Topic 3: SpectrochemicaI methods of AnaIysis

focus: absorption spectrophotometry with visible

radiation (optical spectroscopy).
The amount of light absorbed is proportional
to the concentration of the analyte.
A = f[analyte]: A = m[analyte] + b
Radiation and spectroscopy
#adiation WaveIengths: 2 m transitions
Gamma
rays
5 x 10
-13
- 1.4 x 10
-10
NucIear
X-rays 1 x 10
-9
- 1 x 10
-8
Core eIectrons
UV- visibIe 1.8 x 10
-7
- 7.8 x 10
-7
Bonding eIectrons
infrared 7.8 x 10
-7
- 3 x 10
-4
MoIecuIar
vibrations
microwave 7.5 x 10
-4
- 3.75 x 10
-3
MoIecuIar
rotations
&': 180 350
'is: 350 800
nanometers nm
1nm = 1 x 10
-9
m
Photons or
wave packets
with E = hc2
E
2
SWH: 7
th
ed. Chapter 22 (501 -504;
510-519), Chapter 24 (562 572).
SWHC: 8
th
ed. Chpt 24 (710-728), 25
(771-774), 26 (784-790)
'ogel: 17 (645-651: 671-678)
Topic 3: SpectrochemicaI methods of AnaIysis focus: absorption
spectrophotometry with visible radiation (optical spectroscopy) continued
3.1 nstruments
Wavelength
selector
sample
Detector
& readout
Source
E
P
0(2)
P
(2)
W 350 -2200 nm
D
2
160 380 nm
Cells
Quartz: 180 3500 nm
Si glass: 380 2000 nm
SWH p 564
Topic 3: SpectrochemicaI methods of AnaIysis focus: absorption
spectrophotometry with visible radiation (optical spectroscopy) continued
3.2 Absorption Spectra
Po P
S
a
m
p
l
e
l - the Cell
Path length
T, called the transmittance, s a
measure of the light that passes
through the sample
T = PPo
A, the absorbance, is
defined by
A = Iog
10
(P
o
P)
= -Iog
10
(T)
SWH p 504
Energy is absorbed through electronic
transitions within the bonding orbitals of
the analyte
Eg: Sites of unsaturation in organic
molecules eg: double bonds (-C=C-; -
C=O, -NC=O) 6 6 *, or
d d transitions in metal complexes
or Metal d ligand transitions
2
max
wavelength at which
A is a maximum
KMnO
4
y = 0.094x + 0.005
0.00
0.02
0.04
0.06
0.08
0.10
0.0 0.2 0.4 0.6 0.8 1.0
A.
concentration uM
Phosphate in Marine Water
3.3 The Beer-Lambert Law
A =1 c l
Lambert's Law
A M l
Beer's law
A M c
+
c - concentration
1 the proportionality constant,
is called the extinction
coefficient or molar absorptivity.
1l is the slope of the plot of
A against c.
The magnitude of 1 is
wavelength dependent.
1l
chromophore example 2
max
nm
1
alkene C
6
H
13
CH=CH
2
177 13,000
carbonyl CH
3
(C=O)CH
3
186
280
1000
16
Azo CH
3
N=NCH
3
339 5
aromatic C
6
H
6
204
256
7900
200
? Starch-
5
-
600 25000
Metal-ligand FeSCN
2+
470 4900
&nits of 1
concentration
-1
.length
-1
eg: dm
3
mol
-1
cm
-1
A = m[analyte] + b
Topic 3: SpectrochemicaI methods of AnaIysis focus: absorption
spectrophotometry with visible radiation (optical spectroscopy) continued
3.4 Analyses by Spectrophotometric methods.
a) Qualitative: A =1 c l
Many organic functional groups have well
defined absorption characteristics:
2
max
& 1 (usually in the &')
- prepare a solution of known concentration (c)
- record the spectrum between 180 and 350nm
- identify 2max, calculate 1 & consult texts.
Transition metals often
coloured due to transitions
between d orbitals absorb
in the visible region, the
number of peaks and the
2
max
& 1
identify the metal ion &
geometry - O
h
vs T
d
SWH
p560
Non-coloured analytes
can react to give
coloured compounds
eg PO
4
3-
3.4 Analyses by Spectrophotometric methods continued.
b) Quantitative analyses
i) &se the Beer-Lambert equation A =1 c l
- prepare a solution of analyte and add reagents
- measure the absorbance at known 2
max
and l
- look up 1 and calculate c.
