The Chromatographic Process

1
?????? ????? ??????? ?????????

• ????? ???????????? ?? ??????? ????????????

????' ????? ??????? ?????? ??????????????
????????? ?????????????? ?????????? ?"?
2
The Chromatographic Process
3
??????? ??????? ??????? ????????????? ?? ???????
????????????
ns - No. of moles of Protein x in the Stationary
Phase nm - No. of moles of Protein x in the
Mobile Phase to - Retention time for unretained
compounds tR - Retention time for Protein x u -
Linear velocity of the Solvent ux - Linear
velocity of Protein x
4
??????? ?????????

• ???? ????? ??? a ?????
• ??? ??? ???? ???? ??? ???? ?????? ????? ??????
  ???? ??"? ?????? ?? ?????? ???? ?- a
• ?????? ?? ??????? ?????? ????? ????? ?????? ????
  ???? ???? ?- a

Stm t0
ts
S(tmts) tR
a t0/tR
t0
tm
5
??????? ??????? ??????? ????????????? ?? ???????
????????????

6
The Correlation of the Capacity Factor (k) and
Affinity Constant (Ka)
7
Effect of Sample Load on k
8
The Correlation of the Capacity Factor (k) and
Distribution Constant (KD)
9
Evaluation of Chromatographic Behavior
Theoretical Plate
tR and VR vary from one compound to the other. L
is common to all compounds separated on a column
10
The Theoretical Plate
11
Diffusion
12
Effect of Linear Velocity on Peak Broadening
Van Deemter Equation
13
Peak Broadening
14
Peak Broadening (Cont.)
15
Peak width
16
Peak Broadening (Cont.)
17
Velocity vs. Pressure
18
Spherical and irregular HPLC Packings
19
Peak Tailing
20
Evaluation of Peak Tailing
21
Causes for Peak Tailing

• Overloading
• Poor Column packing (Channeling)
• Mixed Retention Mechanism
• Poor Equilibration and/or Slow Mass Transfer
• Extra-Column Effects
• Micelle Formation

22
Resolution

k1/k1
1
23
Two-Peak Separation
24
Separation Optimization
25
The Correlation between k and tw
tw4to/?N
KtR/to-1
26
Effect of Solvent Strength
27
Effect of Selectivity (a)
28
Effect of Efficiency (N)
29
Effect of temperature on RP
If DGlt0 and T
Then Ka and k
Peaks usually become sharper at higher
temperatures
30
HPLC ?- FPLC
31
The HPLC Chromatographic System
Damp
Back-Pressure Regulator
32
Problems Derived from High Pressure Chromatography

• Need for Pressure Control/Monitoring.
• Stainless Steel Columns.
• High Pressure/Low Stroke Volume Pumps.
• High Pressure Mixing.
• High Pressure Injectors.
• Need for Gas Bubbles Elimination Degassing
  and/or Back Pressure Regulator.

33
The FPLC
Computer Control
Injector
Pump A
Mixer
Fraction Collector
Pump B
Column
Buffer A Buffer B
UV Monitor
Conductivity Meter
34
FPLC vs. HPLC

• HPLC
• High Pressure Operation
• Uses Mostly Reversed and Normal Phase
  Chromatographies Often with Organic Solvents
  Applicable to small proteins, peptides and low MW
  molecules.
• Highest advantage High Resolution.
• FPLC
• Medium Pressure Operation.
• Uses Mostly Ion Exchange, Hydrophobic, Affinity
  and Gel Chromatographies in aquous buffers
  Highly applicable to proteins.
• Highest Advantage Ease of Operation and
  Versatility.