Spectrophotmetry-1

18-08-2023
Biochemistry Laboratory
Modern Theory and Techniques
Second Edition
By: Rodney Boyer
Some of the earliest experimental measurements on
biomolecules involved studies of their interactions with
electromagnetic radiation of all wavelengths, including
X-ray, ultraviolet–visible and infrared
It was experimentally observed that when light impinges
on solutions or crystals of molecules, at least two distinct
processes occur:

light scattering

light absorption
 Spectroscopy

Both processes have led to the development of fundamental

techniques for characterizing and analyzing biomolecules

We now use the term spectroscopy to label the discipline that

studies the interaction of electromagnetic radiation with matter
Absorption of ultraviolet–visible light by molecules is an
especially valuable process for measuring concentration and
for molecular structure elucidation

The absorption process is dependent upon two factors:

the properties of the radiation (wavelength, energy, etc.),
the structural characteristics of the absorbing molecules
(atoms, functional groups, etc.)
The interaction of electromagnetic radiation with molecules is
a quantum process and described mathematically by quantum
mechanics; that is, the radiation is subdivided into discrete
energy packets called photons
In addition, molecules have quantized excitation levels and
can accept packets of only certain quantities of energy, thus
allowing only certain electronic transitions
 Fluorescence

With some molecules, the process of absorption is followed by

emission of light of a longer wavelength

This process, called fluorescence, depends on molecular

structure and environmental factors and assists in the
characterization and analysis of biologically significant
molecules and dynamic processes occurring between molecules
ULTRAVIOLET–VISIBLE ABSORPTION
SPECTROMETRY
Wavelength and Energy
The electromagnetic spectrum, as shown in Figure 7.1, is
composed of a continuum of waves with different
properties defined by wavelength and energy
The electromagnetic spectrum
Several regions of the electromagnetic spectrum are of
importance in biochemical studies, including X-ray (X-ray
crystallography, up to 7 nm), the ultraviolet (UV, 180–340 nm),
the visible (VIS, 340–800 nm), the infrared (IR, 1000–100,000
nm), and radio waves (NMR, 106-1010nm)
 UV-visible Spectroscopy: Spectrophotometry

We will concentrate on the UV and VIS regions. Light in these

regions has energy sufficient to excite the valence electrons of
molecules and thus move the electrons from one energy level
(or state) to a higher energy level (excited state)
Figure 7.2 shows that the propagation of light is due to an
electrical field component E and a magnetic field component
H that are perpendicular to each other. The wavelength of
light, defined by Equation 7.1, is the distance between
adjacent wave peaks, as shown in Figure 7.2
Figure 7.2 shows that the propagation of light is due to an
electrical field component E and a magnetic field component
H that are perpendicular to each other. The wavelength of
light, defined by Equation 7.1, is the distance between
adjacent wave peaks, as shown in Figure 7.2
Light also behaves as though it were composed of energetic
particles. The amount of energy E associated with these
particles (or photons) is given by Equation 7.2:
Note the inverse relationship between wavelength and energy.
In Figure 7.1, X-rays have the shortest wavelength, but the
most energy, and microwaves have long wavelengths, but the
least energy
 Example-1

Assignment Question-1
Calculate the amount of energy (E) associated with light of
wavelength 400 nm and 700 nm
Thank You
Spectrophotometry-2
25-08-2023
Biochemistry Laboratory
Modern Theory and Techniques
Second Edition
By: Rodney Boyer
 Light Absorption

Light Absorption
When a photon of specified energy interacts with a molecule,
one of two processes may occur. The photon may be
scattered, or it may transfer its energy to the molecule,
producing an excited state of the molecule. The former
process, called Rayleigh scattering, occurs when a photon
collides with a molecule and is diffracted or scattered with
unchanged frequency
Light scattering
Light scattering is the physical basis of several experimental
methods used to characterize macromolecules. Before the
development of electrophoresis, light scattering techniques were
used to measure the molecular weights of macromolecules. The
widely used techniques of X-ray diffraction (crystal and solution),
electron microscopy, laser light scattering, and neutron
scattering all rely in some way on the light scattering process
 Light Absorption

The other process mentioned above, the transfer of energy from a

photon to a molecule, is absorption. For a photon to be absorbed,
its energy must match the energy difference between two energy
levels of the molecule
Molecules possess a set of quantized energy levels
Although several states are possible, only two electronic states
are shown in Figure 7.3, a ground state, G, and the first excited
state S1
These two states differ in the distribution of valence electrons
Energy Level Diagram
When electrons are promoted from a ground state orbital in G to
an orbital of higher energy in S1 an electronic transition is said
to occur
The energy associated with ultraviolet and visible light is
sufficient to promote molecules from one electronic state to
another, that is, to move electrons from one quantized level to
another
Within each electronic energy level is a set of vibrational levels
These represent changes in the stretching and bending of
covalent bonds
Transitions between the vibrational levels are the basis of
infrared spectroscopy
The electronic transition for a molecule from G to S1
represented by the vertical arrow in Figure 7.3, has a
high probability of occurring if the energy of the photon
corresponds to the energy necessary to promote an
electron from energy level E1 to energy level E2

A transition may occur from any vibrational level in G to

some other vibrational level in S1, for example, v = 3;
however, not all transitions have equal probability
The probability of absorption is described by quantum
mechanics
Energy Level Diagram
 The Absorption Spectrum

