5
–
Troubleshooting
Thermo Scientific
Product Manual for IonPac AS31 Columns
Page 35 of 45
155007-01
For Research Use Only. Not for use in diagnostic procedures.
5.4
Poor Peak Resolution
When carrying out separations using gradient analysis the column must be sufficiently
equilibrated with the eluent concentration used at the start of the analysis. The actual equilibration
time depends on the ratio of the strongest eluent concentration to the weakest eluent concentration.
Typically, equilibration takes place in 3-5 column volumes of eluent. Depending on flow rate and
concentration change, equilibration times range from 3 to 10 minutes.
A.
If increased separation is needed for the first group of peaks, reduce the concentration of the
starting eluent (E1). This part of the chromatogram is run isocratically with E1.
B.
Due to different system configurations, the observed gradient profile may not match the
gradient shown in the example. The gradient conditions can be adjusted to improve resolution
or to adjust retention times either by changing the gradient timing or by changing the gradient
eluent proportions.
C.
Keep the concentrations of E1 and E2 (final eluent concentration) constant and adjust the
gradient time. This is the simplest way to compensate for total system differences if resolution
is the problem.
D.
Change the proportions of E1 and E2 and adjust the gradient time. This approach requires
more time to develop and more knowledge in methods development work. The advantage is
that it allows a method to be tailored for a particular application where selectivity, resolution,
and total run time are optimized. Be aware that poor peak resolution can be due to any or all
of the following factors.
5.4.1
Loss of Column Efficiency
When chromatographic efficiency is lost, peak resolution may decrease to an unacceptable level
as the peaks broaden.
A.
Check to see if headspace has developed in the guard or analytical column. This is usually
due to improper use of the column such as exposing it to high pressures. Remove the
column’s inlet end fitting according to
Section 5.1.2
, “Replacing Column Bed Support
Assemblies”. If the resin does not fill the column body all the way to the top, it means that
the resin bed has collapsed, creating a headspace. The column must be replaced.
B.
Extra-column effects can result in sample band dispersion, making the elution of the peaks
broader resulting in reduced efficiency. Make sure connections are made correctly with
PEEK tubing with an ID of no greater than 0.010" for 4 mm systems or no greater than 0.005"
for 2 mm systems. Cut the tubing lengths as short as possible, checking to ensure a smooth,
90
o
cut.
C.
If tubing is not connected properly at the column inlet and outlet, it can cause dispersion
resulting in low efficiency numbers.
a.
When installing the columns, it is recommended to turn off the pump while
making connections. This will avoid any slippage of the ferrule under high
pressure conditions which can result in a void in the fitting. Dispersion results
in chromatograms with tailing peaks as shown in Figure 11 below.
b.
Before connecting the fittings, it is recommended to inject water into the
cavities of the fluidic system using a syringe or a micropipette while the flow
