Spectrophotometry:

An Analytical Tool

PGCC CHM 103 Sinex/Gage

Io I

Cell withPathlength, b,

containing solution

lightsource detector

blank where Io = I

concentration 2concentration 1

b

with sample I < Io

The process of light being absorbed by a solution

As concentration increases, less light is transmitted (more light absorbed).

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Some terminologyI – intensity where Io is initial intensity of

light entering a solution and I is the intensity of light exiting a solution

T – transmission (no units, ratio)T = I/ Io %T = 100 x T

(absorption: Abs = 1 – T or %Abs = 100 - %T)

A – absorbance (no units)A = - log T = -log I/ Io

Remembering the “More Lights, Color, Absorption” lab activity, what factors affect the amount of light that is absorbed by a solution in a spectrometer?

PGCC CHM 103 Sinex/Gage

PGCC CHM 103 Sinex/Gage

Beer’s Law

A = abcwhere

a = molar absorptivity (actually the symbol ε is the correct symbol for this but a is

easier to remember)

b = pathlengthc = molar concentration

See the Beer’s Law Simulator

Molar absorptivity

• Depends on the electronic structure of the substance being analyzed (analyte)

• Varies with the wavelength of light because a compound absorbs different amounts at different wavelengths

• Units = L mol-1 cm-1

(a = A/bc = 1/(mol/L x cm))

PGCC CHM 103 Sinex/Gage

PGCC CHM 103 Sinex/Gage

Analyze at what wavelength?

Scan visible wavelengths from 400 – 650 nm (detector range) to produce an

absorption spectrum (A vs. )Crystal Violet Absorption Spectrum

0

0.2

0.4

0.6

0.8

1

1.2

1.4

200 250 300 350 400 450 500 550 600 650 700 750wavelength, nm

Abso

rban

ce

max

max - wavelength where maximum absorbance occurs

phototube detector range

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The BLANK

The blank contains all substances except the analyte.

Is used to set the absorbance to zero:Ablank = 0

This removes any absorption of light due to these substances and the cell.

All measured absorbance is due to analyte.

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Light source

Grating

Rotating the gratingchanges the wavelength going through the sample

slits

slits

Sample

filter

Phototube

The components of a Spec-20D

occluder

When blank is the sample Io is determined

otherwise I is measured

Separates white lightinto various colors

detects light &measures intensity

- white light of constant intensity

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What does the absorbed light (electromagnetic radiation)

do to the molecule?

high energy UV – ionizes electrons

low energy UV and visible – promotes electrons to higher energy orbitals(absorption of visible light leads to a colored solution)

IR – causes molecules to vibrate (more later)

700 nm 400 nm

IR UV

visibleEnergy increasing

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UV/visible light absorption

In organic molecules, electronic transitions to higher energy molecular orbitals – double bonds: *

In transition metals, hydrated ions such as Cu2+ have splitting of d orbital energies and electronic transitions – weak absorption

In complexed transition metals, charge transfer of electrons from metal to ligand as Cu(NH3)4

2+ – strong absorption

Valence electrons

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Uses of visible spectrophotometry

Analysis of unknowns using Beer’s Law calibration curve

Absorbance vs. time graphs for kineticsSingle-point calibration for an equilibrium

constant determinationSpectrophotometric titrations – a way to

follow a reaction if at least one substance is colored – sudden or sharp change in absorbance at equivalence point, a piece-wise function

(Been there, done that!)

Standard Curves

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Concentration (mol/L or M)0.01 0.02 0.05 0.06 0.070.03 0.04

Absorbance

regression equation

If you know the absorbance of an unknown youcan determine the concentration.

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Kinetics of Crystal Violet Reaction

CV+ + OH- CV-OHpurple colorless colorless

Follow concentration of crystal violet over time as it reacts by measuring its absorbance.How will absorbance change with time?

For a absorbance vs. time plot, how will you determine the rate of the reaction?

Chime structures

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ab

sorb

an

ce

time

Since the absorbance is related to concentration, rate or A/time is the slope of a regression line.

CV+ + OH- CV-OHpurple colorless colorless

Short run times to get initial rates.

STELLA model

This is tracking reaction progress over time.

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Single-point calibration• Standard with measured absorbance

Astd = abcstd

• Unknown with measured absorbanceAunk = abcunk

Ratio the two equationsAunk/ Astd = abcunk /abcstd

Aunk/ Astd = cunk /cstd

• Solve for cunk

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Equilibrium Constant Determination

Fe+3 + SCN- Fe(SCN)++

colorless colorless orange

K = (Fe(SCN)++)/(Fe+3)(SCN-)

Using the reactants, shift reaction based on Le Chatelier’s principle.

Fe(SCN)++ + SCN- = Fe(SCN)2+

We start with a high concentration of Fe+3 and lower its value by dilution.

Interactive Excel spreadsheet

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When calibration curves go bad!

• The linear Beer’s Law relationship starts to show curvature at high concentrations

• Single-point calibration assumes a linear calibration curve

Calibration Curve

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0.2

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0.8

1

0 0.2 0.4 0.6 0.8 1concentration

Abso

rban

ce

linear

curved

Linear (linear)

Non-linear

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Spectrophotometric titration• Let’s consider the analysis of

hydrogen peroxide with potassium permanganate in an acidic solution.

• The potassium permanganate or MnO4

- is the only colored substance in the reaction. (It can serve as its own indicator.)

• How would the absorbance change as titrant was added?

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abso

rban

ce

Volume of titrant (mL KMnO4)

5H2O2 + 2MnO4- + 6H+ 5O2 (g) + 2Mn+2 + 8H2O

purple

Equivalence point

MnO4- reacting,

color disappears xs MnO4-

accumulates

Notice you do not need to have adata point at the equivalence point. Equivalence point located by extrapolation of the two lines.