The Absorption Spectrum

A UV-VIS spectrum is obtained by measuring the light
absorbed by a sample as a function of wavelength. Since only
discrete packets of energy (specific wavelengths) are
absorbed by molecules in the sample, the spectrum
theoretically should consist of sharp discrete lines
 The Absorption Spectrum

However, the many vibrational levels of each electronic

energy level increase the number of possible transitions. This
results in several spectral lines, which together make up the
familiar spectrum of broad peaks as shown in Figure 7.5
The Absorption Spectrum of Hemoglobin
 The Absorption Spectrum

An absorption spectrum can aid in the identification of a

molecule because the wavelength of absorption depends on
the functional groups or arrangement of atoms in the
sample. The spectrum of oxyhemoglobin in Figure 7.5 is due
to the presence of the iron porphyrin moiety and is useful for
the characterization of heme derivatives or hemoproteins
 The Absorption Spectrum

Note that the spectrum consists of several peaks at wavelengths

where absorption reaches a maximum (415, 542, and 577 nm).
These points, called λmax are of great significance in the
identification of unknown molecules
The Absorption Spectrum of Hemoglobin
The Beer-Lambert Law
 Lambert's Law

When a ray of monochromatic light passes through a solution its

intensity decreases exponentially as the path length increases

Beer's Law

When a ray of monochromatic light passes through a solution its

intensity decreases exponentially as the concentration increases
 Experimental measurements are
usually made in terms of transmittance
(T), which is defined as

I
I0 I T =
I 0

where I is the intensity of light after it

passes through the sample and Io is
the initial light intensity
A more useful quantity in performing analyses is the
Absorbance (A) or the negative log of Transmittance (T)

The relation between A and T is

A = -log (T) = -log (I/I0)

The Beer-Lambert Law
Since the absorbance, A, is derived from a ratio it is unitless.

The term E or ε, which is a proportionality constant, defines

the efficiency or extent of absorption. If this is defined for a

particular chromophore at a specific wavelength, the term
absorption coefficient or absorptivity is used
However, in the older biochemical literature, the term

extinction coefficient (E or ε), is often used

The units of E depend on the units of l (usually cm) and c
(usually molar) in Equation 7.5. For biomolecules, E is often

used in the form molar absorption coefficient (E or ε), which is

defined as the absorbance of a 1M solution of pure absorbing
material in a 1-cm cell under specified conditions of wavelength
and solvent
The units of molar absorption coefficient are M-1 cm-1
To illustrate the use of Equation 7.5, consider the following
calculation
Assignment Question-2
Assignment Question-3
The molar absorbance coefficient of ATP is 15.4 X 103 M-1 cm-1
If the light path is 1.0 cm calculate the concentration of a
solution whose absorbance at 260 nm is 0.77

Assignment Question-4
A solution of UTP of concentration 29.3 mg/litre has an
absorbance of 0.25 at 260nm. If the light path is 1.0cm and the
molecular weight of UTP is 586 calculate the molar absorbance
coefficient of UTP
Thank You
Spectrophotmetry-3
01-09-2023
Biochemistry Laboratory
Modern Theory and Techniques
Second Edition
By: Rodney Boyer
 Instrumentation

The spectrophotometer is used to measure absorbance

experimentally
This instrument produces light of a pre-selected wavelength,
directs it through the sample (usually dissolved in a solvent and
placed in a cuvette) and measures the intensity of light
transmitted by the sample
The spectrophotometer has 5 major components

These consist of a light source, a monochromator (including

various filters, slits, and mirrors), a sample chamber, a
detector, and a meter or recorder

All of these components are usually under the control of a

computer
Schematic of a single beam spectrophotometer
A systronics spectrophotometer
Light Source
For absorption measurements in the ultraviolet region, a
high-pressure hydrogen or deuterium lamp is used. These
lamps produce radiation in the 200 to 340 nm range
The light source for the visible region is the tungsten-halogen
lamp, with a wavelength range of 340 to 800 nm
Instruments with both lamps have greater flexibility and can
be used for the study of most biologically significant molecules
Monochromator
Both lamps discussed above produce continuous emissions of all
wavelengths within their range. Therefore, a spectrophotometer
must have an optical system to select monochromatic light
(light of a specific wavelength)
Modern instruments use a prism or, more often, a diffraction
grating to produce the desired wavelengths
It should be noted that light emitted from the monochromator
is not entirely of a single wavelength, but is enhanced in that
wavelength. That is, most of the light is of a single wavelength,
but shorter and longer wavelengths are present
Before the monochromatic light impinges on the sample, it
passes through a series of slits, lenses, filters, and mirrors.
This optical system concentrates the light, increases the
spectral purity, and focuses it toward the sample
Sample Chamber
The processed monochromatic light is then directed into a
sample chamber, which can accommodate a wide variety of
sample holders. Most UV-VIS measurements on biomolecules are
taken on solutions of the molecules. The sample is placed in a
tube or cuvette made of glass, quartz, or other transparent
material
 The Transmission properties of several
materials used in cuvettes

