1. Chromatography

2. Lab Scale
Chromatography System
Large Scale
Chromatography System

3. Chromatography
Solute fractionation technique which relies on the dynamic distribution of molecules between stationary (or binding) phase and a mobile (or carrier) phase
The velocities at which these molecules move through the column depend on their respective interactions with the stationary phase

4. Chromatography Used for the separation of different substances:
proteins, nucleic acids, lipids, antibiotics, hormones, sugars
Analytical chromatography: used for analysis of complex mixtures
Preparative chromatography: used to separate molecules as part of a manufacturing process

5. System mobile phase reservoir/s, column, pump/s, sample injector,
detector/s
Fraction collector.

6. Types of column Packed bed column Packed capillary column
Open tubular column
Membrane
Monolith

7. Separation Mechanism Ion exchange - electrostatic interaction
Reverse phase - solutes partition
Hydrophobic interaction - hydrophobicity
Affinity - specific recognition and binding
Size exclusion – size (inert porous particle)

8. Separation Mechanism

9. Chromatography separation
components move at different velocities through the column
components are segregated into moving bands which appear in the effluent stream as separate peaks at different times

10. Chromatography separation

11. Column Chromatography
Separates molecules by their chemical and physical differences
Most common types:
Size exclusion (Gel filtration): separates by molecular weight
Ion exchange: separates by charge
Affinity chromatography: specific binding
Hydrophobic Interaction: separates by hydrophobic/hydrophilic characteristics 11

13. Size Exclusion Chromatography
separation technique based on the molecular size of the components. 
Separation is achieved by the differential exclusion from the pores of the packing material, of the sample molecules as they pass through a bed of porous particles. 
The principle feature of SEC is its gentle non- adsorptive interaction with the sample, enabling high retention of biomolecular activity 13

14. GFC & GPC
For the separation of biomolecules in aqueous or aqueous/organic mobile phases, SEC is referred to as gel filtration chromatography (GFC)
while the separation of organic polymers in non- aqueous mobile phases is called gel permeation chromatography (GPC). 14

15. Gel Filtration Chromatography15

16. Ion Exchange Chromatography
pH < pI < pH
Relies on charge-charge interactions between the protein of interest and charges on a resin (bead).
cation exchange chromatography: a positively charged protein of interest binds to a negatively charged resin;
anion exchange chromatography: a negatively charged protein of interest binds to a positively charged resin.

17. Ion Exchange Chromatography
using buffers of different pHs to alter the charge on the protein.
the column is washed with equilibration buffer to remove unattached entities.
Then the bound protein of interest is eluted off using an elution buffer of increasing ionic strength or of a different pH.
Weakens the attachment of the protein of interest to the bead and the protein of interest is bumped off and eluted from the resin.
Ion exchange resins are the cheapest of the chromatography media available

18. Affinity Chromatography
separates the protein (antigen) of interest on the basis of a reversible interaction between protein and its antibody coupled to a chromatography bead.
With high selectivity, high resolution, and high capacity for the protein of interest, purification levels in the order of several thousand-fold are achievable.
Biological interactions between the antigen and the protein of interest can result from electrostatic interactions, van der Waals' forces and/or hydrogen bonding.

19. Affinity Chromatography
To elute the protein of interest from the affinity beads, the interaction can be reversed by changing the pH or ionic strength.
The concentrating effect enables large volumes to be processed. The protein of interest can be purified from high levels of contaminating substances.
Making antibodies to the protein of interest is expensive, so affinity chromatography is the least economical choice for production chromatography.

20. Affinity Chromatography
Abs 280nm
Time (min)

21. Hydrophobic Interaction Chromatography (HIC)
the molecular mechanism of HIC relies on unique structural features
Usually HIC media have high capacity and are economical and stable.
Adsorption takes place in high salt and elution in low salt concentrations.

22. HIC Typically used for protein separations
Employs derivatized polymer resins, with phenyl, butyl, or octyl ligand groups
Proteins adhere to the hydrophobic surface under high salt conditions
Redissolved into the mobile phase as the salt concentration is reduced

23. Hydrophobic Interaction Chromatography (HIC)

24. Reverse-phase chromatography (RPC)
Principles are quite similar to hydrophobic interaction chromatography
In RPC, the solid support is highly hydrophobic which allows the mobile phase to be aqueous
The more hydrophobic the molecule the more time it will spend on the solid support and the higher the concentration of organic solvent that is required to promote de-sorption.
To have the best resolution, a gradient elution with organic solvent is needed
Limitation in protein separation  denature

25. Mobile Phases
The thumb-rules for selecting mobile phases in binary chromatography based on different separation mechanisms

26. Theory

27. Theory Capacity Factor (Concentration) “Distribution coefficient”
Ideally between
Capacity factor (Mole)

28. Theory-retention time
time at which the concentration of that solute reaches its maximum value in the effluent
the mobile phase retention time (tM): based purely on hydrodynamic considerations,
the adjusted retention time (tR'): due to solute-stationary phase interaction

29. Theory

30. Theory

31. Theory
Chromatographic separation primarily relies on difference in solute retention times
The solute peaks should appear separately.
The quality of separation depends on how well the peaks are resolved.