Molar absorptivities are not very precisely know, amongst other
things (see later), and so such calculations are not very precise.
Seldom done this way
ii) &se calibration curves the
common method.
-Prepare a set of standards and add
reagents
-Measure their absorbances at 2
max
-Plot A against c:
A = m[analyte]
standards
+ c
-Measure the analyte absorbance
-Calculate analyte concentration
[analyte]
sample
= (A c)/m
y = 0.00756x - 0.00064
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0 5 10 15 20
A
b
s
o
r
b
a
n
c
e
[P] ug (ppb)
P in marine waters
3.4 Analyses by Spectrophotometric methods continued.
3.4.1 The determination of PO
4
3-
in aqueous samples can be present as
inorganic, organic or particulate: understand the Chemistry
Phosphoric acid:
H
3
PO
4
+ H
2
O H
2
PO
4
-
+ H
3
O
+
pK
a1
= 2.12;
H
2
PO
4
-
+ H
2
O HPO
4
2-
+ H
3
O
+
pK
a2
= 7.21;
HPO
4
2-
+ H
2
O PO
4
3-
+ H
3
O
+
pK
a3
= 12.3.
Main species in
aqueous solutions in
the pH 3 11 range
HPO
4
2-
& H
2
PO
4
-
Colourless species
K
a1
= [H
2
PO
4
2-
][H
3
O
+
]/[H
3
PO
4
] The analysis is based on the reaction of PO
4
3-
with molybdate
H
2
PO
4
-
+ 12HMoO
3
+
+ 14H
2
O P(Mo
3
O
10
)
4
3-
+ 14H
3
O
+
.
if pH 7.2 a deep yellow colour (2
max
= 420nm).
n the presence of potassium antimonyl tartrate
(KSbC
4
H
4
O
7
) and at pH < 1 ascorbic acid will reduce
some of the Mo(') centres to Mo(') to give a mixed
oxidation state cluster of intense blue colour.
(Mo blue): 2
max
= 880nm, 1 = 23,000 dm
3
mol
-1
cm
-1
.
P(') in a
tetrahedral
array of
Mo
3
O
10
2-
units.
Cluster
formation is
pH
dependent
structure
and rate.
3.4.1 The determination of PO
4
3-
in aqueous samples continued
The analytical method: from
"Standard Methods for the Examination of Water and Wastewater. 'alidated but
must be verified within your laboratory understand the method options.
sample
filtrate
Suspended
matter
Total P
Filter (0.45 m)
H
2
SO
4
hydrolysis
+ colourimetry
S
2
O
8
6-
digest
+ colourimetry
colourimetry
Soluble reactive
phosphorus (SRP)
(~ orthophosphate)
Soluble acid-
hydrolysable P
+ SRP
Total
dissolved P
The various sample
treatments allow for
the "speciation of
the P in the sample
colourimetry
&nderstand
and ;erify
the method.
The analytical method: from
"Standard Methods for the Examination of Water and Wastewater.
4500-P E. Ascorbic Acid Method read the book.
3.4.1 The determination of PO
4
3-
in aqueous samples continued
a) Principle: Mo-blue via ascorbic acid reduction.
- 3terfere3ces:
AsO
4
3-
at > 0.1 mgAs/L;
Cr(') at > 1 mgCr/L;
NO
2
-
at > 1 mgN/L
S
2-
> 1 mgS/L
SiO
4
4-
> 10 mgSi/L
c) Detection limit 10 gP/L
0.3 2 mgP/L = 0.5 cm
0.15 1.3 mgP/L = 1.0 cm
0.01 0.25 mgP/L = 5.0 cm
&nits:
mgP/L = mgPO
4
3-
/L
eg:
2mgP/L = (2/31)mM
while
2mgPO
4
3-
/L= (2/95)mM
But mM P = mM PO
4
3-
[PO
4
3-
] in tropical
coastal waters > 0.1 M
considered to be
detrimental to corals:
need = 10.0cm
2 mgP/L = 65 M
10 gP/L = 0.3 M
mgP/L or ppm P
1 mM is 1x10
-3
M
1 M is 1x10
-6
M
1 mg is 1x10
-3
g
1 g is 1x10
-6
g
3.4.1 The determination of PO
4
3-
in aqueous samples continued
d) Reagents:
i) 5N H
2
SO
4
70 mL conc. H
2
SO
4
to 500
mL distilled water (DW).
ii) Potassium antimonyl tartrate 1.3715g
K(SbO)C
4
H
4
O
6
. H
2
O in 500 mL DW.
iii) Ammonium molybdate 20 g
(NH
4
)
6
Mo
7
O
24
.4H
2
O in 500 mL DW.
iv) Ascorbic acid 1.76 g ascorbic acid in
100 mL DW.
v) Combined reagent
50 mL a) + 5 mL b) + 15 mL c) + 30 mL d)
in that order with mixing after each addition.
