CHEM 315 Study Guide - Quiz Guide: Trichloroacetic Acid, Monochrome, Cuvette

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FUNDAMENTALS OF SPECTROPHOTOMETRY
Spectrophotometry
Any techniques that uses light to measure concentrations (ex: colorimetry)
Properties of light
Perpendicular and oscillating electric and magnetic fields
Plane polarized light
X-rays (10-11 - 10-8 m): breaks molecules
UV-VIS (10-8 , 400-800nm): electron excitation
IR(10-6 – 10-3m): vibration of molecule
Microwayve (10-3 – 10-1m): rotation of molecules
E = h = hc/ = hc
Absorption of light
Light absorbed by a chromophore in a sample, I (irradiance: intensity of light) is reduced.
Monochromator ( selects certain wavelength)
Light goes through the cell with certain distance
Then the light detector detects the absorbance value; A= log(I0/Itr) =
Highest peak of graph is where the light is strongly absorbed
*any colour that absorb vis-light appears coloured when white light is transmitting through*
Our eyes detect wavelength that are not absorbed
Beer’s law works on dilute (< 0.01M) and monochromatic radiation
Blank compensates for reflection, scattering and absorption by cuvet + solvent
Measuring absorbance
To measure solid sample, IR is usually used
Unknown solid sample is mixed with 1% sample with salt (KBr), apply pressure (~600 bar) and
transform solid into translucent pellet. IR measures absorbance as a function of wavenumber
Absorbance should be measured at max peak (most sensitive)
Curve is flat at max abs -> monochromator drift can be avoided
Most precise/ reproducible when A ~0.3-2 (adjust sample conc. to be in range)
Beer’s law in analysis
To be quantified by spectrophotometry, compound must absorb light, and be distinguishable from
others
Most samples absorb light in UV region (not very useful)
Example: measurement of Fe content in transferrin in blood
1. Fe 3+ is quantitatively reduced to Fe 2+
Add acid to protonate a.a
2. Precipitate proteins (getting rid of matrix)
Trichloroacetic acid centrifugation will leave Fe2+ in acid sol’n
3. Transform Fe 2+ to coloured complex
Supernatant + buffer + ferrozine = purple
4. Measure reagent blank, and use suitable iron standards (ex. Highly pure ferrous
ammonium sulfate)
This contains Cu, the results would be 10% higher, need to mask it
Spectrophotometric titrations
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Document Summary

Any techniques that uses light to measure concentrations (ex: colorimetry) X-rays (10-11 - 10-8 m): breaks molecules. Microwayve (10-3 10-1m): rotation of molecules. Light absorbed by a chromophore in a sample, i (irradiance: intensity of light) is reduced. Light goes through the cell with certain distance. Then the light detector detects the absorbance value; a= log(i0/itr) = Highest peak of graph is where the light is strongly absorbed. *any colour that absorb vis-light appears coloured when white light is transmitting through* Our eyes detect wavelength that are not absorbed. Beer"s law works on dilute (< 0. 01m) and monochromatic radiation. Blank compensates for reflection, scattering and absorption by cuvet + solvent. To measure solid sample, ir is usually used. Unknown solid sample is mixed with 1% sample with salt (kbr), apply pressure (~600 bar) and transform solid into translucent pellet. Ir measures absorbance as a function of wavenumber. Absorbance should be measured at max peak (most sensitive)