Applied Physics

On
UV-vis Spectrophometry

Kuwat Triyana
http://triyana.staff.ugm.ac.id
EM Spectrum
 Ultra Violet (UV)
• Three regions of UV
– UV-A:
• long-wave UV, near-ultraviolet, black light, or Wood’s light
• between 320 and 400 nm
– UV-B:
• medium-wave UV
• between 280 and 320 nm
– UV-C:
• short-wave, far ultraviolet, or germicidal UV
• between 180 and 280 nm
 Absorption Spectroscopy
Introduction

A.) Absorption: electromagnetic (light) energy is transferred to atoms, ions, or molecules

in the sample. Results in a transition to a higher energy state.

E1 (excited state)
h Energy required of photon
to give this transition:
Eo (ground state)
h = DE = E1 - Eo

- Transition can be change in electronic levels, vibrations, rotations, translation, etc.

- Concentrate on Molecular Spectrum in UV/Vis (electronic transition)

- Power (P): energy of a beam that reaches a given area per second

- Intensity (I): power per unit solid angle

- P and I related to amplitude2

Absorption and Emission
 Molecular Absorption and Emission

Atoms:
Electronic states only

Molecules:
Vibrational states
within electronic states
 B.) Terms:

1.) Beer’s Law: A = ebc

The amount of light absorbed (A) by a sample is dependent on the path
length (b), concentration of the sample (c) and a proportionality constant (e
– molar absorptivity)

Amount of light absorbed is dependent on frequency ()

Increasing Fe+2 concentration 

Absorbance is directly proportional to concentration Fe+2

B.) Terms:

1.) Beer’s Law: A = ebc

Transmittance (T) = I/Io %Transmittance = %T = 100T

Absorbance (A) = log10 Io/I

No light absorbed-
% transmittance is 100%  absorbance is 0

All light absorbed-

% transmittance is 0%  absorbance is infinite
Relationship Described in Terms of Beer’s Law

A = Absorbance = ebc = -log(%T/100)

e = molar absorptivity: constant for a compound at a given frequency ()

or wavelength (l)
units of L mol-1 cm-1
b = path length: cell distance in cm

c = concentration: sample concentration in moles per liter.

Therefore, by measuring absorbance or percent transmittance at a given

frequency can get information related to the amount of sample (c) present
with an identified e and l.

Note: law does not hold at high

concentrations, when A > 1
C.) Components of an Instrument for UV/Vis Absorbance Measurements:

1.) Basic Design:

Hitachi Instruments U-3010

Light Source, l selector, Sample cell holder, Detector (amplifier, recorder)

a) Desired Properties of Components of UV/Vis:
Light Source l Selector
Creates Proper l Narrow Bandpass:
Stable: Selects Desired l
Constant P Large Light Throughput:
Good Precision Increase P
Intense:
Increase P
Easier to See Absorbance

Sample Cell Holder Detector

Fixed Geometry: Stable
Constant b Sensitive to l of Interest
Transmits l of Interest:
Increase P
 Molecular Spectra

Absorption Emission
Intensity

400 500 600 700

l (nm)

Bands vs. Lines (atoms)

 Absorbed vs. Observed
R
O RV

V
Y

YG BV

G B
BG

Color absorbed → Complementary color observed

 Spectroscopy Terms Describing
Absorption (Beer’s Law)
• Consider a beam of light
with an (initial) radiant

Concentration (c)
intensity Io
• The light passes through a I0 hv
I
solution of concentration (c)
• The thickness of the
solution is “b” cm.
• The intensity of the light b
after passage through the
solution (where absorption
occurs) is I
 We Define
• Transmittance (T) = I/I0 (units = %)
• Absorbance (A) (units = none)
– A = log (I0/I)
– A = -log (T) = log (1/T)
– A = abc (or εbc) <--- Beer’s Law
• a = absorptivity (L/g cm)
• b = path length (cm)
• c = concentration (g/L)
• ε = molar absorptivity (L/mol cm)
– Used when concentration is in molar units
Other expression of Lambert’s Law of Absorption
Lambert described how intensity changes with
distance in an absorbing medium.
• The intensity I0 if a beam of light decreases exponentially as it passes though a uniform
absorbing medium with the linear decay constant α.
Restatement: In a uniform absorbing medium, the intensity of a beam of light decreases
by the same proportion for equal path lengths traveled.
• The linear decay constant α is a characteristic of the medium. It has units of reciprocal
length. α is the path length over which the intensity is attenuated to 1/e.

