CHEM-E3140 Bioprocess technology II
Chromatography to separate biomolecules
Ion exchange chromatography:
Ruostemaa, Seppo
Välisalmi, Teemu
Yang, Junyuan
Size exclusion chromatography:
Niemeläinen, Mikko
Stubb, Janne
Tamminen, Juho
Hydrophobic interaction chromatography:
Alasuvanto, Elisa
Ruuth, Ida
Thian, Marcus
Report submitted:
5.12.2016
ii
Abstract
Chromatography is an analytical technique that is used to separate molecules from
each other by using their affinity towards two different phases and due to its
separating properties chromatography is often used in downstream processing. In
this report three commonly used chromatography techniques, ion exchange (IEX),
size exclusion (SEC) and hydrophobic interaction chromatography (HIC) and their
properties in separating biomolecules were studied.
IEX separates biomolecules according to differences in their net surface charge. The
benefits of IEX over other chromatography techniques are higher flow rate,
concentrated sample output, high yield and no denaturing buffers but the pH has to
be controlled precisely and the sample is loaded at low ionic strength. HIC has fairly
similar properties with IEX but it separates biomolecules according to their
hydrophobic properties. SEC separates molecules according to their size and it is
applicable to sensitive biomolecules that cannot withstand large changes in pH, metal
ion concentrations or otherwise harsh conditions. Due to low flow rate it is not
suitable for high feed volumes.
In conclusion IEX, SEC and HIC are capable of separating biomolecules under specific
conditions that are different for each method. Thus, they have their own role in
purifying biomolecules, such as metabolic products in bioprocess technology.
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Table of contents
Abstract .........................................................................................................................ii
Table of contents ......................................................................................................... iii
1
Introduction to chromatography ......................................................................... 1
2
Ion exchange chromatography ............................................................................ 2
3
4
5
2.1
General information .................................................................................... 2
2.2
Importance................................................................................................... 3
Size exclusion chromatography ........................................................................... 4
3.1
Size exclusion chromatography ................................................................... 4
3.2
Importance................................................................................................... 5
Hydrophobic interaction chromatography .......................................................... 6
4.1
General information .................................................................................... 6
4.2
HIC medium ................................................................................................. 6
Conclusion ............................................................................................................ 8
References ................................................................................................................... 9
1
1 Introduction to chromatography
Chromatography is an analytical technique commonly used for separating a mixture
of chemical substances into its individual components, so that the individual
components can be thoroughly analyzed. Chromatography techniques are also can be
used in the final stages of purification of a number of products. There are many types
of chromatography e.g., liquid chromatography, gas chromatography, ion-exchange
chromatography, affinity chromatography, but all of these employ the same basic
principles (Peter, et.al. 2016).
Chromatography uses these two phases to separate the components in mixtures. One
of the phases is immobilized, usually by chemically bonding it to a solid particle or the
inside of a tube. This part is generally referred to as the stationary phase, but is also
often called the packing, support, or column. The other phase, typically a liquid, gas
or supercritical fluid, is passed across the stationary phase by applying pressure on the
inlet of the system. This phase is called the mobile phase, or carrier. The samples are
subjected to flow by mobile liquid onto or through the stable stationary phase. The
sample components are separated into fractions based on their relative affinity
towards the two phases during their travel. The fraction with a greater affinity to
stationary layer travels slower and at a shorter distance, while that with a less affinity
travels faster and longer.
Chromatography has numerous applications in biological and chemical fields. It is
widely used in biochemical research for the separation and identification of chemical
compounds of biological origin. In the petroleum industry the technique is employed
to analyze complex mixtures of hydrocarbons (Roy, et.al. 2016).
As a separation method, chromatography has a number of advantages comparing with
the conventional techniques—crystallization, solvent extraction, and distillation, for
example. It can be handled simply and very pure products can be recovered. It is
capable of separating all the components of a multicomponent chemical mixture in
low temperature according to the size or chemical properties, without requiring an
extensive foreknowledge of the identity, number, or relative amounts of the
substances present.
In many fermentation processes, chromatographic techniques are used to isolate and
purify relatively low concentrations of metabolic products. In this report,
chromatography will be concerned with the passage and separation of different
solutes as liquid is passed through a column (Stanbury, et.al. 2003). Ion exchange
chromatography, size exclusion chromatography and hydrophobic interaction
chromatography are mainly discussed in the following chapters.
