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What is Spectroscopy?

The study of molecular structure and


dynamics through the absorption,
emission and scattering of light.

What is Light?
According to
Maxwell, light is an
electromagnetic field
characterized by a
frequency f, velocity v
( c in vacuum), and
wavelength . Light
obeys the
relationship
= c / .
Its energy is E= h

The Electromagnetic Spectrum

E = h

=c/l

X-ray:
core electron
excitation

UV:
valence
electronic
excitation

IR:
molecular
vibrations

Radio waves:
Nuclear spin states
(in a magnetic field)

Spectral Distribution of Radiant Energy


Wave Number (cycles/cm)

X-Ray

UV
200nm

Visible
400nm

IR
800nm

WAVELENGTH(nm)

Microwave

Spectroscopic Techniques
UV-vis

UV-vis region

bonding electrons

Atomic Absorption

UV-vis region

atomic transitions (val. e-)

FT-IR

IR/Microwave

vibrations, rotations

Raman

IR/UV

vibrations

FT-NMR

Radio waves

nuclear spin states

X-Ray Spectroscopy

X-rays

inner electrons, elemental

X-ray Crystallography

X-rays

3-D structure

10-5

10-5
UV = 300 kcal/mol

Molecules have quantized energy levels:


ex. electronic energy levels.

energy

energy

hv

}
= hv

Q: Where do these quantized energy levels come from?


A: The electronic configurations associated with bonding.
Each electronic energy level
(configuration) has
associated with it the many
vibrational energy levels we
examined with IR.

s*
p*
2p

2p

s
s*
2s

2s
s

Ethane

C C

s*

s*
hv

s
s
H

C C

H
H

HH
H

s*

lmax = 135 nm (a high energy transition)

Absorptions having lmax < 200 nm are difficult to observe because


everything (including quartz glass and air) absorbs in this spectral
region.

C C

s*
p*

s*
p*

= hv
=hc/l

hv

p
s

p*

Example: ethylene absorbs at longer wavelengths:


lmax = 165 nm = 10,000

C O

s*
p*

s*
p*

hv

p
s

s
n

p*

The n to pi* transition is at even lower wavelengths but is not


as strong as pi to pi* transitions. It is said to be forbidden.
Example:
Acetone:
ns* lmax = 188 nm ; = 1860
np* lmax = 279 nm ; = 15

C C

s s*

135 nm

C C

p p*

165 nm

n s*

183 nm

weak

p p*
n s*
n p*

150 nm
188 nm
279 nm

weak

H
C O

C O

180 nm

C O

A
279 nm

Conjugated systems:
C

LUMO

HOMO

Preferred transition is between Highest Occupied Molecular Orbital


(HOMO) and Lowest Unoccupied Molecular Orbital (LUMO).

Note: Additional conjugation (double bonds) lowers the HOMOLUMO energy gap:
Example:
1,3 butadiene:
lmax = 217 nm ; = 21,000
1,3,5-hexatriene
lmax = 258 nm ; = 35,000

Lycopene:

lmax = 114 + 5(8) + 11*(48.0-1.7*11) = 476 nm


lmax(Actual) = 474.

Electronic (UV) spectroscopy


Light absorbed electron excited to higher
molecular orbital
Transitions occur from HOMO to LUMO
- Highest Occupied Molecular Orbital
- Lowest Unoccupied Molecular Orbital

E=h

What functional groups are observed


in UV/vis spectrum?
Excitation
LUMO s*

UV/vis (200-600 nm) C-C


Functional groups
HOMO s
Ketone
LUMO p*
Ester
C=C
Amide
Conjugated D.B
HOMO p
Not observed
C-C
p4*
LUMO *
p3
C-H
C=C-C=C
C=C (isolated)
p
HOMO

p1

l =125nm

l =165nm

l =217 nm

Chromophores
Chromophores
- Part of molecule responsible for
absorption
Auxochromes
- Groups that modify absorption of
neighboring chromophores
- Often have lone pairs, e.g. OH, -OR,
-NR2, -halogen
- Bathochromic shift: towards longer
wavelength
- Hypsochromic shift: towards shorter
wavelength

The wavelength and amount of light that a compound


absorbs depends on its molecular structure and the
concentration of the compound used.

