CHAPTER 4

Techniques in
Biochemical Analysis

Learning Outcomes
State, write and explain the concepts
and theories in biochemical analysis.
Adapt the appropriate scientific methods and interpret the
respective data.

Display collaboration among teammates to disseminate

knowledge and awareness
Website (video) can be refer to:
https://www.khanacademy.org/test-prep/mcat/chemicalprocesses#separations-purifications

Topic Outlines
Introduction to biochemical analysis

Safety rules
Extraction methods
Chromatography principles and techniques
Example of chromatography techniques
Spectrophotometry*
Beers Law*

INTRODUCTION
Biochemical analysis techniques refer to a set of methods, assays
and procedures that enable scientist to analyze the substances
found in living organisms and the chemical reaction underlying life
processes.
To perform biochemical analysis of a biomolecule in a biological
system, there is a need to design a strategy to detect the biomolecule
& isolate it in pure form

BIOMOLECULE
Any molecule is produced by a living organism, including large
macromolecules as well as micromolecules such as primary
metabolites and natural products.

APPLICATIONS
Biochemical investigations are involved in every branch of clinical
medicine.
The result from the biochemical tests can be use in:
Diagnosis and in the monitoring of treatment.
Screening for disease or in assessing the prognosis.
Research into the biochemical basis of disease.
Clinical trials of new drugs.

Specialized tests:
Hormones

Specific proteins, Lipids and lipoproteins

Trace elements
Vitamins
Drugs

 DISTILLATION

SOLVENT EXTRACTION
SOLID-PHASE EXTRACTION

EXTRACTION METHOD
A technique to separate compound (analytes) from complex
sample matrices.
In other words - a way to separate a desired substance when it is
mixed with others.

Extractions use to separate the substance from two immiscible

phases.
Typical lab extractions - organic compounds out of an aqueous
phase and into an organic phase.

TECHNIQUES OF EXTRACTIONS:
1. DISTILLATION
2. SOLVENT EXTRACTION
3. SOLID-PHASE EXTRACTION

1. DISTILLATION
DISTILLATION: process of purify liquid by BOILING and
CONDENSING the vapour.
Distillation is a laboratory technique used for
SEPARATING and PURIFYING liquids.
Uses:
To separate volatile from non-volatile substances
To separate two or more volatiles liquids by exploiting
the different BOILING TEMPERATURES of liquids.

DISTILLATION
Instrument Set-up
A process in which a liquid or
vapour mixture of two or
more substances is
separated into its component
fractions of desired purity, by
the application and removal
of heat.

DISTILLATION The Principle

Distillation is based on the fact that the vapour of a boiling
mixture will be richer in the components that have lower
boiling points.
Liquid with lower boiling point will evaporate first and collected
in the collection flask.
When this vapour is cooled and condensed, the condensate
will contain more volatile components. At the same time, the
original mixture will contain more of the less volatile material.
Distillation columns are designed to achieve this separation
efficiently.

Important aspects:
Distillation is the most common separation technique
It consumes enormous amounts of energy, both in terms of
cooling and heating requirements

1. Simple distillation:
Distillation consists of boiling a liquid and
condensing the vapour in such way that the
condensate (distillate) is collected in a separate
container.

Most useful to separate a volatile liquid from a non

volatile impurity such as a dissolved solid.
The simple distillation process works best for liquids
with very different boiling points.

2. Fractional distillation
Has additional FRACTIONATING COLUMN filled with
beads that gives better separation between the
liquids.
Used to separate volatile liquids with a smaller
difference in boiling points.
Has a long tube that allows the liquid to vaporize
and condense SEVERAL TIMES as it hits cooler air
every time it makes its way further up the column.
E.g... - crude oil

Simple Distillation

Fractional Distillation

DISTILLATION - Application
In the fossil fuel industry distillation is a major class of
operation in obtaining materials from crude oil for fuels
and for chemical feedstock.
In industrial use distillation permits separation of air into its
components - notably oxygen, nitrogen, and argon.
In the field of industrial chemistry, large ranges of crude
liquid products of chemical synthesis are distilled to
separate them, either from other products, or from
impurities, or from unreacted starting materials.
Distillation of fermented products produces distilled
beverages with a high alcohol content, or separates out
other fermentation products of commercial value.

