HPLC_quantitaive_methods_prezentacja.pdf

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1. qualitative and
quantitative analysis

4. Russian scientist Tswett in 1906 used a glass columns packed
with divided CaCO
3(calcium carbonate) to separate plant
pigments extracted by hexane. The pigments after separation
appeared as colour bands that can come out of the column one
by one.

Tswett was the first to use the term "chromatography" derived
from two Greek words "Chroma" meaning color and "graphein"
meaning to write.

5. 5
Invention of Chromatography by M. Tswett
Ether
CaCO
3
Chlorophyll
Chromatography
Colors

6. *Definition of chromatography

*Tswett (1906) stated that „chromatography is
a method in which the components of a mixture are
separated on adsorbent column in a flowing system”.

*IUPAC definition (International Union of pure and applied
Chemistry) (1993):
Chromatography is a physical method of separation in which
the components to be separated are distributed between
two phases, one of which is stationary while the other
moves in a definite direction.

7. *Principles of Chromatography

*Any chromatography system is composed of three components :

*Stationary phase
*Mobile phase
*Mixture to be separated



The separation process occurs because the
components of mixture have different
affinities for the two phases and thus move
through the system at different rates.
A component with a high affinity for the
mobile phase moves quickly through the
chromatographic system, whereas one with
high affinity for the solid phase moves more
slowly.

8. *The affinity differences of the components for the stationary or the mobile
phases can be due to several different chemical or physical properties
including:

* Ionization state
* Polarity and polarizability
* Hydrogen bonding / van der Waals’ forces
* Hydrophobicity
* Hydrophilicity

*The rate at which a sample moves is determined by how much time it spends
in the mobile phase.
*Forces Responsible for Separation

9. Chromatographic methods are classified according to:

A – Mechanism of separation:

The mechanism of separation depends mainly on the nature of the
stationary phase. Based on separation mechanisms chromatography can be
classified into:
*Classification of chromatographic methods

10. *1- Adsorption Chromatography:
It is the oldest technique.
Separation is due to
difference in the
adsorption power
of mixture components.
The stationary phase is
a solid with adsorption
characters.
Silica gel and alumina are the
most common stationary
phase in adsorption
chromatography.

11. *2- Partition Chromatography:
Separation is due to difference in solubility of components
in two immiscible liquids.
The stationary phase is a liquid thin film on an inert solid
support. The stationary liquid is usually more polar than
the mobile phase. Cellulose powder and wetted silica gel
are examples of supports in partition chromatography that
carry film of water act as stationary phase.

12. *3- Ion Exchange Chromatography (IEC):
It is used for separation of charged molecules.
The stationary phase is an ion exchange resin to which a cationic or
anionic groups are covalently bonded. Ions of opposite charges
(counter ions) in the mobile phase will be attracted to the resin and
compete with the components of the mixture for the charged group
on the resin.

13. Molecules that are very small in relation to the
pore size all behave similarly and these small
molecules are also not separated.
Medium sized molecules are separated based
on how far they penetrate into the gel beads.
Separation is based on molecular size.
Stationary phase is a material of controlled pore
size.
*4- Molecular Exclusion ( Size Exclusion)
Chromatography:

14. The separation is based on the affinity of proteins to specific ligands
such as enzymes. The ligand is attached to suitable polysaccharide
polymer such as cellulose - agarose – dextran.


*5- Affinity Chromatography:

15. B- According to the nature of the mobile and
stationary phase:

In this regard chromatography is classified into:

1- Liquid Chromatography (LC):
The mobile phase is liquid.

2- Gas Chromatography (GC)
The mobile phase is an inert gas nitrogen or helium.

16. C- According to the technique (methods of holding the
Stationary Phase):

1- Planar or Plane Chromatography:
*In this type the stationary phase is used in the form of
layer. Plane chromatography is additionally classified into:
a- Thin Layer Chromatography (TLC):
The stationary phase is spread on glass or plastic or
aluminum sheets.
b- Paper Chromatography (PC):
A specific type of papers is used as stationary phase.

2- Columnar or Column Chromatography (CC):
The stationary phase is held in to a tube made of glass or
metal (gel – ion exchange – adsorption).

17. D- ACCORDING TO PURPOSE OF USE:
QUALITITATIVE CHROMATOGRAPHY
In this case Chromatography can be used to:
1- Confirm the absence or probable presence of certain constituent in the sample
under investigation
2- Give an idea about the complexity of the mixture and the least number of
compounds present.
3- Check purity and identity of any compound.

