Agilent Technologies ZORBAX Series Manuale utente
for Agilent
Reversed-Phase Columns
COLUMN USER GUIDE
Agilent 反相色谱柱用户指南
Guide d’utilisation des colonnes pour les colonnes à
phase inverse Agilent
Säulenbenutzerhandbuch für Agilent
Umkehrphasensäulen
Manuale utenti per colonne a fase inversa Agilent
Guía de usuario de columna para columnas de fase
reversa Agilent
Agilent 逆相カラム ユーザーガイド
ZORBAX reversed-phase / 反相 / à phase reversa / Umkehrphase /
Fase inversa / Fase inversa / 逆相
HPLC phases / 相/ Phases HPLC / Phasen / Fasi / Fases /カラム
InfinityLab Poroshell 120 / Pursuit / Polaris / HC-C18(2) / TC-C18(2)
This booklet provides general information
for all InfinityLab Poroshell 120,
ZORBAX, Pursuit and Polaris columns.
For additional detailed information
about your specific phase or family, see:
www.agilent.com/chem/columnchoices
Getting Started
A QC Column Performance Report, including a test chromatogram, is
enclosed with every Agilent column. The QC test system has been modified
from a standard system to minimize system dead volume, so it may vary
from the system used in your lab. This allows a better evaluation of the
column and assures a more consistent product. A properly configured
LC system will generate similar results to the chromatogram on your QC
Performance Report.
Modern columns are robust and are designed to operate for long periods
under normal chromatographic conditions. You can maximize column
performance by running it within specifications. Always review the
specifications before putting in place a final method.
Using Your Column
Installation
• The direction of flow is marked on the column.
• 1.8 µm columns (ZORBAX RRHT, ZORBAX RRHD) can only be operated in the
direction flow marked on the column.
• Agilent recommends InfinityLab Quick Connect fittings (5067-5961 for 0.075 x
105 mm) or Quick Turn fittings (5067-5966) for a perfect column connection,
every time. Polyketone fittings (5042-8957) can also be used for columns up to
600 bar
Column conditioning
Every column is tested before shipment and shipped in the test eluent. So, for first
use, rinsing with water is not necessary. If mobile phase additives are used (such as
buffers or ion-pair reagents) it is advisable to do an intermediate flush with a mobile
phase of the correct composition, but without these additions. Flushing with 10 to 20
column volumes should help in transitioning to your mobile phase. For shorter chain
chemistries (C8, Phenyl, CN, for example), care should be taken to make sure the
column has been properly equilibrated prior to use. This will ensure reproducibility
and help prevent retention time drifting.
Important safety considerations
• All points of connection in liquid chromatographic systems are potential sources
of leaks. Users should be aware of the toxicity or flammability of their mobile
phases.
• Because of the small particle size, dry column packings are respirable. Columns
should only be opened in a well-ventilated area.
• Please adhere to operating pressure limits noted for each column (see chart).
Exceeding these limits will compromise chromatographic performance and could
be unsafe.
ENGLISH
Search Agilent.com for “quick connect fitting” to learn more
Polyketone fitting,
p/n 5042-8957
Agilent 1200 bar removable
fitting, p/n 5067-4733
Agilent Quick Connect fitting,
p/n 5067-5961
Other operating tips
• While generally not harmful to the column, reverse flow should be
avoided except to attempt removal of clogged frit (see “column care”).
• Always use high purity reagents and chromatography grade solvent
to prepare your mobile phase. Degas and filter all mobile phase prior
to use.
• Disassembling a column will degrade column performance.
• New columns contain a mixture of organic solvents and water. See
your QC Performance Report for the solvent composition in your
column. Initially, care should be taken not to pass any mobile phase
through the column that may cause a precipitate to form.
• Agilent reversed-phase columns are compatible with water and all
common organic solvents.
• The use of a guard column is recommended to protect your column
and increase its lifetime.
• Columns should not be maintained at elevated pH or elevated
temperature when not in use.
• Avoid use of this column outside of recommended pH ranges for
column phase (see next page). Expect reduced lifetime when operating
outside the recommended pH and temperature ranges.
If you are using InfinityLab Poroshell 120 or ZORBAX RRHT or RRHD
columns, use a Fast Guard for UHPLC to protect your analytical
column.
See www.agilent.com/chem/fastguards for more information.
Column Operating Parameters: pH and Temperature
Phase Recommended
pH range
Maximum operating
temperature
InfinityLab Poroshell 120 EC-C18,
EC-C8 and Bonus-RP.
ZORBAX Eclipse Plus C18 and C8.
ZORBAX Eclipse XDB-C18,
XDB-C8 and XDB-Phenyl ZORBAX
Bonus-RP.
HC-C18(2) and TC-C18(2).
pH 2.0 to 9.0 60 °C
InfinityLab Poroshell 120 PFP and
Phenyl-Hexyl.
ZORBAX Eclipse Plus Phenyl-Hexyl.
ZORBAX XDB-CN.
ZORBAX Eclipse PAH.
ZORBAX ODS and Phenyl.
All Pursuit, Polaris, Microsorb,
Metasil, Monochrom, Hypersil,
OmniSpher, MicroSpher bonded
phases.
pH 2.0 to 8.0 60 °C
ZORBAX Eclipse PAH pH 2.0 to 8.0 60 °C at pH <6.0
ZORBAX TMS pH 2.0 to 7.0 60 °C
InfinityLab Poroshell 120 SB-C18,
SB-C8 and SB-Aq.
