1. Chromatography involves separating mixtures based on differences in how components distribute themselves between a stationary and mobile phase. 2. Common stationary phases include silica gel, alumina, and ion exchange resins. The interaction between the stationary phase and components leads to separation as they travel through the column at different rates. 3. Types of chromatography covered include liquid column chromatography, high performance liquid chromatography (HPLC), and ion exchange chromatography. Key applications are separating pharmaceuticals, proteins, food/industrial chemicals, and pollutants.

1. Modern Analytical
Chemistry
Chromatography

2. CHROMATOGRAPHY
Chromatography basically involves the
separation of mixtures due to differences in
the distribution coefficient (equilibrium
distribution) of sample components between 2
different phases.
One of these phases is a mobile phase and
the other is a stationary phase.

3. Stationary Phase: Alumina
O
Al
O O
Al
O
OH
Al
O
OH
Al
O
OH
Al
OH
O
Acidic: -Al-OH
Neutral: -Al-OH + -Al-O-
Basic: -Al-O-

4. Stationary Phase: Silica (SiO2)
OH
Si
O
OH
Si
O
O
O
OH
Si
O
O
OH
Si
O
O
OH
Si
O O
O
Si
O
O
Si
O
O
Si
O
O
Si
O O
O
Si
O
O
Si
O
O
Si
O O
O

5. Definition:
Different affinity of these 2 components to stationary
phase causes the separation.
Concentration of component A in stationary phase
Concentration of component A in mobile phase
Distribution Coefficient (Equilibrium Distribution )

6. Some Types of Chromatography
1. Liquid Column Chromatography (Reverse
Phase too)
2. High Pressure (performance) Liquid
Chromatograph (HPLC)
3. Paper Chromatography
4. Thin-layer Chromatography (TLC)
5. Gas Liquid Chromatography

7. LIQUID COLUMN CHROMATOGRAPHY
A sample mixture is passed through a column
packed with solid particles which may or may
not be coated with another liquid.
With the proper solvents, packing conditions,
some components in the sample will travel the
column more slowly than others resulting in
the desired separation.

8. A + B + C
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOO OOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOO OOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOO OOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO
Sample
(A+B+C)
Column
Solid Particles
(packing material-
stationary phase)
Eluant (eluate)
DIAGRAM OF SIMPLE LIQUID COLUMN CHROMATOGRAPHY
A
B
C
Solvent(mobile or
moving phase)
Diagram of Simple Liquid Column Chromatography

9. The 4 basic liquid chromatography modes are named according to the mechanism
involved:
1. Liquid/Solid Chromatography (adsorption chromatography)
A. Normal Phase LSC
B. Reverse Phase LSC
2. Liquid/Liquid Chromatography (partition chromatography)
A. Normal Phase LLC
B. Reverse Phase LLC
3. Ion Exchange Chromatography
4. Gel Permeation Chromatography (exclusion chromatography)
BASIC LIQUID CHROMATOGRAPHY

10. LIQUID SOLID CHROMATOGRAPHY

Si- O - H

Normal phase LS
Reverse phase LS
Silica Gel
The separation mechanism in LSC is based on the
competition of the components of the mixture sample
for the active sites on an absorbent such as Silica Gel.

11. LIQUID SOLID CHROMATOGRAPHY
Si - OH
HEXANE
OH
C-CH3
CH3
CH3- C
CH3
CH3
OH
OH
CH3
CH3

12. WATER-SOLUBLE VITAMINS
1. Niacinamide 2. Pyridoxine
N
CONH2
N
CH2OH
CH2OH
HO
H3C
3. Riboflavin
N
N
NH
N
CH2
HOCH
HOCH
HOCH
CH2OH
O
O
H3C
H3C
Cl
N
S
N
N
H3C
CH2
NH2
CH3
CH2CH2OH
4. Thiamin

13. WATER-SOLUBLE VITAMINS
0 5 10 15 20
Column:u Bondapak C18
Solvent: MeOH
Sample: Water-Soluble Vitamins
Inject
1
2
3
4

14. LIQUID-LIQUID CHROMATOGRAPHY
ODPN(oxydipropionylnitrile)
Normal Phase LLC
Reverse Phase LLC
NCCH
3 CH2 OCH2 CH2 CN(Normal)
CH3 (CH2 )16 CH3 (Reverse)
The stationary solid surface is coated with a 2nd liquid (the
Stationary Phase) which is immiscible in the solvent (Mobile) phase.
Partitioning of the sample between 2 phases delays or retains some
components more than others to effect separation.