Figure 7.8 shows the transmission properties of several

transparent materials used in cuvette construction
Glass cuvettes are inexpensive, but, because they absorb UV
light, they can be used only above 340 nm. Quartz or fused
silica cuvettes may be used throughout the UV and visible
regions
Disposable plastic cuvettes are now commercially available in
polymethacrylate (280–800 nm) and polystyrene (350–800 nm)
Sample chambers for spectrometers come in two
varieties—those holding only one cuvette at a time
(single-beam) and those holding two cuvettes, one for a
reference, usually solvent, and one for a sample (double-beam)
In the past, single-beam instruments were usually less
expensive but more cumbersome to use because reference and
sample cuvettes required constant exchange
However, modern single-beam instruments with computer
control and analysis can be programmed to correct automatically
for the reference spectrum, which may be stored in a memory
file
In double-beam optics, the light beam is split into two paths by
directing it through a monochromator. The two beams, which are
of identical wavelength and intensity, pass through the sample
cell (analyte plus solvent) and reference chamber (solvent only)
This allows for the correction of sample absorbance by
continuously subtracting the reference spectrum
Double-beam spectrophotometer
Double-beam spectrophotometer
Detector
The intensity of the light that passes through the sample
under study depends on the amount of light absorbed by the
sample. Intensity is measured by a light-sensitive detector,
usually a photomultiplier tube (PMT)
The PMT detects a small amount of light energy, amplifies this
by a cascade of electrons accelerated by dynodes, and
converts it into an electrical signal that can be fed into a
meter or recorder
Printers and Recorders
Less expensive instruments give a direct readout of
absorbance and/or transmittance in analog or digital form
These instruments are suitable for single-wavelength
measurements; however, if a scan of absorbance vs.
wavelength (Absorption Spectrum) is desired, some type of
device to display the spectrum must be available
Modern, research-grade spectrometers are available that offer the
latest in technology. All of the components discussed above are
integrated into a single package and are completely under the
control of a computer
By simply pushing a button, one can obtain the UV-VIS spectrum
of a sample displayed on a computer screen in less than 1 second
In addition, these modern instruments with computers can be
programmed to carry out several functions, such as subtraction
of solvent spectrum, spectral overlay, storage, difference spectra,
derivative spectra, and calculation of concentrations and rate
constants
Applications of UV–VIS Spectrophotometry
 Applications of UV–VIS Spectrophotometry

Although many different types of operations can be carried

out on a spectrophotometer, all applications fall in one of two
categories:
1. Measurement of absorbance at a fixed wavelength

2. Measurement of absorbance as a function of wavelength

Measurement of absorbance at a fixed wavelength
Measurements at a fixed wavelength are most often used to
obtain quantitative information, such as the concentration of
a solute in solution or the absorption coefficient of a
chromophore

A chromophore is a molecule or part of molecule

responsible for absorption of light
Measurement of absorbance as a function of wavelength
Absorbance measurements as a function of wavelength provide
qualitative information that assists in solving the identity and
structure of a pure substance by detecting characteristic
groupings of atoms in a molecule
Fixed Wavelength Measurements
For fixed-wavelength measurements with a single-beam
instrument, a cuvette containing solvent only is placed in
the sample beam and the instrument is adjusted to read
“zero” absorbance
A matched cuvette containing sample plus solvent is then
placed in the sample chamber and the absorbance is read
directly from the display
The adjustment to zero absorbance with only solvent in the
sample chamber allows the operator to obtain a direct
reading of absorbance for the sample
Fixed Wavelength Measurements
Fixed-wavelength measurements using a double-beam
spectrophotometer are made by first zeroing the instrument
with no cuvette in either the sample or reference holder

Alternatively, the spectrophotometer can be balanced by

placing matched cuvettes containing water or solvent in both
sample chambers
Fixed Wavelength Measurements
Then, a cuvette containing pure solvent is placed in the
reference position and a matched cuvette containing solvent
plus sample is set in the sample position
The absorbance reading given by the instrument is that of
the sample; that is, the absorbance due to solvent is
subtracted by the instrument
An absorbance spectrum of a compound is obtained by
scanning a range of wavelengths and plotting the absorbance
at each wavelength
Most double-beam spectrophotometers automatically scan the
desired wavelength range and record the absorbance as a
function of wavelength

To plot ultraviolet absorption spectrum of DNA

Wavelength Absorbance Wavelength Absorbance

(λ) nm (λ) nm
230 0.569 280 0.564
240 0.658 300 0.272
250 0.786 320 0.078
260 1.021 360 0.013
270 0.986 400 0.011
If solvent is placed in the reference chamber and solvent plus
sample in the sample position, the instrument will continuously
and automatically subtract the solvent absorbance from the total
absorbance (solvent plus sample) at each wavelength; hence, the
recorder output is really a difference spectrum (absorbance of
sample plus solvent, minus absorbance of solvent)
Both types of measurements (fixed wavelength and
absorbance spectrum) are common in biochemistry, and
you should be able to interpret results from each
The following four examples are typical of the kinds of
problems readily solved by spectrophotometry
 Measurement of the Concentration
of an Analyte in Solution

According to the Beer-Lambert law, the absorbance of a material

in solution is directly dependent on the concentration of that
material

Two methods are commonly used to measure concentration. If

the absorption coefficient is known for the absorbing species, the
concentration can be calculated after experimental measurement
of the absorbance of the solution
Measurement of the Concentration of an Analyte in
Solution: Using the Molar Absorption Coefficient

