Chapter 4 bio 300 obe

1

Chapter 4: Techniques in
Biochemical Analysis

BIO 300

BIOLOGICAL TECHNIQUES AND SKILLS
SARINI BINTI AHMAD WAKID
FACULTY OF APPLIED SCIENCE


2

CHAPTER 4
Techniques in Biochemical
Analysis


3

What is Chromatography?
Chromatography is a technique for separating
mixtures into their components in order to analyze,
identify, purify, and/or quantify the mixture or
components.

• Analyze
Separate

• Identify
• Purify

Mixture

Components
Chapter 4: Techniques in Biochemical
Analysis

• Quantify
4

Chromatography









Chromatography is a method of separating a mixture of
molecules depending on their distribution between a
mobile phase and a stationary phase.
The mobile phase (also known as solvent) may be either
liquid or gas.
The stationary phase (also known as sorbent) can be
either a solid or liquid, a liquid stationary phase is held
stationary by a solid.
The solid holding the liquid stationary phase is the
support or matrix.
The molecules in the mixture to be separated are the
solutes.
Analysis

5

Uses for Chromatography
Chromatography is used by scientists to:
• Analyze – examine a mixture, its components,
and their relations to one another

• Identify – determine the identity of a mixture or
components based on known components

• Purify – separate components in order to isolate
one of interest for further study

• Quantify – determine the amount of the a mixture
and/or the components present in the sample


6

Uses for Chromatography
Real-life examples of uses for chromatography:
• Pharmaceutical Company – determine amount of each
chemical found in new product

• Hospital – detect blood or alcohol levels in a
blood stream

patient’s

• Law Enforcement – to compare a sample found at a crime
scene to samples from suspects

• Environmental Agency – determine the level of
in the water supply

• Manufacturing Plant
make a product

– to purify a chemical

Analysis

pollutants
needed to

7

Definition of Chromatography
Detailed Definition:

Chromatography is a laboratory technique that
separates components within a mixture by using the
differential affinities of the components for a mobile
medium and for a stationary adsorbing medium through
which they pass.

Terminology:

• Differential – showing a difference, distinctive
• Affinity – natural attraction or force between things
• Mobile Medium – gas or liquid that carries the components
(mobile phase)

• Stationary Medium – the part of the apparatus that does
not move with the sample (stationary phase)
Analysis

8

Definition of Chromatography
Simplified Definition:
Chromatography separates the components of a
mixture by their distinctive attraction to the mobile
phase and the stationary phase.

Explanation:
•
•
•
•

Compound is placed on stationary phase
Mobile phase passes through the stationary phase
Mobile phase solubilizes the components
Mobile phase carries the individual components a
certain distance through the stationary phase,
depending on their attraction to both of the
phases
Analysis

9

Illustration of
Chromatography
Stationary Phase

Separation

Mobile Phase

Mixture

Components

Components

Affinity to Stationary
Phase

Affinity to Mobile
Phase

Blue

----------------

Insoluble in Mobile Phase

Black









Red
Yellow


        

10


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Types of Chromatography
• Liquid Chromatography – separates liquid samples
liquid solvent (mobile phase) and a column
(stationary phase)

with a
composed of solid beads

• Gas Chromatography – separates vaporized samples with a
carrier gas (mobile phase) and a column
of solid beads (stationary
phase)

composed of a liquid or

• Paper Chromatography – separates dried liquid
with a liquid solvent (mobile phase) and a
phase)

samples
paper strip (stationary

• Thin-Layer Chromatography – separates dried liquid
samples with a liquid solvent (mobile phase) and a glass
covered
with a thin layer of alumina or silica gel
(stationary phase)
Analysis

plate

12

Types of chromatography
•
•
•
•

Partition chromatography
Adsorption chromatography
Gel filtration
Ion exchange chromatography


13

(A) uses charge, (B) uses pores, and
(C) uses covalent bonds to create the
differential affinities among the mixture
components for the stationary phase.

14

Partition chromatography
• The distribution of solutes between
two immiscible phases.
• The solute will distribute it self
between the two phases according to
its solubility in each phase, this is
called partitioning.


15

Examples of partition
chromatography









The two most common types of partition chromatography are thin
layer chromatography and paper chromatography.
In both cases the stationary phase is a liquid bound to a matrix.
In paper chromatography the stationary phase are water molecules
bound to a cellulose matrix.
In TLC, the stationary phase is the solvent added to the support to
form the thin layer so the solvent gets bound to the matrix
(support).
Partition chromatography is mainly used for separation of
molecules of small molecular weight.

Analysis

16

Paper chromatography
• The cellulose support contains a large
amount of bound water.
• Partitioning occurs between the
bound water which is the stationary
phase and the solvent which is the
mobile phase.