Distillation of Crude Oil

Distillation of Air

2. SOLVENT EXTRACTION
A method for separating mixtures by using the
differences in the SOLUBILITY of the components.
To extract substance from an aqueous phase, organic
solvent that immiscible with water must be used.
Among widely used solvent extraction are:
a) Soxhlet (solid-liquid)
b) Liquid-liquid extraction

Soxhlet Extraction
Solid-liquid extraction is often used to extract a solid natural
product from a natural source, such as plant.
SOXHLET EXTRACTOR is most commonly used as solid-liquid
extraction apparatus.
Soxhlet extraction involves placing sample in a
PAPER THIMBLE and placing the thimble in the SOXHLET
FLASK.

Soxhlet Extraction - Operation

The solid (sample) to be extracted (containing some of the
desired compound) is placed inside a THIMBLE made from
thick filter paper, and is loaded into the central chamber of
the Soxhlet extractor.
Solvent in the reservoir (round bottomed distilling flask) is
gently boiled to reflux.
The vapor rises through the sidearm into the water-cooled
CONDENSER where it liquefies. The condensation state (liquid)
drips into the thimble containing the solid (sample).
The hot solvent begins to fill the thimble and EXTRACT the desired
compound from solid.

Soxhlet Extraction - Operation

When the SIPHON tube level is REACHED, solvent in the
thimble is RETURNED to the reservoir (distillation flask).
The solvent flask now contains solvent and solute.
The VAPORIZATION-CONDENSATION-EXTRACTION-SIPHONING
process is repeated hundreds of times.

The desired PRODUCT is concentrated in the flask because the

product has a higher boiling point than solvent (solid state)

Soxhlet Instrument Setup

Soxhlet
Flask

Liquid-liquid Extraction
When product is very soluble in water, it is often difficult
to extract the desired compound.
Therefore, the aqueous solution need to be extracted
numerous times with immiscible organic solvent in order
to remove desired product from water.
Liquid-liquid extraction (LLE) involves ADDING A SOLVENT
to the sample that is immiscible, followed by
SEPARATING the desired compound (product/analytes)
between the two phases.
Separation occurs when the solutes have different relative
solubility in the two solvents used.
Organic solvent must have high distribution coefficient for
the product.

LLE - Operation
This extracting solvents is added to the sample, then the two
phases are AGITATED by vortexing or shaking.
After agitation, the phases are allowed to SEPARATE.

It is necessary to use an adequate amount of extracting

solvent to capture all of the desired compound (product)
from the original sample.
Since most pharmaceutical samples are aqueous, the
extracting solvent typically non-polar organics.
Usually applied in production of perfumes, fragrances and
essential oils.

LLE - Theoretical

LLE Instrument Setup

Limitations of SOLVENT EXTRACTION

Only for aqueous samples.
Relatively used large volume of solvents that
generate waste disposal problem.
Expensive and toxic solvents

Often manually performed and may require a back

extraction.

3. SOLID PHASE EXTRACTION

Solvent was replaced by SOLID PARTICLE
SURFACES (exp. powdered silica) that has
hydrophobic functional groups and act as the
EXTRACTING PHASE.
Has many advantages over SOLVENT
EXTRACTIONS such as use less solvent and more
efficient.

SOLID PHASE MICROEXTRACTION (SPME)

Is a solvent-less extraction
technique.
Suitable for analyte collection
for determination by GC.
Equipped with silica fiber for
solid, liquid or gaseous sample

 WHAT IS CHROMATOGRAPHY
TYPES OF CHROMATOGRAPHY

CHROMATOGRAPHY
DEFINITION
CHROMATOGRAPHY is a process whereby the
Purity of a sample may be determined.
The components of a mixture may be separated.