Full details of preparations
given in Standard Method
reference.
Standards:
$tock 219.5 mg anhydrous
KH
2
PO
4
in 1000 mL DW
1.00 mL = 50.0 g PO
4
3-
- P.
$ta3/ar/ phosphate solutio3
50 mL stock to 1000 mL DW
1.00 mL = 2.5 g P PO
4
3-
- P
&nits:
1.00 mL = 50.0 g PO
4
3-
- P ???.
219.5/(39.1 + 2 + 31 + 64)/L = 1.613 mM
1.613 mM = 1.613 x 31 mgP/L = 50.0 mgP/L
1.613 mM = 1.613 x 95 mgPO
4
3-
/L
= 153 mgPO
4
3-
/L
&nderstand the units
i.e. 50.0 g PO
4
3-
- P
mea3s 50.0 g P present as PO
4
3-
.
Storage conditions, shelf
lives also given.
1 week at 4C
4 hours
y = 0.0076x - 0.0006
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0 2 4 6 8 10 12 14 16 18
A
b
s
o
r
b
a
n
c
e
[P] ug (ppb)
P in marine waters
Calibration curve:
5 standards plus blank over range required
3 possible operating ranges
0.3 2 mgP/L = 0.5 cm
0.15 1.3 mgP/L = 1.0 cm
0.01 0.25 mgP/L = 5.0 cm
sta3/ar/ 1.00 mL = 2.5 pg P (or 2.5 mgP/L
standard - 2.5 mgP/L
40 mL standard to 50 mL 2.0 mgP/L
30 mL standard to 50 mL 1.5 mgP/L
20 mL standard to 50 mL 1.0 mgP/L
10 mL standard to 50 mL 0.5 mgP/L
0 mL standard to 50 mL 0.0 mgP/L
For 2
nd
(3
rd
) range dilute standard by 2 (10)
and then as above.
Prepare standards from stock daily.
3.4.1 The determination of PO
4
3-
in aqueous samples continued
e) Procedure:
Sample add 50 mL sample to a
125 mL erlenmeyer flask and add 1
drop phenolphthalein indicator.
f red add 5N H
2
SO
4
dropwise until
just colourless.
Add 8 mL mixed reagent, wait at
least 10 minutes but no more than
30 minutes,
Measure absorbance at 880nm
using 0 mgP/L standard as
reference solution.
Calibration standards treat as for
sample.
Follow procedure carefully, use clean
glassware, proper pipette and
volumetric flask techniques a good
calibration curve suggests adequate
care. 'erify the method using
solutions of known concentration.
3.4.1 The determination of PO
4
3-
in aqueous samples continued
AppIication of the method to marine waters.
Expected concentrations are about 0.1 0.3 M (~3 - 9 gP/L) and
Need a detection limit of about 0.03 M (~1 gP/L) and
a standards range of detection limit to about 15 gP/L (~ 0.5 M) in order to
span the expected concentrations and have the samples having absorbances
that are close to the middle of the calibration curve..
How should the standards be prepared?
A = a c
y = 0.00756x - 0.00064
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0 5 10 15 20
A
b
s
o
r
b
a
n
c
e
[P] ug (ppb)
P in marine waters
y = 0.00756x - 0.00064
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0 2 4 6 8 10 12 14 16 18
A
b
s
o
r
b
a
n
c
e
[P] ug (ppb)
P in marine waters
The preparation of an appropriate set of standards from a stock solution.
What range of concentrations are needed for the analysis?
for tropical marine waters
- from a detection limit of 1 gP/L (~ 0.03 M) to about 15 gP/L (~ 0.5 M).
- Need five to six calibration standards (including a 0) to be prepared from a stock
3.4.1 The determination of PO
4
3-
in aqueous samples continued
How to prepare the calibration standards.
&se pipettes and volumetric flasks to dilute the stock standard.
Pipettes: 5, 10, 15, 20, 25, 50 mL are readily available
'olumetric flasks: 50, 100, 250, 500 mL are readily available
* Note that 25 mL from a pipette diluted into a 100
mL volumetric flask is a fourfold dilution. (or?)