QUANTITATIVE CHROMATOGRAPHY
The development of modern instruments enable the use of chromatography to
determine the amount of any component in a mixture as absolute amount or relative to
another component HPLC/ GC/ HPTLC can be used for there applications.

18. quantitative and qualitative
liqiud chromatography

19. *Thin layer chromatography

20. *qualitative analysis

21. After the sample has been applied on the
plate, a solvent or solvent mixture (known
as the mobile phase) is drawn up the plate
via capillary action.
Because different analytes moved the TLC
plate at different rates, separation is
achieved.

22. The R
f retardation factor
is defined as the distance the center of the spot moved divided by the
distance the solvent front moved (both measured from the origin)
AB CU
x xxx
Solvent Front
Origen
Distance solvent
migrated = 5.0 cm
Distance A
migrated = 3.0 cm
Distance B
migrated = 2.0 cm
Distance C
migrated = 0.8 cm
0.8 cm
3.0 cm
R
f (A) =
R
f (B) =
R
f (C) =
R
f (U
1) =
R
f (U
2) =
2.0 cm
5.0 cm
= 0.40
= 0.60
= 0.16
= 0.60
= 0.16
3.0 cm
5.0 cm
0.8 cm
5.0 cm
3.0 cm
5.0 cm
0.8 cm
5.0 cm
D
x
R
f (D) = = 0.80
4.0 cm
5.0 cm
4.0 cm

24. 1 2 3 sample
The three substances
have the same Rf
standards

25. 1 2 3 sample
The three substances
have the same colour

26. 1 2 3 sample
The three substances
are…invisible

27. *Visualization Method
*Most of the time, the spots won’t show unless
they are visualized!

*Visualization is a method that is used to render
the TLC spots visible.

*A visualization method can be:
*Ultraviolet light
*Colored reagents to stain spots

28. *ULTRAVIOLET LAMP
After developing a TLC plate, the first
analysis technique should always be UV light.
This technique is fast. When using "F
254 silica
gel" TLC plates (silica gel that fluoresces with
a 254 nm absorption) these compounds will
appear as dark spots (because they "block"
the fluorescence by absorbing the UV light)
on a green background.

30. Colored reagents to stain spots

31. A TLC stain is used in TLC development to reveal
compounds that are not visible by UV. Also, selective
detection of compounds is possible by choosing the
appropriate TLC stain.

32. *TLC SCANER

34. Classical densitometry uses monochromatic light and a slit of selectable length
and width to scan the tracks of a chromatogram, measuring the diffusely reflected
light. The CAMAG TLC Scanner uses the entire spectral range from 190 to 900
nm with high spectral selectivity for data acquisition. Absorption spectra for
substance identification and for selection of the most suitable measurement
wavelength can be recorded within this range.

36. Thin-Layer Chromatography:
Qualitative Analysis















1 2 3 sample

37. Caffeine spectrum Paracetamol spectrum
Densytometr can automatically record spectra as soon as all
peak positions are known.

The difference beetwen spectra allows us to confirm the identity of analytes in a sample.
However, some spectra have small differences and cannot be absolute confirmations by
themselves.
Analytical chemists use both retention time and spectra to determine a probability of identifying a
chemical in a sample.

39. The Mass Spectrometer
In order to measure the characteristics of individual molecules, a mass spectrometer converts them to
ions so that they can be moved about and manipulated by external electric and magnetic fields. The
three essential functions of a mass spectrometer, and the associated components, are:
1. A small sample is ionized, usually to cations by loss of an electron. The Ion Source
2. The ions are sorted and separated according to their mass and charge. The Mass Analyzer
3. The separated ions are then measured, and the results displayed on a chart. The Detector

Ionizer
Sample
+
_
Mass Analyzer Detector

40. *Mass Spec Principles
Ionizer
Sample
+
_
Mass Analyzer Detector

42. The versatile instrument to extract compounds from a TLC/HPTLC plate and feed
them into a mass spectrometer for substance identification or structure elucidation.

44. http://www.google.pl/url?sa=i&source=imag
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369251650161134

46. quantitative analysis

48. Classical densitometry uses monochromatic light and a slit of selectable length
and width to scan the tracks of a chromatogram, measuring the diffusely reflected
light. The CAMAG TLC Scanner uses the entire spectral range from 190 to 900
nm with high spectral selectivity for data acquisition. Absorption spectra for
substance identification and for selection of the most suitable measurement
wavelength can be recorded within this range.