The separated components may be identified.

INTRODUCTION
Chromatography is a method for separating the
substances in a mixture.
Used to separate various components of analytical
samples to be determined.

The method was first applied to the separation of

colored substances.
(Greek: chromos = color , graphy = write).

Today, the method is applied to all kinds of mixtures.

CHR Standard
The STANDARD will be used for
comparison with the test sample
and the chromatograms.
STANDARD should contain either:
A single pure compound or
A mixture of known amounts of
several compounds.

CHR Sample (mixture)

A sample that requires analysis is often a mixture of
many components in a complex matrix.
A mixture can be separated using the differences in
PHYSICAL or CHEMICAL properties of the individual
components.
Physical Properties
Based on the states of matter of the two components.
Separations based on density and size.

Chemical Properties
Can be separated by: solubility, boiling point and vapor
pressure.

CHR Features
General, versatile method for separation of
components in a mixture.
Can identify the unknown by comparing with
reference sample (standard)
No restriction on the sample type organic,
inorganic, biological or medical.
High sensitivity detection of small amount g or less
(10-6g)

CHR Essential Components

CHROMATOGRAPHY is a physical separation method of
components in a sample by distribution of the components
between two phases, one that is stationary (STATIONARY
PHASE) and one that moves (MOBILE PHASE).

Stationary Phase

Mobile Phase

Sample Mixture

CHR Essential Components

Stationary Phase
is the SUBSTANCE which is fixed in place for the
chromatography procedure.
is the phase to which SOLVENTS and the ANALYTE
travels through or binds to.
A solid/liquid which does not move.

Mobile Phase
is the ANALYTE and SOLVENT MIXTURE which travels
through the stationary phase.
A gas/liquid that pass through column

CHR Essential Components

The separation process involving the INTERACTION of
one or more solutes and two phases.
SAMPLE COMPONENTS are carried by MOBILE PHASE
through a bed of STATIONARY PHASE.

Individual species are retarded by the stationary

phase based on various INTERACTION such as:
Surface adsorption

Relative solubility
Charge

CHR SP vs. MP
What does it do?
What physical
form?
How does it affect
sample
movement?
Analogous to
Example

STATIONARY PHASE

MOBILE PHASE

Stay put / fix

Fine solid with
lots of surface

Flows through SP
Fluid (Liquid / Gas)

Retain sample by
surface interaction

Moves samples along

An obstacle course
Silica gel (SiO2)x

Motivating force
Ethyl acetate

CHR Analogy

CHR Terms
The ANALYTE is the substance which is to be purified
or isolated during chromatography
A CHROMATOGRAPH takes a chemical mixture
carried by liquid or gas and separates it into its
component parts as a result of differential
distributions of the solutes as they flow around or
over the stationary phase.
The visual output of chromatograph.

Different peaks or patterns on the chromatogram correspond

to different components of the separated mixture.

CHROMATOGRAPHY
PREPARATIVE CHROMATOGRAPHY
Used to nondestructively purify sufficient quantities
of a substance for further use, rather than analysis.
ANALYTICAL CHROMATOGRAPHY
Used to determine the identity and concentration of
molecules in a mixture.

CHR Retention Time

RETENTION TIME
is the characteristic time that takes for a particular
molecule to pass through the system under set
conditions.
Can be calculated using this formula:

8 Types of Chromatography
Paper Chromatography (PC)

Liquid Chromatography
(LC)
Gas Chromatography
(GC)
Ion Exchange Chromatography
(IEC)

Thin Layer Chromatography

(TLC)
High-Performance Liquid
Chromatography (HPLC)
Size-exclusion Chromatography
(SEC)

Affinity Chromatography (AC)

1. Paper Chromatography (PC)

This is the simplest form of
chromatography.
The STATIONARY PHASE is a
special chromatography
paper.
The MOBILE PHASE is a
solvent mixture.
E.g.. water and ethanol.