2
2
Ion exchange chromatography
2.1 General information
Ion exchange chromatography (IEX) is one of commonly used chromatography
techniques and it was introduced to separate biomolecules for the first time in the
1960s. IEX separates biomolecules, such as proteins, peptides and nucleic acids,
according to differences in their net surface charge with high resolution and loading
capacity. For example, two proteins that differ by one charged amino acid can be
separated by using IEX. (GE Healthcare, 2016)
The net surface charge of a protein is composed of its charged groups that have
different acid ionization constants (pKa). Charged groups can be ionized and therefore
the net surface charge is dependable on pH as shown in figure 1. Due to differences in
pKa values each specific protein acts differently under the change of pH which drives
the IEX separation. Each protein has its own isoelectric point where the protein has no
electric charge which can be seen in figure 1 when y axis is zero. Above pH of the
isoelectric point, the protein has a negative charge and binds to positively charged
medium of IEX or anion exchanger. Below pH of the point the protein will bind to
negatively charged medium or cation exchanger. (GE Healthcare, 2016)
Figure 1. Surface net charge of a protein in function of pH. (GE Healthcare, 2016)
IEX separation starts with the equilibration of the stationary phase (figure 2) that
includes the target molecule to the desired start conditions. When it is achieved, all
charged groups are bound with counter ions, such as sodium. In the next step the
sample flows through columns where the slightly charged target proteins bind. The
columns are then washed removing possible impurities. Target proteins are eluted by
increasing the ionic strength of the buffer which can be seen in figure 2 as spikes in
absorbance. Alternatively, the spike can be caused by an impurity with properties
similar to the target protein. Salt ions compete with the bound components and the
proteins with the lowest net charge detaches first. In the final step the columns are
regenerated by increasing ionic strength of the buffer enough to remove any
molecules that are still bound to the columns. (GE Healthcare, 2016)
3
Figure 2. Absorption in function of time/volume during ion exchange
chromatography. Gray/green line originates from the sample solution and the blue
line from the buffer. (Modified from GE Healthcare, 2016)
The distance between the spikes in absorbance during elution stands for selectivity of
the IEX. With bad selectivity the spikes are on top of each other and the target protein
is not purified properly. Selectivity is dependable on properties of functional groups
and experimental conditions. One way to achieve a good selectivity is to select the pH
carefully so that the difference in net charge is maximized between the target protein
and the impurities. (GE Healthcare, 2016)
2.2
Importance
IEX can be used to separate biomolecules without histidine tag that can be added via
recombinant expression. Main advantages of the IEX over other chromatography
techniques is higher flow rate, concentrated sample output, relatively high yield and
none denaturing buffers. Limitations of the IEX is that the sample must be loaded at
low ionic strength and therefore in some cases the buffer needs to exchanged prior to
IEX. Additionally, the pH needs to be controlled very carefully since even small changes
can alter the ion exchanger resulting in loss of capacity and resolution. Resolution is
also affected by particle size. Finally, clusters of positively charged residues can cause
a negatively charged protein to bind a cation exchanger, and vice versa. (Bio-Rad)
As an example for how IEX can be used, in a research “Immunomodulatory effect of
Nigella sativa proteins fractionated by ion exchange chromatography” IEX was used to
purify certain proteins located in seeds of plant Nigella sativa. Soluble extract was
prepared by powdering 100g of dried seeds of N. sativa. The isoelectric point of target
proteins was known prior to this experiment. The extract was mixed with IEX buffer
and purified with DEAE-Sephadex A5 exchanger. Purity of collected fractions were
tested by using SDS-PAGE gel and presence of proteins of interest was confirmed by
introducing fractions in lymphocyte culture and measuring the amount of produced
interleukins. (Afrozul, 1998)
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3
Size exclusion chromatography
3.1 Size exclusion chromatography
Size exclusion chromatography (SEC), sometimes also referred as gel exclusion or gel
filtration chromatography, separates different sized molecules from each other as
they are passing through a packed medium. The principle is that smaller molecules
diffuse faster into media’s pores than larger molecules. During elution, the larger
molecules cannot enter the pores and are thus passing through the column faster than
the small particles. This is depicted in figure 3 (D). In addition to separation and
purification, SEC is also used to analyze molecular-weight distributions. (GE Healthcare
2014.)