If the absorption coefficient for an absorbing species

is known, the concentration of that species in
solution can be calculated as outlined
Measurement of the Concentration of an Analyte in
Solution: Using the Molar Absorption Coefficient
However, there are limitations to this application
Most spectrophotometers are useful for measuring
absorbances up to 1, although more sophisticated instruments
can measure absorbances as high as 2. (If the absorbance is
above 1 dilute sample with the same solvent)
Most absorbance readings below 0.1 are not accurate
Also, some substances do not obey the Beer-Lambert law; that
is, absorbance may not increase in a linear fashion with
concentration
Reasons for deviation from the Beer-Lambert law are many;
however, the majority are instrumental, chemical, or physical
Spectrophotometers often display a nonlinear response at
high absorption levels because of stray light
Physical reasons for nonlinearity include hydrogen bonding of
the absorbing species with the solvent and intermolecular
interactions at high concentrations
Chemical reasons may include reaction of the solvent with
the absorbing species and the presence of impurities
Linearity is readily tested by preparing a series of
concentrations of the absorbing species and measuring the
absorbance of each
A plot of Absorbance vs. Concentration (Standard Curve)
should be linear if the Beer-Lambert law is valid
If the absorption coefficient for a species is unknown, its
concentration in solution can be measured if the absorbance
of a standard solution of the compound is known
Measurement of the Concentration of an Analyte in
Solution: By Direct Ratio
This linear relationship between concentration and
absorbance allows scientists to use spectroscopy for
quantitative measurements of unknown samples

Using a calibration curve prepared

from standard solutions (solutions
of known concentration), the
concentration of an unknown
solution can be interpolated by
linear regression
 Measurement of the Concentration of an Analyte in
Solution: Using a Standard Curve of the Solute

A Typical Standard Curve

Note the linearity, indicating that the Beer-Lambert

law is obeyed over this concentration range of
standard protein
Note the linearity, indicating that the Beer-Lambert law is
obeyed over this concentration range of standard protein
Two different volumes of unknown protein were tested. This
was to ensure that one volume would be in the concentration
range of the standard curve
Since the accuracy of the assay is dependent on identical times
for color development, the unknowns must be assayed at the
same time as the standards
Measurement of the Concentration of an Analyte in
Solution: Using a Standard Curve of the Solute
 Measurement of the Concentration of an Analyte in
Solution: Using a Standard Value (Standard Relation)

A standard solution of double-stranded DNA at a

concentration of 50 μg/ml in a 1-cm quartz cuvette will yield
an absorbance (A260nm) of about 1.0
A solution of single-stranded DNA or RNA that has an
absorbance (A260nm) of 1.0 in a cuvette with a 1-cm path
length has a concentration of about 40 μg/ml

Double-stranded DNA absorbs less light as compared to single

stranded DNA : This phenomenon is called Hypochromicity
When DS DNA is denatured the resulting SS DNA absorbs more
light : This phenomenon is called Hyperchromicity
Assignment Question
Two solutions, one of double-stranded DNA and one of
single-stranded RNA, were transferred to 1-cm quartz
cuvettes and the absorbance (A) at 260 nm of each of the
solutions was found to be 0.15
What are the concentrations of the DNA and RNA solutions
Thank You
Spectrophotmetry-4
01-10-2024
Biochemistry Laboratory
Modern Theory and Techniques
Second Edition
By: Rodney Boyer
Applications of UV–VIS Spectrophotometry
 Applications of UV–VIS Spectrophotometry

Although many different types of operations can be carried

out on a spectrophotometer, all applications fall in one of two
categories:

1. Measurement of absorbance at a fixed wavelength

2. Measurement of absorbance as a function of wavelength

Measurement of absorbance at a fixed wavelength
Measurements at a fixed wavelength are most often used to
obtain quantitative information, such as the concentration of
a solute in solution or the absorption coefficient of a
chromophore

A chromophore is a molecule or part of molecule

responsible for absorption of light
Measurement of absorbance as a function of wavelength
Absorbance measurements as a function of wavelength provide
qualitative information that assists in solving the identity and
structure of a pure substance by detecting characteristic
groupings of atoms in a molecule
Fixed Wavelength Measurements
For fixed-wavelength measurements with a single-beam
instrument, a cuvette containing solvent only is placed in
the sample beam and the instrument is adjusted to read
“zero” absorbance
A matched cuvette containing sample plus solvent is then
placed in the sample chamber and the absorbance is read
directly from the display
The adjustment to zero absorbance with only solvent in the
sample chamber allows the operator to obtain a direct
reading of absorbance for the sample
Fixed Wavelength Measurements
Fixed-wavelength measurements using a double-beam
spectrophotometer are made by first zeroing the instrument
with no cuvette in either the sample or reference holder

Alternatively, the spectrophotometer can be balanced by

placing matched cuvettes containing water or solvent in
both sample chambers
Fixed Wavelength Measurements
Then, a cuvette containing pure solvent is placed in the
reference position and a matched cuvette containing solvent
plus sample is set in the sample position
The absorbance reading given by the instrument is that of
the sample; that is, the absorbance due to solvent is
subtracted by the instrument
An absorbance spectrum of a compound is obtained by
scanning a range of wavelengths and plotting the absorbance
at each wavelength
Most double-beam spectrophotometers automatically scan the
desired wavelength range and record the absorbance as a
function of wavelength