Trial and error establishes

the best solvent for good
resolution.

Paper Chromatography

1. Paper Chromatography (PC)

The mixture under analysis is placed
in a tiny, concentrated dot near the
bottom of the paper.
The paper is hung with the bottom
dipped in solvent, which rises up the
paper to come in contact with the
mixture.
As the solvent rises further up the
paper, the components are
separated as they are swept along.
The strip of paper is called a
chromatogram.

1. Paper Chromatography (PC)

Identification of the components is based on Rf values
a ratio between the distance traveled by the
component to the distance traveled by the solvent
front.

Application
While chlorophyll is the chief pigment responsible for
photosynthesis in green plants, many plants contain other
colored pigments as well.

Before

These chlorophyll and colored pigments may be separated

according to their various chemical charges by a technique
known as chromatography.

In this technique, a mixture of plant pigments is separated

by placing a drop or two of pigment on a special paper
called CHROMATOGRAPHY PAPER which is dipped in a
chemical allowing the different plant pigments to move
based on their charges.
A picture of a completed chromatography (right).

After

2. Thin Layer Chromatography (TLC)

Thin Layer Chromatography

Thin-layer chromatography
(TLC) is useful for separating
organic compounds.
A simple and rapid method
used to monitor the progress
of organic reactions and to
check the purity of products
The MOBILE PHASE is a solvent.
The STATIONARY PHASE is a
solid adsorbent on a flat
support.

2. Thin Layer Chromatography (TLC)

Thin-layer chromatography
consists of a stationary phase
immobilized on a glass or
plastic plate, and an organic
solvent.

Capillary tubing

The sample, either liquid

dissolved in a volatile solvent,
is deposited as a spot on the
stationary phase.

2. Thin Layer Chromatography (TLC)

The constituents of a sample can be
identified by simultaneously running
standards with the unknown.
The bottom edge of the plate is
placed in a solvent reservoir, and the
solvent moves up the plate by
capillary action.
When the solvent front reaches the
other edge of the stationary phase,
the plate is removed from the solvent
reservoir.

Solvent
Reservoir

2. Thin Layer Chromatography (TLC)

The separated spots are visualized with
ultraviolet light or by placing the plate
in iodine vapour.
The different components in the
mixture move up the plate at different
rates due to differences in their
partitioning behavior between the
mobile liquid phase and the stationary
phase.

Glass plate with silica

Magnification 500X

Typical TLC
Chromatograms

Application
Thin-Layer chromatography (TLC) is a very useful technique
for the rapid separation and qualitative analysis of
small amounts of material.
It is often used to check the purity of organic compounds
and to monitor the progress of a reaction.

The analysis of the location, size, color, and form of the

various spots formed on the plate provides information
about the chemical identity of the components of the
sample.

2. Thin Layer Chromatography (TLC)

Silica gel - silicon dioxide (SiO2)x
(common, inexpensive stationary phase)
O
O
O
|
|
|
OSiOSiOSiOH These exposed OH units
|
|
|
give silica gel a
O
O
O
relatively polar surface.
|
|
|
OSiOSiOSiOH
|
|
|
O
O
O

bulk (SiO2)x

surface

TLC plate
5 x 10 cm. TLC slide
250 mm silica gel layer
impregnated with a
fluorescent indicator,
on a plastic backing

2. Thin Layer Chromatography (TLC)

Four Stages in TLC
i.

Sample Application

: Capillary used to spot solution of

each sample.

ii. Development

: This is when the separation

actually occurs.

iii. Visualization

: Our results will be viewed,

e.g. under UV light.

iv. Interpretation of Results : Comparison of retention factors.

i.

Sample Application (spotting)

A. Draw guide lines

lightly with pencil

TLC plate
finishing line

1 cm.