15. MOBILE PHASE LIQUID
Liquid-Liquid
Chromatography
(Partition)
Liquid-Solid
Chromatography
(Adsorption)
Liquid Solid
Normal Phase Reverse Phase Normal Phase Reverse Phase
Mobile Phase -
Nonpolar
Stationary phase -
Polar
Mobile Phase -
Polar
Stationary phase -
Nonpolar
FORMAT
STATIONARY
PHASE
Chromatography Schematic

16. ION-EXCHANGE CHROMATOGRAPHY
SO3
-
Na
+
Separation in Ion-exchange Chromatography is based on the
competition of different ionic compounds of the sample for the
active sites on the ion-exchange resin (column-packing).

17. REMEMBER…
 The stationary phase is POLAR
 The more polar component interacts more
strongly with the stationary phase
 The more polar component moves more
slowly.
 The non-polar component moves more
rapidly.

18. MECHANISM OF ION-EXCHANGE
CHROMATOGRAPHY OF AMINO ACIDS
SO3
-
SO3
-
Na
+
COO
-
H3N
+
Na
+
COOH
H3N
+
pH2
pH4.5
Ion-exchange Resin

19. H3N
+
SO3
-
SO3
-
SO3
-
SO3
-
SO3
-
SO3
-
H3N
+
COOH
OH
COOH
COOH
H3N+
H3N
+
OH
COO
-
Na
+
H3N
+
COO
-
Na
+
Na
+
H
+
OH
-
= H2O
H
+
OH
-
= H2O
Na
+
Na
+
pH3.5
Mobile Phase
Stationary Phase
Exchange Resin
pH4.5
Chromatography of Amino Acids

20. GEL-PERMEATION CHROMATOGRAPHY
Gel-Permeation Chromatography is a mechanical sorting of molecules
based on the size of the molecules in solution.
Small molecules are able to permeate more pores and are, therefore,
retained longer than large molecules.

21. SOLVENTS
Polar Solvents
Water > Methanol > Acetonitrile > Ethanol >
Oxydipropionitrile
Non-polar Solvents
N-Decane > N-Hexane > N-Pentane >
Cyclohexane

22. SELECTING AN OPERATING MODE
Sample Type LC Mode
Positional isomers LSC or LLC
Moderate Polarity Molecules LSC or LLC
Compounds with Similar Functionality LSC or LLC
Ionizable Species IEC
Compounds with Differing SolubilityLLC
Mixture of Varying Sized Molecules GCC

23. 1. Ultraviolet Detector
200-400nm
254 nm
2. Reflective Index Detector
Universal Detector
Detectors

24. Liquid Chromatography Set Up

25. HPLC Chromatography
1. Pump System. Mobil phase pressures up to 6000 psi are
necessary to achieve reasonable column elution times (~ minutes).
Typical flow rates are 0.1 to 10 mL/minute.
2. Injection System. Used to introduce small samples (0.1 to 500
µL) into the carrier stream under high pressure.
3. Reservoirs (Solvents). Multiple solvents are necessary for
performing gradient elution's (i.e. changing the polarity of the mobil
phase during a run).
4. Chromatographic Column. Typically 10-30 cm in length
containing a packing of 5-10 µm diameter. Many types of columns
are available, depending on the type of liquid chromatography
desired.
5. Detector. Many types are available including UV, IR, refractive
index, fluorescence, conductivity, mass spectrometry, and
electrochemical. Diode array detectors are used when wavelength
scans are desired.

26. Schematic of an HPLC System

27. HPLC System

28. Pump System
Desirable Features:
 Must generate pressures
up to 6,000 psi
 To allow for separation in
reasonable time frames
 Flow-rates range from 0.1
to 10 mL/minute
 Limited pulsing in the
system
 Many HPLC systems have a
dual pump system to
minimize pulsing
 Flow control and
reproducibility < 0.5%
 Corrosion resistance

29. Sample Injection System
Used to
introduce small
samples (0.001
to 0.5 mL) into
the carrier
stream under
high pressure

30. HPLC Detectors
 No universal or versatile detector
 Types
 General – respond to mobil phase bulk
properties which vary in the presence of
solutes (e.g. refractive index)
 Specific – respond to some properties of the
solute (not possessed by the mobil phase
(e.g. UV adsorption)
 “Hyphenated” detector – LC-MS

31. Absorbance Detectors
 The UV/Vis source usually comes
from a monochromator so the
wavelength can be selected, or
scanned.
 Absorbance increases as eluate
passes through the cell.
 If wavelength scanning is desired, the
flow is stopped long enough for the
scan to take place.
 It’s possible to have the same setup
using IR light, although not as
common since many useful solvents
are not IR transparent.

