Spectrophotometry: An Analytical Tool. PGCC CHM 103 Sinex/Gage IoIo I Cell with Pathlength, b, containing solution light source detector blank where I.

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).


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?



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))



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


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.


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


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


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


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


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.


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


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.


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


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


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

0

0.2

0.4

0.6

0.8

1

0 0.2 0.4 0.6 0.8 1concentration

Abso

rban

ce

linear

curved

Linear (linear)

Non-linear


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?


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.

Spectrophotometry: An Analytical Tool. PGCC CHM 103 Sinex/Gage IoIo I Cell with Pathlength, b, containing solution light source detector blank where I.

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