‫תהליכי הפרדה למוצרים ביולוגיים‬
‫ניתוח ואופטימיזציה של תהליכים‬
‫כרומטוגרפיים‬
‫פרופ' גדעון פלמינגר‬
‫המחלקה למיקרוביולוגיה מולקולרית ולביוטכנולוגיה‬
‫אוניברסיטת ת"א‬
‫‪1‬‬
The
Chromatograph
ic Process
2
‫פרמטרים המשמשים באנליזה ובאופטימיזציה של‬
‫תהליכים כרומטוגרפיים‬
(1) k ' 
ns
nm
(2)   R 
R
Capacity Factor
nm
1

Re lative Mobility
nm  ns 1  k '
tm
t
u
 o  x
t m  t s t R uo
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
3
‫שיקולים סטטיסטיים‬
‫• עבור חלבון בעל ‪ ‬נתונה‪:‬‬
‫– בכל רגע נתון היחס בין מספר המולים בפאזה הניידת לבין‬
‫סה"כ המולים של החלבון שווה ל‪ -‬‬
‫– הסיכוי של מולקולה מסוימת להיות בפאזה הניידת ברגע‬
‫נתון שווה ל‪ -‬‬
‫‪Stm= t0‬‬
‫‪ts‬‬
‫‪S(tm+ts)= tR‬‬
‫‪ = t0/tR‬‬
‫‪t0‬‬
‫‪tm‬‬
‫‪4‬‬
‫פרמטרים המשמשים באנליזה ובאופטימיזציה של‬
‫תהליכים כרומטוגרפיים‬
‫‪+‬‬
‫‪t R  t o VR  Vo‬‬
‫‪(5) k ' ‬‬
‫‪‬‬
‫‪to‬‬
‫‪Vo‬‬
‫‪5‬‬
The Correlation of the Capacity Factor
(k’) and Affinity Constant (Ka)
6
Effect of Sample Load on k’
7
The Correlation of the Capacity Factor
(k’) and Distribution Constant (KD)
8
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
9
The Theoretical Plate
10
Diffusion
11
Effect of Linear Velocity on Peak
Broadening
Van Deemter Equation
12
Peak
Broadening
13
Peak
Broadening
(Cont.)
14
Peak width
15
Peak
Broadening
(Cont.)
16
Velocity vs. Pressure
17
Spherical and irregular HPLC
Packings
18
Peak
Tailing
19
Evaluation of Peak Tailing
20
Causes for Peak Tailing
•
•
•
•
Overloading
Poor Column packing (Channeling)
Mixed Retention Mechanism
Poor Equilibration and/or Slow Mass
Transfer
• Extra-Column Effects
• Micelle Formation
21
Resolution
+
*k1/k1
1
22
Two-Peak
Separatio
n
23
Separation
Optimization
24
The Correlation between k’ and tw
K=tR/to-1
25
tw=4to/N
Effect of Solvent Strength
26
Effect of Selectivity ()
27
Effect of Efficiency (N)
28
Effect of temperature on RP
If DG<0 and T
Then Ka and k’
Peaks usually become
sharper at higher
temperatures
29
HPLC -‫ ו‬FPLC
30
The HPLC Chromatographic System
Damp
Automatic Control
BackPressure
Regulator
31
Problems Derived from High
Pressure Chromatography
•
•
•
•
•
•
32
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.
The FPLC
Computer
Control
Injector
Pump A
Mixer
Fraction
Collector
Pump B
Column
Buffer A
Buffer B
Conductivity
Meter
33
UV
Monitor
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.
34