32. Resolution
The spatial separation to the two peaks (i.e. whether the peaks overlap or not) is measured in terms of the resolution parameter

33. Resolution The peak width depends on :
the interactions between the solute and the stationary phase,
the mobile phase flow rate and
the solute concentration in the injected sample.
The greater the value of R, the better is the spatial separation of the peaks.
An R value of less than 1 indicates that the peaks overlap, a value of 1 indicates that the peaks are just resolved, while a value greater than 1 indicates there is a gap between the separated peaks

35. Selectivity Parameter
Chromatographic separation can also be quantified in terms of the selectivity parameter (α).
This depends on the difference in interaction of the two solutes with the stationary phase and the mobile phase flow rate
It is independent of the solute concentrations.
The greater the value of α, the better is the separation.

36. Plate Theory
A chromatographic column can be assumed to be made up of a large number of theoretical plates which are analogous to those in a distillation column.
The greater the number of these plates in a column the better is the separation.

37. HETP
The height equivalent of a theoretical plate (HETP or H) is given by:

38. Theory-HETP
The number of theoretical plates in a chromatographic column can be determined by obtaining a chromatogram with a solute.
N depends on the solute retention time and peak width as shown below:

39. HETP Van Deemter equation for plate height
The resulting band shape of a chromatographic peak is therefore affected by the rate of elution.
It is also affected by the different paths available to solute molecules as they travel between particles of stationary phase.
Van Deemter equation for plate height
HETP = A + B / u + C u
U- velocity of mobile phase; A - Eddy diffusion; B - Longitudinal diffusion; C - Resistance to mass transfer

40. A - factor contributed by eddy diffusion, primarily due to dead volume and proportional to resin particle size
B - molecular diffusion factor
C - mass transfer and intraparticle diffusion

41. Resolution
The Resolution for N theoretical plate (RN) can be related to the chromatographic parameters, α, K, and N as:

42. Example

43. Solution
Residence time

44. Solution

45. Solution
The selectivity of separation can be obtained using equation (10):

46. Solution
The peak width can be determined using equation (12):

47. Solution
The resolution can be determined using equation (9):

48. The concentration profile in a peak is given by

50. Yield & Purity
the yield of solute in the effluent collected in the time period t1 to t2 is:
The purity of a solute A in the binary separation of solutes A and B, in effluent collected in the time period t1 to t2 is:

51. Example

52. Example

53. Solution

54. Solution

55. Solution

56. Solution

58. When t’=0

59. Example
We plan a large scale purification of urease using a packed column of polyacrylamide beads. We obtain the following data:
Volume Eluted
Concentration
174
0.0063
190
(maximum)
The bed volume is 20 liters. Find the yield at 190 and 200 liters

60. Solution
When t’=0

61. 0.5907
erf(0.5907)=0.596
Yield at 190 liters = 50% and after 200 liters is 80%

62. Changing Capacity
An efficient way to increase the capacity while maintaining purification is to increase the flow through the column
The concentration C is a function of three parameters: Co, V/Vo or t/to and σ’

63. Changes in Standard deviation- rate constant
V = velocity; l = column length, k =rate constant,
a = area / volume of packed bed particles
Diffusion Control
External mass transfer Control

65. Changes in Standard deviation- dispersion
Dispersion become significant in larger column due to the polydisperse packing and nature of flow
For Laminar flow:
For turbulent flow

66. Example
10 g of the enzyme fumarase are being purified in an ion exchange column. At a velocity of 30 cm/hr, the peak in concentration exits the column in 93 min and the standard deviation of this peak is given as 12 min.
How long must we purify for a 90% yield?
If we increase the flow to 60 cm/hr, how long must we run for this same yield if the process is controlled by diffusion and reaction?

67. Solution (a)

68. Solution (b)
The velocity is doubled but the column length and packing diameter are unchanged. Thus the time to elute the peak is cut in half; to=46.5 min
We must wait (57.39/46.5) or 1.23 times the peak to get a 90% yield

69. Scale Up
Chromatography scale up algorithms typically account for changes in bed height and diameter, linear and volumetric flow rate, and particle size
General approach to scale up is based on keeping the resolution constant

70. Scale Up

71. Scaling Up
For scale up with constant resolution from scale 1 to scale 2 for the same product and the same column void fraction, the scale up equation is

72. Scaling Up
As the particle size increases on scale up, the flow rate relative to the column volume must decrease and/or the gradient slope must decrease to maintain constant resolution
In practice, the gradient and the stationary phase size and chemistry are not changed upon scale up : use the same resin and same gradient
Only the ratio between column volume and flow rate need be addressed

73. Scale Up
When the bed height can be maintained on scale up, the mobile phase linear velocity remains the same, and the column is simply scaled by diameter

74. Example
A column 20 cm long, with an internal diameter of 5 cm, gives sufficient purification to merit scaleup. The column produces 3.2 g of purified protein per cycle, and a cycle takes 6 hr, from equilibration through regeneration. You want a throughput of 10 g/h. What are the new column’s dimensions if linear velocity is held constant?

75. Solution