32. Diode
Array
Detector

33. HPLC Detectors

34. 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

35. Type of HPLC
Depends on:
1. Molecular
weight of solute
2. Water solubility
of the solute
3. Polarity of the
solute
4. Ionic/non-ionic
character of the
solute

36. Separation Principles in HPLC
General Rule of Thumb:
Polarity of analytes ≈ polarity of stationary
phase ≠ polarity of mobile phase
To achieve good separation, the analytes
should interact with the stationary phase,
but not too strongly or the retention time
will become very long

37. Increasing Mobil
phase Polarity,
Decreases
Elution Time
Reversed order
of elution

38. Typical Applications of
HPLC Chromatography
Field of Application Separation
Pharmaceuticals Antibiotics, Sedatives, Steroids, Analgesics
Biochemical Amino acids, Proteins, Carbohydrates, Lipids
Food Products Artificial Sweeteners, Antioxidants,
Preservatives
Industrical Chemicals Condensed Aromatics, Surfactants,
Propellants, Dyes
Forensic Chemistry Drugs, Poisons, Blood Alcohol, narcotics
Clinical Medicine Bile Acids, Drug Metabolites, Urine Extracts,
Estrogens
Pollutants Pesticides, Herbicides, Phenols, PCBs

39. HPLC of Orange Juice Compounds

40. How to Increase HPLC Resolution
1. Increase column length
2. Decrease column diameter
3. Decrease flow-rate
4. Pack column uniformly
5. Use uniform stationary phase (packing material)
6. Decrease sample size
7. Select proper stationary phase
8. Select proper mobile phase
9. Use proper pressure
10. Use gradient elution

41. Separating Proteins from Mixtures
In order to understand and study proteins it is
essential to separate them from the biological fluid.
Proteins can be separated from each other based on
differences in physical properties
Due to different amino acid sequences proteins differ
in solubility, size, charge, and binding affinity and can
be separated on either of these properties.
The inside of a cell. White shapes are
proteins (several 10s of thousands per
cell).

42. Water, Chemical bonds and groups Amino acids, pH dependence
C
COO-
H R
H3N+
Protein primary sequence, peptide bonds, secondary structures

43. Protein studies: Understanding protein
structure and function relationships
All proteins have a distinctive 3D structural
conformation
This unique structure enables its function
Amino acid sequence determines structure
A major goal of biochemistry is to determine how
amino acid sequences specify the 3D conformations
of proteins and to catalogue all proteins in cells.
Characterization
cell

44. Protein purification:
general experimental
setup
Homogenize
Centrifugation
Column
chromatography
Characterization

45. Gel permeation chromatography:
separating on basis of size
Mixture of proteins
1. A mixture of proteins in a small volume
is applied to a column filled with
porous beads
2. Because large proteins cannot enter the
beads, they emerge sooner than do
small ones
3. A detector (e.g. UV) is used to detect
protein fragments
4. Fragments are collected separately
UV
time

46. Affinity Chromatography: separating on
the basis of affinity
X
X X
X
X
X
X
X
To separate proteins that recognize
a chemical group X
1. X is covalently attached to beads that
are packed in a column
2. Sample of proteins is added
3. Washed with buffer to remove non
specifically bound protein
4. Eluted with high concentration of
soluble X
XX
X
X X
X
X X
X

47. Separation on the basis of charge
All proteins are charged. Their charges depend on the
relative number of acid and basic amino acids in their
primary structure.
All proteins have a pH value where they are uncharged:
the isolelectic point (pI)
H2N- Met Ala Asn Cys His Glu Ser Thr Glu Arg-COOH

48. Ionic amino acids

49. Separation on the basis of charge (continued)
H2N- Met Ala Asn Cys His Glu Ser Thr Glu Arg-COOH
His: 6.0
Glu: 4.1
Arg: 12.5
N-terminal amine: 8.0
C-terminal acid: 3.1
For this peptide:
pI=pKa/N= 6.3
Positively charged at pH < 6.3
Negatively charged at pH > 6.3

50. Ion Exchange Chromatography:
separation on basis of net charge
+
+ +
+
+
+
+
+
--
-
+
--
-
+ -
-
-- +
+
+
-
-
--
--
-
-
-
--
--
-
--
--
- -
-
--
--
1. Positive or negatively charged resin can be
used for separation of positive or
negatively charged proteins
2. Sample of proteins is added
3. Washed with buffer to remove non
specifically bound protein
4. Elute with increasing concentration of salt
5. Proteins with highest net charge come of
last

51. Why hydrogels are used for protein separations
1. Correct protein folding in aqueous environment
2. Proteins can denature on surfaces
3. Hydrogels are >90% water, good environment for proteins
1. 2.
3.

