
0149 0
...
2028 0
...
2051

0
...
3817 0
...
3784

0
...
577

0
...
5834

0
...
0052
0
...
Absorbance readings collected from Perking Elmer AAS 800 using
solutions of known Lead concentrations
...
3nm
...
7
y = -0
...
0231x - 0
...
9996

0
...
5

0
...
3

0
...
1

0
0

5

10

15

20

25

30

35

40

45

Lead Concentration (mg/L)

Figure 1
...
The R2 value is 0
...

Sample

Absorbance

Mean Absorbance

Blank

0
...
004

0
...
0052

Plasma 1

0
...
474

0
...
474333333

Plasma 2

0
...
473

0
...
464

Plasma 3

0
...
479

0
...
479333333

Reference Standard (10mg/L)

0
...
2021

0
...
2032

Table 3
...


Using the equation of the calibration graph of Figure 1, it is possible to calculate the
lead concentration in the plasma samples
...
0002

B

0
...
0017

Absorbance y

0
...
0231±√0
...
0002)(−0
...
464)
2(−0
...
0231±√5
...
0002)(−0
...
0004
−0
...
3361x10−4 −4(9
...
0004
−0
...
3361x10−4 −3
...
0004
−0
...
6105x10−4
−0
...
02 or 89
...
Lead concentration in Plasma 2 calculation
...
02mg/L is
applicable in this case
...
However, the linear correlation shown means that the
value of concentration which fits in the polynomial, linear trend line between
absorbance and concentration of Figure 1 is the only value that is valid
...


Plasma Sample

Lead Concentration in Blood (mg/L)

Plasma 1

26
...
02

Plasma 3

27
...
71

Reference Standard

9
...
Lead concentrations in the plasma samples calculated using the quadratic
equation in Figure 1
...
68) / 9
...
7% (3sf)
Precision= 100- [(Standard Deviation/Mean) x 100)]
= 100- [((0
...
71) x 100)]
= 100-2
...
6% (3sf)

Practical 2 – Fluorescence and UV-VIS Spectroscopy
Calibration

Total

Volume of fluorescein stock

Molar

Standard (mg

Volume

solution (1 mg L-1) required

Concentration

L-1)

(mL)

(mL)

(mol L-1)

1x10-3

20

0
...
66x10-9

5x10-3

20

0
...
33x10-9

1x10-2

20

0
...
66x10-8

2
...
5

6
...
33x10-7

Table 6
...


Calibration

Total

Volume of eosin stock

Molar

Standard (mg

Volume

solution (1 mg L-1) required

Concentration

L-1)

(mL)

(mL)

(mol L-1)

1x10-3

20

0
...
45x10-9

5x10-3

20

0
...
23x10-9

1x10-2

20

0
...
45x10-8

2
...
5

3
...
28x10-8

Table 7
...


Compound

Molar Concentration

Intensity (a
...
66 x 10-9

15
...
33 x 10-9

15
...
66 x 10-8

29
...
64 x 10-8

195
...
33 x 10-7

202
...
45 x 10-9

10
...
23 x 10-9

16
...
45 x 10-8

36
...
61 x 10-8

52
...
28 x 10-8

160
...
Intensity readings of Fluorescence for Fluorescein and Eosin using the Van
Cary Eclipse Fluoremeter with excitation at 501
...
89nm
...
705
R² = 0
...
u)

200

150

100

50

0
0
...
00E-08

4
...
00E-08

8
...
00E-07

1
...
40E-07

Molar Concentration (mol/L)

Figure 2
...
A
strong positive, linear correlation is shown whereby as concentration of Fluroescein
increases, so does the intensity
...
8187 which means that the data is
reliable
...
u)

Intensity of Eosin
180
160
140
120
100
80
60
40
20
0
0
...
2247
R² = 0
...
00E-08

2
...
00E-08

4
...
00E-08

6
...
00E-08

8
...
Intensity of fluorescence versus concentration in mol/L for Eosin
...
The R2 value is 0
...


Figure 4 (left)
...
28 x 10-8 mol/L
...
Then a
vertical line was drawn
up the peak and a
horizontal line was
drawn across the y-axis
from the top of the
peak
...
The
peak height was then
multiplied by this result
to get the AU for the
peak
...
66 x 10-9

0

Fluorescein

1
...
66 x 10-8

0
...
64 x 10-8

0
...
33 x 10-7

0
...
45 x 10-9

0
...
23 x 10-9

0
...
45 x 10-8

0
...
61 x 10-8

0
...
28 x 10-8

0
...
Absorbance readings of Fluorescence for Fluorescein and Eosin using the
Shimadzu UV-1650P at 500nm for Fluorescein and 525nm for Eosin
...
003
y = 14763x + 0
...
5688

Absorbance

0
...
002
0
...
001
0
...
00E+00

2
...
00E-08

6
...
00E-08

1
...
20E-07

1
...
Absorbance versus concentration in mol/L for both Fluorescein
...
The R2 value is 0
...


