Principles of the
spectrophotometric methods
mirka.rovenska@lfmotol.cuni.cz
Interaction of light with matter



When a beam of light impinges upon a sample:
 a) some photons may have no interaction with a sample and be transmitted
 b) some photons may be absorbed by a sample
 c) some photons may be scattered
 d) some photons may be reflected
The extent of a) – d) depends on the material of the sample and on the wavelength
of the radiation
In spectrophotometric measurements, c) and d) should by kept to a minimum
I0
absorption
of radiation
transmitted
radiation
I
scatter
The intensity of the transmitted
radiation (I) is lower than
the intensity of the incident
radiation (I0)
Electromagnetic spectrum
λ [m]
10-12
10-10
10-8
10-3
10-1
Absorption of radiation

Molecules of the sample absorb the photons of a suitable wavelength ( λ)
and change their energy level (state):



1) in the microwave and far infrared region, the photons have such a low
energy that, if absorbed, can cause only the changes of the rotational
energy states
2) absorption of photons of the infrared radiation can bring about the
changes of the vibrational energy states
3) energy of photons of UV and visible light (VIS) is sufficient to cause
the transition of electron to a higher electronic energy level
http://uk.video.search.yahoo.com/search/video?rd=r1&p=molecular+vibration&toggle=1&cop=mss&ei=UTF-8&fr=yfp-t-702
energy
Energy levels of a molecule
ΔE = ΔEe + ΔEv + ΔEr ;
ΔE = hν = hc/λ
ΔEe >> ΔEv >> ΔEr
1st excited state
of electron
UV/VIS
ΔEe
electronic levels
ΔEr
ΔEv IR
vibrational levels
far IR
rotational levels
Thus, the change of the electronic state is accompanied by changes of vibrational
as well as rotational states!!!
Colour


Only those substances appear coloured that absorb VIS radiation
The colour is then determined by the reflected light (the colour of the
substance is complementary to that one which has been absorbed):
Chromophores




Absorption of visible light can cause transitions of π or n electrons (for
transition of σ electrons, UV absorption is necessary); thus, only substances
containing π or n electrons can appear coloured
Groups containing unsaturated centres (π electrons) and non-bonding
electrons are called chromophores – e.g..:
A compound will absorb in the visible region (and thus appear coloured) if it
contains at least several chromophores (absorption maximum then moves to
a longer λ, i.e. from the UV to the visible region):
The compounds that absorb only in the UV region are NOT coloured
(saturated hydrocarbons)
Absorption of UV and visible light


Routinely in biochemistry, absorption of UV and VIS
light (that causes electronic transitions) is measured
The UV/VIS absorption is the principle of all the
methods that will be discussed from now on
The Beer-Lambert law
l
I0
absorption
of radiation
transmitted
radiation
I
 due to absorption, the intensity of
the transmitted light is lower than
the intensity of the incident light
scatter

This decrease of the radiation intensity can be expressed as:
T = I/I0


T is called transmittance and varies from 0 to 1 (0 – 100%); it is the ratio
of the transmitted to the incident radiation intensity
Compounds with T (in the VIS region) approaching 100%...transparent;
0%......opaque


Absorbance is then defined as: A = - log T = log I0/I
The Beer-Lambert law states that the absorbance (at a given λ) is directly
proportional to the thickness of the absorbing layer (l) and to the molar
concentration (c) of the absorbing substance:
A = ελ c l
ε…molar absorption coefficient [dm3mol-1cm-1] = [M-1cm-1]
The law is only true for monochromatic light !!!

ε depends on λ and this dependence characterizes the substance
Absorption spectrum




Spectrum is the dependence of the intensity of radiation on its wavelength
or frequency (ν = 1/λ)
Absorption spectrum can be acquired by analysis of radiation (emitted from
the source) that has passed through the analyzed substance (by comparing
the intensities of the incident and transmitted light)
Absorption spectrum of a given substance is often depicted as the
dependence of absorbance or ε on the wavelength of the radiation
Spectrophotometry deals with acquiring and analyzing the absorption
spectra
KMnO4
A
Absorbance (relative units)
Characteristics of an absorption band
λ1max

λ2max
λ [nm]
Absorption band is characterized by the:
 wavelength(s) λmax of its peak(s) – usually, A is measured at this λmax
 by the corresponding εmax
Example 1: the Beer-Lambert law


If we know ε (for the compound and given λ) and the thickness of the
absorbing layer (i.e. the width of a cuvette), we can calculate the
concentration of the absorbing compound (using the B-L law) from the
absorbance measured
E.g.: How many grams of vitamin D2 are solved in 1 liter of solution, if
its absorbance measured in the 2-cm wide cuvette is A264 = 0,4 and ε for
vit. D2 at this λ is 18,4 [dm3mol-1cm-1]. Mr of vitamine D2 is 396.