Transmittance and
Concentration
The Bouguer-Lambert Law

T I / I 0 eConstPathlength

Transmittance and Path Length:


Beers Law

Concentration

T I / I 0 eConstConcentration

The Beer-BouguerLambert Law

A log T logI / I 0 logI 0 / I b c

BEER LAMBERT LAW

Light

I0

Glass cell filled with


concentration of solution (C)

As the cell thickness increases, the intensity of I


(transmitted intensity of light ) decreases.

Beers Law
source

slit

cuvette
detector

A = -logT = log(P0/P) = bc
T = Psolution/Psolvent = P/P0
Works for monochromatic light
Compound x has a unique at different
wavelengths

T- Transmittance
T=

I
I0

I0 - original light intensity


I- transmitted light intensity

% Transmittance = 100 x I
I0
1
Absorbance (A) or optical density (OD) = Log
T
= Log

I0
I

I
Log
is proportional to C (concentration of solution)
and is I0
also proportional to L (length of light path
through the solution).

A CL = KCL by definition and it is called


the Beer Lambert Law.

A = KCL
K = Specific Extinction Coefficient ---- 1 g of
solute per liter of solution

A = ECL
E = Molar Extinction Coefficient ---Extinction Coefficient of a solution containing
1g molecule of solute per 1 liter of solution

E =

Absorbance x Liter
Moles x cm

E differs from K (Specific extinction Coefficient) by


a factor of molecular weight.
UNITS

A = ECL
A = No unit (numerical number only)
Liter
E =
Cm x Mole

L = Cm
C = Moles/Liter
A = ECL = (

Liter
Cm x Mole

)x

Mole
Liter

x Cm

A = KCL

A = No unit

C = Gram/Liter

L = Cm

Liter
K=

Cm Gram

A = KLC = (

Liter
Cm x Gram

)x

Gram
Liter

x Cm

UV-VIS SPECTROMETERS ARE UBIQUITOUS


BEGIN
HERE

P8

Characteristics of UV-Vis spectra of


Organic Molecules
Absorb mostly in UV unless highly
conjugated
Spectra are broad, usually to broad for
qualitative identification purposes
Excellent for quantitative Beers Law-type
analyses
The most common detector for an HPLC

Broad spectra
Overlapping vibrational and rotational
peaks
Solvent effects

P354

Isoprene
A = 0.8
Conc: 4x10-5 M
Path: 1 cm

= 0.8/4x10-5x1
= 2x104
= 20000

P352

P356
(see also 359)

Common UV-vis instuments


Scanning Instrument
monochromator

Tungsten
Filament (vis)

slit

slit

Deuterium lamp
Filament (UV)

Photomultiplier
tube
cuvette

Conventional
Spectrophotometer

Schematic of a conventional single-beam spectrophotometer

Light Sources
Hydrogen Gas Lamp
Mercury Lamp
Tungten lamp (350-2500 nm)

Deuterium (200-400 nm)


Xenon Arc lamps (200-1000 nm)

Dispersion Devices
Non-linear dispersion
Temperature sensitive

Linear Dispersion
Different orders

Dispersion of
polychromatic light with a
prism
Infrared

Polychromatic
Ray

PRISM

Red
Orange
Yellow
Green

monochromatic
Ray

SLIT

Blue
Violet

Ultraviolet

Polychromatic Ray

Monochromatic Ray

Prism - spray out the spectrum and choose the certain


wavelength (l) that you want by moving the slit.

