Question Part 1:

1. What is chromatography (definition)?

Skript:
Chromatography is a physical method of separation in which the components to be
separated are distributed between two phases, one of which is (stationary phase) while the
other (the mobile phase, eluent) moves in a definite direction.
Mine:
Chromatography is a separation method where components are separated by different
strong interactions with the used mobile and stationary phase.
2. What is the fundamental mechanism of chromatography?
The most important one is based on adsorption which uses the different adsorption affinities of
the sample components on the active solid. For Liquid-Liquid chromatography it is based on
different solubilities of the sample components (partition chromatography).
3. What separation procedures can be used for a one-step separation of a multi-component
mixture?
One can use different chromatography methods like Gas-chromatography, liquid
chromatography, supercritical fluid chromatography….
4. What are the differences between gas, liquid and supercritical fluid chromatography
(advantages, disadvantages)?
Gas chromatography offers the best mass transfer leading to well defined peaks but has a low
solvent capacity and uses temperature to control the separation which is not applicable for
temperature sensitive components.
Liquid chromatography offers relatively bad mass transfer but a very good solvent capacity
and is run at ambient pressure and ambient temperature resulting in gentle process conditions
the separation performance is defined by the solvent composition and the stationary phase.
Supercritical fluid chromatography offers good mass transfer and good solvent capacities,
easy solvent removal, high degree of freedom (pressure, temperature, solvent composition
and stationary phase used for optimization).

Skript:
Gas

GC: Has a high efficiency and universal detection however it is only applicable for high
volatile and thermally stable substance.

LC or HPLC: Applicable for low volatile and thermally unstable substances but low efficiency
and no universal detection (not every detector can be used)

SFC: Can be used for low volatile and thermally unstable substances, universal detection,
higher efficiency than HPLC but still lower efficiency than GC

5. What are the three chromatographic methods by classification according to the physical
state of the mobile phase?
Gas chromatography, Liquid chromatography and supercritical fluid chromatography.
6. Please describe three different operation modes for chromatography (discontinuous).
Frontal chromatography – sample is fed continuously into the chromatographic bed no
additional mobile phase is used and only one component can be separated with high purity.
 Displacement chromatography – sample is fed into the system as a finite slug and the
mobile phase contains a compound (the displacer) which is more strongly retained than the
components of the sample.
Elution chromatography – mobile phase is continuously passed through the chromatographic
bed and the sample is fed into the system as a finite slug.
7. Please draw a schematic chromatogram and indicate the characteristic parameters.

8. What is the definition for the retention factor k?

= − = ∙ = ∙ (1 − ) = ∙ (1 − )

9. What does the hold-up volume VM describe?

The holdup volume is the free volume of the column which is not occupied by the solid
parts of the stationary phase.
10. How can the hold-up time tM be determined experimentally?
The hold-up time can be determined by a substance which is detectable by the detector but
does not interact with the stationary phase. However the substance has to be able to fit into
the pores of the porous material.
11. What is normal phase and reversed phase chromatography? Please give an example of
phase combinations for each.
Normal phase: polar stationary phase and unpolar mobile phase Example: silica gel
as stationary phase and hexane 90/10 ethanol as mobile phase.
Reversed phase: unpolar stationary phase and polar mobile phase Example: octadecylsilan
as stationary phase and water 90/10 acetonitril as mobile phase.
Question Part 2:

1. What is linear and nonlinear chromatography?

In linear chromatography the concentration of the sample is quiet low resulting in a linear
adsorption isotherm. In nonlinear chromatography the isotherm is due to the higher sample
concentration not linear anymore resulting in for example Langmuir behaviour.

Linear chromatography:

• Concentration in stationary phase and mobile phase are proportional

• Equilibrium isotherms are linear
• Individual band shapes and retention times are independent of the sample
composition and amount
• Peak height is proportional to the amount of each component
• Phenomena mostly observed in analytical chromatography

The name is given due to the linearity of the adsorption isotherm (Freundlich isotherm)

Nonlinear chromatography:

• The equilibrium concentrations of a component in the stationary phase and mobile

phase are not proportional
• Equilibrium isotherms are not linear (Langmuir, BET)
• Isotherm depends on the concentration of all other components in the mixture
• Band shape and retention time depend on the sample composition and amount
• Phenomena mostly observed in preparative chromatography (high concentrations)
2. What system components do you need for an HPLC apparatus? Please draw a schematic
diagram of the chromatographic apparatus.
Solvent pump with solvent tank, injection valve for the sample, possibly a guard column, then
the actual chromatographic column and then the detector recorder system. In case of a
preparative process also valves to guide the flow and product collecting tanks are needed.

3. Please write 3 detectors suitable for HPLC. What substances can be detected by
them? UV-Vis detector which is able to detect UV-active compounds
FT-IR which is able to detect IR active compounds
Light scattering detector which detects the light scattering of the whole mixture
Fluorescence detector which is able to detect fluorescent.
4. What are the 3 relevant porosities in chromatography?
You have the interstitial porosity which describes the free volume of the column (the space
which is not occupied by the stationary phase)
The particle porosity which describes the free volume of the stationary phase.
The total porosity which is the free volume of the column + the free volume of the particles.
5. How can the total porosity be determined experimentally? Give an equation and explain.
The total porosity can be determined by measuring the hold-up time at a certain flow rate. The
hold-up time can be determined by adding a substance which does not interact with the
stationary phase but is able to pass through all pores of the stationary phase and is detectable
by the detector.
= ∙

6. Define the peak resolution RS and show how RS is dependent on N and α.

2− 1

=2
2− 1

For Gaussian peaks with the same width the resolution can be calculated with. The peak
resolution improves with higher N and higher .
√ −1 2

= ( )( )

1+ 2
4
7. For a HPLC chromatogram (column length L = 25 cm) with a hold-up time of tM = 2.0
min retention times for two components were determined to tR1 = 16.0 min and tR2 = 20.0
min. Both peaks show a standard deviation of σ1= σ2=0.3 min. Calculate the separation
factor, the peak resolution and the effective plate number of the column for component
2.
2 = 20−2 =9

16−2
1 = =7

= 2
= 9

4 =

=
20−16
3.33
=2

4(0.3 + 0.3)

2
=( ) = 4444.44

− 2

2
=( ) = 3600

σ
25
= 3600 = 69.4

8. Please write the van Deemter equation for HPLC and describe the meaning of the
different terms.
For this the Van-Deemter curve can be used to characterize the peak broadening. The
optimization problem is to find the minimum of this curve to minimize the peak broadening.

Van-Deemter equation:
= + +

Flow distribution due to Eddy diffusion (multiple paths lead to different elution times) the
Eddy diffusion can be described with the bed parameters.
=2

as the packing factor and as the particle diameter.

 longitudinal diffusion (back mixing due to undirected diffusion)
=2

labyrinth factor which can be defined with the solid porosity: = 0.45 + 0.55 0 and as the molecular diffusion coefficient.

the mass transfer between the solid and mobile phase depends on the speed at which the
phase equilibrium is reached. (peak broadening increases when the velocity increases because
the time mass transfer to the solid and back to the mobile phase will be slower than the mass
transport through the bed)

9. What is ideal and nonideal chromatography?

Ideal chromatography assumes infinite efficiency (infinite plate number), no axial dispersion
and mass transfer resistances. (No peak broadening effects) This results in an ideal model
Non ideal chromatography is described with a finite column efficiency taking axial
dispersion and mass transfer resistance into account. There are several models for nonideal
chromatography taking different nonideal conditions into account. (normal peak broadening
effects)

https://goldbook.iupac.org/html/N/NT06942.html
https://goldbook.iupac.org/html/I/IT06941.html

10. What are the reasons for band broadening?

The brand broadening occurs due to the uneven flow distribution in the bed, diffusion and the
mass transfer between mobile and stationary phase
Question Part 3:

1. What phenomena does the ideal model for chromatography include?

The ideal model includes the convection and the adsorption equilibrium.

2. What phenomena does the ideal model for chromatography not take into account?
Dispersion, mass transfer resistance, diffusion inside the particle.
3. Please write the model equation for the ideal model and explain the symbols.

For the ideal model a mass balance of the system is done. The adsorption equilibrium is
considered within the change of concentration in the mobile and stationary phase and
the convective transport is also considered.
+ =0
+ (1−)

ℎ + + ℎ

With as the concentration in the fluid, the time, the coordinate for the flow direction,
the velocity in flow direction, the total porosity of the stationary phase and the loading of the
stationary phase.
4. Please name three models for chromatography.

Ideal model only considers the convective transport and the adsorption process
+ =0
+ (1−)

Equilibrium dispersive model extends the ideal model with a dispersion term
2
+ +(1 − ) − =0

General rate model additionally considers dispersion mass transfer and particle diffusion.
2
(1 − ) 3 (1 − )

+ + − + ( − )=0

2
,

5. Please describe two modes for elution chromatography.

Repeated injections: Repeated injections are used to increase the productivity of elution
chromatography. Before the first sample exits the column a new sample is injected resulting in
less unused volume of the column. The time between the injection has to be chosen wisely to
remove any interactions between the different samples.
Peak shaving/Recycling chromatography can be used to improve the separation which is
incomplete within one column volume such that the component which elutes first is collected
until the impurities of the other component elute the parts where the peaks overlap are
recycled and then the other pure component is collected.
6. Please describe three different modes for continuous chromatography.

Annular chromatography uses a stationary phase which is filled into an annular gap. The
eluent is continuously fed across the whole bed interface also the feed is continuously fed at
the top of the stationary however only at a certain point and not a cross the whole bed. The
stationary phase is then rotated with a certain rotation speed. The rotation speed, eluent and
feed flow rates have to be defined precisely such that the collector vessels only collect the
correct substance. The retention times are transformed into the respective retention angles.