52. In Normal Phase Liquid Chromatography:
The column packing in the column is very polar!
Polar compounds are going to be attracted to the polar
column packing by hydrogen bonding or dipole-dipole
attractions. Polar compounds are going to move slowly!
Non-polar compounds are going to come off the column first,
while the polar compounds are going to come off column last.
Usually, one starts will a less polar solvent to remove
the less polar compounds, and then you slowly
increase the polarity of the solvent to remove the more
polar compounds.
Compare Reverse Phase to
Normal Phase Column Chromatography

53. Reverse Phase
Column Chromatography
 The stationary phase (column packing) is
now NON-POLAR
 Non-polar compounds will move more
slowly because they are attracted to the
column packing.
 The more polar component moves more
quickly down the column.
 Polar solvents, such as water and
methanol are used in reverse phase
chromatography
 Used mainly in columns, such as HPLC

54. Reverse phase chromatography
Silica is alkylated with long chain hydrocarbon groups, using 18
carbons long. This is usually referred to as C-18 silica.
O
Si
O
O
Si
O
O
O
O
Si
O
O
O
Si
O
O
O
Si
O
O
O
Si
O
O
Si
O
O
Si
O
O
Si
O
O
O
Si
O
O
Si
O
O
Si
O
O
O
CH2
CH3
17
Si
CH3
CH3
CH2
CH3
17
Si
CH3
CH3
SiCH3)3
SiCH3)3
SiCH3)3

55. Summary of Methodology
One of the main aims of biochemistry is to
characterize and catalogue all proteins in the cell
We have discussed some important tools for
separating proteins based on physical properties
such as size, affinity, charge.
Chromatography methods: ion exchange, affinity,
gel permeation chromatography
Electrophoresis: iso electric focusing, SDS PAGE,
2D gels (in the Biochemistry lecture series)

56. Overview of Paper Chromatography
 Works on principle of
Partition.
 Separates dried liquid
samples.
 Mobile phase is solvent
used.
 Stationary phase is water
bound to surface of
paper.
 Advantage : its cheap!

57. Important Concepts in
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.

58. Paper/TLC
Chromatography
Animation

59. Simple Example of Paper
Chromatography using “Sharpie” Pens

60. Dye Separation in a Black “Sharpie”
Concentration of Isopropanol
0% 20% 50% 70% 100%
1. Dyes separated – purple and black
2. Not soluble in low concentrations of
isopropanol
3. Partially soluble in concentrations of
isopropanol >20%

61. Thin Layer Chromatography
 Works mainly on
principle of
adsorption.
 Mobile phase is the
solvent
 Stationary phase is
the solid on the plate.

62. TLC vs. Column Chromatography
 Thin-layer chromatography and column
chromatography and are different types of
liquid chromatography.
 The mobile (moving) phase is a liquid.
The stationary phase is usually silica or
alumina. This phase is very polar.
 The principle of operation is the same!

63. Thin Layer Chromatography
The surface of a plate consists of a very thin layer of silica on a plastic or
aluminum backing. The silica is very polar. This is the stationary phase. Material
is spotted at the origin (bottom) of the TLC plate.
The plate is placed into a glass jar with a small amount of a solvent in a glass jar.
This solvent acts as the moving phase.
The plate is removed from the bottle when the solvent is close to the top of the
plate.
The spots are visualized (explanation to follow).
Non-polar compounds will be less strongly attracted to the plate and will spend
more time in the moving phase. This compound will move faster and will appear
closer to the top of the plate.
Polar compounds will be more strongly attracted to the plate and will spend less
time in the moving phase and appear lower on the plate.