Cells
UV Spectrophotometer
Quartz (crystalline silica)
Visible Spectrophotometer
Glass
IR Spectrophotometer

NaCl

Cell Types I

Open-topped rectangular standard cell (a)


and apertured cell (b) for limited sample volume

Conventional
Spectrophotometer

Optical system of a double-beam spectrophotometer

Conventional
Spectrophotometer

Optical system of a split-beam spectrophotometer

Diode array Instrument


mirror

Diode array detector


328 individual detectors

Tungsten
Filament (vis)
slit
slit

cuvette
Deuterium lamp
Filament (UV)

monochromator

Advantages/disadvantages
Scanning instrument
High spectral resolution (63000), l/l
Long data acquisition time (several
minutes)
Low throughput

Diode array
Fast acquisition time (a couple of
seconds), compatible with on-line
separations
High throughput (no slits)
Low resolution (2 nm)

STEPS IN DEVELOPING A
SPECTROPHOTOMETRIC
ANALYTICAL METHOD
1. Run the sample spectrum
2. Obtain a monochromatic
wavelength for the
maximum absorption
wavelength.
3. Calculate the concentration
of your sample using Beer
Lambert Equation: A = KCL

Absorbance

2.0

0.0
200

250

300

350

Wavelength (nm)

400

450

Slope of Standard Curve =

A
C

Absorbance at 280 nm
1.0

0.5

4
2
3
Concentration (mg/ml)

There is some A vs. C where graph is linear.

NEVER extrapolate beyond point known where


becomes non-linear.

SPECTROMETRIC ANALYSIS USING


STANDARD CURVE
Absorbance at 540 nm
1.2

0.8

0.4

3
1
2
Concentration (g/l) glucos e

Avoid very high or low absorbencies when drawing a


standard curve. The best results are obtained with 0.1 < A
< 1. Plot the Absorbance vs. Concentration to get a
straight line

Every instrument has a useful range for a


particular analyte.
Often, you must determine that range
experimentally.
This is done by making a dilution series of
the known solution.
These dilutions are used to make a
working curve.

Make a dilution series of a known quantity


of analyte and measure the Absorbance.
Plot concentrations v. Absorbance.

What concentration do you think the


unknown sample is?

In this graph, values above A=1.0 are not linear. If we


use readings above A=1.0, graph isnt accurate.

Calibration Methods
Standard Addition
1.)

Protocol to Determine the Quantity of an Unknown


(i)

Known quantities of an analyte are added to the unknown


- known and unknown are the same analyte
- increase in analytical signal is related to the total quantity of the analyte
- requires a linear response to analyte

(ii)

Very useful for complex mixtures


- compensates for matrix effect change in analytical signal caused by
anything else than the analyte of interest.

(iii) Procedure:
(a) place known volume of unknown sample in multiple flasks

Calibration Methods
Standard Addition
1.)

Protocol to Determine the Quantity of an Unknown


(iii) Procedure:
(b) add different (increasing) volume of known standard to each unknown
sample

(c) fill each flask to a constant, known volume

Calibration Methods
Standard Addition
1.)

Protocol to Determine the Quantity of an Unknown


(iii) Procedure:
(d) Measure an analytical response for each sample

- signal is directly proportional to analyte concentration


Concentrat ion of analyte in initial solution
signal from initial solution

Concentrat ion of analyte plus s tan dard in final solution signal from final solution

Standard addition equation:

X i
S f X f

IX
IS X

Total volume (V):

V Vo VS , Vo unknown initial volume , VS added volume of s tan dard

X f X i Vo
V

S f S i VS
V

Calibration Methods
Standard Addition
1.)

Protocol to Determine the Quantity of an Unknown


(iii) Procedure:
(f) Plot signals as a function of the added known analyte concentration and
determine the best-fit line.

X-intercept (y=0) yields X f


which is used to calculate X i from:

Vo

X i X f V

VIS-Transmission and Color

The human eye sees the complementary color to that which is


absorbed

Absorbance and
Complementary Colors