17

Experimental procedure for paper
chromatography














A small volume of a solution of a mixture to be separated or identified is
placed at a marked spot (origin) on a sheet or strip of paper and allowed to
dry.
The paper is then placed in a closed chamber and one end is immersed in a
suitable solvent.
The solvent is drawn (moved) through the paper by capillary action.
As the solvent passes the origin, it dissolves the sample and moves the
components in the direction of flow.
After the solvent front has reached a point near the other end of the paper,
the sheet or strip is removed and dried.
The spots are then detected and their positions marked.
The ratio of the distance moved by a solute to the distance moved by the
solvent = Rf.
The Rf. is always less than one.
Analysis

18

Chromatogram









Once a sample is applied on TLC or paper, it’s called
chromatogram.
Paper chromatogram can be developed either by
ascending or descending solvent flow.
Descending chromatography is faster because gravity
helps the solvent flow.
Disadvantages : it’s difficult to set the apparatus.
Ascending is simple and inexpensive compared with
descending and usually gives more uniform migration
with less diffusion of the sample "spots".

Analysis

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Detection of spots


1.
2.
3.

4.

Spots in paper chromatograms can be detected in 4
different ways:
By their natural color
By their fluorescence
By their chemical reactions that take place after the
paper has been sprayed with various reagents for
example: during paper chromatography of amino acids,
the chromatograms are sprayed with ninhydrin.
By radioactivity

Analysis

20

Identification of spots
• The spots are usually identified by
comparing of standards of known Rf
values.


21

Thin layer chromatography










Paper chromatography uses paper which can be
prepared from cellulose products only.
In TLC, any substance that can be finely divided
and formed into a uniform layer can be used.
Both organic and inorganic substances can be
used to form a uniform layer for TLC.
Organic substances include: cellulose, polyamide,
polyethylene
Inorganic: silica gel, aluminum oxide and
magnesium silicate
Analysis

22

TLC
• The stationary phase is the solvent
used to form a layer of sorbent
spread uniformly over the surface of
a glass or plastic plate


23

Advantages of TLC over
paper chromatography
• Greater resolving power because
there is less diffusion of spots.
• Greater speed of separation
• Wide choice of materials as sorbents


24

The separation of compounds by
chromatography depends on several factors:
 Partition of a solute between a moving
solvent phase and a stationary aqueous
phase. The solute moves in the direction of a
solvent flow at a rate determined by the
solubility of the solute in the moving phase.
Thus a compound with high mobility is more
attracted to the moving organic phase than to
the stationary phase.

Analysis

25

Cont..




Ion exchange effect: any ionized impurities in
the support medium will tend to bind or attract
oppositely charged ions (solutes) and will
therefore reduce the mobility of these solutes.
Temperature: Since temperature can effect
the solubility of the solute in a given solvent
temperature is also an important factor.

Analysis

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





The molecular weight of a solute also affects the
solubility and hence chromatographic performance.
Adsorption of compound (solute) onto support medium:
Although the support medium (silica gel) is theoretically
inert, this isn't always the case. If a solute tends to bind
to the support medium this will slow down its mobility in
the solvent system.
The composition of the solvent: since some compounds
are more soluble in one solvent than in the other, the
mixture of solvents used will affect the separation of
compounds.

Analysis

27

Expression of the results


The term "Rf" (relative flow) is used to
express the performance of a solute in a
given solvent system /support medium. The
term Rf value may be defined as the ratio of
the distance the compound migrates to the
distance the solvent migrates. Rf value is
constant for a particular compound, solvent
system and insoluble matrix.
Rf= Distance of migration of solute
Distance moved by solvent
Analysis

28

Rf values


qualitative results of TLC






expressed as fractions of 1.0
can be expressed from Rf values (eg Rf x 100)
no more than two decimal places
 due to inaccuracy of physical measurement

may not be reproducible





only give an indication of possible nature of unknown
complete identification only obtained if spot is eluted and
micro-scale physical measurements done (MS, UV, IR)

standard references should always be used on
same plate for comparison



most sprays produce differential colours of fluorescence
colour test provides extra evidence with distance
migration
Analysis

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Analysis

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Principles of Paper Chromatography

• Capillary Action – the movement of liquid within the spaces
of a porous material due to the forces of adhesion, cohesion,
and surface tension. The liquid is able to move up the filter
paper because its attraction to itself is stronger than the
force of gravity.

• Solubility – the degree to which a material (solute) dissolves
into a solvent. Solutes dissolve into solvents that have similar
properties. (Like dissolves like) This allows different solutes
to be separated by different combinations of solvents.
Separation of components depends on both their solubility in
the mobile phase and their differential affinity to the mobile
phase and the stationary phase.


31

Paper Chromatography Experiment

What Color is that Sharpie?


32

Overview of the
Experiment
Purpose:

To introduce students to the principles and
terminology of chromatography and
demonstrate separation of the dyes in
Sharpie Pens with paper chromatography.