64. Thin-Layer Chromatography: A
Two-Component Mixture
More polar!
Less polar!
solvent front
origin mixture
solvent front
component B
component A
origin
solvent front
component B
component A
origin
Increasing Development Time

65. Thin-Layer Chromatography:
Determination of Rf Values
solvent front
component B
component A
origin
dS
dB
dA
Rf of component A =
dA
dS
Rf of component B =
dB
dS
The Rf value is a decimal
fraction, generally only
reported to two decimal
places

66. Thin-Layer
Chromatography:
Qualitative
Analysis
A B unknown

67. Visualization Method
 The previous slide shows colored spots. Most of
the time, the spots won’t show unless they are
visualized!
 Vizualization is a method that is used to render
the TLC spots visible.
 A visualization method can be:
 Ultraviolet light
 Iodine vapors to stain spots
 Colored reagents to stain spots
 Reagents that selectively stain spots while leaving
others unaffected.

68. TLC Advantages
Advantages over paper:
 Its faster
 It gives a better separation.
 It is more versatile as the solid on the
plate can be varied.

69. Uses of TLC
 To determine how many components
there are in a mixture (is it really pure?)
 To determine the best solvent conditions
for separation on a column
 To identify the substances being studied
 To monitor the composition of fractions
collected from column chromatography
 To monitor the progress of a reaction

70. Gas-Liquid Chromatography
 Works on principle of Partition.
 Mobile phase is the carrier gas.
 Stationary phase is oil bound to surface of
beads within the column.

71. Most Common Stationary Phases
1. Separation of mixture of polar compounds
Carbowax 20M (polyethylene glycol)
2. Separation of mixtures of non-polar compounds
OV101 or SE-30 (polymer of methylsilicone)
3. Methylester of fatty acids
DEGS (diethylene glycol succinate)

72. Gas-Liquid Chromatography

73. Gas-Liquid Chromatography
 Retention time is used to identify a
component of a mixture. It depends on:-
 The temperature of the column.
 The length of the column.
 The material used to pack the column.
 The gas pressure.

74. Gas –Liquid Chromatography
 The area under a peak is proportional to the
amount of substance present.

75. Filters/Traps
Air
Hydrogen
Gas
Carrier
Column
Gas Chromatography
 gas
system
 inlet
 column
 detector
 data
system
Data system
Syringe/Sampler
Inlets
Detectors
Regulators
H
RESET

76. Schematic Diagram of Flame Ionization Detector
Collector
Jet
Flame
Detector electronics
 - 220 volts
Column
Chassis ground
Signal output

77. Gas-Liquid Chromatography
 Gas-Liquid Chromatography is often combined
with mass spectroscopy. The GC separates
the components then the MS analyses them.

78. One possible Use of GC:
SEMI- QUANTITATIVE ANALYSIS OF FATTY ACIDS
C
C
C
Detector Response
RetentionTime
14
16
18 Peak Area (cm )
Sample Concentration (mg/ml)
2
4
6
8
10
0.5 1.0 1.5 2.0 2.5 3.0
2
The content % of C fatty acids =
C
C + C + C






= the content % of C fatty acids
14
14

79. Gas Chromatogram of Methyl Esters of Fatty Acids

80. Another GC Use:
TENTATIVE IDENTIFICATION OF UNKNOWN COMPOUNDS
Response
GC Retention Time on Carbowax-20 (min)
Mixture of known compounds
Hexane
Octane
Decane
1.6 min = RT
Response
Unknown compound may be Hexane
1.6 min = RT
Retention Time on Carbowax-20 (min)

81. GLC ADVANTAGES
1. Very good separation
2. Time (analysis is short)
3. Small sample is needed - l
4. Good detection system
5. Quantitatively analyzed

82. DISADVANTAGES OF GAS CHROMATOGRAPHY
Material has to be volatilized at 250º C without decomposition!
R C OH CH3OH H2SO4
O
R C O CH3
O
CH2 O C R
CH O C R
CH2 O C R
O
O
O
CH3OH
O
R C O CH3
CH3ONa
Fatty Acids Methylester
Reflux
+ 3
Volatile in Gas
Chromatography
Volatile in Gas
Chromatography
+ +

83. Summary of Some Chromatographic Techniques
Technique Stationary Phase Mobile Phase Typical Application
Paper Trapped water in
the paper Organic Solvent
 amino acid
mixtures
 food colors or dyes
Thin Layer Oxide Coating
Organic Solvent
 detect amino acids
 composition of dyes
and food colors
Column Oxide packing or
resin Organic Solvent
 preparative
 separation of plant
pigments
Gas-Liquid Oxide or volatile
liquid on a solid
support
Gas
 analysis of oil
mixtures
 detect drugs &
steroids
 fruit esters
High Performance
Liquid
Oxide Packing or
Resin or
Molecular Sieve
Liquid
 analyze foods,
pesticides, etc
detect iron in body
fluids
detect blood alcohol