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17. Does displacement chromatography work with oligonucleotide separations?
18.  What if I need more technical help? 

                                           

1.  Is displacement chromatography more complicated than elution chromatography?

Displacement chromatography is no more complicated than elution chromatography, but operating parameters are different.  In some ways displacement is more straightforward than elution.  However, the considerations for optimization are different.  It is important that you follow the protocols closely when getting started.  Find SACHEM's displacer protocols here. 

2.  Do I need to know binding isotherms?           

Binding-isotherms are important to fundamental understanding of all forms of chromatography.  However there is no more need to do detailed isotherm measurements in displacement than there is in elution.  Optimization is usually carried out empirically (trial-and-error) in optimizing both methods.

3.  Am I limited to certain loadings when using displacement?

Displacement chromatography requires high loadings to set up a good displacement train.  One typically uses loading of 50-80% of the maximum resin loading capacity.  As a starting point, use 60%. If you do not have enough starting material to load at that level, switch to a smaller (narrower) column.

4.  Why are the dimensions of the column important?

Well packed columns of proper length work best.  Short columns also work, but recoveries (yields) can be lower. 

5.  Why would I operate chromatography in displacement mode vs. elution mode?

Owing to the convergence of high loading, high recovery and high purity (high resolution) of displacement chromatographic methods, one can obtain higher throughput per cycle, higher purity and increased concentrations from each use of the column.  This can be useful if you need to quickly purify large amounts of product using smaller "analytical-type" columns. Displacement chromatography is also very useful for trace component analysis and impurity profiling.  If you are trying to analyze or isolate a trace component, displacement methods will allow you to concentrate these components while simultaneously removing the main components. 
Learn more about trace component amplification here.

6.  What concentration displacer should I use?

Good displacers work at low concentrations, typically in the range of 5-25 mM.  The displacer concentration is a parameter that should be optimized for the system under study.  Initially, start at 10 mM if no other information is available.

7.  How do I get the displacer out of my protein?

Good displacers only come into contact with the last few fractions of the last band in the displacement train.  Hence, most fractions contain no displacer.  In contrast, an eluter is found in nearly all fractions in elution chromatography.  Although usually unnecessary, a desired product (protein, oligopeptide, oligonuclteotide) with sufficient molecular weight (>3kD) is readily removed from the low molecular weight displacer (<1kD) via diafiltration.

All of SACHEM's displacers are single component, high purity molecules, and are designed with UV chromophores for easy detection. 

8.  What is the level of detection for Expell™, Isolis™ and Propell ™ Displacers?

The level of detection for SACHEM's displacers using simple HPLC methods (UV detection) are in the range 0.1-1.0 ppm. HPLC.

9.  What are the storage conditions for the displacers?

All of SACHEM's displacers can be stored under ambient conditions.  Once diluted in a buffer mixture, the displacer should be used within 2 weeks.    

10.  Can the displacer be reused?

The displacer should not be reused.  Expell™ and Isolis™ displacers are purified chemical entities and the purity of the displacer has a significant impact on the displacement experiment.  

11.  I have only ever seen displacement done on model systems.  Does it really work on real world proteins?

Until now the unavailability of commercially viable displacers has limited the use of displacement chromatography on real world systems.  You can view some example results of SACHEM displacers on various systems under Displacement Chromatography 101 on our website. 

12.  Can this be used for analytical applications?

Displacement can be a useful technique in analytical applications when trying to enrich trace components in a complex mixture because all impurities appear in the tight transition zones.

13. At what stage is displacement best used?

It is possible to use displacement chromatography as early stage capture, after an affinity purification step, in late stage product polishing or to isolate a trace component.

14.  What if I can't get the displacer off of my column?

In most cases, the stated regeneration protocols will efficiently remove the displacers.  For convenience, SACHEM supplies Regenerate™ Solutions which are available for purchase on www.sachemstore.com.  These are available in various pH's. 

15.  Does displacement chromatography work on monoclonal antibodies?

Displacement chromatography using ion-exchange matrices works great for mAb's, especially following an affinity step (protein A) or as the final polishing step.  It is usually difficult to compare a finishing step (ion-exchange) with an affinity step (protein A) because they operate so differently.  However, we know of cases where the advantages of displacement chromatography (IEC, HIC) together with a cleaner input stream render the earlier affinity step unnecessary.  In these cases, ion-exchange chromatography operated in displacement mode functions well as a capture step.

16. Is column packing important?

The quality of column packing is very important in displacement chromatography, perhaps even more so than in elution chromatography.   In displacement mode operation, all displacement bands, one for each protein component, move down the column next to each other without any "solvent space" between them.  These bands sharpen as they move down the column and the overlap between the bands is minimized.  Poor column packing causes overlap regions to broaden, while purity and recovery suffer. Hence, well packed columns that lead to well-defined, sharp bands are important for successful displacement mode operation.

Similarly, column geometry is also important.  Long, thin columns give better results in elution chromatography because resolution in improved.  Longer columns also work better in displacement chromatography because recovery in improved, not resolution.  Owing to slower flow-rates, issues relating to back-pressure, diffusion and column flow dynamics are less problematic.  Column length is am important parameter for optimization.  Once the column is long enough to get adequate recovery, capacity is increased by increasing the column diameter.

17. Does displacement chromatography work with oligonucleotide separations?

Displacement chromatography is well suited for preparative purification of oligonucleotides using anion-exchange, reversed-phase and ion-pairing reversed-phase chromatographic methods.  Small PCR primers, antisense DNA drugs, miRNA/siRNA compounds and hybridized DNA/RNAs with natural or synthetic bases or backbone chemistry are all suitable candidates for preparative purification by displacement chromatography.  This also includes small oligomers (3-8mer) used in large-scale, block-synthesis of 20-35mers.  Displacement chromatography is ideal for preparative purification of DNA/RNA compounds whose impurities include related deletion and insertion sequences (n-2mer, n-1mer, n+1mer etc) as well as phosphorothiolate compounds (monothiophosphate) that have P=O impurities (S-1mer, S-2mer).  Larger target sequences (40-250mer) can also be purified in displacement mode using RP and IPRP methods.

18.  What if I need more technical help?

Please  This email address is being protected from spam bots, you need Javascript enabled to view it   or call us at 512-421-4900.  We would be happy to answer your questions.