A = ελ.c.l
c = A / (ελ.l) = 0,4 / (18,4 . 2) = 0,01 M

m = c . M . V = 0,01 . 396 . 1 = 4,3 g
Calibration graph

In practice, it can often be more precise to determine the concentration of
the absorbing compound not by calculation according to the Beer-Lambert
law, but by construction of the calibration graph:


we prepare a series of standards of known concentration of the
absorbing compound, measure absorbance for each of them and plot the
absorbance values against their individual concentrations
we measure absorbance of the „unknown“ sample; its concentration can
then be read from the calibration graph
Example 2: determination of
concentration using calibration graph



To determine the protein conc. in the sample, the Lowry protein assay can be used:
by the reaction of proteins with the reagent, a coloured complex is formed that
absorbs light at λ= 750 nm
We prepare several samples of a pure protein, e.g. BSA (bovine serum albumin) so
that the final conc. of BSA is 5, 10, 20, 40, 60, and 80 µg/ml. The reagent is added.
We measure the absorbance values of these standards and plot them against conc.
of BSA (protein):
0,5
y = 0,0054x + 0,0248
0,45
A
0,4
0,35
0,3
0,25
0,2
0,15
0,1
0,05
0
0
10
20
30
40
50
60
70
80
90
konc. proteinu [µg/ml]
protein concentration

Then, we add the reagent to the sample in which we want to determine the protein
concentration, measure its A750 and read the corresponding protein conc. from the
calibration graph (e.g. for A = 0,3, the conc. is 50 µg/ml)
Example 3: monitoring enzymatic reactions

molar absorption coefficient
molární
[M-1cm-1]

Using spectrophotometry, we can monitor the increase of the product
concentration or the decrease of the substrate concentration, respectively
Often, reactions using NAD+/NADH+H+ as coenzymes are monitored, based
on the difference between the absorption spectra of these two coenzymes:

The increase/decrease of NADH concentration
per a fixed period is measured at 340 nm and
compared with calibration
wavelength [nm]
E.g: determination of acetoacetate and -hydroxybutyrate: their ratio in
arterial blood reflects the intramitochondrial redox state and is used to
assess the energy state of the liver after transplantation or failure:
+ NAD+
+ NADH + H+
acetoacetate
-hydroxybutyrate
How can we measure absorbance?

Most often by a spectrophotometer that consists of:
source prism slit
detector
sample
monochromator


readout
system
PC
The source usually emits a polychromatic radiation (contains various λ)
from which monochromator (prism or diffraction grating + slit) separates
the monochromatic light (one λ)
As a source of the visible light, the tungsten lamp is used; the deuterium
lamp can serve as a source of UV
Spectrophotometers




Before we start to analyze the samples, we have to measure the A value of
„blank“, i.e. solution containing the same components as the samples (the
same buffer, coenzymes…) except for the absorbing substance; its
absorbance value must be subtracted from the A values of the samples
The solvent should not absorb at the wavelength used for measurement
The solutions must not be turbid, must not contain bubbles…
The error of measurement is acceptable for A values ranging from 0,1 to 1;
if A > 1, we have to dilute the sample and measure A once again!
Cuvettes

Cuvettes must by made of a material that does not absorb the radiation
used for measurement  cuvettes used for measurement in the VIS region
can be made of glass, cuvettes used for measurement in the UV region
must be quartz
Measuring absorbance in microplates

If we want to measure A values of more samples at once, we can pipette all
the samples into the wells of a microplate that can be analyzed in the
microplate reader:
http://www.moleculardevices.com/pages/instruments/readers_main.html
ELISA – sandwich assay





ELISA=enzyme-linked immunosorbent assay
Wells of the plate are coated with the antigen
(Ag)
We add the sample in which the (primary)
antibody (Ab) against the antigen is to be
determined; if the antibody is present, it binds
the antigen
After the unbound components have been
washed away, the secondary antibody,
specific to the primary antibody, is added
The secondary antibody is enzyme-linked and
after addition of a substrate (S), the enzyme
(E) catalyzes formation of a coloured product
that is measured using spectrophotometer
positive sample
Ab
against
Ag
negat. control
Ag
secondary Ab
wash → measurement of A
enzyme-linked Ab

ELISA can also be performed other way
round: the wells are coated with the antibody
that binds the antigen present in the sample;
then, another, enzyme-linked antibody against
the antigen is added
Ab
against
Ag
Ag
E
S
E
coloured
product
Example: HIV infection screening

Presence of anti-HIV antibodies in patient's serum is assessed:
 the wells are coated with the antigen – viral protein (e.g. protein of the
viral envelope…commercially available) and patient's serum is added;
after infection, the serum contains antibodies against the viral proteins
and these antibodies bind the antigen in the well
 after washing, the enzyme-linked secondary antibody is added that
binds the patient's antibodies; the enzyme then catalyzes formation of
a coloured product
Ab
against
HIV
HIV
protein
secondary
Ab
positive
negative
Turbidimetry and nephelometry




Turbidimetry measures the decrease of the intensity of the TRANSMITTED
light compared to the intensity of the incident light that has been caused
by scatter on the particles of a SUSPENSION (e.g. precipitate)
Turbidance is defined by analogy with absorbance:
S = log I0/I
Under certain conditions and when using the monochromatic light,
turbidance is directly proportional to the concentration of suspended
particles and thickness of the layer (analogy of the B-L law)
Turbidance can be measured by spectrophotometers; the detector is thus
aligned with the cell and the source of radiation:
source
I0
cuvette transmitted
radiation
detector


Nephelometry measures the intensity of the SCATTERED
radiation (scattering is again caused by the suspended
particles) that is emitted from the cuvette in the direction
perpendicular to the path of incident radiation:
The intensity is measured by nephelometers:
cuvette
source
I0
scaterred radiation
detector
Applications of turbidimetry and
nephelometry

These methods can be used to assess clinically relevant proteins in blood:
 an antibody against the protein (antigen) is added and formation of
immunocomplexes antigen-antibody is monitored
 e.g.:
 quantification of CRP (C-reactive protein): its concentration increases
with all invasive bacterial infections, but rarely with viral infections 
it‘s determination is useful to the clinician in evaluating acute phase
response
 quantification of IgG, IgA a IgM: can be helpful in differential
diagnosis of chronic liver diseases and in diagnosis of immunodeficiencies and autoimmune diseases