49. concentration ug/L 0,6 0,9 1,2 1,5
AU 20 30 40 50
0
10
20
30
40
50
60
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6
AU

concentration ug/L

52. HPLC

53. *What does HPLC means?

High Performance Liquid Chromatography

High Pressure Liquid Chromatography

High Price Liquid Chromatography

High Patience Liquid Chromatography

54. Smaller column particle size can improve
chromatographic resolution, but increased
solvent delivery pressure is needed.

57. Mobile phase is the most important parameter in
HPLC. Type of mobile phase used may have a big
effect on the retention. It can promote or
suppress an ionization of the analyte molecules,
and it also can shield an accessible residual silanol
or any other active adsorption centers on the
adsorbent surface.
The most common
solvent reservoirs are as simple as glass bottles
with tubing connecting them to the pump inlet.

58. High-pressure pumps are needed to push the
mobile phase through the packed stationary
phase.

59. An injector for an HPLC system should provide
injection of the liquid sample within the range
of 0.1-100 mL of volume with high
reproducibility and under high pressure (up to
4000 psi). For liquid chromatography, liquid
samples can be directly injected and solid
samples need only to be diluted in the
appropriate solvent.

62. There are many different types of detectors
that can be used for HPLC.

68. The R
f retardation factor
is defined as the distance the center of the spot moved divided by the
distance the solvent front moved (both measured from the origin)
AB CU
x xxx
Solvent Front
Origen
Distance solvent
migrated = 5.0 cm
Distance A
migrated = 3.0 cm
Distance B
migrated = 2.0 cm
Distance C
migrated = 0.8 cm
0.8 cm
3.0 cm
R
f (A) =
R
f (B) =
R
f (C) =
R
f (U
1) =
R
f (U
2) =
2.0 cm
5.0 cm
= 0.40
= 0.60
= 0.16
= 0.60
= 0.16
3.0 cm
5.0 cm
0.8 cm
5.0 cm
3.0 cm
5.0 cm
0.8 cm
5.0 cm
D
x
R
f (D) = = 0.80
4.0 cm
5.0 cm
4.0 cm

69. Each peak is labeled with retention time. Retention time indicates how long it takes
for a compound to come out of the HPLC column.

70. CAPACITY FACTOR (k): is one way to measure sample retention; bands
which come out in the chromatogram at the column dead time have a k-value
of zero. Later bands have k-values that increase with band retention time.
Values of k for each band or compound are constant if experimental conditions
do not change. k does not change when flow rate or column dimensions are
changed. k can change when mobile phase composition, stationary phase
chemistry, or temperature change.

71. Thin layer chromatography
HPLC chromatogram

72. The data-acquisition system of most HPLC
systems is a computer. The computer
integrates the response of the detector to each
component and places it into a chromatograph
that is easy to read and interpret. Other more
advanced features can also be applied to a
chromatographic system. These features
include
computer-controlled automatic injectors, multi-
pump gradient
controllers and sample fraction collectors.

73. The column or stationary phase is the core of any chromatographic system. Columns
are commercially available in different lengths, bore sizes and packing materials.
The use of the correct combination of length and packing material in correlation with the
appropriate mobile phase can assist in the most effective separation of a sample
compound

74. *HPLC Column
Must operate in high pressure
*Usually constructed of metals
Typical dimensions
*10-30 cm long
*1-3 cm ID
Contains packing material which holds
the stationary phase
*Many types exist
*Typical packing materials are 5-10 µm in
diameter
Guard column used to extend life of
main column

76. *As shown in Figure, classes of molecules can be ordered by their
relative retention into a range or spectrum of chromatographic
polarity from highly polar to highly non-polar.
*Molecules with similar chromatographic polarity tend to be
attracted to each other; those with dissimilar polarity exhibit
much weaker attraction, if any, and may even repel one another.
This becomes the basis for chromatographic separation modes
based on polarity.
Separations Based on Polarity

77. To design a chromatographic separation system we create competition for the
various compounds contained in the sample by choosing a mobile phase and a
stationary phase with different polarities. Then, compounds in the sample that are
similar in polarity to the stationary phase [column packing material] will be delayed
because they are more strongly attracted to the particles. Compounds whose
polarity is similar to that of the mobile phase will be preferentially attracted to it and
move faster.

78. Normal-Phase HPLC
In his separations of plant extracts, Tswett was successful using a polar stationary phase
with a much less polar [non-polar] mobile phase. This classical mode of chromatography
became known as normal phase.
The stationary phase is polar and retains the polar yellow dye most strongly. The
relatively non-polar blue dye is won in the retention competition by the mobile phase, a
non-polar solvent, and elutes quickly. Since the blue dye is most like the mobile phase
[both are non-polar], it moves faster. It is typical for normal-phase chromatography on
silica that the mobile phase is 100% organic; no water is used.

