Silica
| Q. |
Which Silica is best to use for a flash column? |
| A. |
Our most popular material for Flash Chromatography is our product 9385 - a 40-63µm
60Å pore size irregular silica. This is the original packing material sited in the
first paper describing Flash chromatography by Still, Kahn and Mitra - J.Org. Chem. 43, 2923
(1978). The material is produced to the same specifications today as then, however, it is now
produced in a modern fully automated plant under ISO 9001 guidelines to meet today's exacting
standards of quality and reproducibility for chromatographic grade silicas. |
| Q. |
Which Silica is recommended for batch adsorption? |
| A. |
Batch adsorption requires a consistent material of large surface area and larger particle
size than normal column chromatography. The recommended materials for this would be 7734 or
7733. Both of which are derived from the same base material as all our Silica gel 60 products.
The 7734 material is a 60Å pore size product with a particle size of 63-200µm.
The 7733 material is the same 60Å pore size base material with a particle size of
200-500µm. All Silica Gel 60 products have a surface area of 480-540m2/g. |
| Q. |
What does the F mean in the Silica Gel 60 F254 |
| A. |
This refers to the presence of a fluorescent indicator in the Silica. In this case it
fluoresces at a wavelength of 254nm. |
| Q. |
What does the G stand for in Silica Gel 60 G? |
| A. |
This indicates that the Silica contains a Gypsym binder (calcium sulfate hemihydrate) |
| Q. |
What does the H stand for in Silica Gel 60 H? |
| A. |
This indicates that the Silica contains no foreign binders. |
Self Packer
| Q. |
What is the maximum pressure that columns can be packed to? |
| A. |
The maximum pressure the columns can be used at is 200 bar for the 25mm and 100 bar for
the 50mm and 100mm columns. In all cases the maximum hydraulic pressure to be used during
packing should not exceed 100 bar. |
| Q. |
Which way round does the distributor plate go in my column? |
| A. |
The distributor plate in the 50mm selfpacker has different sides and the side with the
radial grooves and holes must face the end flange so that the side with the holes only,
faces the frit. |
| Q. |
Which way round should the frit go into the column? |
| A. |
Like the distributor plate, the frit has two different sides. The side with the
course woven mesh is the side that faces away from the packing material. The fine woven
mesh side faces the packing material. |
| Q. |
I have packed my column and started the flow in the normal direction and the pack pressure
if off the scale. What is wrong? |
| A. |
The most likely cause of this is that the outlet distributor is fitted the wrong way
round so that the flat surface with only holes is facing the outlet flange and acts like a
shut-off valve as the pressure starts to build. Switch off the pump, allow the pressure to
reduce to zero and then remove the column from your pumping system. Next, release the
compression pressure on the packing and leave the column for at least 1 hour to allow
the packing bed to relax. Next, remove the outlet flange and remove the frit and distributor
plate. Clean all parts of packing material and reassemble placing the distributor plate the
correct way round with the radial grooves facing the flange. Replace the top flange and
recompress the packing material. The column should then be re-equilibrated and flow packed
as per the normal packing method. |
Fractogel® process media
| Q. |
What are the standard methods for regeneration of ion-exchangers? |
| A. |
Commonly all columns of polymer based ion exchangers are resistant to NaOH concentrations up to 0.5M. |
| Q. |
Are there alternative methods for column cleaning or CIP (cleaning in place)? |
| A. |
The weak anionic detergent SLS (sodium laouroyl sarcosinate) is an appropriate reagent for column
cleaning. Use 2% SLS and 0.25M NaCl for regeneration and isopropanol/HCl (1 liter of 25%
isopropanol and 12.5ml 25% HCl) to remove the detergent. |
| Q. |
What are the pK-values of the different functional groups used for ion exchange chromatography? |
| A. |
The following pK-values were determined:
TMAE group
DEAE group
DMAE group
COO-group
SO3- group |
>
~
~
~
< |
13
11
10
4.5
13 |
The advantages of strong ion exchangers are their high capacity over a wide range of pH-values.
|
| Q. |
What is the recommended packing procedure for Fractogel® EMD Tentacle sorbents? |
| A. |
Please follow the detailed packing instructions. However, for all Fractogel® EMD media,
a certain reduction of the volume (compression) which leads to a reduced interparticle
volume is necessary. |
| Q. |
How many cycles can be run using Fractogel® MED tentacle ion-exchangers? |
| A. |
The life time depends on the sample (i.e. protein content, main contaminations, lipids,
low molecular substances...) and on the regeneration procedures. Injecting standard protein
solution, you can run over more than 100 cycles without any loss of resolution. Due to
the reduced interaction of the Fractogel matrix with biological compounds, many cycles can be
run when separating crude extracts.