B. Dissolve solid
sample in solvent
(e.g. CH2Cl2 )
C. Use TLC capillary
to transfer and spot
dissolved sample

starting line

1 cm.
ACE ASP CAF ACE ASP CAF
#5
#5
#5 Ref. Ref. Ref.

ii.

Development of TLC Plate

A. Place spotted TLC plate

in developing chamber

{keep capped}

TLC plate

B. Developing solution
is drawn up the plate
by capillary action
C. Remove TLC plate when
solvent reaches top line
NOTE: During this ~20 min.
developing stage, compounds
in the original spots are being
pulled through the silica gel.

Developing
solution
(mobile phase)

}
TLC Developing Chamber
(just a glass jar with solvent in it)

iii. Visualization of TLC Results

A. Allow solvent to evaporate
from surface of TLC plate.
B. View results under UV light.
look for grayish spots on the
fluorescent green background

C. Mark spots with a pencil

while viewing under UV.

UV

iv. Interpretation of TLC Results

A. Determine retention factors
(Rf) for each spot detected.
Rf =

Rf =

distance spot ___________

has moved
____________
distance solvent has moved

distance spot has moved

_______________________
distance solvent has moved

X
Y

Z
Y

Y
Z
X

Rf =

distance spot has moved

_______________________
distance solvent has moved

T
Y

iv. Interpretation of TLC Results

Rf =

distance spot has moved

_______________________
distance solvent has moved

X
Y

B. Use Rf s of reference spots to

identify the other components.
Finally:

How do you identify

the unknown tablets?

UNK

asp acet caf

std std std

3. Liquid Chromatography (LC)

LC is an analytical chromatographic technique that is
useful for separating ions or molecules that are
dissolved in a solvent.
Conventional LC is most commonly used in
preparative scale work to purify and isolate some
components of a mixture.
It is also used in ultra trace separations where small
disposable columns are used once and then
discarded.

3. Liquid Chromatography (LC)

Used to separate analytes in solution including metal ions
and organic compounds.

The MOBILE PHASE is a solvent.

The STATIONARY PHASE
is a liquid on a solid
support, a solid, or
an ion-exchange resin.

Application
LC for protein
purification
BioLogic DuoFlow System
from Bio-Rad

Liquid Chromatography

4. High-Performance Liquid
Chromatography (HPLC)
HPLC is a variation of liquid chromatography that
utilizes high-pressure pumps to increase the
efficiency of the separation.
The MOBILE PHASE can be a polar liquid, which
moves slowly through the column.
Suited to prepare relatively large quantities of a
pure compound or identifying the presence of
very small quantities.
HPLC can be used to separate components
unsuited to GC.

High Performance
Liquid Chromatography

4. High-Performance Liquid
Chromatography (HPLC)
HPLC is a form of column chromatography used
frequently in biochemistry and analytical chemistry.
The analyte is forced through a column (STATIONARY
PHASE) by a liquid (MOBILE PHASE) at high pressure.
HPLC distinguished from conventional LC by:
sophisticated instrumentation and high efficiency columns

ADVANTAGES such as:

increased speed
higher resolution
higher sensitivity

better recovery
better reproducibility

5. Gas Chromatography (GC)

Gas Chromatography consists of
Flowing MOBILE PHASE.
An injection port.
A separation column containing the STATIONARY
PHASE.
A detector.

The organic compounds are separated due to

differences in their partitioning behavior between
the mobile gas phase and the stationary phase in
the column.
Applied to volatile organic compounds.

5. Gas Chromatography (GC)

The MOBILE PHASE is a gas.
The STATIONARY PHASE is usually a liquid on a solid
support or sometimes a solid adsorbent.

GC column

5. GC

MOBILE PHASES are generally inert gases such as

helium, argon, or nitrogen.
Injection port: consists of a rubber septum through
which a syringe needle is inserted to inject the
sample.
Used to separate volatiles, non-polar compounds.

5. Gas Chromatography (GC)

The readout shows the:
order of elution (order of components coming off the column)
time of elution (retention time)
relative amounts of the components in the mixture.

