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| For the HPLC separation of proteins and enzymes, usually
reversed phase chromatography, gel permeation chromatography or
ion-exchange chromatography are used. Because ion-exchange
chromatography can achieve separation with a mobile phase of
organic solvents, it is the preferred procedure. However for the
separation of some enzymes which may lose their activity by
contact with metals like, stainless steel or titanium columns,
glass packed columns may be more appropriate. The COSMOGEL glass
column consists of a hydrophilic polymer packed in a glass column,
for separations requiring non-metallic contact. Furthermore, the
high-pressure resistance of the glass makes it possible to use a
10mm polymer particle as the packing material. This way the
COSMOGEL glass column can provide equivalently high-resolution to
stainless steel columns. For ion exchange chromatography solvents
containing halogens such as Cl are often used. In such cases,
stainless steel columns react with the mobile phase, while glass
columns are completely resistant to salts and other corrosive
mobile phases.
|
| totally porous spherical hydrophilic
polymer |
| 5 µm |
| approx. 1000Å |
| Gel type and characteristics |
| Diethylaminoethyl |
N+H(C2H5)2 |
Cl- |
0.6 |
10.8 |
| Quarternary ammonium |
N+(CH3)3 |
Cl- |
0.4 |
11.0 |
| Carboxymethyl |
COO- |
Na+ |
0.3 |
5.7 |
| Sulfopropyl |
SO3- |
Na+ |
0.4 |
2.6 |
| 8mmI.D.×75mm |
<11 |
0.5-1.0 |
1.5 |
1.5 |
10-50 |
| 1-14 |
| >4 |
| 1-14 |
Selection of the mobile phase |
Generally, a mobile phase within the pH range of +/-1 pH unit
away from the isoelectric point (pI) of the sample is used. For
anion exchange columns mobile phase with 1 pH above the
isoelectric point of the sample, and for cation exchange columns
mobile phase with 1 pH below that is used. When Bis-tris HCI salt
of a bivalent ion and Tris HCI salt of a univalent ion are in the
same concentration, the elution force of the former is greater due
to its ionic strength.
|
| pH |
Buffer |
| 4.0 |
Formic Acid |
| 5.0 |
Acetic Acid |
| 6.5 |
Phosphoric Acid |
| 8.0 |
HEPES Na |
| 9.5 |
|
| The initial mobile Phase (A) is 20-50mM of one of the above
mentioned buffer solutions and the final mobile phase (B) is the
mobile phase (A) with an addition of 20-600mM of salt. |
Selection of salts |
In general, a salt containing the same ion as the ion of mobile
phases buffer solution and the counter ion of stationary phase is
used. NaCl is suitable for any column, however when the elution
force is weak, CaCl2, MgCl2 for DEAE and QA
columns and Na2SO4 for CM and SP columns can
be used.
Since the ionic strength of these salts is greater than that of
HCI with the same concentration, the retention is consequently
greater.
|
Selection of organic solvents |
When the retention is not strong enough with only a
chromatography buffer solution and salt, the addition of some
water-soluble organic solvents might improve the retention. With
COSMOGEL Ion Exchange Column up to 20% of acetonitrile or
2-propanol or similar of water soluble organic solvent can be
used.
|
Isoelectric point and elution time of proteins |
| Generally, in cation exchange chromatography, retention
increases as the isoelectric point increases. The opposite is true
in anion exchange chromatography. |
Separation of proteins on anion exchange column |
| With an anion exchange column having a positive charge, with
higher negative charge of the sample the retention becomes longer.
When using a weak alkaline mobile phase with lower isoelectric
point the elution becomes longer. |
Separation of proteins on cation exchange column |
With a cation exchange column having a positive charge, with
higher positive charge of the sample the retention is increasing.
When using a weak acid mobile phase with higher isoelectric point
the retention time becomes.
|
Difference in the separation between anion and cation exchange
column |
Lipoxytase is an acidic protein with an isoelectric point of 5.
Lipoxytase can be purified by anion exchange columns because its
negative charge in mobile phase at pH 8.2. Lypoxitase can be
purified by cation exchange columns because in lower pH (e.g. pH
4.5) than its pI (pI 5) it is charged positively Efficient
separation of crude lipoxytases is achieved by using both anion
and cation exchange column to separate impurities.
As an example, the 50% dissociation pH of the functional group of
the cation exchange column COSMOGEL SP is 2.6, which has
sufficient exchange capacity in a 4.5 pH mobile phase. However the
50% dissociation pH of the same cation exchange column COSMOGEL CM
is 5.7, sufficient exchange capacity is not available in a 4.5 pH
mobile phase and the curve has broad peaks. |
Recovery of protein using an ion-exchange column |
| The-COSMOGEL ion Exchange Packing material is basically composed
of a hydrophilic polymer. Since there is no non-specific
absorption occurring, a high recovery of proteins can be expected. |
Injection volume and resolution |
| For ion-exchange gradient separations, even when a large volume
of diluted sample is injected; sufficient separation can be
obtained because the sample is concentrated at the head of the
column. The injection of a large volume of diluted sample has no
effect on the chromatographic separation. |
Sample loadability and resolution |
| An 8mmI.D. column can resolve a sample up to 1mg in size without
any deterioration of separation characteristics. If the resolution
factor is greater than the example below, a sample load of 10mg
might be achieved. |
Preparation of proteins |
| The COSMOGEL 20mm I.D.×100mm for mass preparation can separate
a large sample volume up to 10mg without deterioration of the
separation characteristics. If the resolution factor is high
enough a sample load of 100mg may be permissible. |
Ordering information
GLASS PACKED COLUMN |
| 8mmI.D.×75mm |
37845-81 |
1/4-28UNF |
| 37846-71 |
| 37844-91 |
| 37847-61 |
| 7.5mmI.D.×75mm |
43371-91 |
Waters type |
| 43373-71 |
| 43375-51 |
| 43377-31 |
|
Kit ontents: empty column, 10ml of packing material, nut,
ferrule, tube
|
37854-61 |
1/4-28UNF |
| 37855-51 |
| 37856-41 |
| 37857-31 |
| Kit contents: empty column, 10ml of
packing material, connecting tube |
37928-01 |
Waters type |
| 37926-21 |
| 37924-41 |
| 37922-61 |
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