CHEM 213
Instrumental Analysis
Lab Lecture – Fe and
Extraction
Determination of Trace Iron (LM, p14-22)
Chem 102 – used Crystal field theory to explain colour of
coordination complexes
Chem 213 – use intensity of colour to determine concentration of
metal ion
+ Fe2+
3
Bipyridyl
(bipy)
Fe(bipy)32+
red
Spectrophotometry – any method using light to measure chemical
composition
2
Fe: Concentration to Colour
Intensity of colour = Absorbance (A)
Measure absorbance of soln to determine the iron
concentration.
Transmitted
P/P0
1
0.1
0.01
%T
100
10
1
A
0
1
2
Transmittance (T) = P/Po
Absorbance A = -log(T) = abc
a = absorptivity
b = pathlength
3
c = concentration
Spectrophotometer
Readout
Readout
Genesys
Spec 20D+
20
pg
pg21
21
(Text p410, LM p19-21)
On/Off
On/Off
Sample comp.
Zero control
4
Spectrophotometer
(Text p410, LM p19-21)
Record spectrometer number
Sample cell matching LM p 19 (1 hour)
- Need 8 test tubes with the same pathlength (b)
- prepare dilute amaranth dye soln
-Set wavelength to 510 nm
-Mark tube with line
-Insert test tube in sample
compartment
-Adjust trans/absorb control
to 90%
- Insert 2nd tube in spec
- Rotate tube to get T=90%
- Put on alignment line
- Repeat until you have 8
matched tubes
5
Determination of Trace Iron (LM, p14-22)
+ Fe2+
3
Fe(bipy)32+
red
Bipyridyl
(bipy)
• Reduce any Fe(III) to Fe(II) so all iron is in the Fe(II)
state, because only Fe(II) forms coloured complex
1. Fe3+acidic + xs NH2OH  Fe2+
hydroxylamine
reducing agent
2. xs
+ Fe2+  Fe(bipy)32+
3. Adjust pH to ~ 5 by adding sodium acetate
6
Determination of Trace Iron(LM, p14-22)
Measure absorption spectrum of the complex to
determine the wavelength of max. absorbance.
Absorbance spectrum –
graph of absorbance of
light vs. wavelength
A = abc
Measure A for most conc standard from 640 to 400 nm
at 20 nm intervals.
The 0 %T (20D Only) and the 100 %T (A = 0) settings
must be reset for each new wavelength.
7
Determination of Trace Iron (LM, p14-22)
Why is 100 %T (A = 0) reset at each wavelength?
Light intensity (Po)
varies with wavelength)
A is max
Best sensitivity
8
Prepare Calibration Curve
Standards = pipet 5, 10, 15 & 20 mL Fe3+ into flasks
Blank = soln with all of the reagents but no analyte
[Fe3+]=0
Zero (set A =0) instrument using blank
Measure absorbance of all standards and samples
Re-zero
Take second absorbance reading for all stds and samples
9
Prepare Calibration Curve
standards
Asample
Blank
Csample
10
Calibration Curves LM, Appendix A
11
Calibration Curves
Calibration Curve – graph of instrument response vs. conc of
analyte
- when response for sample is measured, its conc is determined by
interpolating from the graph
- blank value should be part of the calibration curve, i.e. signal for
[C] = 0.0
-standards must cover the complete range of expected unknowns
- plot signal on the Y axis and conc on the X axis.
-Normally response is linear
or with math transformation can be made linear
Signal = m(conc) + b
m = slope
b = intercept
12
Drawing a Calibration Curve
Manually on graph paper
13
Manual Graphing of Calibration Curve
Inst Response
0.6
0.5
0.4
0.3
0.2
0.1
0
0
1
2
3
4
5
Concentration, ppm
Useful for spotting:
14
Manual Graphing of Calibration Curve
Inst Response
0.6
0.5
0.4
0.3
0.2
0.1
0
0
1
2
3
4
5
Concentration, ppm
Useful for spotting: outliers
15
Manual Graphing of Calibration Curve
Inst Response
0.6
0.5
0.4
0.3
0.2
0.1
0
0
1
2
3
4
5
Concentration, ppm
Useful for spotting: outliers
curvature
16
Manual Graphing of Calibration Curve
Manually on graph paper
- best for spotting outliers, curvature, etc.
- disgard outliers
Calculate least squares line
Mathematically calculate line using least squares
fits data to y = mx + b ?
determines m and b
determines error in m and b?
allows accurate calculation of sample conc
17
Determination of Dextromethorphan in Cough Syrup
Expectorant
Glyceryl guaiacolate
Antihistamines
Chloropheniramine maleate
Diphenyhydramine HCl
Dextromethorphan
cough suppressant
Preservatives
Flavouring
Colouring
Decongestants
Phenylephrine HCl
Analgesics
Acetaminophen
Solvent
extraction
toluene
water
18
B. Solvent Extraction System
Colour = Absorbance @ 400 nm  [BTB-]org  [DMH+]aq
Calibration curve:
(absorbance-blank) vs. [DMH+]aq
Ion-association Extraction
(DMH+ BTB-)org
yellow
toluene
water
+
Dextromethorphan Bromothymol blue
xs BTBDMH+
(DMH+BTB-)
ion pair
19
First week solution preparation (pg 50)
Do NOT add BTB
Do NOT prep sample
20
b) Mohr pipet – use like a buret
d) Fluted filter paper – allows
liquid to pass through quickly
21