To plot ultraviolet absorption spectrum of DNA

Wavelength Absorbance Wavelength Absorbance
(λ) nm (λ) nm
230 0.569 280 0.564
240 0.658 300 0.272
250 0.786 320 0.078
260 1.021 360 0.013
270 0.986 400 0.011
If solvent is placed in the reference chamber and solvent plus
sample in the sample position, the instrument will continuously
and automatically subtract the solvent absorbance from the total
absorbance (solvent plus sample) at each wavelength; hence, the
recorder output is really a difference spectrum (absorbance of
sample plus solvent, minus absorbance of solvent)
Both types of measurements (fixed wavelength and
absorbance spectrum) are common in biochemistry, and
you should be able to interpret results from each
The following four examples are typical of the kinds of
problems readily solved by spectrophotometry
 Measurement of the Concentration
of an Analyte in Solution

According to the Beer-Lambert law, the absorbance of a material

in solution is directly dependent on the concentration of that
material

Two methods are commonly used to measure concentration. If

the absorption coefficient is known for the absorbing species,
the concentration can be calculated after experimental
measurement of the absorbance of the solution
Measurement of the Concentration of an Analyte in
Solution: Using the Molar Absorption Coefficient

If the absorption coefficient for an absorbing species

is known, the concentration of that species in
solution can be calculated as outlined
Measurement of the Concentration of an Analyte in
Solution: Using the Molar Absorption Coefficient
However, there are limitations to this application
Most spectrophotometers are useful for measuring
absorbances up to 1, although more sophisticated instruments
can measure absorbances as high as 2. (If the absorbance is
above 1 dilute sample with the same solvent)
Most absorbance readings below 0.1 are not accurate
Also, some substances do not obey the Beer-Lambert law; that
is, absorbance may not increase in a linear fashion with
concentration
Reasons for deviation from the Beer-Lambert law are many;
however, the majority are instrumental, chemical, or physical
Spectrophotometers often display a nonlinear response at
high absorption levels because of stray light
Physical reasons for nonlinearity include hydrogen bonding of
the absorbing species with the solvent and intermolecular
interactions at high concentrations
Chemical reasons may include reaction of the solvent with
the absorbing species and the presence of impurities
Linearity is readily tested by preparing a series of
concentrations of the absorbing species and measuring the
absorbance of each
A plot of Absorbance vs. Concentration (Standard Curve)
should be linear if the Beer-Lambert law is valid
If the absorption coefficient for a species is unknown, its
concentration in solution can be measured if the absorbance
of a standard solution of the compound is known
Measurement of the Concentration of an Analyte in
Solution: By Direct Ratio
This linear relationship between concentration and
absorbance allows scientists to use spectroscopy for
quantitative measurements of unknown samples

Using a calibration curve prepared

from standard solutions (solutions
of known concentration), the
concentration of an unknown
solution can be interpolated by
linear regression
 Measurement of the Concentration of an Analyte in
Solution: Using a Standard Curve of the Solute

A Typical Standard Curve

Note the linearity, indicating that the Beer-Lambert

law is obeyed over this concentration range of
standard protein
Note the linearity, indicating that the Beer-Lambert law is
obeyed over this concentration range of standard protein
Two different volumes of unknown protein were tested. This
was to ensure that one volume would be in the concentration
range of the standard curve
Since the accuracy of the assay is dependent on identical times
for color development, the unknowns must be assayed at the
same time as the standards
Measurement of the Concentration of an Analyte in
Solution: Using a Standard Curve of the Solute
 Measurement of the Concentration of an Analyte in
Solution: Using a Standard Value (Standard Relation)

A standard solution of double-stranded DNA at a

concentration of 50 μg/ml in a 1-cm quartz cuvette will yield
an absorbance (A260nm) of about 1.0
A solution of single-stranded DNA or RNA that has an
absorbance (A260nm) of 1.0 in a cuvette with a 1-cm path
length has a concentration of about 40 μg/ml

Double-stranded DNA absorbs less light as compared to single

stranded DNA : This phenomenon is called Hypochromicity
When DS DNA is denatured the resulting SS DNA absorbs more
light : This phenomenon is called Hyperchromicity
Assignment Question
Two solutions, one of double-stranded DNA and one of single-
stranded RNA, were transferred to 1-cm quartz cuvettes and
the absorbance (A) at 260 nm of each of the solutions was
found to be 0.15
What are the concentrations of the DNA and RNA solutions
Thank You
Spectrophotmetry-5
01-10-2024
Applications of UV–VIS Spectrophotometry
 Applications of UV–VIS Spectrophotometry

