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Bio Adsorption Chromatography

Bio Adsorption Chromatography

A term mainly used in prep and production scale of biologics using polymeric materials. Resin manufacturers also use more familiar terms including Reverse Phase Chromatography and Solid Phase Extraction (SPE) and Hydrophobic Interaction Chromatography
What type of Molecules can be extracted and purified ?

Bio Adsorption Chromatography is used to extract / isolate a wide range of natural or synthetic compounds from a crude liquor to be purified.

This method is commercially used to purify Saccarides e.g. Stevia based sweetener, Cyclodextrines, Amino Acids, Nucleic Acids, Nucleosides from fermentation broth. Removal of nucleic acids from proteins produced in cell cultures systems is done with this strategy. Alkaloids from plant culture, removal of endotoxines and viruses from cell cultures. Some companies use this method to remove lipids and Prostaglandins from cultures.

In food processing removal bitter principles and dyes from fruit juices is practiced. Broad range of medicinal plant extracts are extracted and purified with bio adsorption LC or SPE.
Bio Adsorption Process Benefits

The function of synthetic adsorbent materials is similar to that of a solvent in a solvent extraction process
Desorption is done with small amounts of organic solvents.
Solid Phase Extraction is less toxic and flammable than liquid liquid extraction
Adsorbents have a molecular sieving function and based on the pore structure fractionation by molecular size can take place
Unbeatable economics with large liquor to small compound ratios.
Mild adsorptivity, thus compounds can be eluted under mild conditions
Repeated use of adsorbent will results in great economic advantage.
Ability to use aggressive mobile phases for selectivity optimization
The poystyrene based resins can be cleaned-in-place easily with strong acid or base
Thermal or chemical sanitation possible
Gradient elution or cleaning with broad range of solvents with minimal change in bed volume.
Enables repeated cycles with assured column stability and reproducible performance
Ability to operate high flow rates, for high through put with moderate back up pressure

Terminology

The terms such as Reverse Phase Chromatography, Solid Phase Extraction or Adsorption Liquid Chromatography is used to describe the process of extracting / isolating with resinous materials hydrophobic organic compounds from aqueous solutions including fermentation broth , food, beverages or effluents. From Laboratory to Production Scale.
The Term Reverse Phase Chromatography (RPC) is generally used in Lab to prep scale when higher pressures are used
Solid Phase Extraction (SPE) is primarily used in sample preparation where polymers and silica is being used.
Adsorption Liquid Chromatography is primarily used in prep and production scale purification of hydrophobic compounds.
The Adsorption / Desorption Process

Explanation

Step 1: Resin is supplied with water on the surface (yellow dots). The ratio of the solvent /water concentration on the adsorbent surface to that in the liquid phase is constant (distribution constant)at low concentrations. Van der Waals Forces,(physicosorption, delta Ha < 50kJ.mol-1 .Adsorbents can be manufactured in a highly porous form. Adsorbents have a surface >400m2/g
Step 2: Solute (Target Comound ) is percolated through the resin. Adsorption occurs when molecules in the fluid phase are held for a period of time by Van der Waal forces. The surface represents a discontinuity in the structure of the solid, and atoms at the surface have a residue molecular forces which are not satisfied by the surrounding atoms like those in the body of the structure. These residual or van der Waals forces are common to all surfaces.
Step 3: Small quantities of eluent (Desorbing Solvent) is added to release compound.

Materials used

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Process Selectivity

 

Reverse Phase Chromatography, Solid Phase Extraction or Adsorption – LC, a very effective and competitive  method to isolated and purify compounds
Page Index
What type of Molecules can be extracted
SPE Process benefits
Terminology
The Adsorption / Desorption Process
Materials used
Process Selectivity
Industrial Application of SPE
Process Parameters Effecting Adsorption Capacity
 
What type of Molecules can be extracted
This technology enables to extract / isolate a wide range of natural or synthetic compounds from a crude liquor to be purified including: Saccarides  e.g. Stevia based sweetener, Cyclodextrines, Amino Acids, Nucleic Acids, Nucleosides from fermentation broth, removal of nucleic acids  from proteins produced in cell cultures systems. Alkaloids from plant culture, removal of endotoxines  and viruses from cell cultures, removal of lipids, Prostaglandins from cultures, In food processing removal bitter principles and dyes from fruit juices, wide range of medicinal plant extracts are extracted and purified with adsorption LC.
 