Figure 3. (A) Three figures of a column used in SEC, depicting different volumes used
in calculations (from left: void volume, total volume, particle volume). (B) One of the
particles inside the column depicted closer. (C) Pores in a particle. Smaller molecules
fit in the pores of particles and their travel is hindered. It takes more time for them
to pass the column. (D) Larger molecules cannot fit in the particles and travel faster
through the column. (Picture modified from GE Healthcare 2014)
As molecules are separated based on their size, they do not bind to the
chromatography medium. SEC media are chemically and physically stable and inert,
meaning that they do not react with sample or have adsorptive properties. This leads
to an important advantage of the method: the conditions in the column can be varied
and optimized for desired purpose. In an SEC process, the selection of a buffer media,
sample volume and the dimensions of the column are the most important parameters.
(GE Healthcare 2014)
The most important characteristics of a SEC buffer media is its pore size, which is
varied depending of the separation area and the wanted resolution. (GE Healthcare
2014.) Crosslinked dextrans and agarose are often used as a gel media (Stanbury et al.
2016). The dosing of the sample into a SEC column greatly affects the separation
efficiency: Smaller dosing normally gives better resolution than larger feed (up to 30
% of the column volume). Sample volume has to always be smaller than separation
volume (distance between two peaks). (GE Healthcare 2014.)
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3.2 Importance
As there is no chemical linking between the media and the particles, SEC is applicable
to sensitive biomolecules that cannot withstand large changes in pH, metal ion
concentrations or otherwise harsh conditions. The most important processes are
purifications of enzymes, nucleic acids, polysaccharides, proteins and other larger
biological molecules. Since the buffer media is inert towards the feed, SEC can be used
after most of the other purification methods. Being also the slowest chromatography
technique and more restricted in feed volumes, SEC is often favoured as a final
polishing step in the purification process. (GE Healthcare 2014.)
SEC can be used to separate smaller and larger molecules from each other. It also
serves as a way for buffer exchange. This can be used for example in protein
purification, where removal of salts is needed as well as a buffer exchange before
storing. (GE Healthcare 2014) One of the early industrial application of the SEC
technique was the purification of tetanus-diphtheria vaccines (Stanbury et al. 2016).
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4
Hydrophobic interaction chromatography
4.1 General information
Hydrophobic Interaction Chromatography (HIC) is a separation technique which uses
the hydrophobic properties of proteins to separate them from one another. This is
based on the reversible interaction between a protein and the hydrophobic ligand
bound to the chromatography matrix. Most of the time, hydrophobic groups in
proteins are hidden within the protein and not on the surface of the protein. However,
there are certain biomolecules whose hydrophobic groups are sufficiently exposed,
allowing them to bind with hydrophobic ligands. These molecules are the targets of
HIC. (GE Healthcare, 2006) (Wikibooks)
The first step in HIC is equilibration; salt is added to the mobile phase, giving it a
greater ionic strength. Salt concentration is dependent on the mixture and the target
protein to be separated. The second step is sample application and washing; mixture
is added into the chromatography column. Biomolecules with sufficiently exposed
hydrophobic groups on their surfaces will bind to the hydrophobic ligands in the
chromatography matrix while the rest of the biomolecules, who are more hydrophilic
in nature, will be washed down the column. The addition of salt, which increases the
ionic strength of the buffer, will bind to the hydrophilic groups and remove them from
the chromatography matrix, leaving only the hydrophobic groups which are bound to
the ligands. The third step is elution; the chromatography matrix will contain not just
the target molecule, but also other molecules with strong hydrophobic properties. The
purpose of elution is to separate these hydrophobic molecules. This is done so by
varying the concentration of salt inside the buffer. When the concentration of salt is
lowered, the buffer loses some of its ionic strength and becomes more hydrophobic.