Time Required:

Prep. time: 10 minutes
Experiment time: 45 minutes


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•
•
•
•
•
•
•
•
•
•
•

6 beakers or jars
6 covers or lids
Distilled H2O
Isopropanol
Graduated cylinder
6 strips of filter
paper
Different colors of
Sharpie pens
Pencil
Ruler
Scissors
Tape

Materials List


34

Preparing the Isopropanol
Solutions
• Prepare 15 ml of the following isopropanol solutions
in
appropriately labeled beakers:
- 0%, 5%, 10%, 20%, 50%, and 100%


35

Preparing the
Chromatography Strips
• Cut 6 strips of filter
paper
• Draw a line 1 cm above
the bottom edge of the
strip with the pencil
• Label each strip with its
corresponding solution
• Place a spot from each
pen on your starting line


36

Developing the
Chromatograms

• Place the strips in the
beakers
• Make sure the solution
does not come above your
start line
• Keep the beakers covered
• Let strips develop until
the ascending solution
front is about 2 cm from
the top of the strip
• Remove the strips and let
them dry


37

Developing the
Chromatograms


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Developing the
Chromatograms


39


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Observing the
Chromatograms

0%

20%

50%

70%

100%

Concentration4:of Isopropanol
Chapter Techniques in

41

Black Dye
1. Dyes separated – purple and black
2. Not soluble in low concentrations
of isopropanol
3. Partially soluble in concentrations
of isopropanol >20%

0%

20%

50%

70%

Concentration of Isopropanol

100%
42

Blue Dye
1. Dye separated – blue
2. Not very soluble in low
concentrations of isopropanol
3. Completely soluble in high
concentrations of isopropanol

0%

20%

50%

70%

Concentration of Analysis
Biochemical Isopropanol

100%
43

Green Dye
1. Dye separated – blue and yellow
2. Blue – Soluble in concentrations
of isopropanol >20%
3. Yellow – Soluble in concentrations
of isopropanol >0%

0%

20%

50%

70%

Concentration of Analysis
Biochemical Isopropanol

100%
44

Red Dye

1. Dyes separated – red and yellow
2. Yellow –soluble in low concentrations of isopropanol and
less soluble in high concentrations of isopropanol
3. Red – slightly
soluble in low
concentrations
of isopropanol,
and more
soluble in
concentrations
of isopropanol
>20%

0%

20%

50%

70%

Concentration of Isopropanol

100%
45

Alternative Experiments
• Test different samples:
– Other markers, pens, highlighters
– Flower pigments
– Food Colors
• Test different solvents:
– Other alcohols: methanol, ethanol,
propanol, butanol
• Test different papers:
– Coffee filters
– Paper towels
– Cardstock
– Typing paper


46



47



48



49

Chromatography Instruments


50

• Chromatography techniques

• Affinity Chromatography (AC)
• Hydrophobic Interaction Chromatography (HIC)
• Ion Exchange Chromatography (IEC)
• Gel Filtration (GF)
• Capillary electrochromatography (CEC)


51

Affinity Chromatography
Surface bound with
Epoxy, aldehyde or aryl ester groups

Metal Interaction Chromatography
Surface bound with
Iminodiacetic acid + Ni2+/Zn2+/Co2+

(Christian G. Huber, Biopolymer

Chromatography, Encylcopedia

52

in analytical chemistry, 2000)

Metal Interaction Chromatography (AC)

Points to Note:
1.

Avoid chelating agents

2.

Increasing incubation time

3.

Slow gradient elution

(www.qiagen.com)

53

Binding Capacity (mg/ml) medium
12mg of histag proteins (MW= 27kDa)
Depends on Molecular weight

Degree of substitution /ml medium
~15µmol Ni2+
Backpressure ~43psi
Change the guard column filter

Analysis

54
(Christian G. Huber, Biopolymer Chromatography, Encylcopedia in analytical chemistry, 2000)

Hydrophobic Interaction Chromatography
Biopolymer (phenyl agarose - Binding Surface)
Driving force for hydrophobic adsorption
Water molecules surround the analyte and the binding
surface.
When a hydrophobic region of a biopolymer binds to the
surface of a mildly hydrophobic stationary phase,
hydrophilic water molecules are effectively released from
the surrounding hydrophobic areas causing a
thermodynamically favorable change in entropy.
Temperature plays a strong role
Ammonium sulfate, by virtue of its good salting-out
properties and high solubility in water is used as an eluting
buffer

Analysis

Hydrophobic region

55
(Christian G. Huber, Biopolymer Chromatography, Encylcopedia in analytical chemistry, 2000)

Ion Exchange Chromatography
Fractogel matrix is a methacrylate resin upon which polyelectrolyte
Chains (or tentacles) have been grafted. (Novagen)

Globular
Protein
Maintenance of conformation while
interacting with tentacle ion exchanger
Deformation due to interaction
with conventional ion exchanger

(www.novagen.com)

56

Gel Filtration

57
(http://lsvl.la.asu.edu/resources/mamajis/chromatography/chromatography.html)

