Analytical Biochemistry
http://www.waters.com
1
How Does Chromatography Work?
Chromatography is a method for
separating the components of a
mixture by differential adsorption
between a stationary phase and a
mobile (moving) phase
Liquid Liquid
Liquid Solid
Partition Adsorption
Stationary Phase chromatography
chromatography
(固定相)
2
Principles of Separation Techniques
AB 3.1
Molecular
Physical property Separation Technique
Characteristic
3
Factors Involved in Separation
Gravitational (Ultracentrifugation)
Impelling Electrokinetic (Electrophoresis)
Force Hydrodynamic (Chromatography, 沖堤液
驅動力)
Retarding Force
Zonal (區帶)
A band (zone) in a solvent system
-- Change in pH, size…etc
Displacement
A band (zone) in two-phase solvent
system
---Different affinity for the solid
support (stationary/mobile phase)
Frontal (前端)
Large sample containing in mobile
phase
5
POLARITY (affinity of like molecules
for each other)
PARTITION BETWEEN TWO PHASES
Adsorption Solubility
Thin-Layer chromatography
• Stationary phase is coated onto glass, metallic or plastic
plate.
8
Liquid-Solid Chromatography (Adsorption)
AB 3.2.1
Adsorption (吸附):
Some substances physically bind to the
surface of a solid polar substances
Polar compound
Large surface for adsorption
Often by OH (hydroxy group) to form H-bonding
9
Polarity of Selected Solutes and Solvents
Silica
Adsorption Solvent
Solute Solvent
Energy Strength
Solvent Strength
the affinity of a solvent with an adsorbent ( vary with adsorbant) 10
Stationary Phase: Alumina
O OH OH OH OH
Al Al Al Al Al
O O O O O O
Acidic: -Al-OH
Neutral: -Al-OH + -Al-O-
Basic: -Al-O-
11
Examples of Absorbents and Applications
12
Thin-layer chromatography and column
chromatography are different types of
liquid chromatography. The principle of
operation is the same!
13
Thin Layer Chromatography (薄層層析法)
1. The surface of the plate consists of a very thin layer of
silica on a plastic or aluminum backing. The silica is very
polar the stationary phase.
solvent front
component B
Rf of component A = component B
dA
dS dS
dB
Rf of component B =
component A
dB
dS dA
origin
1. Only reported to two decimal places
16
Thin-Layer Chromatography:
Qualitative Analysis
O OH
Example:
High Performance Liquid Chromatography
• Partition chromatography
• Adsorption chromatography
• Gel filtration chromatography
• Affinity chromatography
• Ion-exchange chromatography
19
High Performance Liquid Chromatography
Small and regular AB 3.2.2
support media with
stationary phase
porous.
6-port
valve INJECT position
Sample
INJECT
Column
25
HPLC Diode Array Detection Analysis -
Elution Time
pyrithiones
Absorbance is
measured at two or Absorption Wavelength
more wavelengths
Pyrithione is an anti-oxidant 26
Column Chromatographic Separation
Stage 1 2 3 4
8 7
6 5 8 7 8 7 8 Addition of
4 3 Mobile phase
6 5
2 1
6 5 7
4 3 Assume
4 3 5 6
2 1 Partition
coefficient =1
4 3
2 1 2
Stationary
Phase
1
Mobile
Phase
27
Band Broadening Chromatographic
Peak
Stage 1 2 3 4
8 7
6 5 8 7 8 7 8
4 3 6 5
2 1
6 5 7
4 3
4 3 5 6
2 1
4 3
2 1 2
Gaussian
Distribution
1
28
Separation Efficiency: Plate Theory
The plate theory suppose that the chromatographic
column contains a large number of separate layers, called
theoretical plates. Separate equilibrations of the sample
between the stationary and mobile phase occur in these
"plates". The analyte moves down the column by transfer
of equilibrated mobile phase from one plate to the next.
Bad
Resolution
Good
Resolution
tB Peak width at
tA half peak height
W1/2
A B
W
Peak WA WB
broadening Elution Time 30
Theoretical Plate Number—Resolution
A measure of separation efficiency: How many times the
Analyte mobile → Analytestationary equilibrium is achieved
2 2
tR tR tR -σ tR +σ
N= =
σ2 σ
W1/2 =2.355σ
2 Peak broadening may be
tR W1/2
N = 16 expressed by variance
W
2 tR
2.355 × t R
N = W≈4σ
peak width at half peak height
2
retention distance 2
Retention time: measure of
= × 5.54 tR effective column volume for
width at half peak height analyte
W Base width 31
Height Equivalent to a Theoretical Plate
(HETP)
Length of a column necessary
for the attainment of compound
distribution equilibrium (measure
the efficiency of the column).
length of the column HETP Insufficient time for
HETP =
N equilibrium
4.4
Relative retention = = 1.3
3.4 min 3.4
33
Quantitative Analysis
50.5 mm
20.0 mm
Standard
Reference: Propanol
Standard: Ethanol
35
Reference:
Ethanol Peak Ethanol/Propan
Peak height Propanol
Conc. height ol
20 78 1 34 2.29
15 57 1 35 1.63
10 37 1 34 1.09
5 21 1 36 0.58
X 45 1 35 1.29
2.50
Ethanol/Propanol
2.00
1.50 數列1
線性 (數列1)
1.00
0.50
0.00
0 5 10 15 20 25
Ethanol Conc.
36
Partition Chromatography BMB 11.5
37
Normal-Phase HPLC
Adsorption of analytes on the polar, weakly acidic surface
of silica gel
O H
O H
O H
O H
O H S i
S i O
S i O
S i O O
S i O O
O O
O O
O S i S i
O O
S i
S i O O O
S i O O
O
O O
O
S i
S i O
O
O O
O
+ charge
Charge of Analyte
- charge
Anion exchanger.
43
Gel Permeation Chromatography --
Molecular Sieve Chromatography
The separation is based on the molecule
size and shape by the molecular sieve
properties of a variety of porous material
44
Gel Permeation Chromatography (GPC)
45
Mass measurement by Gel Permeation
Chromatography AB 3.4
Small
Large
mass
mass
Elution
time
47
Determination of Mass
The elution volume is approximately a
linear function of the logarithm of the
relative molecular mass
Elution volume
49
Covalent Attachment of Ligand to the Matrix
Derivation of Epoxy-Activated Agarose
50
Affinity chromatography
Substrate analogue affinity chromatography
Affinity Enzyme
ligand
Immunoaffinity chromatography
Antibody Protein epitope
ligand
54
Two-dimensional Chromatography (2D-LC)
First LC
4 fractions
Second
t1 LC
Mixtures
t2
t3
t4
55
Complex Human Proteome
含有許多
標記蛋白質
0-90% 90-99%
Fig. Pie chart representing the relative contribution of proteins within plasma.
Twenty-two proteins constitute 99% of the protein content of plasma
www.plasmaproteome.org
Ref: Molecular & Cellular Proteomics 2:1096–1103, 2003
56
3D LC for Global Analysis of Serum Proteome
IEF-SCX-RP
2071 peptides (identification of
1143 unique proteins).
Iso-electric
Focusing
(IEF)
Strong-Cation
Reverse-Phase Exchange
(RP) (SCX)