Fundamentals of Spectrophotometry

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Ch. 18 Fundamentals of SpectrophotometryDefinitions

•

Spectrophotometry

(spectroscopy) is anyanalytical method that uses light formeasurement of chemical concentrations

Absorption spectrum of O

3

Real-time monitoring of gases in the human airwaywith spectroscopy

Properties of Light

•Light can be thought of as a wave or particle.

–The

wavelength

,

λ

, is the distance between crestsof a wave (m)–The

frequency

,

υ

, is the number of oscillations persecond (Hz)

m/s10998.2

8

×==×

cc

λ υ

sJ10626.6

34

⋅×==

−

υ υ

h E

The EM Spectrum

r)(wavenumbe / 1 ~ ~

λ υ υ

==

hc E

Absorption Process

A molecule that absorbs light photons will end up with increasedenergy. Themolecule will be promoted to an

excited

state. Microwave energy will causerotation of compounds. IR energy is high enough to promote bondstretching.UV/Visenergy promotes electrons into higher orbitals. Short-

λ

UV and X-rayscan ionize molecules or even break bonds.

Energy Example

•How much is the energy of CO

2

increased permole when it absorbs IR at 4700 cm

-1

?

kJ/mol56)10J)(6.022109.33( J109.33cm/m)100)(cmm/s)(470010s)(2.998J10(6.626 ~

2320-20-1-834-

=××= ×=×⋅×= =

υ

hc E

2

Spectrometer Experiment

A light source can be a lamp, laser, or even a light bulb. A

monochromator

(“one color”) is used to select a particular wavelength. This is typically a grating,but can be a prism or filter. The light then passes through thesample, containingthe analyte. Afterwards, the light is detected.

Irradiance inIrradiance outPathlength

Irradiance

,

P

, is measured in W·m

-2

Transmittance

,

T

, is the fraction of original light not absorbed by thesample, or

0

/

PPT

=

Absorbance and Beer’s Law

•

Absorbance

, A, the amount light absorbed bythe sample is related to transmittance:•Beer’s law relates the absorbance of a chemicalto its concentration:

b

is the pathlength, typically in cm, and

c

is theconcentration of the chemical species in M

ε

is the molar absorptivity, the unit that tells howmuch light is absorbed for a given wavelength.

ε

has units of M

-1

cm

-1

T PP A

loglog

0

−=

   

=

bc A

λ λ

ε

=

Beer’s Law Example

•Find the absorbance of a 0.0220 M solutionwith a molar absorptivityof 15.5 M

-1

cm

-1

in a2.00 cm pathlengthcell•If you wanted to find transmittance:•21% of the irradiated light emerges from theanalytesample

68.0M)cm)(0.022000.2)(cmM5.15(

-1-1

===

bc A

λ λ

ε

21.010log

68.0

==−=

−

AT

Wavelengths and ColorCells for SpectrophotometryErrors in Spectroscopy Measurements

There can be many sourcesof error with a spectroscopicmethod. Electronic noise inthe detector is the mostcommon. Mistakes with thecuvetcan also be important--improper positioning,touching with fingers orallowing dust on the cell allcould introduce error.

3

Beer’s Law and Calibration

Iron can be analyzed spectroscopicallyas an iron-ferrozinecomplex. However, some metals, especiallycopper can interfere with the complexationreaction.If copper is masked, then the analysis can be done.Theoretically, the y-intercept should be at 0, but asyou’ve probably seen in lab, error can cause anabsorbance reading at 0 concentration.

The Effects of Absorbing Light

UV-Vispromoteselectrons into higherorbitalsIR promotes bendingand stretching of bonds

Emission of Light

Fluorescence occurs at lower wavelengths (higher energy) thanphosphorescence. Spectroscopy using emission of light is considerably

moresensitive

than absorption of light due to inherently lower background.

Absorption vs. Emission Spectra

Molecules always emitradiation at longerwavelengths than they absorbat.The absorbance and emissionspectra usually resemble amirror image when viewedtogether.

Emission SpectroscopyExperimental Setup

Pretty similar to the absorbance measurements,except that you add in an extra monochromatorafter the sample and before the detector.We typically hold the excitation (absorption)wavelength constant and measure the emissionspectrum resulting from it.

Chemiluminescence

•When a chemical reaction makes a productthat emits light, it is

chemiluminescence

•This is the type of chemistry that takesplace when a firefly gives off light or whenyou have a glow stick •Some biochemical reactions alsochemiluminesce

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