This will allow the buffer to bind to some of the hydrophobic molecules, removing
them from the chromatography matrix. The variation in salt concentration is
dependent on the hydrophobicity of the target molecule. This is done so until the
target molecule is the only molecule left in the matrix. Final step is regeneration; the
target molecule is regenerated by removing it from the matrix. (GE Healthcare, 2006)
4.2 HIC medium
The medium can also affect the selectivity of the separation in many ways. The media
are hydrophobic and they are made of porous materials. By choosing the size of the
pores according to particle size one can reach higher binding capacity. In the case of
large biomolecules open pore structure and high porosity make the separation more
efficient. Pore size also affects flow rate. (GE Healthcare, 2006)
The matrix also needs to be physically stable so that it can endure the changing
conditions, for example changing salt concentration. Chemical stability is also an
important aspect when choosing the material for the matrix. Sometimes the matrix
may require cleaning and chemical cleaning agents shouldn’t affect the matrix. If nonspecific interactions with sample components are undesired, an inert medium is
chosen. (GE Healthcare, 2006)
7
Today agarose-based or polymeric media are popular in HIC processes. One example
of an agarose-based medium is Sepharose. It has a cross-linked structure made of
agarose chains. The structure of Sepharose is presented in figure 4.
Figure 4. The structure of a HIC matrix Sepharose. (GE Healthcare, 2006)
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5 Conclusion
Chromatography techniques separate components by using their affinity towards two
different phases, and three commonly used techniques, ion exchange
chromatography (IEX), size exclusion chromatography (SEC) and hydrophobic
interaction chromatography (HIC) and their capability of separating biomolecules
were studied more closely in this report. IEX separates biomolecules according to
differences in their net surface charge. The benefits of IEX over other chromatography
techniques are higher flow rate, concentrated sample output, relatively high yield and
no denaturing buffers. However, the pH has to be controlled very carefully and the
sample loaded at low ionic strength. HIC separates biomolecules according to their
hydrophobic properties and it has fairly similar properties with IEX. SEC separates
molecules according to their size and it is applicable to sensitive biomolecules that
cannot withstand large changes in pH, metal ion concentrations or otherwise harsh
conditions. Due to low flow rate it is not suitable for high feed volumes.
The main benefits of chromatography over traditional separating techniques, such as
crystallization and distillation, is that it is easy to handle, it produces very pure
products and it is capable of separating target protein from multicomponent mixture.
Thus, it is often used in bioprocess technology to isolate and purify metabolic
products.
9
References
Afrozul, H., Peter, I., Lobo, Mohammed A., Nona R. and Sultan T. 1998.
Immunomodulatory effect of Nigella sativa proteins fractionated by ion exchange
chromatography. International Journal of Immunopharmacology. Vol. 21:4, p. 283295. ISSN 0192-0561.
Bio-Rad. Ion Exchange Chromatography. http://www.bio-rad.com/endz/applications-technologies/liquid-chromatography-principles/ion-exchangechromatography.
GE Healthcare Life Sciences. Hydrophobic Interaction and Reversed Phase
Chromatography: Principles and Methods. E-handbook. 2006. Available at:
http://proteins.gelifesciences.com/knowledge-library/protein-purificationmethods/hydrophobic-interaction-chromatography/.
GE Healthcare Life Sciences. Ion exchange chromatography: Principles and Methods.
p. 7, 11-25. E-handbook. 1/2016. Available at:
http://proteins.gelifesciences.com/knowledge-library/protein-purificationmethods/ion-exchange-chromatography/
GE Healthcare Life Sciences. Size Exclusion Chromatography: Principles and Methods.
p. 15-35, 89-94. E-handbook. 11/2014. Available at:
http://proteins.gelifesciences.com/knowledge-library/protein-purificationmethods/size-exclusion-chromatography/
Roy A. Keller, J.Kalvin Giddings. 2016. Chromatography chemistry. Available at:
https://global.britannica.com/science/chromatography.
Stanbury, Peter F.; Whitaker, Allan & Hall, Stephen J. Principles of Fermentation
Technology. Third edition. Butterworth-Heinemann, Oxford, 2016, Page 666.
Wikibooks. Proteomics/Protein Separations - Chromatography/Hydrophobic
Interaction Chromatography (HIC).
https://en.wikibooks.org/wiki/Proteomics/Protein_Separations__Chromatography/Hydrophobic_Interaction_Chromatography_(HIC).