The order of elution is related to the boiling points and

polarities of the substances in the mixture.
In general, they elute in order of increasing boiling point
but occasionally the relative polarity of a compound will
cause it to elute "out of order".
This is why it is important to run a standard before using
your sample.

Compound

Boiling Point
( C)
0

The observed elution pattern appears.

Notice the reversed elution of toluene and
4-methyl-2-pentanone

pentane

36

hexane

69

cyclohexane

80

isooctane
(2,2,4-trimethylpentane)

99

toluene

110

4-methyl-2-pentanone

117

octane

126

6. Size Exclusion Chromatography (SEC)

GEL FILTRATION CHROMATOGRAPHY
Also known as:
molecular sieving
gel permeation

Achieve rapid separation of molecules

based on size.
GEL:
is in bead form for easy column packing.
consists of an open, cross-linked 3-D
molecular network of pores and
channels into which molecules of less
than maximum pore size may penetrate.

6. Size Exclusion Chromatography (SEC)

GEL FILTRATION CHROMATOGRAPHY
Used to separate mixtures of MACROMOLECULES.
Particularly enzymes, antibodies and other globular
proteins.

For protein purification, it is desirable to have proteins

of interest to be well separated from unwanted
proteins.
The MOBILE PHASE is a solvent.
The STATIONARY PHASE is a packing of porous
particles.

6. Size Exclusion Chromatography (SEC)

GEL FILTRATION CHROMATOGRAPHY
Choice of Buffer:
The most important when choosing a buffer for gel
filtration, make sure it is compatible with target
molecule.
Beware of dissociating agents and detergents that can:
Induce conformational change.
Inactivate biologically active.
Dissociate proteins into subunits.

Sodium phosphate buffer and Tris-HCl are used to

control pH.

6. Size Exclusion Chromatography (SEC)

GEL FILTRATION CHROMATOGRAPHY
Choice of column
Theoretically, resolution increase with column length

Choice of gel matrix (3 Common gel matrix)

Sephadex (Pharmacia, is a cross-linked dextrans)

Polyacrylamide beads (Biogels, Bio-rad)
Agarose (Sepharose)

Pores within beads:

are of such sizes that are not accessible to large
molecules, but smaller molecules can penetrate
all pores.
Extent to which molecules can enter pores
depends on its shape and MW
Hence separation depends on difference in ability
of various molecules to enter (penetrate pores).
As solvent moves through the column, 3 possible
things can happen to solutes in sample:
Can run with solvent front
Be washed out quickly
Totally adsorbed on to gel matrix

Very large molecules that are too large to

enter the pores move through the
chromatographic bed are THE FASTEST.
Smaller molecules which enter the gel pores
move MORE SLOWLY since they go in and
out of the beads as they travel down the
column.
Hence, molecules are eluted in order of their
decreasing size.
Results are monitored as liquid exits from
column, usually by monitoring using UV
absorbance at 280 nm.

Size Exclusion Chromatography

7. Ion Exchange Chromatography (IEC)

Separation based on charges
STATIONARY PHASE: ion-exchange resin, also
depends on buffer pH
Exploits amphoteric character of protein.
Packing materials or support medium have
functional group that is covalently bonded.

7. Ion Exchange Chromatography (IEC)

The test substances are strongly bound by
electrostatic attraction to the ion-exchange resin
on passage through the system, other
components will be eluted.
Two types of IEC:
1. Cation exchanger
2. Anion exchanger

_
_

(-) charged functional groups

attached to support matrices
(packing materials)
(+) charged proteins bind to
cation exchanger

_
_

_
_

_
+

_
_
_

Cation exchanger

CATION EXCHANGER

Application
Water softening process.
Removed magnesium and
calcium ion.

