Welcome to BIO 260
Molecular Techniques
Unit 8 –
Spectrophotometry and Chromatography
Objectives for Spectrophotometry
& Chromatography Unit:
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Explain how a spectrophotometer works.
Contrast the difference between absorption and transmittance.
Explain the difference between using a spectrophotometer and a nanodrop.
Explain how chromatography works
Explain what is chromatography used for.
List three common methods of chromatography
MATC - BIO 260 - U1
Terms to know for the spectrophotometer:
 Transmittance
 Absorption
 Quality Assurance
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Principle of the spectrophotometer
The spectrophotometer is a very simple but
beautiful instrument. It is employed to measure
the amount of light that a sample absorbs. Simply
put, the instrument operates by passing a beam
of light through a sample and measuring the
intensity of light reaching a detector.
MATC - BIO 250 - U1
MATC - BIO 250 - U1
A spectrophotometer consists of two instruments:
 A spectrometer for producing light of any
selected color (wavelength)
 A photometer for measuring the intensity of
light.
MATC - BIO 250 - U1
Beer’s Law
When monochromatic light (light of a specific
wavelength) passes through a solution there
is usually a quantitative relationship (Beer's
law) between the solute concentration and the
intensity of the transmitted light, that is, the
more concentrated the specimen is, the less
light is transmitted through it.
MATC - BIO 250 - U1
Beer’s Law:
The concentration of a substance in directly
proportional to the amount of light absorbed or
inversely proportional to the logarithm of the
transmitted light.
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Transmittance:
% Transmittance is the ratio of the radiant energy
transmitted (T) divided by the radiant energy
incident on the sample (I):
%T=T/I x 100
0% T is all light absorbed
100%T is no light absorbed.
MATC - BIO 250 - U1
Before beginning, the electrical readout (of transmittance)
is set arbitrarily at 100%T with a “blank” in place. (The
blank is the solvent without the constituent).
Next the sample constituent (with the absorbing
molecules) is placed in the light path.
The difference in amount transmitted by the blank and the
sample is due only to the presence of the absorbing
molecules of the constituent.
%T = measurement of transmitted light/ blank measurement.
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Absorbtion:
 Light of a specific wavelength is used to pass
through a solution or suspension
 The light may be absorbed or scattered
 The amount of light that gets through is a
measure of the concentration of the solution or
suspension
 Absorption is the property of a solution to
absorb the light that comes to it
MATC - BIO 250 - U1
Absorbance is measured by a
spectrophotometer.
 The range of absorbance is 0 (no light
absorbed) to around 2 (all light absorbed).
 A spectrophotometer can be set for a specific
wavelength and provides an absorbance value
for a solution at that wavelength
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Light source visible: incandescent bulb
Light source UV: deuterium-discharge lamps
Monochromator : wave selecting device
Cuvette: round or square, come in sets, quartz must be
used for UV
Photoresistor: converts radiant energy to electrical energy
Amplifier:
MATC - BIO 250 - U1
http://viewpure.com/xHQM4BbR040
ncbionetwork a bit boring, but
decent, quick presentation of a Vis Spec
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Quality Assurance
3 important checks:
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Wavelength accuracy - the wavelength on the dial is the
wave length of the light passing through the monochromator.
Standard absorbing solutions or filters are used to check this.
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Stray light – Wavelengths that pass outside band transmitted by
the monochromator (scratches on surface of monochromator or
dust. The effect is absorbance errors. Correct with cutoff filters
which eliminate all radiation other than the one chosen by the
monochromator.
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Linearity - is evidenced by the straight-line calibration curve
from a change in concentration of solute. Linearity is effected by
stray light. Colored solutions can be used as standards.
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Chromatography
The Science of Separation
The separation of molecules based upon their
chemical and physical properties.
 Used for purification, isolation, and
characterization of inorganic, organic, and
biological compounds.
 There are many types of chromatography,
based upon molecular size, charge, and shape.
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Chromatography is commonly used in
biotechnology for purifying biological molecules,
like proteins, for medicine or other uses.
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Chromatography separates individual components
from complex mixtures.
MATC - BIO 250 - U1
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Chromatography consists of two phases:
 mobile phase (solvent and the molecules to be
separated)
 stationary phase either, in paper (in paper
chromatography) or glass beads, called resin, (in
column chromatography), through which the mobile
phase travels.
MATC - BIO 250 - U1
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Molecules travel through the stationary
phase at different rates because of their
chemistry.
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Some Common Types of
Chromatography
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Gel filtration chromatography
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Affinity chromatography
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Ion exchange chromatography
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High Performance Liquid Chromatography
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Principles of Size Exclusion Chromatography (SEC)
The mass of beads within the column is often referred to as the column bed. The
beads act as “traps” or “sieves” and function to filter small molecules which become
temporarily trapped within the pores. Larger molecules pass around, or are “excluded”
from, the beads. This is called fractionating. As the liquid flows through the column,
molecules below the chosen Dalton size enter the beads and pass through the column
more slowly. The smaller the molecules, the slower they move through the column.
Larger molecules pass around the beads and are excluded from the column—also
referred to as the exclusion limit of a column.
Sample
Column bed
Buffer
MATC - BIO 250 - U1
http://viewpure.com/Z54ec_G12QE column
chromatography by Gen Ed
http://viewpure.com/Hb791WsC78s affinity
http://viewpure.com/kz_egMtdnL4 HPLC
urd2urd
Royal Society
of Chemistry
http://www.ncbionetwork.org/chromatography/
interactive tutorial by BioNetwork – could be used as an assignment includes affinity, ion, size
chromatography
MATC - BIO 250 - U1
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The liquid used to dissolve the biomolecules to make
the mobile phase is usually called a buffer.
The mixture of biomolecules dissolved in the buffer is
called the sample.
The sample is placed on the column bed and the
biomolecules within the buffer enter the top of the
column bed, filter through and around the porous beads,
and ultimately pass through a small opening at the
bottom of the column.
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For this process to be completed additional buffer is
placed on the column bed after the sample has entered
the bed.
The mobile phase liquid is collected, as drops, into
collection tubes which are sequentially ordered.
A set number of drops is usually collected into each tube.
The larger molecules which pass quickly through the
column will end up in the early tubes or “fractions”. The
smaller molecules which penetrate the pores of the
stationary phase end up in the later fractions.
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http://viewpure.com/WBTO9skgVP4
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pretty simple, but could be used elsewhere???
MATC - BIO 250 - U1