Agilent Technologies InfinityLab Poroshell 120 User manual

COLUMN USER GUIDE
for Agilent Normal-Phase
and HILIC Columns
Säulenbenutzerhandbuch
für Agilent Normalphasenund HILIC-Säulen
Guide d’utilisation des colonnes
les colonnes phase normale et les colonnes HILIC Agilent
Guía del usuario de columnas
para columnas de fase normal y de HILIC de Agilent
Manuale D’Uso per colonne
per colonne Agilent a fase normale e HILIC
カラム ユーザーガイド
AGILENT 順相 および HILIC カラム
色谱柱用户指南
适用于 Agilent 正相 和HILIC 色谱柱
Руководство пользователя
для колонок Agilent нормальной фазы и HILIC
Guia do usuário de colunas
para colunas Agilent de fase normal e colunas HILIC

This booklet provides general information for all InfinityLab
Poroshell 120, ZORBAX, Pursuit, and Polaris normal-phase
and HILIC columns. Initial sections include information for both
normal-phase and HILIC columns. Because some method
parameters and column care instructions are different for HILIC
versus normal phase, those sections are broken out separately
in the last few pages. For additional information about your
specific column or family,
see www.agilent.com/chem/columnchoices
Getting Started
A QC Column Performance Report, including a test chromatogram,
comes with every Agilent column. The QC test instrument is modified
(optimized) from a standard instrument to minimize extra column
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.
An LC system, optimized for low volume columns, will generate similar
results to the chromatogram on your QC Performance Report.
This booklet provides general information for all InfinityLab

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Installation
• The direction of flow is marked on the column.
• 1.8 µm columns (ZORBAX RRHT, ZORBAX RRHD) can only be
operated in the flow direction marked on the column.
• InfinityLab Poroshell 120 columns may be operated in the opposite flow
direction when cleaning the column. The frit porosity on the outlet of InfinityLab
Poroshell 120 columns is smaller than on the inlet and can plug more easily.
• 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.
Important Safety Considerations for all LC Columns
• All connection points in LC 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.
• Adhere to operating pressure limits noted for each column (see chart on next
page). Exceeding these limits will compromise chromatographic performance
and could be unsafe.
Polyketone fitting,
p/n 5042-8957
Agilent 1200 bar
removable fitting,
p/n 5067-4733
Agilent Quick Connect
fitting, p/n 5067-5961

Column Usage Tips
• Modern columns are robust and are designed to operate for long periods under
normal chromatographic conditions. You can maximize column lifetime by running
it within specifications. Always review the specifications before putting in place a
final method.
• While generally not harmful to the column, reverse flow should be avoided except
to attempt to clear a clogged frit (see “Installation” for exceptions).
• Always use high purity reagents and chromatography grade solvents or better to
prepare your mobile phase.
• Disassembling a column will degrade column performance.
• New columns contain a mixture of organic solvents. 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 or
an emulsion to form.
• Agilent normal-phase columns are compatible with water and all common
organic solvents.
Column Type Particle Size Pressure Limit
InfinityLab Poroshell 120 1.9 µm 1300 bar (19500 psi)
InfinityLab Poroshell 120 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 Definition (RRHT)
1.8 µm 600 bar (9000 psi)
All other ZORBAX, Pursuit,
Polaris
3.0 µm, 3.5 µm, 5 µm,
10 µm
400 bar (6000 psi)
Rx, Metasil, Monochrom,
Microsorb
3.0 μm, 3.5 μm, 5 μm,
10 µm
400 bar (6000 psi)
Maximum Operating Pressures – Columns up to 9.4 mm id

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• T he use of a guard column is recommended to protect your column and increase
its lifetime.
• Columns should not be maintained at high pH or elevated temperature when not
in use.
• Avoid use of this column outside recommended pH range for column phase (see
next page). Expect reduced lifetime when operating outside the recommended
pH and temperature ranges.
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.
• When using normal-phase columns, ensure that your LC instrument’s rotor seals,
pump seals, tubing, and all other components are compatible with normal-phase
solvents.
• Make sure your data collection rate is optimized for your column. Use a higher
collection rate for Fast LC columns ( InfinityLab Poroshell 120, RRHT and RRHD).
• Use Agilent certified lamps in your LC instruments for best performance.
• Use sample filtration or other sample prep as appropriate for your sample. Learn
more at www.agilent.com/chem/sampleprep
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.