Therefore if we had a standard stock solution of concentration four times
that of our required maximum standard four times we could easily prepare
a set of standards for our calibration by diluting 25, 20, 15, 10 and 5 mL
from pipettes to 100 mL volumetric flasks. (or?)
Therefore prepare a stock that is four times the concentration of
our required maximum standard or 15x4 = 60 gP/L.
$o: f we weigh 264 mg KH
2
PO
4
and dissolve that in 1000 cm
3
the stock
standard solution will be 1000 times more concentrated than required.
(132 mg KH
2
PO
4
in 500 cm
3
would save on chemicals and solvent )
Therefore prepare that stock standard solution and the do 1 in 1000 dilution to
get the intermediate stock standard solution
Note: Small pipettes can lead to large errors so do the 1000 fold in 2 steps
eg: a 100 (5 cm
3
to 500cm
3
) and then a 10 (10 cm
3
to 100 cm
3
) fold dilution.
Prepare the calibration standards from this intermediate stock standard solution
(25. 20. 15, 10, 5 mL to 100 mL; or?)
But: need to weigh at least 100 mg (0.1g) KH
2
PO
4
to minimize
weighing errors and therefore 0.264 mg is too small.
What mass of the KH
2
PO
4
primary standard would be required to prepare the
stock standard solution? Note 1 P per KH
2
PO
4
.
60 gP/L = 60/31 x 136.1 g{KH
2
PO
4
}/L = 264 g{KH
2
PO
4
}/L
= 0.264 mg{KH
2
PO
4
}/L = 0.000264 g{KH
2
PO
4
}/L
The preparation of an appropriate set of standards from a stock solution cont.
( Preparation of standards:
Weigh accurately about 0.13 g
KH
2
PO
4
and dissolve in 500 mL DW.
~ 60 mgP/L. $tock A
5 mL to 500 mL ~ 600 gP/L. Stock B
25 mL to 250 mL ~ 60 gP/L. Stock C
[stock]
pipette
'ol.
flask
[standard]
mgP/L
[actual]
mgP/L
60 25 100 15 16.34
60 20 100 12 13.07
60 15 100 9 9.81
60 10 100 6 6.54
60 5 100 3 3.27
Distilled water 0
f actual weight = 0.1435g KH
2
PO
4
to 500 cm
3
then [stock A] = 65.37 mgP/L
and [stock B] = 653.7 gP/L
and [stock C] = 65.37 gP/L
3.4.1 The determination of PO
4
3-
in aqueous samples continued
The preparation of an appropriate set of standards from a stock solution cont.
Need 5 standards plus blank 0 standard
y = 0.00756x - 0.00064
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0 5 10 15 20
A
b
s
o
r
b
a
n
c
e
[P] ug (ppb)
P in marine waters
y = 0.00756x - 0.00064
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0 5 10 15 20
A
b
s
o
r
b
a
n
c
e
[P] ug (ppb)
P in marine waters
3.4.1 The determination of PO
4
3-
in aqueous samples continued
Develop the colour,
wait 10 15 minutes,
measure absorbance at
880 nm 10 cm cell)
~[std] [std] Abs.
15 16.34 0.126
12 13.07 0.096
9 9.81 0.070
6 6.54 0.051
3 3.27 0.024
0 0 0.001
Plot the actual standard concentrations
Points don't fall exactly on the best fit line experimental uncertainties
Slope 1 = 0.0076 (gP/L)
-1
(10 cm)
- - -1 1 1
= 0.00076 (gP/L)
-1
(cm)
- - -1 1 1
= 0.00076x31 (mol/L)
-1
cm
-1
. = 0.02356 (mol/L)
-1
cm
-1
.
or 1 = 23,560 (mol/L)
-1
cm
-1
.
[PO
4
3-
]
sample
= (A
sample
(-6.4x10
-4
))/0.00756 or read from graph
Applications of &'/'isible (optical) spectroscopy:
1. Clinical, inorganic, organic, biochemical applications.
2. 'ery high sensitivity slope of the calibration curves (1 l) and molar
absorbtivities generally large (1 = 23,300 (mol/L)-1 cm
-1
for the P method):
sensitivity relates to the ability to distinguish between two concentrations.
3. Low detection limits: often M (cf titrations ~ mM).
4. Reasonable selectivity few interferences in most methods.
5. Good precision 5% of calculated concentrations.
6. Convenient rapidly performed, minimal costs
single beam, visible ~ &S$1000 through double beam, &'/vis, &S$5000.