Capillary Electrochromatography
•
•
•
•

CEC is an electrokinetic separation technique
Fused-silica capillaries packed with stationary phase
Separation based on electroosmotically driven flow
Higher selectivity due to the combination of chromatography
electrophoresis

and

Fused silica tube filled with porous methacrylamide-stearyl methacrylatedimethyldiallyl ammonium chloride monolithic polymers, 80 x 0.5mm i.d.,
5.5kV. High Plate count ~ 400,000

Height Equivalent to a Theoretical Plate /Plate Count (HETP) H = L/N
number of plates N = 16(t/W)2
where L = column length, t = retention time, and W = peak width at baseline
Analysis

(http://www.capital-hplc.co.uk)

58

CEC columns

AC, IEC columns

CEC column

NP, RP columns


59

Schematic of a Multi-dimensional Separation System

Analysis

60

Fast Protein Liquid Chromatograph (FPLC)
• No air bubbles
(Priming)
• Use degassed buffers

Injector Module
2

1

Column Inlet
3
Detector 4
Fraction 5
Collector

Analysis

(www.pharmacia.com)

61

Chromatography systems

ÄKTAprime:
simple automated purification

ÄKTAFPLC:
high
performance
purification of proteins &
other biomolecules

ÄKTApurifier: high
performance
purification and characterization

ÄKTAexplorer:
for fast method
development and scale-up

ÄKTApilot
ÄKTAxpress:
:
for high
rapid
throughput
process
tagged
development and pilotprotein purification
scale
Analysis

62

High Performance Liquid
Chromatography (HPLC)



What is HPLC?
Types of Separations
Columns and Stationary Phases
Mobile Phases and Their Role in Separations
Injection in HPLC
Detection in HPLC



Variations on Traditional HPLC



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
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Ion Chromatography
Size Exclusion Chromatography

Analysis

63

What is HPLC?


High Performance Liquid Chromatography



High Pressure Liquid Chromatography (usually true]



Hewlett Packard Liquid Chromatography (a joke)



High Priced Liquid Chromatography (no joke)



HPLC is really the automation of traditional liquid
chromatography under conditions which provide for enhanced
separations during shorter periods of time!



Probably the most widely practiced form of quantitative,
analytical chromatography practiced today due to the wide
range of molecule types and sizes which can be separated
using HPLC or variants of HPLC!!
Analysis

64

Analysis

65

Analysis

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Types of HPLC Separations (partial list)


Normal Phase: Separation of polar analytes by partitioning onto a
polar, bonded stationary phase.



Reversed Phase: Separation of non-polar analytes by partitioning
onto a non-polar, bonded stationary phase.



Adsorption: In Between Normal and Reversed. Separation of
moderately polar analytes using adsorption onto a pure stationary
phase (e.g. alumina or silica)



Ion Chromatography: Separation of organic and inorganic ions by
their partitioning onto ionic stationary phases bonded to a solid
support.



Size Exclusion Chromatography: Separation of large molecules
based in the paths they take through a “maze” of tunnels in the
stationary phase.
Analysis

67

Analysis

68

Analysis

69

Analysis

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What does the analyst do?






Select the correct type of separation for the analyte(s) of
interest, based on the sample type (among other factors).
Select an appropriate column (stationary phase) and
mobile phase
Select an appropriate detector based on whether universal
or compound-specific detection is required or available



Optimize the separation using standard
mixtures



Analyze the standards and sample

Analysis

71

Analysis

72

Columns and Stationary Phases.


HPLC is largely the domain of packed columns




some research into microbore/capillary columns is going
on.
Molecules move too slowly to be able to reach and
therefore “spend time in” the stationary phase of an open
tubular column in HPLC.





In solution, not the gas phase
Larger molecules in HPLC vs. GC (generally)

Stationary phases are particles which are usually about 1 to 20
µm in average diameter (often irregularly shaped)




In Adsorption chromatography, there is no additional
phase on the stationary phase particles (silica, alumina,
Fluorosil).
In Partition chromatography, the stationary phase is
coated on to (often bonded) a solid support (silica,
alumina, divinylbenzene resin)
Analysis

73

Analysis

74

Stationary Phases


Polar (“Normal” Phase):





Silica, alumina
Cyano, amino or diol terminations on the bonded phase

Non-Polar (“Reversed Phase”)



C18 to about C8 terminations on the bonded phase
Phenyl and cyano terminations on the bonded phase



Mixtures of functional groups can be used!!



Packed particles in a column require:





Frits at the ends of the column to keep the particles in
Filtering of samples to prevent clogging with debris
High pressure pumps and check-valves
Often a “Guard Column” to protect the analytical column
Analysis

75

Optimization of Separations in HPLC









Correct choice of column so the above equilibrium has
some meaningful (non-infinity, non-zero) equilibrium
constants.
Correct choice of mobile phase
Decision on the type of mobile phase composition
 constant composition = isocratic
 varying composition = gradient elution
Determination if flow rate should be constant
 usually it is
Decision on heating the column

heating HPLC columns can influence the above
equilibrium….

Analysis

76

Analysis

77

The Mobile Phase in HPLC...