Compact disc-HPLC (CD-HPLC):

Compact disc-HPLC utilizes two stationary phases in form of two discs which are connected
with a channel. The discs are rotated and the mobile phase is continuously fed all over the
bottom disc. The feed can be continuously fed with a fixed capillary such that due to the
rotation off the disc the feed is fed at unused places of the disc. This process can be considered
a true moving bed.
True moving bed:

The true moving bed pumps the eluent and the adsorbent phase in a counter current manner.
The pumping speeds of the eluent and the stationary phase have to be adjusted in such a way
that one component is dragged with the desorbent phase and one component is dragged with
the stationary phase such that both components can be collected at different places within the
TMB. Additionally two cleaning zones are needed one for the desorbent and one for the
adsorbent.

Simulated moving bed:

In SMB multiple columns are used which are connected with multi position valves. The
process is divided into 4 zones:

Zone 1: Cleaning of the adsorbent (stationary phase)

Zone 2: Enrichment of A (Extract)

Feed is fed between zone 2 and 3.

Zone 3: Enrichment of B (Raffinate)

Zone 4: Cleaning of the desorbent

The number of columns per zone can be optimized and (2/2/2/2; 1/2/2/1; 1/3/2/1..)

The amount of columns per zone can also be varied within the process:

First using (1/3/1/1) then using (1/2/2/1) and then going back to the first one result in overall
(1/2.5/1.5/1).

After a certain switching time the eluent, feed inlet and the raffinate extract outlet gets rotated
by 1 column. Example: Between eluent and extract is zone 1, between extract and feed is zone
2, between feed and raffinate is zone 3 and raffinate to eluent is zone 4.
7. Please draw a schematic figure of the classical SMB process with a column configuration
of 2/3/3/2 and indicate the different functions of the zones.

Eluent Zone 1 Zone 1 E

Zo
ne
2
Zo
ne
4

Zo
ne
2
Zo
ne
4

Liquid Zo
ne
2

Zone 3 Zone 3
Zone 3
Feed
Raffinate
Zone 1 is the cleaning zone for the adsorbent (desorption of stronger retained component)
Zone 2 is the enrichment zone for the stronger retained component
Zone 3 is the enrichment zone for the weaker retained component
Zone 4 is the cleaning zone for the eluent/desorbent (adsorption of the weaker retained
component)
8. What are the advantages and disadvantages of SMB chromatography?

Advantages:

1. Continuous process
2. High concentrated products
3. High yield (no waste fraction)

Disadvantages:

1. Technically complex
2. Separation of binary problems only
3. Complicated process design

9. Please name at least three applications for SMB chromatography

Fine chemicals, enantiomer separation, sugar separation and hydrocarbon separation, organic
acids, enzymes, proteins, peptides….
10. Please show the region of complete separation in a triangle diagram for a
linear adsorption isotherm.

11. What is the ParexTM process?

The Parex process is used for the separation of p-xylene from C8 aromatic isomers. For the
separation one column with different chambers for the adsorption is used to perform the
simulated bed function. The inlet for the eluent and the feed and the outlet for extract and
 raffinate are controlled by a rotary valve. The eluent is circulated through the whole column.
The extract and raffinate are concentrated in additional columns to recycle the eluent.

12. What is the maximal specific productivity of a chiral SMB separation found
in literature?
The maximum productivity is 10
∙

Question Part 4:

1. What is adsorption?

Adsorption is the process when a molecule from a fluid phase binds to the surface of the
solid phase.

Desorption is the process where a molecule bound to the solid phase is released to the fluid phase.

Adsorptiv is the molecule within the fluid phase before it adsorbs. The molecule is called
adsorpt when it is in contact with the solid phase (adsorbent). The layer with the adsorpt and the
solid phase is called adsorbate.
Physisorption:

• Van der Waals forces

• Electrostatic interactions
• Hydrophobic interaction
• Hydrogen bonding

In physiosorption multilayer formation is possible.

Chemisorption:

The adsorpt is bond to the adsorbent by a chemical bond. Chemisorption usually forms only
a single layer. (mixed adsorption is possible as well)

2. What is an adsorption isotherm?

An adsorption isotherm shows the equilibrium between loading of the solid phase and
the concentration in the fluid phase at a certain temperature and pressure.

3. Which interactions are possible in physisorption?

a. Van der Waals forces
b. Electrostatic interactions
c. Hydrophobic interaction
d. Hydrogen bonding
e. Affinity specific interactions

4. What are adsorption isotherms needed for?

Adsorption isotherms are needed to describe the equilibrium between the solid phase
concentration and the fluid phase concentration. They are needed to simulate the
chromatography behaviour thus needed for the design of chromatographic processes.
5. Please write three different isotherm models for pure components and illustrate
the shape of the isotherms in a diagram.
Linear, Freundlich,Langmuir,BET,Hills
Freundlich & linear:

Langmuir:
BET

Hill isotherm
6. Please show the influence of increasing concentration of a chromatographic peak with
Langmuir behaviour. Draw a figure and explain!

When the peaks have Langmuir behaviour then the column has a limited loading capacity. In
case of an overloading the interaction between the sample in the mobile phase and stationary
phase cannot take place where the column is already fully loaded leading to a reduction of the
average retention time and also causes tailing in the chromatogram because the
adsorption/desorption is slow for high loading/mobile phase concentrations and fast for small
concentrations/loading.

7. What is the IAS theory?

IAST – Ideal adsorbed solution theory can be used to describe the adsorption isotherms of
mixtures. This method is analogous to Raoult’s law for vapour liquid equilibrium. For the
prediction the pure component isotherms are needed and following assumptions are made:

Ideal mixture – no interactions of the different components

Distribution on the adsorbent of the different adsorpts regarding their adsorption strength

No mixture coefficients are needed

8. Please name three different methods for experimental determination of adsorption

isotherms.

There are different static and dynamic methods.

Static: Gravimetry, Adsorption-desorption-method and circulation method

Dynamic: Integration of breakthrough curves/frontal analysis, peak maximum method, elution

by characteristic points (ECP) and perturbation method.
 9. What are the advantages of the ECP method?
The ECP is fast and does not need a lot of substances however it needs a detector calibration, a
high number of theoretical stages and is limited by solubility.
10. What are the advantages of the perturbation method?
The perturbation analysis does not need a detector calibration, can directly analyse mixtures
and the number of theoretical plates are not important but it needs a high amount of
material and is rather slow.

Question Part 5:

1. What is an eluotropic series?

The eluotropic series ranks the mobile phases regarding their eluting power in respect of their
dielectric constant, dipole moments and hydrophobic-hydrophilic properties. It gives a broad
overview about which solvents can be used and how the chromatographic process can be
improved. Additionally to the eluotropic series the miscibility of the solvents with eachother
has to be take into account when optimizing the mobile phase. The eluotropic series always
has to be done for a certain stationary phase.

For a polar stationary phase (Normal phase chromatography) like alumina or silica gel the
unpolar solvents have a low elution strength and the polar solvents have a strong eluent
strength.

2. Please sort benzene, water, n-hexane and acetonitrile according to increasing eluting
power on alumina stationary phase.
n-hexane -> benzene -> acetonitrile -> water (n-hexane smallest water highest)
3. Please write three common chemically bonded stationary phases for normal phase
chromatography

Diol, Cyano/Nitrile groups and Amino groups modified silica.

4. What are the advantages (at least three) of chemically bonded stationary phases in
comparison with unmodified phases

Advantages:

Similar separation as normal silica but shorter equilibration times and suitable for gradient
elution and suitable for both NP and RP.

5. Please name three common chemically bonded stationary phases for reversed phase
chromatography

Octadecyl (Octadecylsilan), Octyl, Phenyl

6. Please name three parameters that influence separations in ion

exchange chromatography.
pH, ionic strength, type ion exchanger and counterion in the mobile phase.
7. What is the basic separation principle in molecular exclusion chromatography? Molecular
exclusion chromatography utilizes a poly porous stationary phase which causes molecules which
fit into the different pores to take longer elution paths resulting in higher retention times. This
way the sample is separated only by the different sizes of the molecules.

8. What is the difference between gel filtration and gel permeation chromatography?
In gel filtration an aqueous mobile phase is used and in gel permeation an organic mobile
phase is used.
9. What parameter can be optimized in development of a chromatographic method?
Parameters used for the optimization are: Purity, Yield, specific productivity =

, specific solvent consumption = and the most important one costs.

10. What is the influence of modifier concentration on the separation factor in

SFC? With an increase of modifier the separation factor decreases. and the peak
resolution increases
 11. What in general is the influence of production capacity on the production costs per kg
product?
By increasing the production capacity the minimum price for the product decreases. A
tenfold increase in production capacity can decrease the price to a third.

Question Part 6:

1. How can the flow rates and the injectable mass for a preparative chromatographic
process be calculated from analytical flow rates and injectable mass at constant column
length? Comment on run time.

When you keep the length constant you can use the following equation:
2

2 2 2
( ) = =

1 1 1

~2 ~

Note: By keeping the length constant the retention times and productivity will be constant as
well.

When the length is not kept constant then the following equation can be used.
2
2 2
2
( ) =

1 1 1

and
22 2
( )=

Note: By increasing the length of the column the productivity decreases because of the higher
retention times.

2. What is the minimal column length according to column diameter?

The minimum column length is equal to the column diameter.
3. What materials can be used for constructing chromatographic columns?
Glass, stainless steel, acrylic and other resistant polymers.
4. What are typical particle sizes for spherical silica used in chromatography?
5,10,12,15 micrometers
5. Please name three different column packing methods.

