Aqueous SEC (GFC) Columns
Column Specifications
Column Information
Eluent conditions for SB-800 HQ series and SB400 series columns
Some nonionic sample can be analyzed with the use of deionized water as an eluent.
As the eluent, either a saltaqueous solution or buffer solution with or without a polar organic solvent added to it. The followings are how to select the eluent to be used.
1) Ionic and nonionic hydrophilic samles.
Salt solutions or buffer solutions are generally used as the eluent. Typical aqueous salt solutions are as follows:
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(Aqueous salt solutions)
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(Buffer solutions)
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|---|---|---|
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Sodium chloride aqueous solution
Sodium nitrate aqueous solution Sodium sulfate aqueous solution Potasim sulfate aqueous solution Ammonium sulfate aqueous solution |
Phosphoric acid buffer solution
Trishydrochroric acid buffer solution Acetic acid buffer solution Citric acid buffer solution |
CAUTION
(1) Use of salt concentration in the range of 0.05M to 0.3M is recommended. Maxinium usable salt concentration is 0.5M.
(2) pH of eluent should be 3.0 to 10.0.
(3) pH of eluent should be higher than 6.0 when the eluent contains chloride ions.
(4) Boric acid buffer solution is not recommended because di-ol groups of packing material and boric acid make a complex.
2) Nonionic and ionic hydrophobic samples
Addition of polar organic solvents in the eluent is recommended to decrease the hydrophobic adosorption. Addition of salts in the eluent is also recomended when the sample is ionic.
Upper limit of concentration of polar organic solvents are as follows: Polymer-based GFC Columns: OHpak SB-800 HQ, High Perfonmance Semi-micro Polymer-based Aqueous SEC (GFC) Columns: SB400
CAUTION
Flow rate should be slower than 0.5mL/min when in-column solvent is changed to an eluent containing polar organic solvent.
In the case of DMSO, flow rate should be slower than 0.3mL/min and column temperature should be 50deg-C or higher.
3) Protein samples
Urea and 6M guanidine aqueous solutions, which are commnly used as protein modifier, can be used as the eluent. The eluent containing surfactants, such as SDS or Brij-35, is recoummended for the samples, such as membrane proteins, of which solubility in water is poor.
CAUTION
Frequent replacement of the in-column eluent from normal solvent to the above solvent will make the column life short. In such case, it is recommended to use one colunm specifically for the purpose.
Eluent conditions for KW400-4F series and KW-800 series columns
1) Typical eluent
The following solutions are generally used as the eluent.
Phosphate buffer solution
Tris hydrochloric solution
Acetate buffer solution
2) Salt to be added
Salts such as Na2SO4, K2SO4 and (NH4)2SO4 are usually added to the above mentioned buffer solutions. The suitable salt concentration is 0.1 to 0.3M.
CAUTlON
(1) The pH range of the eluent should be 3.0 to 7.5 range.
(2) Use of salts containing chlorine ions should preferably be avoided, as chlorine ions are corrosive to the column or piping of the instrument.
3) Use of urea or guanidlne hydrochloride
Urea or 6M guanidine hydrochloride solutions, which are often used as modifier of proteins, can be used as the eluent.
CAUTION
Since the replacement of the eluent takes a fairly long time, it is recommended to use one column specifically for such an eluent.
4) Use of surfactant
Aqueous solutions of surfactant such as SDS and Brij can also be used.
5) Use of polar organic solvent
Polar organic solvents, such as acetonitrile, methanol and ethanol can also be used as the eluent.
SEC is a method which uses the principle that a larger molecular size(three dimensional volume) sample elutes faster. In fact, the actual molecular size of the sample is different according to the solvent in which the sample is dissolved. In the case of polymers which have ionic functional groups, the ionic functional groups repel each other and the apparent molecular size of the polymers becomes larger. To prevent such ionic dissociation, the addition of salts such as NaNO3 or NaCl is known to be effective.
Especially, in the case of hydrophilic polymers, the existence of small amounts of ionic functional groups affect the peak pattern and measurement results. Addition of salts to the eluent is recommended even when it is considered that the sample polymer have no ionic functional groups.
(Left): Principle of SEC
(Right, above): Large apparent size because of repelling
(Right, below): Ordinary size