The Wrench
Vol 2 Issue 3
November, 2005
System Backpressure
We’ve all seen it. Turn
on the pump and the pressure in the system starts to rise.
300psi…800psi…1500psi…3000psi…3800psi…system shut
down.
It seems every chemist eventually encounters this situation, where
the system shuts down because the max pressure has been exceeded or a connection begins to
leak. This article addresses how to diagnose
and solve a system pressure problem.
If a method or system has been operating normally prior to when a
pressure increase occurs, it is generally due to a clog or restriction in the flow path. The first step to solving the problem is to find
out where the restriction is located. If a new
method is being developed you may need to go back to the drawing board and look at what
types of solvents you are using, what kind of samples are being injected, mobile phase
composition, etc. In this article we will
focus on systems with problems that occur while running established methods that have not
been problematic.
Remove the column from the system and join the column inlet and
outlet lines with a union. Turn the pump back
on. If the pressure rises in the system
without a column in place the clog is in the system. If
the pressure is low and stable then your column is the culprit.
Let’s take a look at a system clog first. To locate the problem you want to work from the
back off the flow path towards the front. With
the pressure still high, begin loosening each connection from the waste line up to the
pump. While loosening each connection check to
see at what point the pressure in the system drops. The
section of the instrument that is just past where the pressure drops is where the
restriction lies. If the clog is in a length
of tubing simply remove that section and replace it with one of similar length and i.d. Another area that can see clogs is in the 6 port
valve of the autosampler. This may require
replacing the rotor seal. While the valve is
apart it is a good idea to run cleaning wire through each of the holes to remove any pump
seal or other material that may have lodged there. Finally,
pre-column or autosampler frits are another common source of high backpressure. If these areas are where the problem is located,
simply replace the frit. One last side thought
about frits. Although it is recommended to
work from the back of the system towards the front, frits are major culprits for clogs. They are just past the pump seals and so can become
fouled with seal material. They are also right
in line to create a perfect place for salting out of buffers used in conjunction with
organic solvents. Finally, they are relatively
simple to check and replace, so it may be worth taking a quick look at them right at the
start.
If the column is clogged the easiest solution is to simply replace it
with a new column of the same type and dimensions from the same vendor. Alternatively, you can try and salvage the column
by flushing it. To get started, attach the
outlet end of the column to the inlet end of the HPLC system. The outlet end should not be connected to the
system, but simply drained to waste (you do not want to hook the column into the system,
because anything you manage to flush off will immediately now be going to the detector). Once the system is set up you can begin flushing
the column with a series of solvents of different strengths/polarities. The recommended solvents and order of flushing are
dependent on whether you had been running normal phase, or reverse phase, the use of
buffers, the type of material the column is made of, and type of sample injected onto the
column. For any procedure you want to flush
with at least 10 column volumes of each solvent. The
column volume can be calculated by the equation Vol= p*r2*L. Typically, the progression is through a series of
solvents of increasing strength with care being taken to ensure that each subsequent
solvent is miscible with the previous solvent. The
flow rate during this process can be set at 20-80% of the normal column flow rate. Outlined below is a select series of washes for the
regeneration of a bonded silica, reverse phase column.
HPLC Grade Water
Methanol
Acetonitrile
Isopropanol/Acetonitrile
Isopropanol
Methylene Chloride
Hexane
Isopropanol
Mobile Phase
In this series the water is used to remove any remaining buffer from
the system or column before switching to a high organic solvent environment. Isopropanol is used as a transition solvent. The Methanol and Acetonitrile are organic solvents
to remove polar compounds, while the Methylene Chloride and Hexane work to dissolve
non-polar contamination. For other types of columns, modes of separation, or types of
samples, column manufacturer web sites can be an excellent resource for troubleshooting
and column care.
Once the column has been returned to its mobile phase, flip the
column back around and reconnect the inlet end of the column to the tubing from the
autosampler and hook the outlet end back into the detector. A
test run should be performed to ensure that pressure, as well as retention times and
theoretical plates are back within an acceptable range.
For help with backpressure or other HPLC system problems please give us a
call at 800-682-6480.
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