l
α
I = I 0e  l The distance traveled through the medium is
called the path length.
I0 I dI
I ( x) = I 0e  x
d I =  I d x =  I
dx
I(x) I  x
I = I 0e  x
=e
x
I0
Lambert’s Law of Absorption (base 10)
Typically base 10 is used in photometry.

I = I 0e  x
= I 010 kx
k =  ln 10
I  x kx
= e = 10
I0
k is the path length over which the intensity is attenuated to 1/10.

I
= 10  k x
I0
 Lambert’s Law Example
If one slab of absorbing material of thickness l reduces the intensity of a beam of light
to half.
l
α I 1
I0 I = 10 k l =
I0 2
Then two slabs of the same absorbing material will then reduce the
intensity of a beam of light to one quarter.
l l
α α 2
I 1 1
I0 I = 10 k 2l =   =
I0 2 4
And three slabs will reduce the intensity of a beam of light to one eight.
l l l
α α α I
3
1 1
 k 3l
I0 I = 10 =  =
I0 2 8
 Beer’s Law
Beer found that Lambert’s linear decay constant k for a solution
of an absorbing substance is linearly related to its
concentration c by a constant, the absorptivity ε, a
characteristic of the absorbing substance.
Restatement: The linear decay constant k is linear in concentration
c with a constant of proportionality ε.
(August Beer, 1825-1863)
k =ec
Typical units are: k cm−1; c M (moles/liter); ε M−1cm−1

A colored absorber has an absorptivity that is dependent on wavelength of the

light ε(λ).
The absorptivity is the fundamental property of a substance. This is the property
that contains the observable spectroscopic information that can be linked to
quantum mechanics (also see absorption cross section.)
 Photometric Quantities
In photometry we measure the intensity of light and characterize its
change by and object or substance. This change is typically
expresses as percent transmittance or absorbance.

Transmittance (T) I
T= usually given in percent
Frequently when your primary I0
interest is the light beam

Absorbance (A) (AKA optical density, O.D.)

Used almost exclusively when your interest
concerns the properties of the material

I 
A =  log  =  log T by convention, base 10 logs are used
 I0 
 Beer-Lambert Law
Lambert’s and Beer’s Laws are combined to describe the
attenuation of light by a solution. It is easy to see how the two
standard photometric quantities can be written in terms of this law.

e c x
I = I 010

Transmittance Absorbance

I I 
T= A =  log  =  log T
I0  I0 
T = 10 e c x A = e cx
 Cross-Sections and Absorptivity
the connection to single particles and molecules

The absorption of light by particles (and single molecules) is

characterized by an absorption cross section C. In this model the
particle is replaced by a perfectly absorbing sphere with a cross
sectional area C. This cross section is a property of the particle
and is not related to its geometric cross sectional area. The
concentration of particles per unit volume is N.

 = NC
typical units are: C cm2; N cm−3
k = NC ln 10

 3 liter  The cross section can be directly related to the

e = N A C ln 10  10  molar absorptivity. NA is Avagadro’s number.
  units
3 are: C cm2; N cm−3; NA mole−1; ε
cm
M−1cm−1
 Efficiency
The absorption efficiency Q of a particle is the ratio of its absorption
cross section C to its geometric cross section Cgeo.
Absorption efficiency is dimensionless.