(+) functional group act as

anion exchanger
(-) charged proteins bind to
anion exchanger by reversible
electrostatic interactions

_
_

+ +
+

+
+

Anion exchanger

Application

ANION EXCHANGER

8. Affinity Chromatography (AC)

This technique in general make use of a bio-specific
interactions that results in a change in properties of
protein such that it can be separated from other
proteins.
When protein solution is passed through such column,
only the proteins that can bind the ligand will be
retained on the column, while the other mixture will
passes through the column.
Then, the conditions can be adjusted to release
proteins of interest from the ligand. Often this can be
done simply by eluting with the soluble version of the
ligand.

Example: purification of
staphylococcal nuclease by
affinity chromatography on
bisphosphothymidine-linked
agarose.

8. Affinity Chromatography (AC)

The goal : is to separate molecules of a particular specificity in
a mixture such as a blood serum.
E.g..: Antibodies in a serum sample specific for a particular
antigenic determinants.
Step 1
An immunoadsorbent is prepared. Consists: solid matrix to which
the antigen (shown in blue) has been coupled (usually
covalently). Agarose, sephadex, derivatives of cellulose, or other
polymers can be used as the matrix.

Step 2
The serum is passed over the immunoadsorbent. Antibodies in the
mixture specific for the antigen (shown in red) will bind
(non-covalently) and be retained.
Antibodies of other specificities (green) and other serum proteins
(yellow) will pass through unimpeded.

Step 3

Elution.
A reagent is passed into the column to release the antibodies from the
immunoadsorbent.
Buffers containing a high concentration of salts and/or low pH are often used
to disrupt the non-covalent interactions between antibodies and antigen.
A denaturing agent, such as 8 M urea, will also break the interaction by
altering the configuration of the antigen-binding site of the antibody
molecule.

Another, gentler, approach is to elute with a soluble form of the antigen.

These compete with the immunoadsorbent for the antigen-binding sites of
the antibodies and release the antibodies to the fluid phase.

Step 4
Dialysis. The eluted antibodies is then dialyzed against, for example, buffered
saline in order to remove the reagent used for elution.

SPECTROPHOTOMETRY
SPECTROPHOTOMETRY
is measuring intensity of light in a part of the spectrum
specially as transmitted or emitted by particular substances

This is measured using a spectrophotometer.

SPECTROSCOPY
is the study of matter and its properties by investigating
light, sound, or particles that are emitted, absorbed or
scattered by the matter under investigation.

Often used for the identification of substances through

the spectrum emitted from them or absorbed in them.

Spectrum
A spectrum is a plot of the intensity of energy
detected versus the wavelength of the energy.
(or mass or momentum or frequency, etc.)

SPECTRO - Application
A spectrum can be used to obtain information about:

Atomic and molecular energy levels

Molecular geometries
Chemical bonds
Interactions of molecules and related processes
The components of a sample (qualitative analysis)
Measure the amount of material in a sample
(quantitative analysis)

SPECTRO - Instrument
An energy source
(commonly a laser, ion source or radiation source).

A device for recording a spectrum

(often a spectrophotometer or spectrometer).

Spectrometer or Spectrophotometer
1. Spectrophotometer:
Instrument used to measure the intensity of wavelengths
in a spectrum of light compared with the intensity of
light from a standard source.
An instrument which measures the amount of light of a
specified wavelength which passes through a medium.
An instrument that will resolve polychromatic (white
light) radiation into different wavelengths.
Different materials will absorb different amounts of light
depending on the wavelengths of the light.

1. Spectrophotometer:
Basic Components of Instrument
Power
Supply

Analytical
Signal
Signal
Generator

Flame

Light

Detector /
Transducer
Photomultiplie
r Tube

Electrical /
Mechanical Signal

Current

Signal
Processor

Signal
Output

Recording
Device

1. Amplify
2. Filter
3. Current to
Voltage

DC
voltage

1. Strip chart
recorder
2. Integrator
3. PC

1. Spectrophotometer
A spectrometers contain:
A source of continuous radiation over the wavelengths
of interest.

A monochromator for selecting a narrow band of

wavelengths from the source spectrum.
A sample cell.

