CONTENTS:
- Column
Cleaning Protocol (Baseline drift and spike
peaks)
- No
Retention of Biomolecules in HYDROCELL
Columns
- High Back
Pressure
1. HYDROCELL
Column Cleaning Protocol
If the peaks have broadened and the baseline has
drifted when you use a HYDROCELL Ion Exchange or
Hydrophobic Interaction Column, the column needs to be
reactivated and regenerated. The procedure for
reactivating and regenerating a 4.6 mm HYDROCELL column
is as follows:
1. Flush with 80% methanol in water at a flow rate
of 3 mL / minute for 10
minutes.
2. Flush with water, and then 20% methanol in 1 M
NaCl solution at a flow
rate of 3 mL / minute for 10 minutes.
3. Flush with B Buffer at a flow rate of 3 mL /
minute for 10 minutes.
4. Equilibrate with the starting buffer at a flow
rate of 2 mL / minute.
5. Run a 10 minute blank gradient from 0 - 100 % B
buffer at a flow rate of
2 mL / minute.
Repeat this process, if necessary, until the
baseline has stabilized. Adjust the flow rates and
volumes for other I.D. columns proportionately.
Alternative Procedure: Elute column with 1.0 M
NaOH aqueous solution continuously, at a flow rate of
1 mL / minute for 1 hour or leave 1.0 M NaOH inside
the column overnight.
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2. No
Retention of Biomolecules in HYDROCELL Ion Exchange or
Hydrophobic Interaction Columns
HYDROCELL columns with the proprietary hydrophilic
oligomers coating are very hydrophilic and do not have
non-specific adsorption and binding. These columns do not
need detergents, surfactants or organic solvents in the
buffers to circumvent the non-specific binding that might
occur in the silica-based or other polymeric packings in
the separation of biomolecules. If you have problems of
retention of your samples in HYDROCELL columns, please
check the preparation of your samples, buffers, and the
equilibrium time with the first buffer before injection
of your samples.
(A) Sample Preparation:
1. Solid samples: The samples are prepared by
dissolving about 3 mg of samples in 1 ml of the first
buffer. It is necessary, likewise, in hydrophobic
interactive chromatography, that a small volume of
secondary buffer can be added to increase the
solubility of the samples.
2. Liquid samples: The liquid samples should have
detergents, surfactants, and solvents removed that
will affect the retention of the samples in the
separation, and adjust their pH value and salt
concentration as close as possible to the first
buffer. The concentration of samples should be high
enough to be detected by the detector.
3. Preparative samples: In preparative
separations, the size of the sample loop should be
increased to increase the loading capacity. The
technique using the dilute solution and repeatedly
loading the samples should be applied instead of
using a high concentration of samples.
(B) Buffer Preparation:
Salt concentrations and pH value are two factors
that are greatly affected in the separation of
biomolecules. A slight change of pH value of the
buffer might affect the retention of biomolecules in
the HYDROCELL column. The pH value of buffers needs
to be adjusted to fit the specific separations in
method development.
(C) Equilibrium Time
HYDROCELL columns are very hydrophilic. If the
equilibrium time of the first buffer is not long
enough, the samples may not have retention or the
retention time are too short in the separation of biomolecules. The equilibrium volume must be at least
10 times column volume.
For example, a HYDROCELL column, 150 x 4.6 mm, has
a column volume ( x 15) = 2.45
ml. The equilibrium volume of the column needs at
least 24.5 ml. If the column is equilibrated at 1 ml
/ minute, it takes 24.5 minutes to equilibrate the
column.
However, HYDROCELL columns are produced from rigid
polymeric supports and have their mechanical
strength. These columns can be equilibrated at high
flow rates. The maximum flow rate for a 150 x 4.6 mm
analytical column is 6 ml / minute. If the column is
equilibrated at 3 ml / minute, it will take 8.16
minutes to equilibrate the column.
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3. High
Back Pressure
When HYDROCELL columns show back pressure higher than
1000 psi in the normal operation of flow rate, the
columns are clogged. The columns need to be cleaned up
using the procedure of the cleaning protocol or eluting
with 1.0 M NaOH aqueous solution. After column cleaning,
if the problems still persist, then the inlet frit of the
column might be clogged and probably needs to be
replaced. We always recommend the use of guard
columns or precolumn filters to
protect both analytical and preparative columns.
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· Please call 812-234-2558 for
additional technical support ·
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