CHEM-E3140 Bioprocess technology II

Adsorption chromatography

Biotechnology

Authors of the report: Lauri Huitula

Juha Laitinen
Pablo Terroba-Seara

Report submitted: 5.12.2017

Table of contents
Abstract .............................................................................................................................................. 1
Background ........................................................................................................................................ 1
Theory ................................................................................................................................................ 2
Case study .......................................................................................................................................... 3
Conclusion .......................................................................................................................................... 8
References ......................................................................................................................................... 8
Abstract

When making drugs in the medical industry, it is really important to be sure that the
product only contain the molecules that there are meant to be. Other ways it might
risk the health of the patient. Chromatography is widely used method for analytical
and purification purposes. Adsorption chromatography, and especially HPLC (High
Performance Liquid Chromatography) remains as the workhorse of analytical
methods to analyze what molecules a mixture consists of. The adsorption
chromatography divides the mixture in fractions, which can then be analyzed with
e.g. the MALDI-TOF analysis. In the adsorption chromatography the molecules
separate due to some of the molecules in the mobile phase interact with the surface,
they are adsorbed by the surface.

Background

Chromatography is a central tool used in all fields of research and industry. It is used
for analytical purposes of detection and identification of compounds as well as
purification of products. Absorption chromatography is the traditional form of
chromatography and is also known as liquid solid chromatography.

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Picture 1. Schematic picture of how adsorption separates a mixture.

Picture 1. shows generally how a chromatography system works. The stationary

phase interacts with some of the molecules in the mixture and therefore, slow them
down. Because some particles move slower than other, that lead to separation.

Theory

Chromatography is based on separation of between two phases, stationary phase

and mobile phase. It could be described that chromatography system has a dynamic
equilibrium between the phases. Compounds attach into stationary phase and
detach based on their properties leading to separation of different compounds. The
technique used for the separation determines the type of chromatography that is
used. (Jaarinen and Niiranen 2005,)

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Adsorption chromatography is based on attachment and detachment of materials
to and from the surface of the adsorbent material. When considering the meaning
of adsorption chromatography, it is good to clear the difference between
absorption and adsorption is that absorption means the material is moved inside
the absorbing material, whereas adsorption means the material is attached into the
surface of the adsorbing material. In adsorption chromatography separation of
materials is based on retention between solid stationary phase and liquid or
gaseous mobile phase. For biotechnological applications in most cases liquids
chromatography is used due to lack of volatility of biological materials. Binding to
stationary phase is caused by van der Waals forces and polarity of materials.
Selection of phase material is done based on required compounds, normally used
phase materials are polar, such as silica (SiO2). If a non-polar phase (reverse phase)
is required, it is usually done by coating a polar material with non-polar functional
groups, e.g. octadecylsilane (-Si-O-Si-C18H37). In comparison, in partition
chromatography separation is based on solubility of components between phases,
e.g. liquid-liquid chromatography where both stationary and mobile phase are
liquid, and samples form an equilibrium between phases. (Jaarinen and Niiranen
2005, Molnár and Horváth 1976, Wohlgemuth 2011)

Case study

Technically, the principle of the adsorption chromatography is the analytical

separation of a chemical mixture (gas or liquid) passing it over an adsorbent bed that
has the function of adsorbing different compounds at different rates [Modern
Chemical Techniques]. Hence, first of all, we are going to introduce different
examples of adsorbents in relation with the stationary phase. In general terms the
idea adsorbent must accomplish with the following requirements [Lloyd R. Snyder,
1968]:

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  Insoluble in the mobile phase.

 Colourless specially when in the study are used coloured mixtures.

 Suitable particle size in order to obtain a good separation and suitable flow
rate.

 Inert to solutes (adsorptive).

Once, we know the different special properties for the adsorbent we are going to
introduce the most suitable for this technique:
• Silica gel

It is the most universally used adsorbent in column and thin layer

Chromatography as well. It is prepared by acidification of sodium silicate with
sulphuric acid followed by washing with water and drying. The active sites of silica
gel are the hydroxyl groups attached to silicom atoms “Silanol groups”. This type
of adsorbent reaches its maximum power when is heated between 150-250°C for
removing the water. This is an important requirement since if the silica gel
contains water it is acting by partition and not by adsorption. In this type of
studies decreasing the particle size, increasing the surface area and, thus,
increasing the separation power.
All of the derivates from the silica gel are based on reaction with the Si-OH groups
(Silanol groups) to block them. One of the most important commercial examples
is the Reversed Phase Silica Gel (RP) in which straight chain aliphatic groups are
attached to the OH of silica gel by silylation.

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 Figure 1: Reversed phase silica [OChemOnline]

• Alumina
Its common name is aluminium oxide (Al2O3). It is activated by heating at 400°C
overnight. It has a wide variety of positive properties such as its large capacity, is
insoluble, relatively inert and easy availability. The mechanism is different from
silica because of the strong positive field of Al+3 and the influence of basic sites
which affect easily the polarized compounds. It is one of the best adsorbents of
aromatics from olefins. Three important disadvantages are that it may cause
rearrangement and ring expansion of unsaturated compounds, it is not for base
labile compounds and it could react chemically with acidic compounds.

