ISPPP 2008

WORKSHOPS

To learn about various aspects of separation technologies four scientific workshops will be held on Sunday preceeding the symposium

 

Sunday, September 21, 2008           14:30 – 16:00

Workshop 1                                                                                     Workshop 2

Sample clean up and interfacing of LC separation methods

LC of biopolymers

Chair : Egidijus Machtejevas, Merck KGaA, Darmstadt/D Chair : Friedrich Birger Anspach, HAW - Hamburg University of Applied Sciences/D
Sample clean up including sampling, sample treatment and prefractionation
is the most decisive and essential step in biopolymer analysis. The aim is to
selectively enrich the target compounds, to deplete the undesired constituents
and to make the procedure robust and reproducible. A survey on the classical
sample clean up methods reveals that these bear severe deficiencies. For this
reason fully automated and highly selected procedures are preferred over off-line
modes. Some of these methods are presented and discussed.

For the separation of proteins and peptides in biofluids multidimensional (MD)
LC coupled with mass spectrometry is the method of choice. One of the major
issues in MD-LC/MS is the interfacing of LC methods not only in technical terms
(switching valves, sampling rate, number of fractions, trap columns) but also in
view of the choice of phase systems and the separation (gradient) conditions.
Illustrative case studies are presented in the field of peptidomics.

A basic introduction into the subject will be provided, including the most often used chromatographic modes as well as binding-elution techniques using various media. Insights will be provided on the influence of mobile phase compositions on tertiary structure. The effect of diffusion and adsorption phenomena will be addressed.
   

Sunday, September 21, 2008          16:30 – 18:00

Workshop 3 Workshop 4

Multidimensional LC/MS

Basics of bioprocessing

Chair : Peter Schoenmakers, Polymer-Analysis Group, University of Amsterdam/NL Chair : Alois Jungbauer, University of Natural Resources and Applied Life Sciences, Vienna/A
In this course the main fundamental and practical aspects of comprehensive
two-dimensional liquid chromatography (LC×LC) will be discussed. The most-
important question that will be addressed involves the value of LC×LC in practice.
The main strengths of the technique will be discussed, viz. the possibility of generate
high peak capacities and structured chromatograms. Different ways to approach
(nearly) orthogonal separation systems will be discussed, including various
combinations of size-exclusion chromatography, reversed-phase liquid
chromatography, and ion-exchange liquid chromatography. Special attention will
be paid to applications of LC×LC for the separation of proteins and peptides.
The course will focus at the contemporary version of LC×LC, which typically
uses two different columns and a switching valve. The common way to perform
LC×LC involves a first-dimension separation that is relatively slow, with a typical
analysis time of an hour or longer. The result of an LC×LC separation is a series
of many (e.g. 100) fast second-dimension chromatograms. These are usually
displayed as a plot that shows peak intensity as a function of the retention times
in the first- and second-dimensions.
In truly comprehensive separations, such a two-dimensional chromatogram
is representative for the entire sample.
The peak capacity of any separation can be defined as the number of peaks
that can be separated with a specific resolution. The peak capacity of high-
resolution one-dimensional LC is somewhere in the hundreds. Using standard commercially
available columns and instruments, LC×LC allows us to achieve peak capacities
in the thousands.
The concept of sample dimensionality is extremely useful when sicussing LC×LC
se-parations. A dimension of a sample can be seen as a property that sets the
molecules apart from each other. If we manage to align the most important
dimensions of the sample with the separation mechanisms we may obtain
structured separations
Orthogonal separations – in which the retention time in the second dimension
is com-pletely independent of that in the first dimension – are ideal for LC×LC.
They allow the entire “separation plane” to be filed with peaks, implying that the
entire peak capacity may – in principle – be used.
There are a number of ways to perform two-dimensional separations of proteins
and peptides. The benchmark technique for proteins is 2D gel electrophoresis
(2D-CE), which offers excellent orthogonality and a great peak capacity. However,
the technique is also laborious and slow, especially when peaks need to be
identified by mass spec-trometry. Potentially, LC×LC are faster and more reliable,
while they also allow on-line interfacing with MS. The most common liquid-
chromatographic technique for the two-dimensional separation of proteins uses
ion-exchange chromatography (IEC) in first dimension and RPLC in the second. An alternative approach combines aqueous SEC with RPLC.
Various opportunities and challenges faced in LC×LC will be discussed, including
the types of detectors that may be used, the possibility to focus the analytes between
the two separation stages, and the availability of suitable software.
In biopharmaceutical industry chromatography is the workhorse for purification proteins, and plasmids. In this workshop an overview on the separation principles of preparative and industrial chromatography will be given. Scale up issues will be addressed in detail. The concept of constant residence time will be elaborated. Also models for scale down of chromatography will be discussed in context of spiking experiments for clearance studies of adventitious agents. Here the focus will be on the extra column peak dispersion and packing of small chromatography columns.

The second part of the workshop addresses the large scale purification of plasmids, virus-like particles and viruses. Due to the restricted pore diffusion into chromatography beads other concepts of chromatographic separation have been developed. These media are based on convection. The separation principles of such media are discussed and examples are given.

 


 

For attending the workshops registration is necessary . As the number of participants is limited, please register early to avoid disappointment.
Ticket: 50 EUR per workshop for regular participants and 25 EUR for students (only in combination with a regular conference ticket).


 

 

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