79. Reversed-Phase HPLC
The term reversed-phase describes the chromatography mode that is just the opposite
of normal phase, namely the use of a polar mobile phase and a non-polar [hydrophobic]
stationary phase.
Now the most strongly retained compound is the more non-polar blue dye, as its
attraction to the non-polar stationary phase is greatest. The polar yellow dye, being
weakly retained, is won in competition by the polar, aqueous mobile phase, moves the
fastest through the bed, and elutes earliest like attracts like.

80. Today, because it is more reproducible and has broad applicability,
reversed-phase chromatography is used for approximately 75% of all HPLC methods.
Most of these protocols use as the mobile phase an aqueous blend of water with a
miscible, polar organic solvent, such as acetonitrile or methanol. This typically ensures
the proper interaction of analytes with the non-polar, hydrophobic particle surface.
A C18–bonded silica [sometimes called ODS] is the most popular type of reversed-
phase HPLC packing.
Si
-O-Si

C
18 (ODS)
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
2
CH
3

81. Figure. (A) 30% MeCN: 70% 20mM
Phosphate, pH 7. (B) 50% MeCN:
50% 20mM Phosphate, pH 7. (C) 80%
MeCN: 20% 20mM Phosphate, pH 7.
MOBILE PHASE

82. STATIONARY PHASE

83. HPLC used for Qualitative
Analysis

86. HPLC used for Quantitative
Analysis

87. concentration ug/L 0,6 0,9 1,2 1,5
AU 20 30 40 50
0
10
20
30
40
50
60
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6
AU

concentration ug/L

88. *Electrophoresis
qualitative and quantitative
analysis

90. Cathode (-)
Anode (+)
One of the key elements in a gel
electrophoresis system is the gel itself.
Polyacrylamide and agarose gels formed as
layers are the principal mediums for
electrophoresis.
The gel matrix acts as an medium reducing
diffusion, so that separated sample
components remain positioned
in sharp zones. In addition, the gel acts as a
molecular sieve, separating molecules
according to their size.
Second key element is buffer. The
electrical current in an
electrophoresis cell is carried
largely by the ions supplied by
buffer, which also maintain proper
pH and provide a medium for heat
dissipation.
Third key is voltage.

91. high OH
- High H
+

92. *Factors Affecting
Electrophoresis

93. Charge of molecule depends on its dissociation at pH of the buffer
solution used.
As a charge of molecule increases, its migration rate also
increases. Note that depending on the nature of the net charge, the
charged particles will migrate to the cathode or to the anode.

Electrophoresis of positively charged particles (cations)
is called cataphoresis, while electrophoresis of negatively charged
particles (anions) is called anaphoresis.

94. As a charge of molecule increases, its migration rate also increases.

catode anode
start

95. The size of molecules influence their migration rate: small
molecules travel faster, large molecules travel slower.
catode anode
start

96. The shape of molecules influence their migration rate.
Globular molecule exhibit a different (higher) migration rate
from that of fibrous one.
+ –
DNA       

97.  Buffer Properties
 Electrical Field Characteristics
 Temperature Effects

98. *Qualitative analysis

99. Can be used to separate the size of:

*DNA

*RNA

*Protein

100. *


A technique used by scientists to distinguish
between individuals of the same species using
only samples of their DNA

101. *
*The process of DNA
fingerprinting was
invented by Alec
Jeffreys at the
University of Leicester
in 1985.

102. DNA molecules are very long
They may consist of millions of base pairs
In order to study the structure of DNA,
the molecules are broken up into smaller fragments
by enzymes called restriction enzymes
Restriction enzymes do not break up the DNA
molecule randomly but ‘cut’ it at particular sites

103. For example, a restriction enzyme called EcoR1*
‘recognises’ the base sequence CAATTC and cuts it
between the two As
--C-C-G-C-A-G-C-T-G-T-C-A-A-T-T-C-T-C-T-C-C-G-G-A-T-C-C-A
recognised
cut
--C-C-G-C-A-G-C-T-G-T-C-A
Other restriction enzymes cut the DNA in different places
and so produce fragments which are easier to analyse
--C-C-G-C-A-G-C-T-G-T-C-A
--C-C-G-C-A-G C-T-G-T-C-A A-T-T-C-T-C-C-G G-A-T-C-C-C-A-
A-T-T-C-T-C-T-C-C-G-G-A-T-C-C-C-A-
A-T-T-C- T-C-T-C-C-G-G-A-T-C-C-A-
3