Must do the following:





solvate the analyte molecules and the solvent they are in
be suitable for the analyte to transfer “back and forth”
between during the separation process

Must be:






compatible with the instrument (pumps, seals, fittings,
detector, etc)
compatible with the stationary phase
readily available (often use liters/day)
of adequate purity




spectroscopic and trace-composition usually!

Not too compressible (causes pump/flow problems)


Free of gases (which cause compressability problems)
Analysis

78

Typical HPLC Pump (runs to 4,000+ psi)

Analysis

79

Analysis

80

Polarity Index for Mobile Phases…..


The polarity index is a measure of the relative polarity of a
solvent. It is used for identifying suitable mobile phase
solvents.
 The more polar your solvent is, the higher the index.
 You want to try to choose a polarity index for your solvent (or
solvent mixture) that optimizes the separation of analytes










usually the index is a starting point
the polarity of any mixture of solvents to make a mobile phase can
be modeled to give a theoretical chromatogram
Usually, optimization of solvent composition is experimental

A similar number is the Eluent Strength (Eo]
Increasing eluent strength or polarity index values mean
increasing solvent polarity.
Remember, the analyte(s) and samples must be mobile phase
and stationary phase compatible!
Analysis

81

Analysis

82

Analysis

83

Optimization of Mobile Phase Polarity…
Changing the mobile phase composition alters the separation.

Analysis

84

Isocratic versus Gradient Elution


Isocratic elution has a constant mobile phase composition

Can often use one pump!

Mix solvents together ahead of time!

Simpler, no mixing chamber required

Limited flexibility, not used much in research

mostly process chemistry or routine analysis.



Gradient elution has a varying mobile phase composition

Uses multiple pumps whose output is mixed together

often 2-4 pumps (binary to quarternary systems)

Changing mobile phase components changes the polarity index

can be used to subsequently elute compounds that were previously
(intentionally) “stuck” on the column

Some additional wear on the stationary phase

Column has to re-equiluibrate to original conditions after each run
(takes additional time).

Analysis

85

Analysis

86

Analysis

87

Analysis

88

Injection in HPLC










Usually 5 to 1000 µL volumes, all directly onto the column
 not much worry about capacity since the columns have a large
volume (packed).
Injector is the last component before the column(s)
A source of poor precision in HPLC
 errors of 2-3 %RSD are due just to injection
 other errors are added to this
 due to capillary action and the small dimensions/cavities inside
the injector
6-PORT Rotary Valve is the standard manual injector
Automatic injectors are available
Two positions, load and inject in the typical injector
Injection loop internal volume determines injection volume.

Analysis

89

LOAD (the sample loop)

Inject (move the sample
loop into the mobile
phase flow)
Analysis

90

Analysis

91

Detection in HPLC












Numerous Types (some obscure)
Original HPLC Detectors were common laboratory
instruments such as spectrophotometers, etc.
Must be solvent -compatible, stable, etc.
Universal
 respond to all analytes
Analyte Specific
 respond to specific properties of analytes
Non-destructive
 most
Destructive
 ELSD, MS and a few others.

Analysis

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Analysis

93

Standard Absorbance Detector….




Single Beam UV-VIS instrument with a flow-through cell
(cuvette)
Can use any UV-VIS with a special flow cell




Extra connections lead to band-broadening if UV-VIS is far from
HPLC column exit.

Usually utilize typical UV-VIS lamps and 254 nm default
wavelenth



Can be set to other wavelengths (most)
Simple filter detectors no longer widely used




Non-destructive, not-universal





adjustable wavelength units are cost-effective

not all compounds absorb light
can pass sample through several cells at several different
wavelenghts

Usually zeroed at the start of each run using an electronic
software command. You can have real-time zeroing with a
reference cell.
Analysis

94

Analysis

95

SPECTROSCOPY


96

Definition
• Spectroscopy - The study of the
interaction of electromagnetic
radiation with matter


97

Introduction
• Spectroscopy is an analytical
technique which helps determine
structure.
• It destroys little or no sample.
• The amount of radiation absorbed by
the sample is measured as
wavelength is varied.

98

Major Types of Spectroscopy








Infrared (IR) spectroscopy measures the bond vibration
frequencies in a molecule and is used to determine the
functional group.
Mass spectrometry (MS) fragments the molecule and
measures the masses.
Nuclear magnetic resonance (NMR) spectroscopy
detects signals from hydrogen atoms and can be used
to distinguish isomers.
Ultraviolet (UV) spectroscopy uses electron transitions to
determine bonding patterns.

Analysis

99

Introduction of Spectrometric
Analyses
The study how the chemical compound
interacts with different wavelenghts in a given
region of electromagnetic radiation is called
spectroscopy or spectrochemical analysis.
The collection of measurements signals
(absorbance) of the compound as a function of
electromagnetic radiation is called a spectrum.
Analysis

100

Energy Absorption
The mechanism of absorption energy is different in
the Ultraviolet, Infrared, and Nuclear magnetic
resonance regions. However, the fundamental
process is the absorption of certain amount of energy.
The energy required for the transition from a state of lower
energy to a state of higher energy is directly
related to the frequency of electromagnetic radiation
that causes the transition.