Figure 2: Aluminium oxide [Santa Cruz BioTechnology: CAS 1344-28-1]

Types of commercial alumina:

o Neutral alumina: pH=7.5

o Acidic alumina: pH=4. It is prepared by washing aluminium oxide with

HCl then with distilled water.

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 o Basic alumina: pH=10. It is prepared by washing NaOh then distilled
water.

After of mentioning the most important adsorbents, we are going to explain the
importance of the adsorption chromatography techniques in the industry. Its
relevance becomes greater and greater in terms of scientific advances due to the
purification and separation of intermediates in multi-stage syntheses [Modern
Chemical Technique]. One of the major advances has been the development of
efficient columns capable of separating specific chiral compounds from a mixture.
Basically, in the chiral chromatography, it is able to separate enantiomers using chiral
stationary phase that react with one enantiomer more than the other, thus, the
separation can be carried out. It is exactly a variant of a column chromatography
where the two enantiomers of the same analyte compound differ in affinity to the
single-enantiomer stationary phase and they exit the column at different times [W.A.
Bonner, 2007].
The Chiral Stationary Phase (CSP) is created by the attachment of a suitable chiral
compound to the surface of an achiral support for instance silica gel. The most
common CSP are based on oligosaccharides for example cellulose or cyclodextrin,
specifically, with β-cyclodextrin. This principle can be used in the fabrication of
monolithic HPLC columns or gas chromatography columns [W.A. König, 2001].
In the next figure, it is possible to observe the results of an experiment carried out
using the technique explained previously [F. Zaera , 2008]:

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 Figure 3: Results between Cd versus Cn [F. Zaera, 2008]

The results show a comparison on the chiral modification of Pt catalysts between

cinchonidine (Cd) versus cinchonine (Cn), which facilitates the production of opposite
enantiomers in catalytic hydrogenation reactions with a-ketoesters. The interesting
of this study was to demonstrate that even though the two cinchona alkaloids are
close enantiomers, we can observe difference in their enantioselectivity. This
dissimilarity was accounted by a combination of two key properties of these chiral
modifiers such as their solubility and adsorption equilibrium on the Pt surface.
Currently, there are only a handful of examples of this effect, hence they will be
needed more studies in the future in order to understand better what drives these
compositional changes between the gas or liquid phases and the adsorbed states [E.
Schmidt, C. Bucher, G. Santarossa, 2012]. It is a phenomenon with a relevance
potential in relation to the design of enantioselective crystallization processes and a
possible explanation for the dominance of one enantiomer in the chiral biochemistry
of life. Finally, it would be interesting to increase these studies of chirality to more
realistic surface area for better understanding the chemistry of chiral systems on
surfaces.

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Conclusion

Adsorption chromatography is a widely used method to analyse the chemical

compounds of a mixture. Generally, adsorption chromatography is a really old
method, it has been studied and used since the early 20th century. HPLC, which is a
more sophisticated and advanced method for analysing mixtures, was developed
during the 70s and has been used a lot and improved since then. There are a few
different materials which are often used in the stationary phase. By choosing the
adsorbing material according to the particles that need to be separated, better
results can be achieved. Properties that are important for a good adsorbent include
insolubility with the mobile phase, inertity and polarity/non-polarity.

References

Imre Molnár And Csaba Horváth, Reverse-Phase Chromatography of Polar Biological

substances: Separation of Catechol Compounds by High-Performance Liquid
Chromatography, Clinical Chemistry, 22 (1976) 1497-1502.

Soili Jaarinen and Jukka Niiranen, Laboratorion analyysitekniikka, 5th edition, Edita,
Helsinki 2005, p. 140-142.

R. Wohlgemuth, Comprehensive Biotechnology, 2nd edition, Academic Press, 2011,

p. 591-601.

Modern Chemical Techniques, The Royal Society of Chemistry, Unilever, 5.

Chromatography, p.116-159.

Lloyd R. Snyder, Principles of Adsorption Chromatography: The Separation of

Nonionic Organic Compounds, Marcel Dekker, INC. (1968), New York.

W.A. Bonner, in Topics in Stereochemistry, ed. E.L. Eliel and S.H. Wilen, John Wiley
& Sons, Inc., Hoboken, NJ, USA, 2007, vol. 18, ch.1, pp. 1-96.

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W.A. König, in Chirality in the Natural and Applied Science, ed. W.J. Lough and I.W.
Wainer, Blackwell Science, 2001, pp. 261-284.

F. Zaera, J. Phys. Chem. C, 2008, 112, 16196-16203.

E. Schmidt, C. Bucher, G. Santarossa, T. Mallat, R. Gilmour and A. Baiker, J. Catal,

2012, 289, 238-248.