Although many different types of operations can be carried

out on a spectrophotometer, all applications fall in one of two
categories

1. Measurement of absorbance at a fixed wavelength

2. Measurement of absorbance as a function of wavelength

Measurement of absorbance as a function of wavelength

Identification of Unknown Biomolecules by Spectrophotometry

Measurement of absorbance as a function of wavelength
Absorbance measurements as a function of wavelength provide
qualitative information that assists in solving the identity and
structure of a pure substance by detecting characteristic
groupings of atoms in a molecule
Identification of Unknown Biomolecules by Spectrophotometry
The UV-VIS spectrum of a biomolecule reveals much about its
molecular structure
Therefore, a spectral analysis is one of the first experimental
measurements made on an unknown biomolecule. Natural
molecules often contain chromophoric (color-producing)
functional groups that have characteristic spectra
The procedure for obtaining a UV-VIS spectrum begins with the
preparation of a solution of the species under study
A standard solution should be prepared in an appropriate
solvent. An aliquot of the solution is transferred to a cuvette
and placed in the sample chamber of a spectrophotometer
A cuvette containing solvent is placed in the reference holder.
The spectrum is scanned over the desired wavelength range and
an absorption coefficient is calculated for each major λmax
Absorption Spectrum of DNA
Absorption Spectrum of Flavin mononucleotide (FMN)
and its reduced form FMNH2
Absorption Spectrum of NAD+ and NADH
Absorption Spectrum of GMP
Absorption Spectrum of Thymine
Kinetics of Biochemical Reactions
Kinetics of Biochemical Reactions
Spectrophotometry is one of the best methods
available for measuring the rates of biochemical
reactions. Consider a general reaction as shown

Enzyme
A + B C + D

If reactants A or B absorb in the UV-VIS region of the

spectrum at some wavelength λ1, the rate of the
reaction can be measured by monitoring the decrease
of absorbance at λ1 due to loss of A or B
Alternatively, if products C or D absorb at a specific wavelength
λ2 the kinetics of the reaction can be evaluated by monitoring
the absorbance increase at λ2
According to the Beer-Lambert law, the absorbance change of a
reactant or product is proportional to the concentration change
of that species occurring during the reaction
This method is widely used to assay enzyme-catalyzed
processes. Since the rates of chemical reactions vary with
temperature, the sample cuvette containing the reaction
mixture must be held in a thermostated chamber
 Assay of Enzyme Lactate Dehydrogenase (LDH)

LDH
Pyruvate + NADH Lactate + NAD+

Absorption Spectrum of
NAD+ and NADH
Progress of the enzyme
catalyzed reaction can be
followed by monitoring
absorbance at 340 nm
The Pyridine Nucleotides-NAD and NADP
Limitations and Precautions in Spectrophotometry

The use of a spectrophotometer is relatively straightforward

and can be mastered in a short period of time
There are, however, difficulties that must be considered
A common problem encountered with biochemical
measurements is turbidity or cloudiness of biological samples
Limitations and Precautions in Spectrophotometry
This can lead to great error in absorbance measurements
because much of the light entering the cuvette is not absorbed
but is scattered. This causes artificially high absorbance
readings
Occasionally, absorbance readings on turbid solutions are
desirable (as in measuring the rate of bacterial growth in a
culture), but in most cases turbid solutions must be avoided or
clarified by filtration or centrifugation
A difficulty encountered in measuring the concentration of
an unknown absorbing species in solution is deviation from
the Beer-Lambert law
Some absorbing species do not demonstrate an increase in
absorbance that is proportional to an increase in
concentration
(In reality, most compounds follow the Beer-Lambert
relationship over a relatively small concentration range)
When measuring solution concentration, adherence to the
Beer-Lambert law must always be tested in the concentration
range under study
 Beer's Law is not obeyed at higher concentrations

Reasons for the deviation from Beer's law at

high concentrations

➢Light may not be monochromatic

➢Solute may undergo dimer/multimer

formation at high concentrations

➢Solute may form aggregates at high

concentrations

➢For every solute there is a threshold

concentration above which it shows
deviation
Thank You
Spectrophotmetry-6
08-10-2024
Flourescence Spectrophotometry
 Flourescence Spectrophotometry

In our discussion of absorption spectroscopy, we noted that the

interaction of photons with molecules resulted in the promotion
of valence electrons from ground state orbitals to higher energy
level orbitals
The molecules were said to be in an excited state to the more
stable ground state
With most molecules, the relaxation process is brought about by
collisional energy transfer to solvent or other molecules in the
solution
Some excited molecules, however, return to the ground state by
emitting the excess energy as light
This process, called fluorescence, is illustrated in Figure 7.11. The
solid vertical arrow in the figure indicates the photon absorption
process in which the molecule is excited from G to some
vibrational level in S
The excited molecule loses vibrational energy by collision with
solvent and ground state molecules
Energy level Diagram showing the phenomenon of flourescence
This relaxation process, which is very rapid, leaves the
molecule in the lowest vibrational level of S, as indicated by
the wavy arrow
The molecule may release its energy in the form of light
(fluorescence, dashed arrow) to return to some vibrational
level of G
Energy level Diagram showing the phenomenon of flourescence
 Stokes Shift

The Stokes shift is defined as the difference between the

positions of the band maxima of the absorption and emission
spectra of the same electronic transition
Two important characteristics of the emitted light