101

Spectral Distribution of Radiant Energy

Wave Number (cycles/cm)
X-Ray

UV
200nm

Visible
400nm

IR

Microwave

800nm

Wavelength (nm)


102

Electromagnetic Spectrum


103

Electromagnetic Spectrum


104

Electromagnetic Radiation
V = Wave Number (cm )
-1

λ = Wave Length
C = Velocity of Radiation (constant) = 3 x 1010 cm/sec.
υ = Frequency of Radiation (cycles/sec)
V =

υ 1
=
C
λ

The energy of photon:
h (Planck's constant) = 6.62 x 10- (Erg×sec)
27

E = h υh
=

C
λ

υ=

C


λ

C = υλ
105

Equation Definitions

• E = energy (Joules, ergs)
• c = speed of light (constant)
• λ = wavelength
• h = Planck’s constant
• ν = “nu” = frequency (Hz)
• nm = 10-9 m
• Å = angstrom = 10-10 m

106

Spectral Properties, Application and Interactions of
Electromagnetic Radiation
Wave
Number V

Energy

Kcal/mol
9.4 x 107

9.4 x 103

9.4 x 101

eV
4.9 x 106

4.9 x 102

4.9 x 100

Wavelength
λ

cm-1

cm

3.3 x 1010

3 x 10-11

3.3 x 106

3.3 x 104

3 x 10-7

3 x 10-5

Frequenc
y
υ

Type
Radiation

Type
spectroscopy

Type
Quantum Transition

Hz
1021

Gamma
ray

1017

X-ray

1015

Ultra
violet

Gamma ray
emission

Nuclear

X-ray
absorption,
emission

Electronic
(inner shell)

UV absorption

Electronic
(outer shell)

Visible
IR absorption

9.4 x 10-1

4.9 x 10-2

3.3 x 102

3 x 10-3

1013

Infrared

9.4 x 10-3

4.9 x 10-4

3.3 x 100

3 x 10-1

1011

Microwave

Microwave
absorption

Radio

Nuclear
magnetic
resonance

9.4 x 10-7

4.9 x 10-8

3.3 x 10-4

3 x 103

107

Analysis

Molecular
vibration

Molecular
rotation

Magnetically
induced spin
states

107

Analysis

108

Spectrum of Radiation


109

Visible Light


110

Visible Light
Red

R

700 nm

Orange

O

650 nm

Yellow

Y

600 nm

Green

G

550 nm

Blue

B

500 nm

Indigo

I

450 nm

Violet

V

400 nm


111

Dispersion of Polymagnetic Light with a Prism
Prism - Spray out the spectrum and choose the certain wavelength
(λ) that you want by slit.
Infrared

Polychromatic
Ray

PRISM

Red
Orange
Yellow
Green

monochromatic
Ray

SLIT

Blue
Violet

Ultraviolet

Polychromatic Ray

Monochromatic Ray

112

Ultra Violet
Spectrometry
The absorption of ultraviolet radiation by molecules is
dependent upon the electronic structure of the molecule.
So the ultraviolet spectrum is called electronic spectrum.


113

INTRODUCTION TO
SPECTROPHOTOMETRY


114

Spectrophotometry
• Spectrophotometry: An analytical
method using several spectra (lights).
(State each spectrum used in
spectrophotometry.)
• Spectrophotometer: An instrument
for measuring absorbance that uses a
monochromator to select the
wavelength.

115

Spectrophotometry
-Advantages of spectrophotometers
i. relatively inexpensive
ii. inexpensive
iii. easy to maintain
iv. portable


116

BACKGROUND
white light is observed, what is actually seen is a
mixture of all the colors of light
Why do some substances appear colored?
When this light passes through a substance, certain energies (or
colors) of the light are absorbed while other color(s) are allowed to pass
through or are reflected by the substance.
If the substance does not absorb any light, it appears white (all light is
reflected) or colorless (all light is transmitted). A solution appears a
certain color due to the absorbance and transmittance of visible light.
For example, a blue solution appears blue because it is absorbing all of
the colors except blue.


117

BACKGROUND


118

BACKGROUND
• The amount of light absorbed by a solution is
dependent on the ability of the compound to
absorb light (molar absorptivity), the distance
through which the light must pass through the
sample (path length) and the molar concentration
of the compound in the solution.
• If the same compound is being used and the path
length is kept constant, then the absorbance is
directly proportional to the concentration of the
sample.


119

Spectrophotometer
• A spectrophotometer is used to
provide a source of light of certain
energy (wavelength) and to measure
the quantity of the light that is
absorbed by the sample.