Absorbance of Eosin
0
...
9748

0
...
006
0
...
004
0
...
002
0
...
Absorbance versus concentration in mol/L for Eosin
...
The R2 value is 0
...


A = ECL
A = absorbance

0
...
66 x 10-8

in solution (mol L-1)
Therefore E = A/(CxL) = 0
...
66 x 10-8 x 1) = 75187
...
Molar Absorptivity calculation example of fluorescein using Beer-Lambert
Law
...
(Dr
Tim Bates, 2014)

Mean Molar Absorptivity for Fluorescein

Mean Molar Absorptivity for Eosin

23617
...
76 (2dp)

Table 11
...


Sensitivity
Fluorescein

Gradient of

2 x 109/14763

x135473
...
92 (2dp)

(Intensity/Absorbance)
Eosin

Gradient of
(Intensity/Absorbance)

Table 12
...
Fluorometry
is 135473 times more sensitive than photometry for Fluorescein, while Fluorometry is
20549 times more sensitive than photometry for Eosin
...
92/20549
...
59 (2dp)
This suggests that the sensitivity ratio is 6
...


Discussion
Based on the analyses performed for the AAS, the data shown in the calibration
curve is not only reliable as shown by the R2 values (Figure 1), but it is also very
accurate (96
...
6%)
...
26mg/L
(closest to mean value) concentration of Lead in the plasma as portrayed in Table 5,
which is significantly higher than the life threatening blood lead level of 1mg/L
(Goddard, 2014)
...
This would have been caused by the several activities
that the patient carried out including painting, removing waste from industrial sites
and drinking
...


Lead-based painting has been a hazard for several years including in the US
(Jacobs, 2002), while industrial sites are commonly known to have several lead
machinery (Greenberg et al
...
, 2013)
...
This
could possibly relate to this case since drinking alcohol would lead to dehydration
and thus the patient could have drunk a lot of water
...


On the other hand, the analyses performed for the Fluorescence and UV/VIS
spectroscopy conveyed a positive correlation between absorbance, intensity and
concentration for both Fluorescein and Eosin (Figure 2, 3, 5 and 6)
...

This describes the extent to which lead absorbs light at 500nm and 525nm using
Fluorescein and Eosin respectively
...


Both the data for Fluorescence and UV/VIS were collected to identify which of the
two procedures was most sensitive
...
The
patient being a ‘social’ drinker could be an understatement as it is possible that he
was binge drinking
...


Conclusion
 The concentration of lead in the plasma sample of the patient using atomic
absorption spectroscopy was investigated to be 27
...

 Eosin has a greater mean molar absorptivity of 159873 as compared to 23617
of Fluorescein
...
Hence, fluoremetry is clearly more sensitive and
should be used to test the liver biopsy of the patient
...
(1978)
...
[Norwalk, Conn
...

David E Jacobs, W
...
The prevalence of lead-based paint hazards in U
...

housing
...
A599
...
ncbi
...
nih
...

2014]
...
(2014)
...
Environmental Science &
Technology, 48(1), pp
...

Erxleben, A
...
Atomic Absorption Spectroscopy
...

Gell, C
...
and Smith, A
...
Handbook of single molecule
fluorescence spectroscopy
...

Goddard, A
...
Lectures
...
, Bozzelli, J
...
, Forstner, E
...
, Stout, D
...
(1981)
...
Environmental Science &
Technology, 15(5), pp
...

Perkampus, H
...
UV-VIS spectroscopy and its applications
...

Rehm, J
...
(2013)
...
The
American Journal of Medicine, 126(12), p
...

Richards, S
...
Calculating final, or working, concentrations for journal‐style
scientific papers
...
Bates College, Department of Biology
...
(2001)
...
Hyderabad, India: Universities Press
...
and Slavin, W
...
Atomic absorption spectroscopy
...

Www2
...
ac
...
University of Warwick Business Facilities - Analytical
Equipment - Fluorescence Spectroscopy
...
warwick
...
uk/services/rss/business/analyticalguide/fluorescence/
[Accessed 10 Dec
...

Word Count - 1473



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