You can use the dry packing method where the column is filled with the particles and by
shaking/vibration the particles are distributed. This method can be used for low pressure
applications and diameters above 25 micrometers for example SEC.

For HPLC, SFC and other high pressure applications the compression method or the
pressure filtration method can be used.

6. Please write three different types of pumps used in chromatography.

Diaphragma pumps, piston pumps and rotary lobe pumps (gear pumps).
 7. How can the fixed-capital investment be calculated?
The fixed capital investment can be calculated by multiplying the costs for the equipment with
the factor 4.3.
8. What are the three major “Purchased equipment delivered” investment costs in
chromatography?
Columns, pumps and separation unit (pervaporation/precipitation)

Question Part 7:

1. What are enantiomers?

Enantiomers are stereoisomers which are mirror images of each other. That means every chiral
carbon atom has the reverse orientation (S/R) of its mirror image.
2. What is chirality?
A carbon atom is considered a chiral center if it is bond to 4 different groups. A molecule with
multiple chiral centers does not have to be chiral. If the molecule has a plane of symmetry the
overall molecule is achiral and called a meso compound.
3. What is a racemate?
A racemate contains equal amount of both enantiomers thus the net rotation in polarimetry is
0. Typically when a compound reacts into an optical active compound a racemic mixture is
formed. Racemic mixtures can have different boiling and melting point from the enantiomers.
When they crystallize they can form separate + and – crystals or form one racemic crystal
structure.
4. What are the different properties of enantiomers?
Enantiomers typically have the same boiling point, melting point, density and refractive index.
However they have a different rotation direction in polarimetry (same value though) and
interact different with other chiral molecules such that one enantiomer can be medicine and
another toxic or one is sweet one is bitter etc. . There is no relationship between (R) (S) and +
clock wise and – counter clockwise rotation.
5. Why is it important to separate enantiomeric drugs?
Chiral molecules interact with other chiral molecules when the chirality of the drug is the
wrong one it can cause the drug to not have the function it should have which can result
into the drug being useless or in worst cases even toxic.
6. What are the different ways for processing single enantiomers?
Resolution of a mixture of enantiomers, using pure chiral substrates and preserve this chirality
to form the right product and asymmetric synthesis of the product with an enantioselective
catalyst.
7. What resolution methods for racemic mixtures are available?
Separation of diastereomers, kinetic resolution, chromatographic methods, membrane
separations and crystallization.
8. Please name at least three types of chiral stationary phases.
Cellulose and amylase derivatives, synthetic polymers, cylcodextrins, macrocyclic
glycopeptides, proteins, brush type, crown ether and chiral stationary phases for
ligand exchange chromatography.
9. What crystallisation methods are used for enantioseparations?
The racemate can be transformed into diastereomer salts and then separated. One can use
chiral solvents to keep one of the enantiomers solved and crystallize the other. It is also
possible to use preferential crystallization where the correct enantiomer is added as a seed to
guide the formation of the right enantiomeric crystal.
Crystallisation of diastereomers
 Crystallisation with chiral solvents
Preferential crystallisation (direct) by using sodium ammonium tatrate
Selective crystallisation from enriched solutions
10. What is a kinetic resolution of racemates?
In a kinetic resolution you start with a mixture of the two enantiomers often as a racemic
mixture. By using an enantioselective catalyst only one of the two enantiomers will react and
form a product. This way you can get a high enantiomeric excess for the substrate and a
relatively good one for the product.

Question Part 8:

1. What is downstream processing?

Downstream processing are all the unit operations after the fermentation/reaction unit. The
downstream process in bio applications contains Cell disruption, Cell removal, Dewatering… .
2. Please name at least three examples for application fields of enzymes.
Proteins are used in medical applications (Insulin, factor VIII), detergents, as catalysts for
reactions, food processing, textiles and leather goods
3. Please name at least three examples for primary unit operations in downstream
processing.
Cell disruption (Sonication, Pressure cells and enzymic lysis)
Cell recovery/removal (centrifugation and filtration)
Dewatering/Concentration step (ultrafiltration, precipitation, spray drying, dialysis)
4. Please name at least three different chromatographic methods for protein purification.
Explain the mechanisms of separation for each method.
SEC – size exclusion chromatography separates the molecules by their size using a
poly porous stationary phase with a certain pore size distribution.
IEC – ion exchange chromatography separates the molecules by using a charged stationary
phase which interacts with ions of the opposite charge in the mobile phase.
HIC – hydrophobic interaction chromatography an unpolar stationary phase is used to bind
hydrophobic proteins at the surface. To achieve a high binding the salting out effect of
ammoniumsulfate is used to force the binding of the protein by removing the water needed
for hydration.
AC – affinity chromatography uses specific interactions of a protein with ligands which are
added to the surface of the stationary phase. These interactions can be due to structural affinity
between the ligand and the protein.
5. What characteristics of proteins are used for separation in SEC, IEC, HIC and AC?
Write out the abbreviations.
SEC – Uses the size of the proteins to separate them
IEC – Uses the charge and size of the proteins to separate them
HIC – Uses the different hydrophobicity of the proteins to separate
them AC – Uses specific affinities of proteins with the used ligands.
6. What molecules elute faster in size exclusion chromatography, smaller or larger and
why?
Larger molecules elute faster in SEC because they do not fit into some pores or any pores
leading to smaller elution paths thus shorter retention times/smaller retention volumes.
 7. By what chromatographic method can the molecular weight of proteins be determined?
Please explain the procedure.
The molecular weight of proteins can be determined by size exclusion chromatography. First
the SEC is run with a mixture of known molecules with known sizes one protein is used which
fits into all the pores to determine the total volume and one protein is used which does not fit
into any pores to determine the interparticle volume. For the other proteins the retention
volumes are determined and with it the distribution constant is calculated. The distribution
constants are then plotted versus the log( molecular weight). After this preparation the SEC is
run with the protein of unknown size where the retention volume and the distribution constant
are determined. With the calibration curve and the distribution constant of the protein of
unknown size the molecular weight can be derived.
8. What is the charge of the matrix in anion exchange chromatography?
The resin of the anion exchange matrix is positive such that anions in the mobile
phase interact with the stationary phase.
9. At which pH should a cation exchanger be operated for protein purification? Compare
with pI and explain why.
When the pH of the proteins is below the proteins then the surrounding phase is a stronger
acid than the protein leading to the protonation of the protein which causes a positive
charge. For cation exchange a pH below the pI should be chosen and in anion exchange a
pH above the pI should be chosen.
10. Please give the group names of common anion and cation exchangers.
Anion exchanger: Diethylaminoethyl (DEAE), Quaternary ammonium (Q),
Quarternary aminoethyl (QAE)
Cation exchanger: Carboxylmethyl (CM), Methyl sulphonate (S) and sulphopropyl (SP)
11. What elution strategies in hydrophobic interaction chromatography are possible?
By decreasing the salt concentration the hydrophobic interactions decrease leading to
elution of the proteins (gradient elution by gradually decreasing the sulphate concentration).
Increasing the concentrations of detergent or changing the pH.
12. Please name at least three different kinds of affinity chromatography. Biospecific
affinity chromatography, Immunoaffinity chromatography, Protein A chromatography,
Lectin affinity chromatography, Dye-ligand chromatography and Metal-chelate
chromatography.

Question Part 9:

1. What are the three stages involved in a liquid-liquid extraction?

First the feed mixture and the solvent have to be brought into contact. Afterwards the two phases
have to be separated. Finally the solvents are removed and recovered from each phase.
2. What are the disadvantages of liquid-liquid extraction? What are the alternatives?
Liquid-liquid extraction only offers one theoretical stage, can only separate binary mixtures
and is discontinuous. An alternative is countercurrent extraction which offers a continuous
multistage separation or countercurrrent chromatography which offers a multistage and
multicomponent separation.
3. What is Droplet Countercurrent chromatography? What is the concept of DCCC? The
countercurrent chromatography uses columns which are filled up with one liquid phase and
then the other liquid phase is fed into the stationary phase in form of droplets. These travel
due to gravity effects upwards if the density of the mobile phase is smaller (ascending mode)
or downwards if the density of the mobile phase is larger (descending mode). The flow is only
 driven by gravity forces such that this method takes a lot of time (days). Additionally
relatively poor mixing reduces the separation efficiency.

4. What are two important requirements for countercurrent chromatography?

Efficient phase mixing and reliable retention of the stationary phase.
5. Please name three examples of typical two-phase partition solvents. What kinds of
solvents are needed for chiral separations in modern countercurrent chromatography?
Hexane and acetonitrile are quiet immiscible (sample is unpolar and water insoluble)
Chloroform/methanol/H2O (for polar water insoluble substances)
Butanol/acetic acid/H2O (for ionic
substances) Butanol/ethanol/H2O (for sugars
or peptides) Dextran/PEG (for proteins)
For chiral separations a chiral selector can be added to the stationary phase which should
have a very high solubility in the stationary phase and a low one in the mobile phase (to keep
it from bleeding out).
6. What operation modes off modern countercurrent chromatography do you know?
Single mode: one direction separation (normal or reversed phase)
Dual mode: mobile phase is switched in the middle of the
run Gradient mode
Elution extrusion mode
pH zone refining: employs basic organic phase and acidic aqueous phase (or vice versa)
for separation of organic acids and bases according to their pK and hydrophobicity.
7. Please give three examples of typical applications of countercurrent chromatography.
Saponins, Alkaloids,Chlorophylls, Tannins, Carotenoids, Phospholipids, fat soluble vitamins,
mono-/oligo-saccharides, anthocyanins, lignans, phenolic compounds, synthetic compounds,
chiral separations and protein separations.
 8. Please compare modern countercurrent chromatography with HPLC.