Light Bulb
Sample

Prism
Detector

Filter

Slit


120

Spectrophotometer
•

The basic operation of the spectrophotometer includes a white light
radiation source that passes through a monochromator. The
monochromator is either a prism or a diffraction grating that
separates the white light into all colors of the visible spectrum. After
the light is separated, it passes through a filter (to block out unwanted
light, sometimes light of a different color) and a slit (to narrow the
beam of light--making it form a rectangle). Next the beam of light
passes through the sample that is in the sample holder. The light
passes through the sample and the unabsorbed portion strikes a
photodetector that produces an electrical signal which is proportional
to the intensity of the light. The signal is then converted to a readable
output that is used in the analysis of the sample.

Light Bulb
Sample

Prism
Detector

Filter

Slit


121

Spectrophotometer
An instrument which can measure the absorbance of a
sample at any wavelength.

Light

Lens
Sample

Slit

Monochromator

Detector


Slits

Quantitative Analysis

122

The process of light being absorbed by a solution
concentration 2
concentration 1
blank where Io = I

with sample
I < Io

light
source

detector

Io

I
b

PGCC CHM 103 Sinex

Cell with
Pathlength, b,
containing4:solution in
Chapter
Techniques

As concentration
increased, less
light was
transmitted (more
light absorbed).
123

Beer – Lambert Law

Light

I0

I

Glass cell filled with
concentration of solution (C)

As the cell thickness increases, the transmitted intensity
of light of I decreases.

124

R- Transmittance
R=

I
I0

I0 - Original light intensity
I- Transmitted light intensity

I
I0

% Transmittance = 100 x

Absorbance (A) = Log

1
T

= Log
Log

I0
= 2 - Log%T
I

I
is proportional to C (concentration of solution) and is
I0
also proportional to L (length of light path
125
through the Analysis
solution).

A ∝ CL = ECL by definition and it is called the Beer
- Lambert Law.
A = ECL

A = ECL
E = Molar Extinction Coefficient ---- Extinction
Coefficient of a solution containing 1g molecule of
solute per 1 liter of solution
Analysis

126

E =

Absorbance x Liter
Moles x cm

UNITS
A = ECL
A = No unit (numerical number only)
E =

Liter
Cm x Mole

L = Cm
C = Moles/Liter

A = ECL = (

Liter
Cm x Mole

)x


Mole
Liter

x Cm
127

The BLANK








The blank contains all substances expect
the analyte.
Is used to set the absorbance to zero:
Ablank = 0
This removes any absorption of light due
to these substances and the cell.
All measured absorbance is due to analyte.

PGCC CHM 103 Sinex

Analysis

128

Beer’s Law

A = abc
where a – molar absorptivity, b – pathlength,
and c – molar concentration
See the Beer’s Law Simulator

PGCC CHM 103 Sinex


129

Spectrophotometer
The spectrophotometer displays this quantity in one of two
ways:
(1) Absorbance -- a number between 0 and 2
(2) Transmittance -- a number between 0 and 100%.
The sample for a spectral analysis is prepared by pouring it into a
cuvette which looks similar to a small test tube. A cuvette is made
using a special optical quality glass that will itself absorb a minimal
amount of the light. It is also marked with an indexing line so that it
can be positioned in the light beam the same way each time to avoid
variation due to the differences in the composition of the glass


130

Fundamentals of Spectrophotometry
Absorption of Light
Beer’s Law


The relative amount of a certain wavelength of light absorbed (A) that passes
through a sample is dependent on:
distance the light must pass through the sample (cell path length - b)
amount of absorbing chemicals in the sample (analyte concentration – c)
ability of the sample to absorb light (molar absorptivity - ε)

Increasing [Fe2+]

Absorbance is directly proportional to concentration of Fe+2

131

Absorption of Light
3.) Beer’s Law




Absorbance is useful since it is directly related to the analyte concentration, cell
pathlength and molar absorptivity.
This relationship is known as Beer’s Law

A = abc
where:

Beer’s Law allows compounds to
be quantified by their ability to
absorb light, Relates directly to
concentration (c)

A = absorbance (no units)
α = molar absorptivity (L/mole-cm)
b = cell pathlength (cm)
c = concentration of analyte (mol/L)


132

Absorption of Light
4.) Absorption Spectrum


By choosing different wavelengths of light (λA vs. λB) different compounds
can be measured

λA

λB


133

Spectrophotometer
1.) Basic Design


An instrument used to make absorbance or transmittance measurements is
known as a spectrophotometer


134

Single Beam Spectrophotometer


135

Dual Beam Spectrophotometer


136

Spectrophotometer
1.) Basic Design


Light Source: provides the light to be passed through the sample
Tungsten Lamp: visible light (320-2500 nm)

Low pressure (vacuum)
Tungsten Filament

-

- based on black body radiation:
heat solid filament to glowing, light emitted will be
characteristic of temperature more than nature of
solid filament

Deuterium Lamp: ultraviolet Light (160-375 nm)
In presence of arc, some of the electrical energy is
absorbed by D2 (or H2) which results in the
disassociation of the gas and release of light

D2 + Eelect  D*2  D’ + D’’ + hν (light produced)
Excited state


137

Spectrophotometer
1.) Basic Design


Wavelength Selector (monochromator): used to select a given wavelength
of light from the light source
Prism:

-

Filter:


138

Spectrophotometer
1.) Basic Design


Wavelength Selector (monochromator): used to select a given wavelength
of light from the light source
Reflection or Diffraction Grating:


139

Spectrophotometer
1.) Basic Design


Sample Cell: sample container of fixed length (b).
-

Usually round or square cuvet
Made of material that does not absorb light in the wavelength range of
interest
1.