1. The emitted light is of longer wavelength (lower energy) than

the excitation light
This is because part of the energy initially associated with the S
state is lost as heat energy, and the energy lost by emission may
be sufficient only to return the excited molecule to a higher
vibrational level in G
2. The emitted light is composed of many wavelengths, which
results in a fluorescence spectrum as shown in Figure 7.12
Absorbance and flourescence spectra of Tryptophan
This is due to the fact that fluorescence from any particular
excited molecule may return the molecule to one of many
vibrational levels in the ground state
Just as in the case of an absorption spectrum, a wavelength
of maximum fluorescence is observed and the spectrum is
composed of a wavelength distribution centered at this
emission maximum
Quantum Yield
A molecule in the excited state can return to lower energy
levels by collisional transfer or by light emission
Since these two processes are competitive the fluorescence
intensity of a fluorescing system depends on the relative
importance of each process
Quantum Yield
The fluorescence intensity is often defined in terms of quantum
yield, represented by Q. This describes the efficiency or
probability of the fluorescence process. By definition, Q is the
ratio of the number of photons emitted to the number of
photons absorbed (Equation 7.7)
Instrumentation
The basic instrument for measuring fluorescence is the
spectrofluorometer. It contains a light source, two
monochromators, a sample holder, and a detector. A typical
experimental arrangement for fluorescence measurement is
shown in Figure 7.13. The setup is similar to that for
absorption measurements with two significant exceptions
Schematic diagram of a Spectrofluorometer
Instrumentation
First, there are two monochromators, one for selection of the
excitation wavelength and another for wavelength analysis of
the emitted light
Second, the detector is at an angle to the excitation beam.
This is to eliminate interference by the light that is
transmitted through the sample. Upon excitation of the
sample molecules, the fluorescence is emitted in all
directions and is detected by a photocell at right angles to
the excitation light beam
Schematic diagram of a Spectrofluorometer
Energy level diagram
The lamp source used in most instruments is a xenon arc
lamp that emits radiation in the ultraviolet, visible, and near-
infrared regions (200 to 1400 nm)
The light is directed by an optical system to the excitation
monochromator, which allows either preselection of a
wavelength or scanning of a certain wavelength range
The exciting light then passes into the sample chamber,
which contains a fluorescence cuvette with dissolved sample.
Because of the geometry of the optical system, a typical
fused absorption cuvette with two opaque sides cannot be
used; instead, special fluorescence cuvettes with four
translucent quartz or glass sides must be used. When the
excitation light beam impinges on the sample cell, molecules
in the solution are excited and some will emit light
Light emitted at right angles to the incoming beam is analyzed
by the emission monochromator. In most cases, the wavelength
analysis of emitted light is carried out by measuring the
intensity of fluorescence at a preselected wavelength (usually
the wavelength of emission maximum). The analyzer
monochromator directs emitted light of only the preselected
wavelength toward the detector.
A photomultiplier tube serves as a detector to measure the
intensity of the light. The output current from the
photomultiplier is fed to some measuring device that indicates
the extent of fluorescence. The final readout is not in terms of
Q, but in units of the photomultiplier tube current
(microamperes) or in relative units of percent of full scale.
Therefore, the scale must be standardized with a known
standard.
Thank You
Spectrophotmetry-7
13-10-2023
Flourescence Spectrophotometry
Applications
 Applications of Fluorescence Spectroscopy

There are many and highly varied applications of

flourescence despite the fact that relatively few compounds
exhibit the phenomenon
The effect of pH, solvent composition and the polarization
of flourescence may all contribute to structural elucidation
 Applications of Fluorescence Spectroscopy

Non-flourescent compounds are often labelled with flourescent

probes to enable monitoring of molecular events. This is
termed as extrinsic flourescence as distinct from intrinsic
flourescence where the native compound exhibits the property
Some flourescent dyes are sensitive to the presence of metal
ions and can thus be used to track changes of these ions in
vitro as well as in whole cells
 Two types of measurements are most common
in fluorescence experiments

✔Measurements of relative fluorescence intensities

✔Measurements of the quantum yield

Measurement of Flourescence Intensity
Most experiments require only relative fluorescence
intensity measurements, and they proceed as follows
The fluorometer is set to “zero” or “full scale” fluorescence
intensity (microamps or %) with the desired biochemical
system under standard conditions
Measurement of Flourescence Intensity
Some perturbation is then made in the system (pH change,
addition of a chemical agent in varying concentrations, change
of ionic strength etc.) and the fluorescence intensity is
determined relative to the standard conditions
This is a straightforward type of experiment because it consists
of replacing one solution with another in the fluorometer and
reading the detector output for each. For these experiments,
the excitation wavelength and the emission wavelength are
preselected and set for each monochromator
Measurement of Quantum Yield
The measurement of quantum yield is a more complicated
process. Before these measurements can be made the
instrument must be calibrated. A thermopile or chemical
actinometer may be used to measure the absolute intensity
of incident light on the sample
Measurement of Quantum Yield
Alternatively, quantum yields may be measured relative to
some accepted standard. Two commonly used fluorescence
standards are quinine sulfate and fluorescein
The quantum yield of the unknown, is then calculated by
Equation 7.8
Measurement of Quantum Yield
Intrinsic Fluorescence
Intrinsic fluorescence
Some biomolecules are intrinsic fluors; that is, they are
fluorescent themselves. The amino acids with aromatic
groups (phenylalanine, tyrosine, and tryptophan) are
fluorescent; hence, proteins containing these amino acids
have intrinsic fluorescence
Intrinsic fluorescence
The purine and pyrimidine bases in nucleic acids (adenine,
guanine, cytosine, uracil, thymine) and some coenzymes
are also intrinsic fluors