9. What are the differences between Countercurrent Chromatography (CCC) and

Simulated Moving bed Chromatography (SMB)?

Extra questions:
Which separation classes are there and how can they be characterized?

Mechanical separation: The driving force is Gravity/centrifugal force and pressure the mixtures
are typically heterogeneous

Membrane seperations: Pressure, electrical field and concentration gradient as possible driving forces
the mixture can be homogeneous and heterogeneous.

Electrical separation: Electrical field and the mixture can be heterogeneous or homogeneous
Thermal separation: Concentration gradient and temperature gradient the mixture is
usually homogeneous.

Which separation class does chromatography belong to?

It belongs to the thermal separations and utilizes two auxiliary components (column + eluent)

How is chromatography defined?

Skript:

Chromatography is a physical method of separation in which the components to be separated are

distributed between two phases, one of which is not moving (stationary phase) while the other (the
mobile phase, eluent) moves in a definite direction.

Mine:

Chromatography is a separation method where components are separated by different

strong interactions with the used mobile and stationary phase.

Why should you use chromatography?

1. Chromatography is highly selective because of the high amounts of phase equilibria (up to
thousand theoretical plates)
2. Can separate highly complex mixtures into the single components which can be
collected individually
3. Ideal method for analysis because only small sample sizes are needed (depends on the
detector sensitivity etc.)
4. Usually the process is quiet simple and fast
5. Easy up-scaling (Higher volume flow rate -> higher diameter)
6. Typically used to achieve the highest possible purity

How did chromatography develop?

Tswett a botanist used chromatography to separate the plant pigments, hence the name
chromatography (chroma for color and graphein for writing).

How does an open column liquid chromatography work?

First the stationary phase is suspended within a column. The stationary phase is held back by a porous
disk. The mixture is then inserted at the top of the column. By adding additional mobile phase the
mixture solves in the mobile phase and gets eluted by it. Due to the different interaction with the
stationary phase the components have a different residence time in the column. (High interaction long
residence time)
On which scales is chromatography used?

Chromatography is used on all usual scales.

Analytical:

Determination of chemical composition

Column: 0.05-10 mm inner diameter and flow rates of 0.1 -30 ml/min

Amount of components: µg - mg

Preparative:

Purify and collect one or more components

Column: 7-50 mm inner diameter and flow rates of 20 – 1200 ml/min

Amount of components: mg – g (lab scale), g – kg (pilot scale), kg – t (production scale)

Production scale chromatography:

Commercial production of one component (kg to tons) usually used for the
final isolation/purification step

Columns: Inner diameter up to 1600 mm and flow rates larger than 1000 ml/min

Amount of components: kg – t
How can you scale up a chromatographic column?

The separation performance of a column is defined by the residence time and the length of the column
thus resulting theoretical plates. To scale up a chromatographic column without changing the
separation performance the superficial velocity should be kept constant:

2 2

=( )

1 1

The mass is the respective added substrate mass.

The volume flow rate has to be adjusted as well:

2
= 2

1 1

Which shapes can be used for chromatography?

Planar chromatography utilizes a plate of the stationary phase for example thin layer chromatography
or paper chromatography.

Column chromatography where a tube is used to keep the stationary phase in place. The stationary
phase can be coated on the tube wall, inserted as particles and hold in place by disks or you can use
centrifugal forces to hold a liquid phase in place.

Chromatography can be separated in the physical state of the mobile phase and solid phase.
Which chromatography classes are there?

Gas-liquid chromatography (GLC), Gas-solid chromatography (GSC or GC), Liquid-liquid

chromatography (LLC), Liquid-solid chromatography (LSC or LC),…, Supercritical fluid-solid
chromatography (SFSC or SFC)

Usually the S for the solid phase is left out because the most common state of the stationary phase
is the solid state.
What is the back pressure?

The back pressure is the pressure at the end of the column.

Which are critical properties of the mobile phase?

The density of the mobile phase is important to characterize its solvent power (high density higher
solvent power), the dynamic viscosity is important to characterize the pressure drop and the
diffusion coefficient is important for the mass transfer.

Gases typically have bad solvent power (low density) but a very good mass transfer (high
diffusion coefficient)

Liquids have a good solvent power but a comparably bad mass transfer.

Supercritical fluids have a liquid like density but a diffusion coefficient which is two magnitudes
better than the ones of liquid (still two magnitudes smaller than the ones of gases)

Good mass transfer: High efficicency of the column (a lot of separations in a small time)

High solvent power: Less solvent is needed to perform the separation

How can you optimize GC, LC and SFC?

In all the chromatographies you can choose different stationary phases to change the separation
performance. Additionally you can change the important operating parameters which are:

GC: Temperature

LC: Solvent composition

SFC: Solvent composition, Temperature and back pressure

What are advantages and disadvantages of GC,LC and SFC?

GC: Has a high efficiency and universal detection however it is only applicable for high volatile
and thermally stable substance.

LC or HPLC: Applicable for low volatile and thermally unstable substances but low efficiency and no
universal detection (not every detector can be used)

SFC: Can be used for low volatile and thermally unstable substances, universal detection, higher
efficiency than HPLC but still lower efficiency than GC

Common detector: Flame ionization detector

Which are the three operation modes for discontinuous chromatography?

Frontal chromatography: The sample is fed continuously into the chromatographic bed and no
additional mobile phase is used. (Only one product can be separated)

Displacement chromatography: The sample is fed as a finite slug and the mobile phase contains a
substance (displacer - V) which is retained stronger than the sample components.
Elution chromatography: The mobile phase is continuously pumped through the column and the
sample is fed as a finite slug.

Chromatography is typically classified by the separation mechanism which classes are there and
which mechanism do they use?

The most common one is the Adsorption chromatography which utilizes the different adsorption
equilibria of the components with the solid stationary phase.

There is also the partition chromatography which uses the different solubilities of the components
in the sample in the mobile and stationary phase (used in Liquid-Liquid chromatography..)

Often used for a rather unselective protein separation is Ion-exchange chromatography which is based on
the differences in ion exchange affinities of the components in the sample and the size of the molecules.
Cation echange resin is negatively charged and anion exchange resin is positively charged.

Another chromatography is exclusion chromatography/size exclusion chromatography (Gel

filtration/Gel permeation chromatography) which is comparable to a filtration method utilizing
different molecular sizes/shapes for the separation.

Affinity chromatography uses affinity ligands which have unique interactions with certain
components. This is very important for the separation of enantiomers.

Adsorption chromatography can be divided into normal and reverse phase. Give examples and
explain the difference.

In Normal-phase chromatography the stationary phase is polar and the mobile phase is unpolar.

Example: Stationary phase: Silica gel and mobile phase: Hexane or Hexane/Ethanol 90/10

In Reverse-phase chromatography the stationary phase is unpolar and the mobile phase is polar.

Example: Stationary phase: Octadecylsilane (silica gel modified with C-18 aliphatic residues) and
mobile phase: Water or Water/Aacetonitrile (90/10)

What is the difference between linear and non linear chromatography?

Linear chromatography:

• Concentration in stationary phase and mobile phase are proportional

 • Equilibrium isotherms are linear
• Individual band shapes and retention times are independent of the sample composition and
amount
• Peak height is proportional to the amount of each component
• Phenomena mostly observed in analytical chromatography

The name is given due to the linearity of the adsorption isotherm (Freundlich isotherm exponent = 1)

Nonlinear chromatography:

• The equilibrium concentrations of a component in the stationary phase and mobile phase
are not proportional
• Equilibrium isotherms are not linear (Langmuir, BSA)
• Isotherm depends on the concentration of all other components in the mixture
• Band shape and retention time depend on the sample composition and amount
• Phenomena mostly observed in preparative chromatography (high concentrations)

Sketch a chromatogram and characterize all the important parameters.

is the hold-up time which is defined as the time a component needs to pass through the
column without interacting with the solid (residence time of the eluent)
∙
= =

the holdup volume is the volume which is not taken by the solid phase, is the flow rate of the
eluent and the total porosity of the bed (particle porosity and solid porosity ).
= +(1− )

Experimental porosity can be calculated with the hold-up time:

 =

Red marked: Free volume of the particles due to pores (porous volume)
′
, is the retention time of the component i and the adjusted retention time , = , −

The retention factor can be defined with the distribution constant:

, ,

= ∙ = ∙ = ∙ =

, ,
, ,

And with the adjusted retention time/volume follows

′ ′
′
, , ,
= = ∙ =

And the separation factor follows as 2

= ; >1

The resolution of the peak can be defined as (Gaussian peak)

′
′ ′
= − =2 ′ − =2 −

,2 ,1 ,2 ,1 ,2 ,1

2 + 1 + +

2 1 2 1
2

It can also be defined with the peak width at half of the height −
,2 ,1
= 1,177
ℎ2 + ℎ1
Explain the importance of the separation factor and the peak resolution?

The separation factor describes the distance of the two peak maxima from each other and the peak
resolution additionally uses the peak geometries to identify if the two components are completely
separated. A high separation factor is good for an efficient separation the effectiveness of the
separation is given by the peak resolution. The peak resolution should be above 1 but not too high to
keep the efficiency high.

How can you define the plate number for a chromatographic separation with Gaussian peaks?
2 2

= 16( ) =( )

′ ′
2 2

= 16 ( ) = ( )

The plate height can then be defined with the column length as such =
=

How is the linear velocity defined?

