ver.220208E
SHOWA DENKO K.K. (https://www.shodex.com/)
3
· Shear degradation is more likely to occur in larger molecular weight compounds. When
shear degradation occurs, the measured molecular weight may be lower than the actual
value or it may influences the repeatability of the analysis. If shear degradation is
suspected, use a lower flow rate.
5. Eluent Preparation
(1) Degas the eluent fully to prevent the formation of air bubbles.
(2) Presence of small debris or insoluble substances may result in deterioration of the column and/or they appear
as noise on the chromatograms. Filter the eluent with a 0.45-
μ
m disposable filter to prevent the problems.
Attention!
· Whenever water is required, use ultra-pure water freshly generated by a water purification
system or water from a newly opened HPLC grade distilled water bottle. Use HPLC grade
organic solvent whenever possible. Solvents left in an opened bottle for a long time should
not be used. The content may have been changed, absorbed moisture, or has been
contaminated.
· Always use freshly prepared solvents. Solvents stored for a long time may have changed
their compositions and may influence elution patterns and/or damage the column.
Note
· Use of on-line degasser is recommended.
6. Sample Preparation
(1) If possible, use the eluent for analysis to dissolve or dilute samples. If this is difficult, use a solvent which
has a composition that is as close as possible to the eluent's composition, but which fully dissolves or dilutes
the sample.
(2) Filter the sample solution using disposable 0.45-
μ
m filter to prevent the column from clogging or deteriorating.
(3) Recommended injection volume is less than 1 mL per column.
(4) Viscosity of high molecular weight compound is largely influenced by its molecular weight and concentration.
Samples with high viscosity cause peak broadening and elution delay, and this makes it difficult to obtain
their accurate molecular weight distributions. In general, the larger the molecular weight of the compound,
the higher its viscosity becomes. To suppress the influence from high viscosity, it is recommended to lower
the sample concentration. Please use the below table as a reference when preparing samples for molecular
weight distribution analyses.
Molecular Weight Range
Optimal Concentration (w/v)
≤
5,000
≤
1.0 %
5,000 - 25,000
≤
0.5 %
25,000 - 200,000
≤
0.25 %
200,000 - 2,000,000
≤
0.1 %
Note
· Use of guard column is recommended to protect the analytical column.
7. Column Usage Procedure
7.1 HPLC System Preparation
Wash entire LC system prior to the column installation, including all flow-lines and sample loop by switching the
valve, and then replace the washing solution with the eluent to be used. If desired new eluent has low
miscibility/solubility to the eluent of previous analysis, first use the eluent that is miscible/soluble to both eluents,
and then replace it with the desired eluent.
e.g. When replacing chloroform to water, first run methanol and then replace it with water.
e.g. When replacing highly concentrated buffer solution or salt solution to water/acetonitrile, first run water and
then replace it with water/acetonitrile.
Attention!
· If the eluent left in the system is not compatible with the column to be used, it may damage
the column.
· A drastic change in the eluent compositions may remove substances adsorbed on the
system and they may enter and deteriorate the column.