SPE Process benefits
The function of synthetic adsorbent materials is similar to that of a solvent in a solvent extraction process
Desorption is done with small amounts of organic solvents
Solid Phase Extraction is less toxic and flammable than liquid liquid extraction
Adsorbents have a molecular sieving function and based on the  pore structure fractionation by molecular size can take place
Unbeatable economics with large liquor to small compound ratios
Mild adsorptivity, thus compounds can be eluted under mild conditions
Repeated use of adsorbent will results in great economic advantage
Ability to use aggressive mobile phases for selectivity optimization
Can be cleaned-in-place easily with strong acid or base
Thermal or chemical sanitation possible
Gradient elution or cleaning with broad range of solvents with minimal change in bed volume
Enables repeated cycles with assured column stability and reproducible performance
Ability to operate high flow rates, for high through put with moderate  back up pressure
 
Terminology
The terms such as  Reverse Phase Chromatography, Solid Phase Extraction or  Adsorption Liquid Chromatography  is used to describe the process  of extracting  / isolating with resinous materials hydrophobic organic compounds from aqueous solutions including fermentation broth , food, beverages or effluents. From Laboratory to Production Scale.
The Term Reverse Phase Chromatography (RPC) is generally  used in Lab to prep scale when higher pressures are used
Solid Phase Extraction (SPE) is primarily used in sample preparation where polymers and silica is being used.
Adsorption Liquid Chromatography is primarily used in prep and production scale purification of hydrophobic compounds.
 
The Adsorption / Desorption Process
Resin supplied with water on the surface
Fig.1
Target molecules in water are adsorbed
Fig.2
Eluent is applied to release target compounds Fig.3

Explanation
yellow dots: Solvent (Water), red  dots: Solute (Target Compound),  blue dots: Eluent , Desorbing Sovent
Fig.1 The ratio of the solvent /water concentration on the adsorbent surface to that in the liquid phase is constant (distribution constant)at low concentrations. Van der Waals Forces,(physicosorption, delta Ha < 50kJ.mol-1 .Adsorbents can be manufactured in a highly porous form. Adsorbents have a surface >400m2/g
Fig.2 Adsorption occurs when molecules in the fluid phase are held for a period of time  by Van der Waal forces. The surface represents a discontinuity in the structure of the solid, and atoms at the surface have a residue molecular forces which are not satisfied by the surrounding atoms like those in the body of the structure. These residual or van der Waals forces are common to all surfaces.
  
Materials used
Macroporous Polystyrene
Pore radius > 5 nm or >500 A
Will adsorb various macromolecular compounds e.g. Proteins (MW ca. 105 Da) Not usually recommended as Protein purification Process because of denaturation
Mesoporous Polystyrene
 Pore radius  20 – 500 A
Suitable for small organic compounds (MW<103) having a large surface area and large adsorption capacity
Hyper-cross-linked  Polystyrene
Hyper cross-linked non modified Polystyrene -Divinyl-benzene Resin  is done by extensive post cross-linking  of the long polystyrene chains. It is an expanded rigid  3D cross-linked network. They have a extremely large surface area (of 1000 to 1500 sqm/g) and no change in solvent uptake in both polar and non-polar resins. the whole interior is well accessible to small and medium sized analytes thanks to the network composition formed by small micro pores of about 2 to 4 nm in diameter.
Mesoporous Brominated Polystyrene
Offers larger adsorption capacity than unmodified PS resins. Excellent for treating large volume of feed solution or for removing organic impurities from the liquor. Have high density and thus can be applied to dense liquors or to up-flow applications.
Polymethacrylate Resins
Somewhat more hydrophilic in nature compared to Polystyrene thus they are more suitable for extraction of relatively hydrophilic organic compounds that are not adsorbed on polystyrene.
Also for sorption of highly hydrophobic compounds which are irreversibly adsorbed on Polystyrene
Silica with C18, C8 and C 4 ligands
 
