Multimode and Aqueous/Organic SEC Columns
Column Specifications
Column Information
Although Asahipak GS-HQ series is basically SEC columns, they permit different types of separation and analysis from conventional ones by use of a multimode which combines the hydrophobic interaction (RPC) and ionic interaction (IEX: ion exclusion). The GS-320 HQ column performs well in separating hydrophillic peptides, particularly acidic and basic peptides. As the columns can be used over a wide range of eluent pH because of being polymer-based column, they can be washed with alkaline solution leading to a longer column life, which is another feature of the column.
Sample : 20micro-L
1. Glu-Ala-Glu 0.025%
2. Arg-Asp 0.05%
3. Gly-His-Lys 0.025%
4. Arg-Pro-Lys-Pro 0.025%
| MW | pI | Σf | |
|---|---|---|---|
| Glu-Ala-Glu |
347
|
3.12
|
0.39
|
| Arg-Asp |
289
|
6.75
|
0.68
|
| Gly-His-Lys |
340
|
9.95
|
0.29
|
| Arg-Pro-Lys-Pro |
497
|
11.44
|
3.24
|
pI : Isoelectric point
Σf : Hydrophobic parameter
Column : Shodex Asahipak GS-320 HQ (7.5mmID*300mm) Eluent : 50mM Ammonium acetate buffer(pH6.7) Flow rate : 0.5mL/min Detector : UV(220nm) Column temp. : 30deg-C
1) When a neutral/acidic eluent is used.
| Acidic samples | As it is the ion exclusion mode which is in operation, substances are eluted relatively quickly. |
| Neutral samples | As substances are separated by reversed phase mode, they are eluted moderately. |
| Basic samples | As it is the ion exchange mode which is in operation, substances are eluted relatively slow. |
2) When a basic eluent is used.
| Acidic samples | Basically, substances are excluded together at the Vo position but, with acidic samples of large molecular weights, there are cases in which substances are retained in the column. |
| Neutral samples | Substances are separated by reversed phase mode. |
| Basic samples | If the eluent has a higher pH than the pKa of the sample, substances are separated by reversed phase mode as the dissociation of basic samples is suppressed. |
As the eluent for Asahipak GS-HQ series and GF-HQ series, either organic solvent or aqueous solution can be used.
Organic solvents: Please refer to Multimode Columns and Aqueous/Organic SEC Columns.
Aqueous solutions: Please refer to the followings:
1. Strong electrolytic salts
Total salts concentration should be 0.5 M or less. Aqueous solutions of NaCl, KCl, Na2SO4, K2SO4, (NH4)2SO4 and other such salts may be used as eluents. Phosphoric acid, acetic acid, citric acid, boric acid, and tris-HCI buffer solutions may also be used, either independently or in combination with these salts.
2. Aqueous uric acid and guanidine hydrochloride
These solutions are widely used as protein denaturalization agents. As their concentration is generally high, it is recommended that they be used in columns set aside exclusively for this purpose, as their frequent interchange with eluents of lower concentration would tend to shorten the column service life.
3. Use of surfactants
Aqueous solutions containing SDS, Brij-35 or other surfactants can also be used as eluents. The eluent interchange time following the use of such an eluent will generally be longer than for ordinary eluents. Approx. 50% methanol may be used to shorten the replacement time.
4. Solutions with high salt content
After passage of a high-concentration salt solution, its removal from the flow lines by flushing with water is sometimes necessary to prevent salt precipitation at the pump and the injector. In such cases, column life is best preserved by first removing the column, before flushing the LC system flow lines.