The linear velocity is the average velocity in the fixed bed:

 = ∙

With
∙ ∙∙
= = =

follows
=
1
=

When scaling up the linear velocity is usually kept constant.

Why does band broadening occur?

The brand broadening occurs due to the flow distribution in the bed, diffusion and the mass
transfer between mobile and stationary phase.

How can the performance of a chromatographic column be optimized by choosing an

appropriate flow rate?

For this the Van-Deemter curve can be used to characterize the peak broadening. The optimization
problem is to find the minimum of this curve to maximize the number of theoretical stages per
column length.

Van-Deemter equation:
= + +

Flow distribution due to Eddy diffusion (multiple paths lead to different elution times) the Eddy
diffusion can be described with the bed parameters.
=2

as the packing factor and as the particle diameter.

longitudinal diffusion (back mixing due to undirected diffusion)
=2

labyrinth factor which can be defined with the solid porosity: = 0.45 + 0.55 0 and as the molecular diffusion coefficient.

the mass transfer between the solid and mobile phase depends on the speed at which the phase
equilibrium is reached. (peak broadening increases when the velocity increases because the time
mass transfer to the solid and back to the mobile phase will be slower than the mass transport through
the bed)
The plate height can also be plotted as the function of the linear velocity if the total porosity is known.

What are the different operating conditions for GC, HPLC and SFC?

GC uses an inert gas as the mobile phase (typical only one gas) elevated temperatures and no back
pressure.

HPLC uses a liquid as a mobile phase (typically contains a mixture of different solvents) usually run at
ambient temperature and no back pressure

SFC uses a supercritical fluid and sometimes adds other solvents to adjust the performance. The
temperature is elevated and the pressure as well; to keep the fluid in the supercritical state a certain
backpressure is needed.

Sketch the typical structure of the GC system

The GC contains of a gas supply vessel, a pressure controller for the gas, a port for the sample
injection, the column (typically wound up as a spiral due to the small diameter and high length), the
oven where the column is placed in and the detector recorder system. As the mobile phase inert gases
like helium, hydrogen and nitrogen are used.

Note: Oxygen in the systems has to be strongly avoided to keep the substances from oxidizing at
the elevated temperatures.
Which detectors can be used for GC?

Flame ionization (FID) – combustion of the sample and measuring the signal; Detection limit 100 pg

Electron capture (ECD) – radioactive detection, good to detect halogens and nitrogenoxides; Detection
limit 1 pg

Thermal conductivity (TCD) – change in resistance of heated wire; Detection limit 100 ng

Fourier transform infrared (FTIR); Detection limit 1 ng

Mass spectrometry (MS); Detection limit 100 fg

Pictogram = 10−12

Femtogram −15
= 10

Sketch the typical structure of the (HP)LC system

You have one or multiple vessels for the solvents a pump which pumps the eluent into the column
passing through the injection valve where the sample is injected. Usually a guard column is used
between the injecting valve and the actual column to protect the expensive column from chemical
impurities. After the column there is the detector and recorder system.
How do injection valves work?

The injection valve loads the sameple into a port with a fixed volume which is kept inside until the
valve is switched such that the eluent flows through the injection valve port.

Which detectors can be used for HPLC?

Light scattering detector: detects every component

Refraction index detector: detects all components

FT-IR: only detects the infrared active components

UV/-Vis detector: Detects UV-active components

Diode array detector: UV active components, scans for a wavelength range instead of one
certain wavelength

Conductivity detector (CD): ions

Electrochemical detector: electroactive

Fluorescence detector: fluorescent

HPLC-MS coupling: detects every component

Which law can be used to calculate the concentration of a component in a mixture after
detecting the component with UV-Vis.
The absorbance of the mixture is up to a certain value proportional to the concentration of the
species absorbing at this wavelength. This relationship is described by the Lamber-Beer law.
( )= ( )∙ ∙

as the length through the sample cell, ( ) as the molar absorptivity at the wavelength and the concentration of the species i.

Sketch the typical structure of the SFC system

The SFC system uses a very similar structure compared to GC however it needs a restrictor because
of the high backpressure and a high pressure pump to achieve elevated pressures.
What is the difference between non ideal and ideal chromatography?

Ideal chromatography assumes infinite efficiency, no axial dispersion and mass transfer resistances.
This results in an ideal model

Non ideal chromatography is described with a finite column efficiency taking axial dispersion and
mass transfer resistance into account. There are several models for nonideal chromatography taking
different nonideal conditions into account.

Which theories can be used to describe a chromatographic process?

You can use all combinations of linear/non linear and ideal/nonideal

The simplest but very unrealistic model is the linear ideal model:

Each component pulse moves without changes in profile, its motion does not depend on the other
pulses.

Linear non ideal model:

Typically used for analytical chromatography

Influence of kinetic phenomena is taken into account. The components do not influence each other
such that the chromatogram of the mixture resembles the sum of the chromatograms of the single
components. Relatively simple to solve.

Non-linear ideal model

The nonlinearity of the phase equilibrium is considered and the dependency of the sample
composition is also taken into account.

Most realisitic models:

Non-linear non ideal model:

Different nonideal behaviour is taken into account additionally nonlinear isotherms dependent
isotherms are used. These models have to be solved numerically.
Show the model equation for the ideal model and explain the terms.

For the ideal model a mass balance of the system is done. The adsorption equilibrium is considered
within the change of concentration in the mobile and stationary phase and the convective transport is
also considered.
+ =0
+ (1−)

ℎ + + ℎ

Name three models and explain what they consider.

Ideal model only considers the convective transport and the adsorption process
+ =0
+ (1−)

Equilibrium dispersive model extends the ideal model with a dispersion term
 2

+ +(1 − )− =0
2

General rate model additionally considers dispersion mass transfer and particle diffusion.
2
(1 − ) 3 (1 − )

+ + − + ( − )=0

2
,

How can you operate your preparative chromatographic process?

Discontinuous:

Elution chromatography, repeated injections and recycling chromatography

Continuous:

Annular chromatography, Compact disc (CD) HPLC, Simulated moving bed chromatography (SMB)
and other multicolumn processes.

How can you increase the productivity of a discontinuous chromatography?

The productivity is defined as the amount of feed processed per time and per stationary phase amount.
=

Thus increasing the amount of feed added in a certain time interval increases the productivity. This can
be done by repeated injections where you inject another sample before the first sample left the column.
When to inject another sample can be derived from the retention times of the components.
= , − , +

With the retention time of the fastest component being , and the retention time of the slowest component being ,

Your chromatographic column is not able to separate all the components what can you change
to increase the efficiency?

You can collect the parts of pure components where no peak overlapping occurs and then recycle the
unresolved mixture. This way one can save costs for a longer column. This method is called
recycling chromatography or peak shaving chromatography.

How does annular chromatography work?

Annular chromatography uses a stationary phase which is filled into an annular gap. The feed is
continuously fed at the top of the stationary at a certain point. The stationary phase is then rotated with
a certain rotation speed. The eluent is continuously fed across the whole bed interface. The rotation
speed, eluent and feed flow rates have to be defined precisely such that the collector vessels only
collect the correct substance. The retention times are transformed into the respective retention angles.
How does the true moving bed process work?

The true moving bed pumps the eluent and the adsorbent phase in a counter current manner. The
pumping speeds of the eluent and the stationary phase have to be adjusted in such a way that one
component is dragged with the desorbent phase and one component is dragged with the stationary
phase such that both components can be collected at different places within the TMB. Additionally
two cleaning zones are needed one for the desorbent and one for the adsorbent.
Instead of actually moving the bed a simulated moving bed can be used instead. How does SMB
work and what has to be taken care of?

In SMB multiple columns are used which are connected with multi position valves. The process is
divided into 4 zones:

Zone 1: Cleaning of the adsorbent (stationary phase)

Zone 2: Enrichment of A (Extract)

Feed is fed between zone 2 and 3.

Zone 3: Enrichment of B (Raffinate)

Zone 4: Cleaning of the desorbent

The number of columns per zone can be optimized and (2/2/2/2;1/2/2/1;1/3/2/1..)

The amount of columns per zone can also be varied within the process:

First using (1/3/1/1) then using (1/2/2/1) and then going back to the first one results in overall
(1/2.5/1.5/1).

After a certain switching time the eluent, feed inlet and the raffinate extract outlet get rotated by 1
column. Example: Between eluent and extract is zone 1, between extract and feed is zone 2, between
feed and raffinate is zone 3 and raffinate to eluent is zone 4.

How can the switching time be modelled?

When the dispersion is negligible and mass transfer resistance are negligible (ideal case) then you can
use the triangle theory to calculate the switching time.

The switching time has to fulfil these conditions:

 ≤
1 ℎ
≤
2 ℎ≤ 2
3≤ ℎ≤ 3
ℎ≤ 4

< ̇<∞
1

< ̇<

< ̇<

̇<
̇=
4

Henry adsorptions isotherme:

=
= [1+(1− )/

is the Henry coefficient.

Regarding the productivity the mass flow rates in zone 3 and 2 can be optimized by plotting a
respective diagram. It is assumed that the feed flow is positive such that the mass flow in zone 3
is greater than the one in zone 2. The best operating point is indicated by w in the diagram below.
Considering non linear isotherms yields a different operating window depending on the
feed concentration.

How can elution chromatography and SMB be optimized and which parameters can be used for
the optimization?

Elution chromatography can be optimized by modifying:

1. Column length
2. Injection volume
3. Injection concentration
4. Flow rates
5. Fraction times

SMB can be optimized by modifying:

1. Column length
2. Column configuration
3. Feed concentration
4. Flow rate
5. Switch time
Parameters used for the optimization are: Purity, Yield, specific productivity = ,
specific solvent consumption = and the most important one costs.