C
Q=
C geo
 Extension to Scattering and
Extinction
Attenuation of light by absorption and scattering both obey
Lambert’s Law. Thus we can extend our treatment of absorption to
scattering and extinction. (Recall that extinction is the effect of
absorption + scattering.)
Cext = Cabs  Csca The scattering efficiency can be much larger than unity.
Extinction paradox: Qext = 2 (Qabs = 1; Qsca = 1)
Qext = Qabs  Qsca for an perfectly absorbing particle very large compared
to the wavelength of light.
e ext = e abs  e sca
A = e ext cx = e abs  e sca cx
Note:
•All of these quantities are in general wavelength dependent.
•Our discussion has not included the mechanism (cause) of absorption and scattering.
•There are many different mechanisms that cause of absorption and scattering.
 Instrumentation
• Spectrometer: measures I vs λ.
Simply measures the spectrum of the light (e.g. emission spectroscopy).
• Spectrophotometer: measures I/I0 vs λ.
Measures how the sample changes the spectrum of the light (e.g.
transmission, reflection, scattering, fluorescence).
All spectrophotometers contain a spectrometer.
• -meter: the detector is electronic
• -graph: light intensity recorded on film
• photometer: measures I/I0 without λ selection.
 The Spectrophotometer
Measures absorbance as a function of wavelength
Components: light source, monochromator, sample cell, detector,
optical system.

monochromator sample cell

detector
slit
diffraction grating

light source
OU NanoLab/NSF
NUE/Bumm & Johnson
 Cary 50 UV-Vis Spectrophotometer
Computer controlled monochromator
acquisition
of absorption spectra balance the
forces:
detector

sample

Can you find the

light diffraction grating and the
source slit?

www.varianinc.co
Making a Measurement with the Cary 50
• First, measure the baseline using a blank sample. This is raw I0.
The blank sample is the cuvette with deionized water
(everything but your nanoparticles). This corrects for any
absorption due to the cuvette, water, and variations of the light
intensity of the light source, monochromator, etc.
• Second, measure the zero by inserting the beam block. This
corrects the instrument for the detector background.
• Third, measure your sample. This is the raw I. The Cary 50
automatically calculates the corrected intensities (I and I0) by
subtracting the zero from each of the raw intensities.
• Subsequent measurements do not require re-measuring the
blank and zero, simply repeat step 3.
I rawI  zero
T= =
I 0 rawI 0  zero
A =  log T
 Transmittance
T => transmittance
I
T = -----
Io
b

Io I
 Example

I0 = 10,000 I = 5,000

-b-
P 5000
T = = = 0.5
P0 10000

A = -log T = -log (0.5) = 0.3010

 Beer’s Law
A = abc = ebc
where T => transmittance
%T => percentage transmittance
I => transmitted Iower of radiation
Io => incident power of radiation
A => absorbance
a => absorptivity
b => path length
c => concentration
e => molar absorptivity, extinction coefficient
 Beer’s Law
A = abc = ebc

c
 Beer’s Law A = ebc
Path Length Dependence, b
Readout
Absorbance

0.82

Source

Detector
 Beer’s Law A = ebc
Path Length Dependence, b
Readout
Absorbance

0.62

Source
b

Detector Sample
 Beer’s Law A = ebc
Path Length Dependence, b
Readout
Absorbance

0.42

Source

Detector Samples
 Beer’s Law A = ebc
Path Length Dependence, b
Readout
Absorbance

0.22

Source

Detector Samples
 Beer’s Law A = ebc
Wavelength Dependence, a
Readout
Absorbance

0.80

Source
b

Detector
 Beer’s Law A = ebc
Wavelength Dependence, a
Readout
Absorbance

0.82

Source

Detector
 Beer’s Law A = ebc
Wavelength Dependence, a
Readout
Absorbance

0.30

Source
b

Detector
 Beer’s Law A = ebc
Wavelength Dependence, a
Readout
Absorbance

0.80

Source
b

Detector
 Non-Absorption Losses

"Reflection and
scattering losses."
 Limitations to Beer’s Law

• Real
– At high concentrations charge distribution effects
occur causing electrostatic interactions between
absorbing species
• Chemical
– Analyte dissociates/associates or reacts with solvent
• Instrumental
– ε = f(λ); most light sources are polychromatic not
monochromatic (small effect)
– Stray light – comes from reflected radiation in the
monochromator reaching the exit slit.
 Chemical Limitations
A reaction is occurring as you record
Absorbance measurements
Cr2O72- + H2O 2H+ + CrO42-
CrO42-

