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CHEM 213Instrumental Analysis
Lab Lecture – Fe and Extraction
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Chem 102 – used Crystal field theory to explain colour of coordination complexes
Chem 213 – use intensity of colour to determine concentration of metal ion
3 + Fe2+ Fe(bipy)32+
Bipyridyl(bipy)
red
Determination of Trace Iron (LM, p14-22)
Spectrophotometry – any method using light to measure chemical composition
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P/P0 %T A 1 100 0 0.1 10 10.01 1 2
Fe: Concentration to Colour
Intensity of colour = Absorbance (A)
Measure absorbance of soln to determine the iron concentration.
Transmitted
Transmittance (T) = P/Po
Absorbance A = -log(T) = abc
a = absorptivity
b = pathlength
c = concentration
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Spectrophotometer (Text p410, LM p19-21)
Spec 20D+pg 21
On/Off Zero control
Readout Mode
Sample comp.
Readout On/Off
Genesys 20pg 21
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Spectrophotometer (Text p410, LM p19-21)
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
- Insert test tube in sample compartment
- Adjust trans/absorb control to 90%
- Insert 2nd tube in spec
- Put on alignment line
- Rotate tube to get T=90%
- Repeat until you have 8 matched tubes
Record spectrometer number
- Mark tube with line
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• Reduce any Fe(III) to Fe(II) so all iron is in the Fe(II) state, because only Fe(II) forms coloured complex
Determination of Trace Iron (LM, p14-22)
3 + Fe2+ Fe(bipy)32+
Bipyridyl(bipy)
red
1. Fe3+acidic
2. xs + Fe2+ Fe(bipy)32+
3. Adjust pH to ~ 5 by adding sodium acetate
+ xs NH2OH Fe2+
hydroxylamine reducing agent
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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 = abc
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.
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Why is 100 %T (A = 0) reset at each wavelength?
Determination of Trace Iron (LM, p14-22)
Light intensity (Po) varies with wavelength)
A is maxBest sensitivity
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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
Prepare Calibration Curve
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Prepare Calibration Curve
Blank
standards
Asample
Csample
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Calibration Curves LM, Appendix A
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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
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Drawing a Calibration Curve
Manually on graph paper
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0 1 2 3 4 50
0.1
0.2
0.3
0.4
0.5
0.6
Concentration, ppm
Inst
Res
po
nse
Useful for spotting:
Manual Graphing of Calibration Curve
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0 1 2 3 4 50
0.1
0.2
0.3
0.4
0.5
0.6
Concentration, ppm
Inst
Res
po
nse
Useful for spotting: outliers
Manual Graphing of Calibration Curve
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0 1 2 3 4 50
0.1
0.2
0.3
0.4
0.5
0.6
Concentration, ppm
Inst
Res
po
nse
Manual Graphing of Calibration Curve
Useful for spotting: outliers curvature
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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
Manually on graph paper
- best for spotting outliers, curvature, etc.
- disgard outliers
Manual Graphing of Calibration Curve
Calculate least squares line
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Solvent extraction
Dextromethorphancough suppressant
Expectorant Glyceryl guaiacolate
Antihistamines Chloropheniramine maleate Diphenyhydramine HCl
Decongestants Phenylephrine HCl
Analgesics Acetaminophen
Preservatives
Flavouring
Colouring
Determination of Dextromethorphan in Cough Syrup
toluenewater
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19Dextromethorphan
DMH+
Bromothymol blueBTB-
+ (DMH+BTB-)
ion pair
(DMH+ BTB-)org
yellow
B. Solvent Extraction System
Ion-association Extraction
water
toluene
Colour = Absorbance @ 400 nm [BTB-]org
xs
[DMH+]aq
Calibration curve: (absorbance-blank) vs. [DMH+]aq
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First week solution preparation (pg 50)
Do NOT add BTBDo NOT prep sample
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b) Mohr pipet – use like a buret
d) Fluted filter paper – allows liquid to pass through quickly