What are advantages of the elution and SMB process?

Elution process:

Advantages:

1. Flexible
2. Can separate multiple components
 3. Simple

Disadvantages:

1. Discontinuous process
2. Diluted products and high solvent consumption
3. Waste fractions

SMB process

Advantages:

1. Continuous process
2. High concentrated products
3. High yield (no waste fraction)

Disadvantages:

1. Technically complex
2. Separation of binary problems only
3. Complicated process design

What are processes using SMB/Sorbex?

Sorbex is simulating a moving bed by using a multichamber adsorption column where the inlet and
outlets into this column are controlled by for example a rotary valve switching at which chamber each
stream enters/exits.

Parex: separation of para-xylene from C8 aromatic isomers

Molex: separation of linear paraffins from branched and cyclic hydrocarbons.

What are typical applications for adsorption?

Gases:

Removal of toxic gases, chlorinated hydrocarbons and water

Liquids:

Removal of phenolics and other toxins; Water purification, fractionation, ion exchange separation
(proteins nucleic acids) affinity separations

Explain important terms when talking about adsorption.

Adsorption is the process when a molecule from a fluid phase binds to the surface of the solid phase.

Desorption is the process where a molecule bound to the solid phase is released to the fluid phase.

Adsorptiv is the molecule within the fluid phase before it adsorbs. The molecule is called adsorpt
when it is in contact with the solid phase (adsorbent). The layer with the adsorpt and the solid phase is
called adsorbate.
Which interactions keep the molecules adsorpt?

Physisorption:

• Van der Waals forces

• Electrostatic interactions
• Hydrophobic interaction
• Hydrogen bonding

In physiosorption multilayer formation is possible.

Chemisorption:

The adsorpt is bond to the adsorbent by a chemical bond. Chemisorption usually forms only a
single layer. (mixed adsorption is possible as well)

How can you enhance the selectivity of your adsorption process?

Adsorption strongly depends on steric phenomena which depend on the solid phase structure and the
adsorptive. Additionally chemical properties have a strong influence on the adsorption namely polarity and
electrostatic charge. Hence by modifying the surface of adsorbents one can adjust the selectivity.

Name a few adsorbents.

The most common one used in chromatography is silica gel. For general adsorption application
activated carbon and charcoal were used quiet often. Besides these zeolites and caoline can be used
as well.

What are the most important properties of an adsorbent?

Polarity, particle diameter, pore size distribution, pore volume and the specific surface area.

What is the reason for a hysteresis in an adsorption desorption diagram?

The reason for a hysteresis is the formation of multilayer adsorption within mesopores which lead to
capillary condensation. This leads to the different adsorption and desorption mechanism.

Describe the Langmuir and the Anti-Langmuir type isotherms.

The typical Langmuir isotherm has a relatively linear form for low concentration in the fluid and at
high concentration a maximum loading is achieved. The Anti-Langmuir isotherm does not have such a
limit and the loading strongly increases at higher concentrations.
You experience fronting and tailing in your chromatogram which isotherms can be used to
describe the respective process?

Tailing is typically caused by the usual Langmuir type isotherm and fronting is caused by the
Anti-langmuir type isotherm.

Which models to describe adsorption isotherms do you know?

The most basic one is the linear (Henry) isotherm. Another one is the Freundlich isotherm which adds
an exponent to the concentration. One of the most often used isotherms is the Langmuir isotherm. A
quiet complex isotherm is the BET isotherm which can be used to describe the multilayer formation.

Show the model equation for the Freundlich isotherm.

1
= ∙

With P as the pressure and a/m as the fitting parameters. (m=1 linear isotherm)

Or
= ∙

q as the loading, c as the concentration, K and n as the fitting parameters.

Show the model equation for the Langmuir isotherm.

=
1+

With as the maximum molecular layer loading and K as the other fitting parameter.

Show the model equation for the cubic Hill isotherm.

2 3
+2 +3

1 2 3

= 3 1+ +2 2
+3 3

1 2 3

Note: the Hill isotherm with n=1 equals the Langmuir isotherm.
Which theory can be used to predict the isotherms of a mixture?

For this the IAST – Ideal adsorbed solution Theory can be used. This method is analogous to Raoult’s
law for vapour liquid equilibrium. For the prediction the pure component isotherms are needed and
following assumptions are made:

Ideal mixture – no interactions of the different components

Distribution on the adsorbent of the different adsorpts regarding their adsorption strength

No mixture coefficients are needed

How can you determine adsorption isotherms?

There are different static and dynamic methods.

Static: Gravimetry, Adsorption-desorption-method and circulation method

Dynamic: Integration of breakthrough curves/frontal analysis, peak maximum method, elution by

characteristic points (ECP) and perturbation method.

What are advantages and disadvantages of the ECP method?

The ECP is fast and does not need a lot of substances however it needs a detector calibration, a high
number of theoretical stages and is limited by solubility.

Which unmodified stationary phases can be used for normal phase chromatography?
Aluminiumoxide Al2O3 or Aluminium hydroxide Al(OH)3 Florisisl – natural Mg-Al-silicate
SiO2 from silica sand (silica gel)

Which interactions bind the adsorpt to Aluminiumoxide and silica gel?

Typical polar interactions of the hydroxyl groups with other polar groups of the adsorpt (hydrogen
bond, dipole interactions …)

Stationary phases can be modified by chemically binding molecules on it. Which are typical
molecules used for normal and reversed phase and the respective advantage?

Normal phase:

Diol, Cyano/Nitrile groups and Amino groups

Advantages:

Similar separation as normal silica but shorter equilibration times and suitable for gradient elution
and for both NP and RP.

Reverse phase:

Octadecyl (Octadecylsilan), Octyl, Phenyl

Higher retention times for less polar molecules.

Shortly explain ion pair chromatography.

Ion pair chromatography is used to separate charged molecules on an unpolar stationary phase. To
achieve this separation an organic counterion is added to the mobile phase. The target ion binds due to
ionic interaction with the counterion forming an uncharged pair which can strongly interact with the
unpolar stationary phase. This method can be alternatively used to ion exchange chromatography.

Which stationary phase is commonly used for ion exchange chromatography and how does the
separation work?

Typically organic resins are used like polystyrene-di-vinylbenzene and these are modified with for
example quarternary ammonium groups for anion exchange or sulfonic acid/carbonic acid groups for
cation exchange. The aqueous mobile phase usually contains an inorganic counterion such that it can
compete with the sample ion for the binding spot. Without the competition the sample ion might bind
on the stationary phase and never leave it.

The separation is influenced by pH, ionic strength, type of ion exchanger and the counterion in
the mobile phase.
Which stationary phase is commonly used for Gel permeation chromatography/Size
exclusion chromatography and how does the separation work?

As stationary phases a poly porous material with a certain pore size destribution is used. Therefore
cross-linked polymers like Sephadex (glucose/glycerol polyemer) and polyacrylamide gel are used.
The pore size distribution of the stationary phase strongly influences the range of molecular weights
which can be separated and also the elution time of the different molecules in the sample. The smallest
molecule which does not fit into any of the pores determines the molecular weight at which no
separation occurs. It is typically possible to separate molecules with atleast a difference of 10% in
molecular weight.

The smallest molecules have the largest retention times and the molecules which do not fit into any
pores have the smallest elution time.

Give a brief overview of affinity chromatography.

Affinity chromatography uses with ligands modified stationary phases. These ligands can for example
be cosubstrates, substrates for enzymes or antigens for antibodies etc. . These ligands have selective
interactions with only a certain protein or antibody or a small group of these (depending on the used
ligand) such that mostly the target component binds to the stationary phase and the impurities elute
quiet fast with (next to) no retention. The bond target component can then be eluted by pH change by
adding a component with an even higher affinity or by increasing the ionic strength of the mobile
phase.

What is the eluotropic series and what is it used for?

The eluotropic series ranks the mobile phases regarding their eluting power in respect off their
diaelectric constant, dipole moments and hydrophobic-hydrophilic properties. It gives a broad
overview about which solvents can be used and how the chromatographic process can be improved.
Additionally to the eluotropic series the miscibility of the solvents with eachother has to be take
into account when optimizing the mobile phase. The eluotropic series always has to be done for a
certain stationary phase.

For a polar stationary phase (Normal phase chromatography) like alumina or silica gel the
unpolar solvents have a low elution strength and the polar solvents have a strong eluent strength.
What is the difference between isocratic elution and gradient elution?

When performing an isocratic elution the solvent mixture composition is kept constant for the
whole process. For gradient elutions the solvent composition is changed after a certain time or
continously for a certain time depending on the method chosen. With gradient elutions the
chromatography process can be sped up and still have a good enough peak resolution.

What are advantages of SFC compared to other mobile phases?

Supercritical fluids offer good mass transfer due to decently high diffusion coefficient and nearly as
good solvent capacity as liquid mobile phases. Additionally the solvent is usually cheap and no
organic solvents are needed. For the optimization the temperature, pressure, stationary phase and
modifier amount can be adjusted leading to a great amount of degress of freedom. The critical point of
CO2 is for example at 31°C such that even temperature sensitive substances can be separated by this
method. Another important advantage is the separation of the solvent from the components which can
be easily done by reducing the pressure resulting in a highly pure product.

How can you optimize your SFC process?

Repetition - Optimization criteria: Separation factor & peak resolution, solubility, productivity, solvent
consumption and costs.

SFC is normal phase chromatography (CO2 is unpolar) such that the migration rate can be increased
by adding polar modifiers to CO2 increasing the density or temperature and a decrease of polarity
of the stationary phase can increase the migration rate as well. By changing these operating
parameters the optimization criteria can be used to decide on the best operating conditions.
How can the column length influence your productivity and solvent consumption?