Cr2O72-
A550 A446

300 400 500 concentration concentration

wavelength
 Instrumental Limitations - ε = f(λ)
• How/Why does ε
vary with λ?
• Consider a
wavelength scan for
a molecular
compound at two
different wavelength
bands

• In reality, a
monochromator can
not isolate a single
wavelength, but
Larger the Bandwidth – larger deviation
rather a small
wavelength band
 Instrumental Limitations – Stray Light

• How does stray light effect Absorbance

and Beer’s Law?
• A = -log I/Io = log Io/I
• When stray light (Ps) is present, the
absorbance observed (Aapparent) is the sum
of the real (Areal) and stray absorbance
(Astray)
 Instrumental Limitations – Stray Light

 I o  Is 
• Aapp = Areal + Astray = log  
 I  Is 

• As the analyte concentration increases

([analyte]↑), the intensity of light exiting the
absorbance cell decreases (I↓)
• Eventually, I < Is
 Instrumental Limitations – Stray Light

• Result – non-linear
absorption (Analyte
vs. Conc.) as a
function of analyte
concentration
– Similar to
polychromatic light
limitations
 Solution too Concentrated
• Refractive index changes with larger
concentrations

A  e en n
n2 + 2
 Allura Red

2Na+

Formula: C18H14N2Na2O8S2
Molar mass: 496.42 g/mol
 During Absorption…
Starting e- arrangement: After photon absorption:
 Absorption is random
•Photons collide with molecules
•Certain probability of absorption
 Transmittance, T
T: ratio of light “in” vs. “out”
= fraction of light passing through
Depends on:
# of molecules b&c
molecules’ identity e
Pathlength, b

I0 I1 I1
=T
Concentration,c
I0
Transmittance and Pathlength
For cell with same pathlength: constant T

I0 I1 I2 I1 I 2
=
I 0 I1
I1 I2
I0 I1
 Transmittance and Pathlength
For cells with same pathlength: constant T
Double pathlength: square T
#1 #2
2
I0 I2 I2 I1 I 2  I1 
=  =  
I0 I 0 I1  I 0 

I1 I2
Ttotal = T1  T2 = T1 
2
I0 I1
 T and Pathlength
For b cells (b pathlengths = 1cm)  Tb
#1 #b

I0 Ib I b  I1 
b
I1 Ib
… =   = 
I0  I0  I0 I b-1

I1 Ib

Ttotal = T1  = T1  Tb

I0 I b-1 b

b in power
 Concentration
• # photons absorbed depends on #
molecules in path

I0 I1
 Transmittance and
Concentration
1.0 M reference solution gives

I0 I1 I2 b
… T =  I1 cm 
 I 
I1 I2  0 
I0 I1

2.0 M: Double concentration,c→“Double pathlength, b”…

bc
 I1 cm, 1M 
T = T1  =  
bc
bc in power
 I0 
pathlength and
concentration
 Molar Extinction Coefficient, e
T: concentration c & pathlength b
What about: molecular identity?

e Probability of absorption
–Specific to molecule
–Function of l

lmax Most efficient absorption

–Peak of absorption curve
–“Best l” for experiment
 b, c, and e
 I1 cm, 1M 
ε =  log 
bc  I0 
 I1 cm, 1M 
T = T1  =    I1 cm, 1M 
bc
  = 10 ε
 I0   I0 
bc in power e in power
pathlength and concentration molar extinction coeff.

bc
 I1 cm, 1M 

Therefore: T =  
 = 10 -ε
  bc
= 10 -εcb

 0  I
 Absorbance
Beer’s Law: Defines absorbance, A

A =  log T 
A =  log 10  εbc

A = εbc
A: Directly proportional to concentration
high A ≡ low T
Part 1 Spectral Profile of Allura
Red, lmax
•Use stock solution (record conc.) cstock

•Record %T, 400 – 700 nm %T

– 10 nm intervals near lmax
– 20 nm intervals elsewhere
•Record cell width, b = pathlength b

