1. General information
1)The following factors affect the separation by RSpak columns:
(a) Composition of the eluent
(b) pH of the eluent
(c) Concentration of salt
(d) Column temperature
2) Chromatographic data, obtained by use of column packed with silica-base gels (silica-base column), are helpful in determining the eluent conditions for the RSpak D-series columns. Generally speaking, it is necessary that the eluent condition of RSpak D-series columns should be a slightly faster condition in elution than that of the silica-base column.
3) In case the eluent is a mixture of water and a polar organic solvent, the sample can be best separated in most cases only by adjusting their mixing ratio. If the separation is still unsatisfactory, the pH of the eluent should be adjusted or ionization of the sample suppressed by adding acid or base to the eluent. In case the sample is acidic, its ionization must be suppressed by adding phosphoric acid or the like, and ammonium hydroxide or a similar substance in case it is basic.
4) It should be noted that in reversed-phase chromatography, if the sample has a long alkyl chain, the longer the chain, the slower the elution. Aromatic compounds are also slow in eluting, being firmly adsorbed to the packing material.
5) The eluent must not contain salt in a quantity larger than 0.5M.
The column is used principally in reversed-phase chromatography, in which the eluent is a mixed solution of water and a polar organic solvent or a mixed solution of the same components plus salt, acid or base.
The following solvents are used as eluent:
Water: In mixture with another solvent except chloroform
THF: In mixture with another solvent except chloroform
Chloroform: In mixture with another solvent except water and THF
Methanol: Singly or in mixture with another solvent
Ethanol: Singly or in mixture with another solvent
Acetonitrile: Singly or in mixture with another solvent
(1) Under no circumstances must water exceed 90% in the eluent, and THF, chloroform and toluene 50%, since they expand the packed gel.
(2) The eluent must be in pH range of 2.0 to 12.0. If it contains any chloride ions, its pH must be 4.0 or more.
The eluent conditions of C18-silica gel columns are helpful for the determination of the eluent condition.
Water, methanol, ethanol, acetonitrile or their mixture is used as the eluent.
(1) Nonpolar organic solvents such as benzene, chloroform, n-hexane and THF must not be used as the eluent.
(2) The eluent must be in a pH range of 2.0 to 12.0. If it contains any chloride ions, its pH must be 4.0 or more.
The eluent condition of C8 and C4-silica gel columns are helpful for the determination of the eluent conditions.
A mixture of water and a polar organic solvent such as acetonitrite, methanol and ethanol, or such mixture with salt added to it is most commonly used as the eluent.
An aqueous solution alone may also be used.
(1) Nonpolar organic solvents such as n-hexane, benzene and chloroform cannot be used as the eluent.
(2) The pH value of the eluent must be in the range of 2.0 to 10.0. In case the eluent contains chloride ions, the pH value must not be below 4.0.
(3) Do not use any strong acids, strong bases or perchlorates as the eluent.
The eluent consists of water and a polar organic solvent such as acetonitrile and ethanol can be used. Sodium salt is the only substance that can be added to the eluent.
A mixed solvent of 50/50 to 90/10 acetonitrile/water or ethanol/water is used as the eluent for the separation of saccharides. The higher the water content, the faster the elution of saccharides.
In the event metal ions are present in the eluent, they will in most cases exchange with the sodium ions of packing, thereby deteriorate the performance of the packing material. To prevent such ion exchange, EDTA-4Na is added in the amount of 50 to 100 ppm to the eluent to mask the metal ions.
(1)Do not use more than 95% of polar organic solvent in preparation of the eluent; otherwise the packing will shrink and cause the column performance to deteriorate.
(2) Use deionized and distilled water in preparation of the eluent.
(3) Do not inject any basic sample.
(4) Do not inject any sample containing heavy metal ions; otherwise, Na+ ions of the ion exchange resin will be exchanged with the heavy metal ions, thereby sometimes causing the column’s separation performance to change.