ZORBAX StableBond SB-C18, SB-
C8, SB-Aq, SB-C3 and SB-Phenyl
ZORBAX Rx-C8.
pH 1.0 to 8.0 90 °C (StableBond C18
and Poroshell 120 SB-C18)
80 °C (Rx-C8 and SB-Aq,
SB-C3 and SB-Phenyl)
InfinityLab Poroshell HPH-C18 and
HPH-C8
pH 3.0 to 11.0 60 °C
ZORBAX Extend C18 pH 2.0 to 11.5 60 °C (40 °C at high pH)
Note: All silica-based packings have some solubility in pH >6 aqueous mobile
phases. When using silica-based columns at pH >6, best column lifetime is
obtained at lower temperatures (40 °C max) using low buffer concentrations in the
range of 0.01 to 0.02 M. Operating at extreme ends of pH and temperature ranges
will have a significant impact on column lifetime.
ENGLISH
Maximum Operating Pressures –
Columns up to 9.4 mm id
Column type Particle size Pressure limit
InfinityLab Poroshell 120,
InfinityLab Poroshell HPH
1.9 µm 1300 bar
(19500 psi)
InfinityLab Poroshell 120,
InfinityLab Poroshell HPH
2.7 µm, 4 µm 600 bar
(9000 psi)
ZORBAX Rapid Resolution High
Definition (RRHD)
1.8 µm 1300 bar
(19500 psi)
ZORBAX Rapid Resolution High
Throughput (RRHT)
1.8 µm 600 bar
(9000 psi)
All other ZORBAX, Rx, Pursuit,
Polaris, HC, TC, Microsorb,
Metasil, Monochrom, Hypersil,
OmniSpher, MicroSpher
3.0 µm, 3.5 µm,
5 µm, 10 µm
400 bar
(6000 psi)
Mobile Phase Selection and Operating Temperatures
The bonded stationary phase is nonpolar in nature and is best used with
polar mobile phases such as methanol/water or acetonitrile/water
mixtures. Increasing the amount of organic component typically reduces
the retention time of the sample.
Recommended Starting Gradients
Stationary phase Usage notes
Most reversed-phase
columns
5% methanol or acetonitrile initially and 100%
methanol or acetonitrile as the final solvent.
ZORBAX Eclipse PAH* 30 or 40% acetonitrile initially, to 100%
acetonitrile as final solvent. It may be necessary
to cool column to 15 to 20 °C for improved
resolution.
ZORBAX and InfinityLab
Poroshell Bonus-RP
Lower concentration of organic mobile phase
modifier is required for compound elution,
compared to traditional long-chain alkyl
stationary phases.
*Best column lifetime is achieved at < 40 °C.
ENGLISH
Column Care
The inlet frit on columns with a particle size of 2.7 µm or greater has a
nominal porosity of 2 µm. Samples that contain particulate matter will plug
the column inlet frit. Guard columns and hardware kits (as required) are
recommended for use with such samples, and are generally recommended
for all column use.
See www.agilent.com/chem/fastguards for more information about
guard columns.
Cleaning Your Column/Extending Column Life
For columns that can be backflushed (InfinityLab Poroshell 120, ZORBAX
columns with particles >1.8 µm and all Pursuit and Polaris columns), start
with a stronger (less polar) solvent.
1. Disconnect column from detector and run wash solvents into a beaker.
2. Start with your mobile phase without buffer salts (water/organic).
Run at 10 to 20 column volumes through.
3. Next, use 100% organic (methanol or acetonitrile).
4. Check pressure to see if it has returned to normal; if not, then,
5. Discard column or consider stronger conditions,
e.g., 75% acetontrile/25% isopropanol
6. Increase to 100% isopropanol, 100% methylene chloride or 100%
hexane (if you use methylene chloride or hexane, you will need to flush
the column with isopropanol prior to use and before returning to your
reversed-phase mobile phase).
For columns with particles < 1.8 µm, do not backflush the column - replace
the column.
Storage Recommendations
Long-term storage of silica-based, bonded phase columns should be in
a pure organic solvent. If the column has previously been used with a
buffered mobile phase, the buffer should first be removed by purging the
column with 20 to 30 column volumes of a 50:50 mixture of methanol or
acetonitrile and water, followed by 20 to 30 column volumes of the pure
solvent. Before storing, end-fittings should be tightly capped with end-
plugs to prevent packing from drying out. Columns may be safely stored for
short periods in most mobile phases. To protect equipment, is it desirable
to remove salts from the instrument and column by purging the column
with the same mobile phase without the buffer (e.g. using 60:40 ACN/
H20 to remove a 60:40 ACN/0.02 M phosphate buffered mobile phase).
Re-equilibration is rapid with the original mobile phase when using this
approach and any danger of corrosion from the salts is eliminated.
ENGLISH
Tips for Getting the Best Chromatographic Results
• Optimize your instrument by minimizing tubing lengths between
components, to reduce extra column volume and band broadening.
Use 0.12 mm id red or 0.075 mm id black tubing for Fast LC/high
efficiency columns. Learn about capillary options at
www.agilent.com/chem/lccapillaries
• Ensure the data collection rate is optimized for your column. Use a
higher collection rate for Fast LC columns (Poroshell 120, RRHT, RRHD).
• Use sample filtration or other sample prep as appropriate for your
sample. Learn more at www.agilent.com/chem/sampleprep
• Use Agilent certified lamps in your LC instruments for best
performance.
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