•Calculate A A
•Plot A vs. l
•Determine lmax lmax
 Part 1: lmax
Find l where
%T is lowest Absorption
Transmittance
A is highest
Intensity

This is not
necessarily
Allura Red
(but would
appear red)
400 500 600 700
l (nm)
l (nm)
400
Absorbance
0.078
Spectral Profile
420 0.130
Spectral profile - Allura Red
440 0.268
460 0.555 1.60
480 0.966
1.40
490 1.189
500 1.383 1.20

510 1.485 1.00

Absorbance
520 1.515
0.80
530 1.459
0.60
540 1.217
560 0.395 0.40
580 0.063 0.20
600 0.019
0.00
620 0.015
400 450 500 550 600 650 700
640 0.010
wavelength (nm)
660 0.009
680 0.006
700 0.000
 Part 2 Absorbance for Various
Concentrations
•Stock solution, 4 dilutions, and blank
• n1 = n2
• M1 . V1 = M2 . V2
•
•Determine %T at lmax for each
%T
•Calculate A
A

•Plot A vs. conc (Beer’s Law plot)

•Slope = eb
 Part 2 Concentrations
•1 blank: pure DI water M0 = 0.00 M allura red
•1 stock: 0.002% allura red M5 = 4×10-5 M

0.002% = 0.002g allura red

100g solution  1mol allurared
496.42g allurared  1g solution
0.001L solution = 4 105 M

•4 dilutions, each by ½:
M concentrated Vconcentrated = M dilute Vdilute  nconcentrated = ndilute

– (4×10-5 M)(25.00 mL) = (M1)(50.00 mL) M1 = 2×10-5 M

– (2×10-5 M)(25.00 mL) = (M2)(50.00 mL) M2 = 1×10-5 M
– (1×10-5 M)(25.00 mL) = (M3)(50.00 mL) M3 = 5×10-6 M
– (5×10-6 M)(25.00 mL) = (M4)(50.00 mL) M4 = 2×10-6 M
 Beer’s Law
e,b constant c varied
%T εbc A
T= = 10 = 10
100%
A =  log T 
A = εb  c
y = m x
 Beer’s Law Plot
0.50

0.45 Dy
slope = = εb
0.40
Dx
A = εb  c
0.35
Absorbance
0.30

Dy
y = m x
0.25

0.20

0.15

0.10
Dx
0.05

0.00

Molarity
y = 1.9 x
 Beer’s Law Plot example
Dilution Factor M (mol/L) T A = -logT Absorbance vs. Concentration - Allura Red
0 0 1.00 0.000
1/16 2.E-06 0.87 0.060 1.200
1/8 5.E-06 0.73 0.137 y = 24943x
1/4 1.E-05 0.58 0.240
1.000 2
R = 0.9995
1/2 2.E-05 0.32 0.495
0.800

Absorbance
1 4.E-05 0.10 1.000
0.600
Stock M 4E-05 mol/L

0.400
A = e×b×c
0.200
Slope = DA/Dc
0.000
Here: e×b = 24943 0.E+00 1.E-05 2.E-05 3.E-05 4.E-05

Molarity (mol/L)

A 1-cm cell: e= 24943 cm-1M-1

 Part 3 Allura Red Concentration in
Mouthwash
•Use 1:25 dilution
•
•Determine %T at lmax %T
•Calculate A
A
•Measure b b
•Find concentration, c c

A = εbc so
A
c=
eb
 Part 3 Example
Mouthwash trials (1:25 dilution):
T A Mdilute Mconcentrated
0.68 0.167 7E-06 1.7E-04
0.70 0.155 6E-06 1.6E-04
0.65 0.187 8E-06 1.9E-04
0.69 0.161 6E-06 1.6E-04

Mdilute= A/(b×e) if cell length b is constant

Average Mconcentrated = 1.7×10-4 M = 2×10-4 M
 Report
• Abstract
• Data/Results
• Sample calculations including:
– Absorbance from transmittance
– Dilution
– Slope and extinction coefficient
– [Allura red]cuvette and [Allura red]mouthwash
– # allura red molecules in 1 mL mouthwash

• Discussion/review questions