Intrinsic fluorescence is most often used to study protein

conformational changes (protein folding) and to probe the
location of active sites and coenzymes in enzymes
Intrinsic Protein Fluorescence
Proteins possess three intrinsic flourophores: tryptophan,
tyrosine and phenylalanine, although the latter has a very
low quantum yield and its contribution to protein
flourescence is thus negligible
Of the remaining two residues tyrosine has the lower
quantum yield and its flourescence emission is almost
entirely quenched when it becomes ionized or located near
an amino or carboxyl group or a tryptophan residue
Intrinsic Protein Fluorescence
Intrinsic protein flourescence is thus usually determined by
tryptophan flourescence which can be selectively excited at
295-305 nm
Excitation at 280 nm excites tyrosine and tryptophan
flourescence and the resulting spectra might therefore contain
contribution from both types of residues
The main application of intrinsic protein flourescence aims at
conformational analysis
Extrinsic Fluorescence
External flourophore or Extrinsic Flour
Frequently molecules of interest for biochemical studies are
non-flourescent. In many of these cases an external flourophore
(Extrinsic Flour) can be introduced into the system by chemical
coupling or non covalent binding
Three criteria must be met by the flourophore in this context
External flourophore or Extrinsic Flour
Three criteria must be met by the flourophore in this context
1. Firstly it must not affect the property of the system under
investigation
2. Secondly its flourescence emission needs to be sensitive to
environmental conditions
3. And lastly the flourophore must be tightly bound at a unique
location
Extrinsic Fluorescence
Valuable information can also be obtained by the use of
extrinsic fluors. These are fluorescent molecules that are
added to the biochemical system under study
Many fluorescent dyes have enhanced fluorescence when
they are in a nonpolar solution or bound in a rigid
hydrophobic environment
Some of these dyes bind to specific sites on proteins or
nucleic acid molecules and the resulting fluorescence
intensity depends on the environmental conditions at the
binding site
Extrinsic fluorescence is of value in characterizing the
binding of natural ligands to biochemically significant
macromolecules. This is because many of the extrinsic fluors
bind in the same sites as natural ligands
Extrinsic fluorescence has been used to study the binding of
fatty acids to serum albumin, to characterize the binding sites
for cofactors and substrates in enzyme molecules, to
characterize the heme binding site in various hemoproteins,
and to study the intercalation of small molecules into the
DNA double helix
Extrinsic fluors that have been of value in studying
biochemical systems
ANS, dansyl chloride and fluorescein are used for protein
studies whereas ethidium, proflavine and various acridines
are useful for nucleic acid characterization
Ethidium bromide has the unique characteristic of
enhanced fluorescence when bound to double-stranded
DNA
Aminomethyl coumarin (AMC) is of value as a fluorogenic
leaving group in measuring peptidase activity
 Extrinsic Flours for proteins

ANS dansyl chloride and fluorescein are used for protein studies
 Extrinsic Flours for Nucleic Acids

Ethidium, Proflavine and various acridines are

useful for nucleic acid characterization

Proflavine
Aminomethyl Coumarin (AMC)

Peptide----AMC

Peptidase

Peptide + AMC
 Examples

1. Properties of Heme binding sites in Hemoglobin

2. Flourescence Microscopy

3. Flourecent Antibody Technique

4. Enzyme Assays

5. Quantitative Estimation of DNA

 1. Properties of Heme binding sites in Hemoglobin

Hemoglobin/Apohemoglobin are incubated with an

extrinsic flour for proteins (ANS) and analysed for
flourescence

Hemoglobin = Apohemoglobin + Heme

 1. Properties of Heme binding sites in Hemoglobin

Hemoglobin + ANS No Flourescence

Apohemoglobin + ANS Flourescence

Apohemoglobin –ANS + Heme No Flourescence

 2. Flourescence Microscopy

Sample is incubated with a specific flour, washed to remove

unbound flour and then illuminated with excitation
wavelength and observed through a filter that excludes
excitation wavelength

Acridine Orange
Binds to DNA and flouresces Green
Binds to RNA and flouresces Orange
DAPI (4′,6-diamidino-2-phenylindole) is a blue-fluorescent
DNA stain that exhibits ~20-fold enhancement of
fluorescence upon binding to AT regions of dsDNA

DAPI - 4′, 6-diamidino-2-phenylindole

Endothelial cells stained with DAPI (Blue) and through
immunoflourescence via an antibody bound to flourescein
isothiocyanate (FITC) Green
 3. Flourescent Antibody Technique

Flourecent Antibody Technique can be used to locate

tumor antigens and intracellular viruses

Antibody is linked to a flourescent

tag and added to cells

Incubation

Washing

Flourescent Microscope
 4. Flourometric Enzyme Assays

Fluorometric assays use a difference in the fluorescence of

substrate from product to measure the enzyme reaction
These assays are in general much more sensitive than
spectrophotometric assays

Flourescein-di-β-D-galactopyranoside

β –galactosidase

Flourescein + β -D -galactopyranoside
 5. Quantitative Estimation of DNA

DNA sample is incubated with Ethidium Bromide

The Quantum Yield of Ethidium Bromide increases upon
binding with DNA
Flourescence is measured with different amount of known
DNA concentrations and a standard curve is plotted
The flourescence of the unkown DNA sample is measured
and its concentrtion is determined from the standard curve
5. Quantitative Estimation of DNA

Standard curve of DNA

Thank You