• 10 •
The analytical column is the heart of your HPLC
system. Taking proper care of your column ensures
that you get reproducible results for amaximum
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 themobile phase. If you are running amobile
phase gradient, sample solubility should be verified
at the low and high extremes of organic content.
Mobile Phases
Filtermobile phases (especially buffers) through a
0.45µmmembrane. 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 columnwith end
plugs and label the columnwith the storage solvent,
to avoid inadvertently combining incompatible sol-
vents in the columnwhen 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
columnmaintenance: clean the column periodical-
ly. If youwait for column performance to signifi-
cantly deteriorate before cleaning, youmay have to
repeat your analyses. Themost 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 amobile 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 fromweaker to
stronger solvent to prevent the accumulation of
strongly retained impurities. When cleaning
columns, flushwith aminimum of ten column vol-
umes.
Avoid Extremes
Follow the columnmanufacturer’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 bemaximized if the pH can bemain-
tained between 3 and 7. Similarly, althoughmost
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.
Columnmanufacturers 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, youwill 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 Teamwill be happy to
help you—call 814-353-1300 or 800-356-1688,
ext. 4, or contact your local Restek representative.
by Greg France, HPLCProduct MarketingManager, and Terry Reid, HPLCApplications Chemist
Tips forMaximizingHPLC
Column Lifetime
✔
Extend the lifetime of your analytical HPLC column.
✔
Achievemore 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 GuardColumn 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 forWaters
®
-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" IDNatural
3m
25320
PEEK
®
Tubing,
1
/
16
" OD x 0.005" IDRed Stripe
3m
25065
PEEK
®
Tubing,
1
/
16
" OD x 0.007" IDYellowStripe
3m
25066
PEEK
®
Tubing,
1
/
16
" OD x 0.010" IDBlue Stripe
3m
25067
PEEK
®
Tubing,
1
/
16
" OD x 0.020" IDOrange Stripe
3m
25068
for
more
info
For additional information about Trident
™
guard columns,
request the Trident
™
Fast Facts (lit. cat.# 59314 and 59896).