Glass – visible region

2.

Quartz – ultraviolet

3.

NaCl, KBr – Infrared region


140

Cuvettes (sample holder)
• Polystyrene
– 340-800 nm

• Methacrylate
– 280-800 nm

• Glass
– 350-1000 nm

• Suprasil Quartz
– 160-2500 nm

141

Spectrophotometer
1.) Basic Design


Light Detector: measures the amount of light passing through the
sample.
-

Usually works by converting light signal into electrical signal

Photomultiplier tube

Process:
a) light hits photoemissive cathode and e- is emitted.
b) an emitted e- is attracted to electrode #1
(dynode 1), which is 90V more positive.
Causes several more e- to be emitted.
c) these e- are attracted to dynode 2, which is
90V more positive then dynode 1, emitting
more e-.
d) process continues until e- are collected at
anode after amplification at 9 dynodes.
e) overall voltage between anode and cathode
is 900V.
f) one photon produces 106 – 107 electrons.142
g) current is amplified and measured

Applications of
Spectrophotometry
Quantitative Applications
• Usually using UV-Vis
• IR can be used

- Environmental applications; analysis waters & waste waters
- Clinical applications: analysis of glucose
- Industrial analysis; analysis of iron content in food
- Forensic applications: Determination of blood alcohol
Analysis

143

Advantage of spectrophotometer over
colorimeter






can be used to profile printers & scanners, measure colors "in
the wild", measure your illumination
colorimeter measures only 3 points on the specturm (RGB),
while a spectrophotometer measures many points across the
entire spectrum
colorimeters use a single type of light (such as incandescent or
pulsed xenon) Spectrophotometers can compensate for this shift,
making spectrophotometers a superior choice for accurate,
repeatable color measurement.

Analysis

144

Sample Cells
UV Spectrophotometer
Quartz (crystalline silica)

Visible Spectrophotometer
Glass


145

Light Sources
UV Spectrophotometer
1.

Hydrogen Gas Lamp

2.

Mercury Lamp

Visible Spectrophotometer
1.

Tungsten Lamp


146

Chemical Structure & UV Absorption
Chromophoric Group ---- The groupings of the
molecules which contain the electronic system which
is giving rise to absorption in the ultra-violet region.


147

UV Spectrometer Application
Protein
Amino Acids (aromatic)
Pantothenic Acid
Glucose Determination
Enzyme Activity (Hexokinase)


148

Flurometric Application
Thiamin (365 nm, 435 nm)
Riboflavin
Vitamin A
Vitamin C


149

Visible Spectrometer Application
Niacin
Pyridoxine
Vitamin B12
Metal Determination (Fe)
Fat-quality Determination (TBA)
Enzyme Activity (glucose oxidase)

150

Major Types of Light Spectroscopy


Absorption spectroscopy






Atomic emission spectroscopy







Samples fluoresce when they emit at higher λ than what they absorb
Measures solvent interactions, distances, molecular shape, and motion

Circular Dichroism spectroscopy





Measures light emitted from burned sample
Elemental analysis

Fluorescence spectroscopy




Measures amount of light absorbed
Most common, non-destructive
Concentration, pH measures, purity, ID

Absorption of circular polarized light
Chiral compound identification

Transmission spect. (colorimetry)
Analysis

151

Introduction


Atomic absorption is the absorption of light by free
atoms. An atomic absorption spectrophotometer is
an instrument that uses this principle to analyze the
concentration of metals in solution. The substances
in a solution are suctioned into an excited phase
where they undergo vaporization, and are broken
down into small fragmented atoms by discharge,
flame or plasma.

Analysis

152

Atomic Emission Spectroscopy


By exposing these atoms to such temperatures they
are able to “jump” to high energy levels and in
return, emit light. The versatility of atomic
absorption an analytical technique (Instrumental
technique) has led to the development of
commercial instruments. In all, a total of 68 metals
can be analyzed.

Analysis

153

Advantages of AA









Determination of 68 metals
Ability to make ppb determinations on major components of a
sample
Precision of measurements by flame are better than 1% rsd.
There are few other instrumental methods that offer this
precision so easily.
AA analysis is subject to little interference.
Most interference that occurs have been well studied and
documented.
Sample preparation is simple (often involving only dissolution in
an acid)
Instrument easy to tune and operate

Analysis

154

Atomic emission spectrometer

Analysis

155

NEXT CLASS:
Chapter 5
DNA Technology

THANK YOU


156

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