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The analytical column is the heart of your HPLC
system. Taking proper care of your column ensures
that you get reproducible results for a maximum
number of sample injections. By following the rec-
ommendations listed below, you can extend the life-
time of your column and improve the accuracy and
reproducibility of your results.
Sample Preparation
The cleaner your samples, the longer your column
will last. Obviously, there will be times when you
must compromise column lifetime in order to
reduce sample preparation efforts. At the very least,
though, you should filter samples through a 0.45µm
syringe tip filter to ensure they are free of particles.
Restek offers a range of syringe filters, from
0.20µm to 1.00µm, in either nylon or PTFE. Also,
make sure that all samples are completely soluble
in the mobile phase. If you are running a mobile
phase gradient, sample solubility should be verified
at the low and high extremes of organic content.
Mobile Phases
Filter mobile phases (especially buffers) through a
0.45µm membrane. A 0.2µm filter removes bacteria
as well as other particles. If you are running a gradi-
ent with a buffer, make sure the buffer salt remains
in solution even at the highest percent organic.
Column Storage
Store columns in an appropriate solvent that con-
tains at least 10% organic solvent and is free of
salts or buffers. Seal a stored column with end
plugs and label the column with the storage solvent,
to avoid inadvertently combining incompatible sol-
vents in the column when using it later. If a column
is dedicated to a particular analysis, the analyte or
method name should be recorded on the column.
Column Protection
Use a guard filter and guard column. Change filters
and guard columns regularly or at the first signs of
impaired performance (e.g., increased pressure,
loss of efficiency or peak symmetry). The Trident™
guard system allows you to choose a configuration
that best suits the needs of your particular applica-
tion: a Trident ™ in-line guard cartridge, the
Trident ™ Direct system, or the Trident™ Integral
guard system. (For illustrations of Trident ™ systems,
see page 5.) Each system can be built from a par-
ticulate filter frit, a filter frit with a 1cm guard col-
umn, or a filter frit with a 2cm guard column.
Alternatively, a Trident ™ in-line or direct system can
be configured as a guard cartridge holder without
the filter frit.
Column Cleanup
Ben Franklin said, “An ounce of prevention is
worth a pound of cure.” The same rings true for
column maintenance: clean the column periodical-
ly. If you wait for column performance to signifi-
cantly deteriorate before cleaning, you may have to
repeat your analyses. The most effective cleanup
technique is to backflush the column to remove the
strongly adsorbed impurities that tend to accumu-
late at the head of the column. Do not flush into the
detector because particles from the inlet frit could
damage the detector. To clean the column, flush it
with a strong solvent (e.g., high organic for
reversed phase columns). If you are using a buffer,
make sure it is completely flushed from the column
and the system before switching to a mobile phase
with a high percentage of organic solvent. This will
prevent salts from precipitating. If you are doing
repetitive isocratic analyses, periodically (i.e., every
ten samples or so) use a gradient from weaker to
stronger solvent to prevent the accumulation of
strongly retained impurities. When cleaning
columns, flush with a minimum of ten column vol-
umes.
Avoid Extremes
Follow the column manufacturer’s recommenda-
tions for usable pH and temperature ranges. Most
silica-based columns will have a recommended pH
range of around 2.5-7.5. Lifetimes for these
columns will be maximized if the pH can be main-
tained between 3 and 7. Similarly, although most
silica-based columns can be operated at tempera-
tures up to 80°C, lifetime generally will be greatest
if the column temperature does not exceed 40°C.
Column manufacturers usually do not specify pres-
sure limits, but higher pressures decrease column
lifetime, especially as pressure exceeds 2000psi
(~140 bar). Pressures above 3000psi (~200 bar)
should be avoided if at all possible.
Conclusion
By following these recommendations, you will pro-
long the life of your analytical column and reduce
the chances for unpleasant surprises during your
routine analyses. If you have any questions, the
Restek Technical Service Team will be happy to
help you—call 814-353-1300 or 800-356-1688,
ext. 4, or contact your local Restek representative.
by Greg France, HPLC Product Marketing Manager, and Terry Reid, HPLC Applications Chemist
Tips for Maximizing HPLC
Column Lifetime
✔ Extend the lifetime of your analytical HPLC column.
✔ Achieve more reproducible analyses.
✔ Protect your chromatographic system.
Filter Diameter Porosity qty. Nylon PTFE
13mm 0.20µm 100-pk. 26066 26068
13mm 0.45µm 100-pk. 26067 26069
25mm 0.20µm 50-pk. 26070 26072
25mm 0.45µm 50-pk. 26071 26073
25mm 1.00µm 50-pk. — — 26074
Resprep™ SPE Syringe Filters
Trident™ Direct Guard Column System
Description qty. cat.# price
High-pressure filter ea. 25082
1cm guard cartridge holder with filter ea. 25084
2cm guard cartridge holder with filter ea. 25086
Connection tip for Waters®-style end fittings ea. 25088
PEEK® tip standard fittings ea. 25087
Replacement Cap Frits: 4mm, 2.0µm 5-pk. 25022
Replacement Cap Frits: 4mm, 0.5µm 5-pk. 25023
Replacement Cap Frits: 2mm, 2.0µm 5-pk. 25057
Inert PEEK® Tubing
Description qty. cat.# price
PEEK® Tubing, 1 /16" OD x 0.0025" ID Natural 3m 25320
PEEK® Tubing, 1 /16" OD x 0.005" ID Red Stripe 3m 25065
PEEK® Tubing, 1 /16" OD x 0.007" ID Yellow Stripe 3m 25066
PEEK® Tubing, 1 /16" OD x 0.010" ID Blue Stripe 3m 25067
PEEK® Tubing, 1 /16" OD x 0.020" ID Orange Stripe 3m 25068
for moreinfo For additional information about Trident™ guard columns,
request the Trident™ Fast Facts (lit. cat.# 59314 and 59896).