 
Process Selectivity
Process  Contaminants

Liquid Phase contaminants
Hydrophylics:
Inorganics   =  too polar, can not be adsorbed
Small Saccarides =  too polar, can not be adsorbed
High Molecular Weight Compounds:
Proteins or Polysaccarides = cannot diffuse into the adsorbent particles
Some Proteins may be adsorbed on the surface of the solid phase material
Pore Criteria
Specific Surface Area
is measured by nitrogen adsorbtion (BET Method) and reflects the contact ratio of the sorbent-liquid interface
Pore radius (Pore distribution) is measured by the nitrogen adsorption / desorption (BJH Method) and concerned with the diffusion space(reflects the molecular sieving effect of an adsorbent)
Don’t hesitate to ask for help in the solid phase material selection
 
Industrial Application of SPE
A.  Large RPC, SPE Plant Concept
Attention: every application requires different components and connection of component. We custom assemble plants and we guide you to achieving success. Please complete questionnaire as basis for quotations

Components
A: Crude Tank, Feed, Target molecules
B: Eluent  Solvent
C: Selection valve
A1: Tubing to selection valve
B1: Tubing to selection valve
C1: Tubing to Pump
D: Pump , HPLC Pump
E: Flow Switching Valve
F: In-line Filter or Pre Column
D1: Tubing to Pump inlet
E1: Drain into waste
G: Column with sorbents
H: Flow Switching Valve
H1: Column By-Pass Tubing
G1: Column Exit Tubing to Flow Switching Valve
H2: Drain to waste
I. Detector e.g UV-Filter, DAD etc
J: Multi port valve
K: Process Controller
J1: Tubing to fraction vessel L
J2 : Tubing to fraction vessel M
J3 : Tubing to fraction vessel N
 J4: Tubing to waste O
Q: Shelf, Rack
 
B.   Resin Choice
Go to Chromalite_RPC
 
C. Resin Pre-treatment
Organic solvent wash (propanone or methanol)
Weigh and suspend adsorbent into propanone in a baeker
Stir mixture generously to remove bubbles and clot up particles. Do not use magnetic stirrer to avoid grinding down particles
Transfer the adsorbent slurry to a column with frit and tap
Ad 3 Batch Volume (3BV) of propanone to the column
Displacement with water
Wash column with 6 BV pure water to remove propanone
Keep adsorbent moist or in a beaker with water.
 
D.  Batch – Testing (convenient for selecting type of adsorbent)
Examine adsorbent volume to liquor (phase ratio), pH, temperature and contact time (bring amphoteric compounds to the isoelectric point.)
Adsorption testing:
1. Measure a certain volume of adsorbent in a graduated cylinder with water
2. Filter adsorbent and remove free water by centrifuge.
3. Transfer to Erlenmeyer and add measured volume of test liquor.
4. Seal flask and shake gently in incubator. Analyse the concentration of target compound in supernatant after several hours.
Calculate adsorption capacity C = (g/L adsorbent )= 1000(Wt – Ws) / VWt = weight (g) of Target Compound in the liquor
Ws = weight (g) of Target Compound in the supernatant
V = Volume (ml) of resin tested
Collect time dependent data to evaluated kinetics of adsorption
 
E.  Column Testing
For evaluating
a. effect of the type of eluent on recovery
b. effect of the type of eluent on the elution profile(Chromatogram)
c. effect of loading on recovery,
d. effect of flow rate(if adsorption or desorption rate is low)
When target compound is neutral a non-ionizable and water miscible organic solvent is used (lower alcohols or propanone)
When target compound is ionizable use aqueous buffer or buffer / solvent mixture

 
at low velocity have low counter-pressure thus have poor liquid distribution
Too high speed does not allow diffusion into  beads. It increases delta P and the possibility of channelling.
 
