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 3 + Fe2+ Fe(bipy)32+ Bipyridyl (bipy) red Spectrophotometry – any method using light to measure chemical composition

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) = abc a = absorptivity b = pathlength c = concentration P/P0 %T A 1 100 0 0.1 10 1 0.01 1 2

Spectrophotometer (Text p410, LM p19-21) Spec 20D+ pg 21 On/Off Zero control Readout Mode Sample comp. Readout On/Off Genesys 20 pg 21

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 Rotate tube to get T=90% Set wavelength to 510 nm Insert 2nd tube in spec Mark tube with line Put on alignment line Insert test tube in sample compartment Adjust trans/absorb control to 90% Repeat until you have 8 matched tubes

Determination of Trace Iron (LM, p14-22) 3 + Fe2+ Fe(bipy)32+ Bipyridyl (bipy) red 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

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.

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

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

Prepare Calibration Curve standards Asample Blank Csample

Calibration Curves LM, Appendix A

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

Drawing a Calibration Curve Manually on graph paper

Manual Graphing of Calibration Curve Useful for spotting:

Manual Graphing of Calibration Curve Useful for spotting: outliers

Manual Graphing of Calibration Curve Useful for spotting: outliers curvature

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

Determination of Dextromethorphan in Cough Syrup Preservatives Flavouring Colouring Expectorant Glyceryl guaiacolate Antihistamines Chloropheniramine maleate Diphenyhydramine HCl Decongestants Phenylephrine HCl Analgesics Acetaminophen Solvent extraction Dextromethorphan cough suppressant toluene water

+ B. Solvent Extraction System Colour = Absorbance @ 400 nm  [BTB-]org  [DMH+]aq Calibration curve: (absorbance-blank) vs. [DMH+]aq (DMH+ BTB-)org yellow water toluene Ion-association Extraction Dextromethorphan DMH+ Bromothymol blue BTB- + (DMH+BTB-) ion pair xs

First week solution preparation (pg 50) Do NOT add BTB Do NOT prep sample

b) Mohr pipet – use like a buret d) Fluted filter paper – allows liquid to pass through quickly