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Separation modes
High resolution mode is used to separate individual macromolecules. This can only be achieved for molecules with partial access to the pores of the gel filtration medium. To help in selection of the proper medium for any given problem, gel filtration media are categorized according to fractionation range. The fractionation range reflects the pore size distribution of a given gel filtration medium by stating the range of molecular masses with partial access to the pores. A wide variety of gel filtration media are available today and together they cover a molecular mass range from 100 to 8000000 i.e. from small peptides to very large proteins.
Applicable sample volume is restricted in high resolution mode, since no concentrating effect is active in gel filtration. Sample volumes of ~0.5 -5 % of the column volume will provide sample zones narrow enough to avoid unacceptable overlap between closely spaced peaks.
Flow rate is kept low to avoid peak broadening due to incomplete mass transfer and columns are long to provide optimum resolution (see under Resolution in gel filtration below!).
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Fig 3.1. High resolution mode -
High resolution run of Peptides on Superdex Peptide.
Group separation mode is used to remove small molecules from macromolecules.
The fractionation range is chosen to elute the macromolecules in the void volume and the small molecules as late as possible. Being completely excluded from the pores, macromolecules will not separate from each other but elute as a group at an elution volume equal to the void volume.
GF will not discriminate between small molecules having full access to the pores (neutral salts, buffer salts, low Mr additives etc.). These molecules will instead elute as a group after approximately one column volume of eluent has passed the column. The effect of this is that the macromolecules "move" into the buffer used for equilibrating the column, while the low Mr components of the original sample buffer lag behind. In other words a buffer exchange has been achieved.
The large difference in elution volumes for macromolecules and for "salts" allows sample volumes up to 30% of the column volume to be applied.
In a group separation like this the degree of overlap caused by zone broadening effects is far less important than in the high resolution mode.
Higher flow rates can consequently be applied and a broader and shorter column may be used.
Simple small gravity-driven columns are normally used to desalt samples with volumes ~1ml. So-called spin columns, used e.g. for post-PCR clean-ups or for clean up after labelling oligos are examples on chromatography driven by centrifugation (Fig 3.2). The great advantage with centrifugation-driven columns is that a very large number of samples can be dealt with in parallel
Fig 3.2. Group separation mode -
Desalting albumin on a PD-10 column.
Fig 3.2 Scetch of a MicroSpin column |
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