Spectrophotometry

Properties of Light

• The wave-particle
duality.
light (and, indeed,
all other subatomic
particles) sometimes
act like a wave, and
sometimes act like a
particle

Light is also viewed as particles or packets of energy called photons.

 Light Absorption and Light Emission

• When light is absorbed Excited

States
by a molecule, the hn E
energy level of the
Ground
molecule is increased. State
(lowest
energy
state)

• When light is emitted by

E a molecule, the energy
hn level of the molecule is
decreased.
 Spectrophotometry

The wavelength of light determines how it interacts with matter.

We use these interactions as a probe to obtain chemical
information about samples.
Spectrophotometry is the use of “light” in chemical
measurements

UV IR

400 nm 750 nm
 Spectrophotometry

 The quantitative measurement of the

reflection or transmission properties
of a material as a function of
wavelength
Spectrophotometry: Absorbance

Absorption
 Spectrophotometry

• Instrument is called a Spectrophotometer

• Measures the light that passes through a liquid
sample
• Spectrophotometer gives readings in Percent
Transmittance (%T) and in Absorbance (A)
 Spectrophotometry

• Transmittance is a measure of the amount

of light transmitted through the sample
• Low conc. Sample  High Transmittance I0 I
• Percent transmittance (%T) :
• %T = (I/I0) x 100

• Absorbance is a calculated measure of the amount of light

absorbed by the sample
• Low conc. Sample  Low Absorbance
• Absorbance (A) :
• A = log10(1/T)
 Beer-Lambert Law
A = Ecl
where:
“A” is Absorbance
“E” is Molar Extinction coefficient of solute
“c” is solute concentration
“l” is the length of the light path
 For a given solute and spectrophotometer, “E” and
“l” are constants
 Let E x l = k (k is a constant)
 Beer-Lambert Law becomes A = kc

Absorbance proportional to solute concentration

Lowry Assay
 Spectrophotometry to Measure
Protein Concentration?
 Proteins do absorb light
 Problem 1: Contaminating molecules
 Problem 2: “E” different for different proteins
 A colorimetric reaction can be done
 A reaction that produces a colored product
 Amount of color proportional to concentration of one
of the reactants (protein)
 Spectrophotometry used to detect colored
product
 Lowry Assay

 A widely-used method of measuring

protein concentration
 A colorimetric assay
 Amount of blue color proportional to
amount of protein
 Absorbance read using 500-750nm light
 Lowry et al, 1951
 Chemistry of the Lowry Assay

Two reactions make the blue color develop:

 Reaction 1

 Cu2+ + peptide bonds  Cu1+-peptide bond complex

 Produces purple-blue color
 Reaction 2
 Folin reagent + Cu1+-complex  reduced Folin reagent
 Produces blue-green color
Standard Curves
 It turned blue... now what?
 You do the reaction
 You get blue color
 You stick it in the spectrophotometer
 You get an Absorbance reading of 0.434
(or whatever)
 How much protein is that?
 To find out, must do a “standard curve”
 Standard Curve
 A graph that correlates Absorbance
with protein concentration
 Standard Curve generated by doing a
Lowry Assay on protein solutions of
known concentration
 Standard Curve must be done each time
unknowns are being tested
 Standard Curve
Protein
Lowry Assay Standard Curve standards:
Protein amount vs. Absorbance
Protein
0.6
(g) A750

0.4
0 0.000
A750

20 0.212
0.2 0
40 0.354
60 0.372
0.0
0 20 40 60 80 100 80 0.480
Protein (g) 100 0.601
 Using Standard Curve

 Do Lowry Assay on unknowns

 Locate A750 value of unknown on graph y-axis
 Draw horizontal line from y-axis to curve
 Draw vertical line from curve to x-axis
 Read amount of protein from x-axis
 Using Standard Curve
Protein
Lowry Assay Standard Curve unknowns:
Protein amount vs. Absorbance
Protein
0.6
(g) A750

0.4
33 0.320
A750

92 0
0.550
0.2

0.0
0 20 40 60 80 100
Protein (g)