However, after 50 up to 100 cycles, one should regenerate the column using 0.5M NaOH. |
| Q. |
What are recommended flow rates for tentacle phases? |
| A. |
The binding of proteins by the functional gorups located on the flexible polymer chains is
very rapid. Therefore, even at high flow rates, an efficient protein binding can be obtained.
The anion exchangers can be run with linear flow rates up to 6.5cm/min (and cometimes more).
In the case ofS-type media with particle sizes between 20-40µm the linear flow rate
(which is the actual flow in ml or cm3 per cross section of the column in
cm2) should not exceed values of 3, 5cm/min. |
| Q. |
What are the elution conditions for thiophilic adsorption chromatography? |
| A. |
In most cases a decreasing gradient of salt (ammonium sulfate or potassium sulfate) is used.
Sometimes one can optimize resolultion and recovery by ading 0.5M NaCl or ethylenglycol
(up to 80%) in buffer B. |
| Q. |
Some of our lots of Fractogel have different shelf lives. Why is this? |
| A. |
We have ongoing stability studies for the various types of Fractogels® and as time
passes we review the stability data and update shelf lives accordingly. Obviously if an
older lot is marked with a 5 year shelf life and a newer lot is marked with a longer shelf
life, it is safe to assume that the older material is also good for the longer shelf life
from the date of its manufacture |
| Q. |
What is the format of the Lot numbers for Fractogel®? |
| A. |
The format is as shown below:
| Example lot number |
K12345683 |
123 |
| |
AXXXXXXYY |
WZZ |
| A = Alpha character - part of the sequential lot number (K) |
| X = Sequential lot number - 6 digits (123456) |
| Y = Last two numbers of the part or item number (83) this makes the part -
Fractogel DEAE (M) material part number 1.16883 |
| W = Year number of manufacturing - 2001 |
| Z = Week number of manufacturing - week 23 |
|
Benzonase®
| Q. |
Which quality/quantity of Benzonase® has to be applied? |
| A. |
This question cannot be answered in general. We tried to give you an idea on the properties
of Benzonase® and the parameters which are influencing its activity. For a certain
problem the optimal conditions have to be determined experimentally. The examples given
may help you to work out a strategy for process optimization. For viscosity reduction
Benzonase® purity grade II (90%) will often be sufficient, especially when the target
protein has been expressed in E. coli, too. |
| Q. |
Do you offer an immobilized Benzonase®? |
| A. |
No. Unfortunately all attempts to immobilize Benzonase® covalently in an active form
have failed so far. But, we do not give up. |
| Q. |
Do you offer an ELISA kit for the assay of traces of Benzonase®? |
| A. |
Yes. An ELISA kit is available: Cat.No. 1.01671.0002.
New: ELISA kit II now available: Cat.No. 1.01681.0002 |
| Q. |
Is it possible to inhibit Benzonase®? |
| A. |
Yes, but only under extreme conditions (NaOH, heat), which usually will be harmful to
your end-product too. There is no specific irreversible inhibitor known for endonucleases.
A reversible inhibition can be achieved by complexation of Mg2+ ions with EDTA, high
concentration of monovalent cations and/or phosphate. |
| Q. |
How to remove Benzonase®? |
| A. |
During downstream processing Benzonase® is removed from the target product by
chromatographic separation methods. Depending on the characteristics of the desired
protein ion exchange, hydrophobic interaction, hydroxyl apatite gel permeation
chromatography can be used. Assuming a significant difference in molecular size between
Benzonase® and the target protein, ultrafiltration may be used successfully.
Benzonase® binds to strong anion exchangers and to hydroxyl apatite matrices, it
does not bind to hydrophobic gels. Cation exchangers are not suitable since Benzonase®
binds poorly.
In addition, Benzonase® can be removed by ammonium sulfate precipitation during the
initial processing steps. 50% of Benzonase® will be precipitated at 50% ammonium
sulfate saturation at pH 7 or pH 8. With 60% saturation at pH 7.0 90% of Benzonase®
can be precipitated.
It is recommended to optimize the separation protocol in a special case using the above
mentioned results as a guideline.
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