For elution chromatography an increase in column length lets you inject more sample (proportional).
However at a certain length the productivity will reach its optimum.

In SMB an increasing column length decreases the productivity (longer elution times) but the
solvent consumption is decreased. SMB typically offers better/smaller solvent consumption
compared to elution chromatography.
How does the feed concetration and column configuration influence your SMB process?

The feed concentration highly influences the solvent consumption and the productivity, such that both
curves have an optimum which is usually not at the same feed concentration. The column
configuration has a strong influence on the productivity because the columns in the washing zones 1
and 4 are basically dead volume not used for the separation such that lowering the number of the
columns in these zones without reducing the purity is a good way to increase the productivity.

What are the major equipments needed for chromatography (elution and smb)?

Elution: Column, Pumps for feed and the eluent, heat exchanger, valves, separator (in SFC e.g.
cylcones) and an analytical unit (detector + recorder)

SMB: Columns, pumps for feed and the eluent, heat exchanger, valves (connecting the columns and
fraction collector), fraction separator (raffinate and extract recycling) and and an analytical unit
(detector + recorder).

How can you scale up your analytical to a preparative column?

When you keep the length constant you can use the following equation:
2

2 2 2
( ) = =

1 1 1
 2
~ ~
Note: By keeping the length constant the retention times and productivity will be constant as well.

When the length is not kept constant then the following equation can be used.
22 2 2

( ) =

and
2
2 2
( )=

Note: By increasing the length of the column the productivity decreases because of the
higher retention times.

How does the column cost change for increasing diamters of the column (stainless steel)?

The costs more or less linear increase with the diameter.

Note: These are the costs without stationary phase just the stainless steel cylinder.

Which materials can be used to construct a chromatographic column?

The most common one in industry is stainless steel however also acrylic, glass and other resistant
synthetic materials can be used.

Which methods can be used to insert the packing into the column?

You can use the dry packing method where the column is filled with the particles and by
shaking/vibration the particles are distributed. This method can be used for low pressure applications
and diameters above 25 micrometers for example SEC.
For HPLC, SFC and other high pressure applications the compression method or the pressure filtration
method can be used.

Explain how a packing is filled with the axial compression method.

First the packing material is suspended in the mobile phase and then filled into the the column. Parts
of the mobile phase are then removed at the bottom and a piston for the compression with an inlet for
for example CO2 is added on top. By transferring CO2 into the column the packing the solvent is
removed and the packing dried. While drying the packing the piston is used to compress the bed to
the final bed height.

What are requirements for pumps in chromatography?

The pumps have to be very accurate, simply cleaned and have a low pulsation (pulsation causes the
retention times to variate leading to peak broadening).

Which pumps can be used for chromatography?

Diaphragma pumps which use a membrane which is pulled up and pressed down by a piston to
expand/compress the volume within the pump leading to the pump flow.

Piston pump: A piston is used for the compression expansion of the volume of the

Rotary pump: Two gears are within a casing rotating entraping the fluid between the gear teeth and
transferring. Rotary pumps need precise machining and have a pulsating output (the pulsation
increases with decreasing gear teeth numbers)
Compare SMB and elution chromatography.

SMB and elution chromatography can achieve similar productivities however SMB does perform better if
the separation factor is low. The solvent consumption in SMB is quiet low and additionally the product is
higher concentrated. For both processes the solvent consumption is dependent on length and separation
factor. Even tho SMB does perform better than elution chromatography in a lot of ways the costs for SMB
are because of the high amount of columns needed way higher than the ones for elution chromatography
(800,000 SMB vs 320,000 elution or 270,000 SMB vs 110,000 elution(SFC)).

Note: The initial investment costs increase with increasing plant capacity however the production
costs for the product typically decrease with increasing capacity.

How can you estimate the capital investment for your process?

One method is to use the purchased equipment costs and use different percentage to estimate the costs for
other factors. The total direct plant costs result in 2.86 times the costs of the purchased equipment. This
method is called the percentage of devilvered-equipment cost. Furthermore the fixed-capital
investment results in 4.3 (2.86 direct plant costs and 1.44 indirect plant cost) times the delivered
equipment costs. The error done by this method is typically 20-30 %.

What are direct and indirect costs?

Direct plant costs: Purchased equipment delivered, installation, instrumentation & controls,
piping, electrical systems, buildings, yard improvements, service facilities,..

Indircet plant costs: Engineering and supervision, construction expenses, legal expenses,
contractors fee and contingency

What are the main costs factors for the cost of investment of a chromatographic process?

The columns are typically the reason for 50% of the investment costs, another big factor are separation
devices and the evaporator and last but not least the pumps do cost quiet a bit as well. In SMB the
costs for valves can be quiet high as well.

How does the fixed-capital investment distribute for SMB and elution-SFC?

SMB – Columns 60%, Pumps 15%, Product precipitation/evaporator 15% and Miscellaneous 10%

Elution – Columns 20%, Pumps 30%, Product precipitation/evaporator 40% and Miscellaneous 10%

Main costs factors columns pumps and product precipitation/evaporator

Chirality and chiral separation techniques.

What types of isomers are there?

The most basic one are constitutional isomers where the same atoms are used to form the molecule but
the binding sequence is different.

There are also stereoisomers which have the same binding sequence with the same atoms however the
spatial arrangement is different.

Within the stereoisomers one can find enantiomers which are stereoisomers which are mirror images
of eachother and one can also find diastereomers which are stereoisomers which are not mirror
images of eachother.

When is a molecule considered chiral?

A molecule is considered chiral when it does not have a plane of symmetry and one or more
atom (chiral center) has 4 different binding partners.
Which properties do enantiomers share and which are different?

Enantiomers have the same boiling point, melting point, vapor pressure, density and refractive index.
However they differ in the rotation direction in polarimetry, interact differently with chiral molecules
(Enzymes and taste buds, scent..) and also have different physiological effects e.g. toxicity.

What is polarimetry and what can you do with it?

Polarimetry uses monochromatic light, usually sodium (λ=589.6 nm), which is filtered twice and polarized. The polarized
light will be sent through the sample cell with the analyte and the detector detects the rotation of the light. Only chiral
molecules will rotate the light. The rotation of a pure solution of one chiral molecule will rotate the light depending on
the specific rotation of this molecule[ ] , the concentration and the length of the sample cell.
[ ] = ( )

With ( ) the observed rotation, the concentration of the molecule in g/ml and the length of the path through the sample in dm. A
counterclockwise rotation is – (L) and a clockwise rotation is + (D).

How can you get an enantiomerically pure product?

Stereoselective synthesis/asymmetric snythesis:

The best case is to perform a enantioselective syntehsis where the catalyst performs the reaction
and strongly favors the reaction of one of the enantiomers (ee above 99%). The enantioselective or
stereoselective synthesis can be done chemically in some cases and in most cases biochemically.

Chiral pool:

Another way is to perform the reaction with chiral substances which are all enantio pure leading to a
preservation of the chirality of the substrates resulting in a enantiopure product.

Resolution:

The last method is to perform a resolution of a mixture of the enantiomers. The resolution can be
performed as a kinetic resolution where one of the enantiomers reacts and the other stays more or
less untouched, it can be done by chromatographic methods where one enantiomer interacts with the
stationary phase more prominent than the other (affinity chromatography), the enantiomers can be
reacted with another substrate to form diastereomers which can be separated due to the different
boiling points (or different crystallization behaviour) and it is also possible to perform membrane
separations (chiral membranes).

How can you separate a racemic mixture by crystallization?

First the racemic mixture is reacted to a mixture of diastereomers using a chiral base or acid. These
diastereomer salts can then be separated by crystallisation and afterwards the bond is cleaved by
hydrolysis. The crystallization can be done by cooling, evaporation of the solvent or by adding a
salting out salt like ammoniumsulfate. The crystallization can be coupled with a chromatographic
step to separate the enantiomers before hand.
When should you use a chromatographic process for a chiral resolution?

It should be used when other methods are too expensive or not possible to execute. If it is used one
should try to use it as early as possible to reduce the quantity of undesired products which would
be carried through the other unit operations.

Which types of chiral stationary phases (CSP) do you know?

The chiral stationary phase can be based on different polymers etc. some examples are:

Cellulose and amylose deravitives

Synthetic polymers like Chiraspher, CHI-DMB

Cyclodextrins for example ChiraDex, Cylcobond

Macrocyclic glycopeptides for example Chirobiotic

Proteins

Crown ether

What are reasons for the increasing importance of chiral chromatography?

Since it is known that the wrong enantiomer can cause problems such as being toxic or just being
inactive reducing the overall product effectiveness especially the pharmaceutical industry aims for
high ees with low costs. Chiral chromatography offers these high chiral purities can be operated at all
scales can be easily scaled up, performed continuous or discontinuous, sationary phases are
commercially aviable and a larger variety is present, the development time for a chromatographic
process compared to kinetic resolution asymmetric synthesis etc. is very short. Additionally by using
chiral SFC SMB unharmful solvents, low solvent consumption (low waste) and mild process
conditions are used resulting in low or no enzyme deactivation.
Protein purification
What is a protein?

A protein is a polymer consisting of more than 100 amino acids which are connected by peptide
bonds.

Which protein properties can be used to separate different proteins?

Proteins can have different size, charge (isoelectric point), binding properties, chirality, solubilities,
surface hydrophobicity, stability … . Therefore the protein structure and its properties has to use these
properties to separate it from other proteins.

What are primary unit operation in downstream processing?