F. Column operations
Apparatus and solution should be thermostated
Column conditioning: Resin is swelled with water and pH adjusted
Column loading: Feed liquor at constant flow rate.
Set volume of feed solution so that amount of target compound is less than adsorbent capacity of column
Monitor effluent (percolate) to ensure target compound is not leached from the column.
Wash column with pH adjusted water (0,5 – 1 BV) to remove contaminants in the intra-particle voids.
Elution (Accelerate or separate impurities with gradient elution)
Wash column and re-equilibrate at correct pH. 
After several cycles rejuvenate column
 
G. Cycle Testing and Regeneration Methods
After operating conditions have been established , the next stage is to estimate average throughput of purification process and the life of adsorbent.
The desired average purification capacity of the process determines the number of cycles between rejuvenation processes
The adsorbent is regenerated to be reloadable in each cycle.
The regeneration condition depending on the eluant used. If target compound is eluted chromatographically a and followed by slower running components, eluent should be supplied until all components have eluted. A multi-step elution process may be appropriate
After elution of target a less polar solvent is used to complete elution and than aqueous regenerant is used
 
H. Column Rejuvenation Methods
Repeated column use leads to irreversible adsorption of contaminants(e.g. Proteins)
Rejuvenation means treating column with stronger conditions.  The effect is dependent on temperature, concentration of agents, contact time etc. and must be judged from the degree of contamination.
In case of severe fouling sodium hypochlorite may be used, however with great caution as it may degrade the resin
Example of various rejuvenation condition on surface area LCC-MAC-DVB-600-800 resin
 
Important Process Parameters (for lab and production scale operation)
General
The smaller the bead diameter the better the separation,  the higher the pressure build-up, the more complex the process technology
Standard approach for production scale operation is to use 0,3 – 1 mm particle diameter
Particles < 0,3 mm require pulsation free, high pressure, pumping systems ( HPLC Chromatography)
Moisture content / stability of resins
Porous resins are sold in their water swelled state .
Moisture content is expressed as % of water retained in the particles.
Moisture content is correlated to pore volume.
Moisture content changes if  adsorbent is contaminated or deteriorated
Resins are chemically (pH 1 – 13) and temperature (< 100°C) stable and insoluble in organic solvents
They are damaged by oxidizing agents
Flow rate
Optimal flow rate : 3 – 7 m3 / h / m2  (m / h)
Linear speed = 0 = same results like in batch operation 
Columns working ressure does not influence quality of adsorption
Pressure difference is a measure of liquid distribution and therefore an influence on adsorption
Pressure greater 3Kg / cm2 leads to breakage of beads
Normal conditions:
Flow rates of 3-7 m/h, 
Resin diameter of  300 – 350 um,
Solvent: water,
Temperature: 10 – 20 °C, 
Delta P: 0.2 – 0.4 kg / m2 per meter bed depth
Higher viscosity solvent will increase Delta P
Higher Temperatures will decrease Delta P
Pressure P
Absolute pressure does not influence quality of adsorption
Pressure difference is a measure of liquid distribution and therefore an influence on adsorption
Pressure greater 3 Kg / cm2 leads to breakage of beads
Normal conditions:
Flow rates of 3-7 m/h, 
Resin diameter of  300 – 350 um,
Solvent: water,
Temperature: 10 – 20 °C, 
Delta P: 0.2 – 0.4 kg / m2 per meter bed depth
Higher viscosity solvent will increase Delta P
Higher Temperatures will decrease Delta P
Temperature T
Max. adsorption is obtained at low temperatures
High temperature promote desorption
Particle Size
Low uniformity coefficient , ideal is 1.6
High uniformity coefficient increases DeltaP and performance
pH

Salt concentration

Molecular Weight

Organic Solvent Concentration