Adsorption-LC,RPC &SPE

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

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



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 Chromatograph
y is primarily used in prep and production scale purification of hydrophobic compounds.


The Adsorption / Desorption Process

Resin supplied with water on the surface

Target molecules in water are adsorbed

Eluent is applied to release target compounds Fig.3



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

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
s 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



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)


  • 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 (< 100C) 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



Salt concentration


Molecular Weight


Organic Solvent Concentration




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