A detector for converting radiant energy into electrical

energy
A device to read out response of detector.

1. Spectrophotometer:
SOP
1. In spectrometric methods, the sample solution
absorbs electromagnetic radiation from an
appropriate source.
2. The amount absorbed is related to the
concentration of the analyte in the solution.
3. The colour of an object we see is due to the
wavelengths transmitted of reflected. The other
wavelengths are absorbed.

1. Spectrophotometer:
Application (Biological Sample)
1. Spectrophotometer for semen counting.
2. To count quantity of DNA and proteins.

3. To determine concentration of compounds in plant

samples.
E.g. Vitamin C (ascorbic acid), Anthocyanin etc.

2. Spectroscopy:
Types
Different types of spectroscopy use different
measurement processes.
3 main types of spectroscopy:
1. Absorption spectroscopy
Uses the range of electromagnetic spectra in
which a substance absorbs.
The method can be automated and is widely
used to measure concentrations of ions.
Such as sodium and calcium in blood.
Example: Ultraviolet/Visible (UV/Vis) Absorption
Spectroscopy and Infrared (IR) Spectroscopy.

2. Spectroscopy:
UV Spectrometer

SmartSpec 3000
from Bio-Rad

From left to right: the housing

for the infrared laser
interferometer arrangement
(EM source); the open sample
compartment; monitor;
computer console.

Perkin Elmer Paragon 5000 FT-IR

spectrophotometers.

2. Spectroscopy:
Types
2. Emission spectroscopy
Uses the range of electromagnetic spectra in which a
substance radiates.

3. Scattering spectroscopy
Measures certain physical properties by measuring the
amount of light that a substance scatters at certain
wavelengths, incident angles, and polarization angles.
The scattering process is much FASTER than the
absorption/emission process.

Example of light scattering spectroscopy is

Raman Spectroscopy.

CCD Raman Spectrophotometer

Spectronic 20

cuvette for sample

Spectrophotometer 22

2. Spectrometer:
SOP Setup
1. Check that the instrument is turned on.
The left-hand knob should be turned
clockwise. Allow 10 - 30 minutes for
warming up.
2. Set the wavelength to the desired value
using the knob on the top. The colour of
an object we see is due to the
wavelengths transmitted of reflected.
The other wavelengths are absorbed.

2. Spectrometer:
SOP Calibrate
3. Set 0 transmittance with empty, closed
sample compartment, turn the left-hand
knob to obtain a reading of 0% T.
4. Use the mirror behind the needle to
avoid parallax error.

5. Wipe the cuvette with a dry Kim wipes to

remove drops of solution or finger prints.

2. Spectrometer:
SOP Calibrate
6. Line up the mark on the cuvette with the
line on the sample compartment.

7. Insert cuvette filled with solvent in the

sample compartment

8. Close the cover. Turn the right-hand

knob to obtain a reading of 100% T.

2. Spectrometer:
SOP Analyzing

9. To analyze your sample, insert sample cuvette and

read the Absorbance value on the scale.
Use the mirror behind the needle to avoid parallax
error.

Beers Law

Beers Law
Beers Law quantifies the relationship between
color and concentration.

Beers Law states that the absorbance of light

by a solution is directly proportional to:
Emissivity
Cell width
Concentration

Beers Law - Quantitative Spectroscopy

Beers Law
Al1 = el1bc

where;
A is absorbance

e is molar absorptivity (unique for a given

compound at l1)
b is path length

c is concentration

Beers Law
slit

cuvette

source
detector

A = -log T = log(P0/P) = ebc

T = Psolution/Psolvent = P/P0
Works for monochromatic light
Compound x has a unique e at different wavelengths

Beers Law - Characteristics

One wavelength
GOOD plots have a range of absorbance from
0.010-1.000.

Absorbance over 1.000 are NOT VALID and should

be avoided.
2 orders of magnitude