These are the first unit operations after the reaction/fermentation. The aim is to remove a great amount
of impurities and strongly increase the concentration of the solution. After the fermentation usually the
cells are disrupted unless the cells are the product (yeast). The disruption can be done by sonication
(lab scale), enzymic lysis (lab scale) or pressure cells (industrial scale). After the disruption the cell
proteins etc. are removed by a typical mechanical unit operation like filtration or centrifugation. The
solution is then concentrated by removing the solvent (water) in a dewatering step. The dewatering can
be achieved by ultrafiltration, precipitation, spray drying and dialysis.

What is the cause for salting in and salting out effects?

The salting in happens when low concentrations of a salt are added to the solution. The salt
coordinates itself around the proteins and increases the hydration layer due to its charge. This
increases the solubility of the protein. When the salt concentration is increased further at some point
the salt binds too much water leading to reduced hydration of the protein which then leads to the
precipitation/aggregation of the protein. Salting in and salting out depends on the salt, solvent
additives, pH and temperature.
What are secondary unit operations in downstream processsing?

Secondary unit operations are used for the purification for example chromatography. Additionally the
protein processing to immobilised enzymes, stabilisation or beading/prilling is a possible step. The last
step is the protein packaging, steilisation, bottling etc.

Which methods can be used to isolate proteins?

Precipitation using the different solubilites

Size exclusion (gel filtration) chromatography using the mass (shape) of the proteins

Ion-exchange chromatography or chromatofocusing using the charge/isoelectric point and mass

Hydrophobic interaction chromatography using the hydrophobicity of the proteins

Affinity chromatography using specific binding due to chirality etc.

How does size exclusion or gel filtration chromatography work?

This chromatography type uses a poly porous sationary matrix which causes small molecules to take
longer elution paths than larger ones. The fractionation range is determined by the pore size
distribution. This method can be used to estimate the molecular weight of a protein by using a protein
standard with different molecular weights. The separation does not utilize any adsorption.

How can you determine the total mobile-phase volume and the interparticle volume?
The total mobile phase volume can be determined by using a small dye which is able to fit into all the pores leading to the longest possible retention
time. The interparticle volume 0 can be determined by using a detectable large polymer like blue dextran which does not fit into any pores resulting in
the shortest retention time. The exclusion limit is between these two volumes.
How is the distribution constant for size exclusion chromatography defined?
= ( − 0)/( − 0)
0< <1

Small proteins distribution constant close to one and large proteins distribution constant close to 0.

How can you determine the molecular weight of your protein using SEC?

First you run the SEC with a standard mixture containing proteins of a known molecular weight.
The total volume is determined with a small dye like flavolin and the interparticle volume is
determined with a large protein like blue dextran. The retention volumes of the other proteins like
Chymotrypsionogen A, Ovalbumin, BSA are used for the calibration.
For each of the standard proteins the distribution constant is calculated and then plotted versus the known molecular weights. ( = ( ))
The molecular weight is plotted at a logarithmic scale to achieve a linear relation.
Afterwards the retention volume of the protein of the unknown molecular weight is determined. With
this retention volume the distribution coefficient is calculated and the molecular weight can be
determined from the calibration curve.

What are advantages and disadvantages of SEC?

Advantage: Simple and predictable

Disadtanges: Low capacity and load has to be non-viscous

Useable for purification from 2x to 6x.

Which ions are used for the resin in ion exchangers and how does IEC work?

For anion exchange typically positively charged groups with nitrogen are used (quarternary
ammonium groups)

For cation exchange either deprotonated carbonic acid groups or sulfonic acid groups are used.

In ion-exchange chromatography the proteins in the mobile phase which have the opposite charge of
the resin interact with the resin due to ion-ion interactions. The proteins in the mobile phase typically
compete with salts added to the mobile phase to make the proteins elute. The separation is dependent
on ionic strength, pH, the type of ion exchanger and the counterion in the mobile phase.

What are advantages and disadvantages of IEC?

Advantages: large sample volumes possible, large amounts of protein possible (10-50g/L) packings
are very robust and flexible conditions – huge number of variations

Disadvantages: protein has to be at same pH and salt concentration as column (buffer exchange
step/pH change) and inconvenient for larger volumes due to high salt concentrations (dilaysis,
desalting,dilution)
How does chromatofocusing work?

Chromatofocusing uses a similar method compared to IEC however instead of using a salt
concentration gradient a pH gradient is used to elute the proteins by different isoelectric points. First
the column is loaded at a pH where the proteins bind to the resin (anion exchange resin pH above
the pIs and cation exchange resin pH below the pIs). The column needs to have a high charge and
high binding capacity. After the binding the pH is slowly increased (cation exchange) or decreased
(anion exchange). When the pI of a protein is reached the net charge of the protein is 0 resulting in a
elution of the proteins with this pI.

Which advantages and disadvantages does chromatofocusing offer?

Advantages: excellent resolution and variety of ranges can be used

Disadvantages: rather expensive need to know pH to avoid waste and low capacity

Should be used as a later purification step.

How does hydrophobic interaction chromatography work?

A typical reverse phase chromatographic column can be used which is first loaded at high ionic
strength using a salting out salt. The molecules which still do not interact with the resin elute first
(high amount of polar residues on the surface). The bound proteins/molecules can then be released by
decreasing the salt concentration, increasing the concentration of detergents or changing the pH.

What are advantages and disadvantages of HIC?

Advantages: large sample volumes possible, large amounts of protein possible, packings are very
robust, flexible conditions (huge number of varitions), equilibration is easy and gradients are
very stable.

Disadvantages: high salt concentrations necessary for binding sometimes causes precipitation.
How does affinity chromatography work?

Affinity chromatography uses special stationary phases which have specific interaction with a certain
molecule type because of multiple non covalent interactions.

In which groups can the modifications of the stationary phase of AC be divided in?

Biospecific affinity chromatography is used for enzyme separation where a affinity ligand is used
which strongly interacts with the target enzyme (for example a cofactor can be used)

Immunoaffinity chromatography uses antibodies as the ligand which interact with the protein epitope.

Other groups are Protein A chromatography, Lectin affinity chromatography, Dye-ligand

chromatography and metal-chelate chromatography.

How can you elute the bond protein in AC?

The binding of the protein can be removed by adding the ligand into the mobile phase, adding a
reagent which competes with the protein for the binding spot or changing the pH if the charge of the
protein changes the interaction with the resin.

What are advantages and disadvantages of AC?

Advantages: High affinity with binding in micro mole nano mol or even pico mole, binding all or none
excellent recovery and lots of variations possible

Disadvantages: Steric hindrance often lowers capcaity, elution sometimes harsh, specific eluent
sometimes needed and application is limited by the selection of available affinity chromatography
beads.
When should you use which chromatographic method?

For high purities often a combination of multiple chromtographic steps is needed resulting in a
relatively low yield.

Liquid-liquid chromatography
What is liquid liquid chromatography based on?

Liquid-liquid chromatography uses liquid-liquid extraction equilibria for the separation of the sample.
Therefore two imiscible liquids are needed for the process each component has a certain partition
coefficient describing the concentration at the equilibrium in both phases.

What is CCD and how does it work?

CCD stands for countercurrent distribution and is a liquid-liquid system which uses multiple tubes
which all offer one theoretical stage. At first all the tubes are filled with the “stationary” phase and
then the “mobile” phase is added with the feed into the first tube. After a certain shaking time the
“mobile” phase/lighter phase is transferred into the next tube and fresh solvent which was used for
the feed is fed into the first tube. After a certain shaking time the upper phase is transferred to the
next tube again and fresh solvent is added to the first tube.

Explain droplet countercurrent chromatography (DCCC).

In DCCC multiple columns are connected with eachother and filled with the sationary phase liquid.
The mobile phase liquid is then flowing through the stationary phase in form of droplets because of
gravity effects.

DCCC typically takes a long time per separation. How can it be improved?

The improvement which was made was using the centrifugal force as a dirving force allowing for a
lot higher flow rates. This method is called centrifugal partition chromatography (CPC).

CPC uses a spinning rotor which introduces a centrifugal field, it is still considered a hydrostatic
separation and has one axis of gyration, it is much faster than DCCC and has rotor volumes from 50
mL to 20 L.

What are advantages of CPC?

CPC offers high recoveries without irreversible adsorption, the scale-up is easy, high versatalitiy for
the stationary and mobile phase, good biocompatibility with mild operating conditions and quiet rapid
due to high mobile flow rates.

Where is CPC used?

CPC is used in Pharamceutical, Bio-medical, Fine chemicals, Biotechnology and Fermentation.

What does High speed counter current chromatography use?

HSCCC uses two rotary axes thus using a hydrodynamic equilibrium which improves mixing.
Each column is rotated around its own axis and the whole system of columns is rotated as well.

What are advantages of CCC?

CCC is a rather quick separation process with low costs (only solvent costs), gentle and versatile
process conditions, applicable for different feed ranges on the same instrument, able to switch between
reverse and normal phase at will and free of irreversible adsorption such that 100% of the sample can
be recovered.

How do you setup and run a CCC?

1. Choosing the instrument size (1g sample per 100 ml column volume)
2. Optimizing the solvent system
3. Preparing the solvent system
4. Filling the columns with the stationary phase
5. Equilibration wih mobile phase
6. Inecting the sample/separation
7. Cleaning the columns

Which solvent systems can be used for CCC?

Hexane and acetonitrile are quiet immiscible (sample is unpolar and water insoluble)

Chloroform/methanol/H2O (for polar water insoluble substances)

Butanol/acetic acid/H2O (for ionic substances)

Butanol/ethanol/H2O (for sugars or peptides)

Dextran/PEG (for proteins)