104. Genetic fingerprinting
90% or more of DNA does not carry nucleotide triplets that code for proteins
The non-coding DNA is often called ‘junk DNA’ but this only means that its
functions have not yet been discovered
Some of the non-coding regions consist of repeated sequences of nucleotides
For example -C-A-T-G-C-A-T-G-C-A-T-G-C-A-T-G- *
The number of repeats in any one section of DNA varies from one individual
to the next
Since these sections do not code for proteins (and, therefore are not genes)
there is no observable difference in these individuals

105. Particular repeat sequences can be ‘cut out’ by restriction enzymes
For example
-CATCCACGACATGCATGCATGCATG CCACATCCA-
restriction enzyme cuts
here……………and…..….…..here
or
-CCACGACATGCATGCATGCATGCATGCATG CCACAT-
here…….…..…..………and…...….…………..here

106. The separation takes place in a sheet of a firm but jelly-like substance (a ‘gel’)
Samples of the DNA extracts are placed in shallow cavities (‘wells’) cut into one
end of the gel
A voltage is applied to opposite ends of the gel
DNA has a negative charge and moves slowly towards the positive end
The shorter fragments travel through the gel faster than the longer fragments
The fragments can be separated using
gel electrophoresis

107. gelatinous sheet
well
solution
DNA extract
added

108. Voltage supply
negative electrode
DNA samples placed in
wells cut in gel
positive
electrode
thin slab of
gel
+
DNA fragments
Move from negative
To positive

109. A sample with the
shorter DNA fragments
travels through the gel
faster than a sample
with the larger fragments

110. *

111. Appearance of separated fragments on gel
These bands will
contain the shorter
DNA fragments
These bands will
contain the longer
DNA fragments
starting positions
© Prof. E. Wood
© Prof. E.J.Wood

115. *
*After the electrophoresis is complete, the molecules in the gel
can be stained to make them visible.
DNA may be visualized using ethidium bromide which, when
intercalated into DNA, fluoresce under ultraviolet light.






*Photographs can be taken of gels, often using a Gel Doc system.

116. Genetic fingerprinting
DNA analysis can be used for catching criminals, establishing
parentage, finding how closely organisms are related and many other
applications.
The pattern of bands in a gel electrophoresis is known as a
genetic fingerprint or a ‘genetic profile’
If a genetic fingerprint found in a sample of blood or other tissue
at the scene of a crime matches the genetic fingerprint of a suspect,
this can be used as evidence
A DNA sample can be obtained from the suspect using blood, cheek
epithelial cells taken from the mouth lining or even the cells clinging
to the root of a hair

117. ….there is a chance of 1 in 10 that this
fragment occurs in many individuals…
Suppose that…………
…and.there is a chance of 1 in 20 that this
fragment occurs in many individuals…
…and.there is a chance of 1 in 10 that this
fragment occurs in many individuals…
…and.there is a chance of 1 in 30 that this
fragment occurs in many individuals, but…

118. …the probability of all 4 bands matching in any person other than
the suspect is

1 in 10 x 1 in 20 x 1 in 10 x 1 in 30
= 1 in 10 x 20 x 10 x 30 That is 1 in 60,000
When a larger number of bands is involved, the probability that
the suspect is not guilty becomes one in many thousands*

119. *

120. *Comparing blood samples on
defendant’s clothing to determine if it
belongs to victim

*Uses: Criminology

121. *Uses: Paternity
+
DNA

child Mom F1 F2
–
Genetic fingerprint of …
1 mom 4 child
2 possible father 1
3 possible father 2
There is a match between 3 of the child’s
restriction fragments and one of the
mother’s.

There is also a match between the child’s
fragments and one from possible father 2.
Neither of the child’s restriction fragments
match those of possible father 1
*

122. *Uses: Medical diagnostic
*Comparing normal allele to disease allele
chromosome with
disease-causing
allele 2
chromosome
with normal
allele 1
Example: test for Huntington’s disease
DNA analysis of Huntington disease. Each lane shows a different person's DNA: two
bands in the normal (N) range show someone is unaffected. One band in the H range
predicts the person will get Huntington disease.

123. *Uses: Evolutionary relationships
*Comparing DNA samples from different organisms to
measure evolutionary relationships
–
+
DNA

1 3 2 4 5 1 2 3 4 5
turtle snake rat squirrel fruitfly

124. *Capillary
electrophoresis