WORKING WITH NORMAL-PHASE COLUMNS
Every normal-phase column is tested before shipment and shipped in a normal-
phase test eluent, with the exception of ZORBAX Eclipse XDB-CN and, InfinityLab
Poroshell 120 EC-CN which are shipped in reversed-phase solvents and
InfinityLab Poroshell 120 HILIC and ZORBAX HILIC Plus which are shipped in
HILIC solvents. ZORBAX carbohydrate columns are shipped in acetonitrile. Care
should be taken to make sure the column has been properly equilibrated prior
to use. This will ensure reproducibility from analysis to analysis and help prevent
retention time drifting.
Phase Recommended
pH Range
Maximum
Operating
Temperature
Suggested
Operating
Temperature
InfinityLab Poroshell
120 EC-CN
pH 2.0 to 8.0 60 °C 30 °C
ZORBAX Eclipse
XDB-CN
pH 1.0 to 9.0 60 °C 30 °C
ZORBAX CN pH 2.0 to 8.0 60 °C 30 °C
ZORBAX
Carbohydrate
pH 2.0 to 8.0 60 °C 30 °C
ZORBAX NH2pH 2.0 to 7.0 60 °C 30 °C
ZORBAX Rx-SIL,
ZORBAX SIL
pH 1.0 to 8.0 Limited only by the
temperature limits of
the mobile phase
Pursuit, Po-
laris, Metasil, Mono-
chrom, Microsorb
pH 2.0 to 8.0 60 °C 30 °C
InfinityLab Poroshell
120 HILIC, ZORBAX
HILIC Plus
pH 1.0 to 8.0 Limited only by the
temperature limits of
the mobile phase
Normal-Phase Column Operating Parameters:
pH and Temperature
Operating at extreme ends of pH and temperature ranges will have a significant impact
on column lifetime.

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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.
Normal-Phase Column Method Development Notes
The stationary phase is polar in nature and is best used with nonpolar mobile
phases such as methylene chloride/hexane or isopropanol/hexane mixtures.
Increasing the amount of the polar component in these mixtures typically reduces
the retention time of the sample.
Column Type Method Development Notes
ZORBAX NH2
Polaris NH2
Many column volumes of mobile phase are necessary
for equilibration to occur after changes in mobile phase
composition. The number of column volumes required
depends on the system in use and can be as high as
50 to 100 column volumes.
ZORBAX Rx-SIL
ZORBAX SIL
Pursuit XRs Si
Polaris Si-A
Microsorb Si
Unbonded silica columns are very sensitive to trace
amounts of water in solvents and sample, so care
must be taken to maintain consistency for method
reproducibility. When gradient elution is employed, both
primary and secondary solvents should be modified
with alcohol or acetonitrile and at least 30 columns of
solvent should be allowed to flow through the column
after completion of each run.
When switching between solvents, first rinse the
column with a mutually miscible solvent such as
isopropyl alcohol.
InfinityLab Poroshell 120
EC-CN
ZORBAX Eclipse XDB-CN
ZORBAX CN
InifintyLab Poroshell 120 EC-CN and ZORBAX Eclipse XDB-
CN columns are shipped with reversed-phase solvents,
but can also be used in normal-phase applications after
flushing with isopropanol.
Retention decreases as the polarity of the mobile phase
increases.
The rapid equilibration of this bonded phase with mobile
phase often makes it more convenient to use this column
than a silica column, especially for gradient elution.

Cleaning Your Column/Extending Column Life:
Normal-phase Columns
For normal phase, we recommend use of organic solvents. Use at least 20 column
volumes of each solvent, which is 50 mL if your column is the traditional analytical
4.6 x 250 mm. Try these solvents in order of increasing strength:
1. 50% methanol: 50% chloroform
2. 100% ethyl acetate
Cleaning a column used in normal-phase mode may depend on the sample type. If
these solvents do not work, contact Agilent so we can recommend a solvent that
may be more effective with your application and sample matrix.
Storage Recommendations: Normal-Phase Columns
Normal-phase columns can typically be stored in their own mobile phase for long
periods. Before storing the column, the end-fittings should be tightly capped with
end-plugs to prevent the packing from drying out.
WORKING WITH HILIC COLUMNS
HILIC is a method used for the separation of polar analytes inadequately retained
on typical reversed-phase columns. Agilent ZORBAX HILIC Plus and InfinityLab
Poroshell 120 HILIC columns are non-bonded silica columns, shipped in
acetonitrile:water or acetonitrile and ready to use for HILIC separations. ZORBAX
Carbohydrate columns are shipped in acetonitrile also ready for use in HILIC
mode. Like normal-phase columns, HILIC columns require more equilibration than
reversed-phase columns. Note that NH2, unbonded silica and CN columns can also
be used for HILIC applications. To change these columns to HILIC mode, flush first
with isopropyl alcohol.

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ZORBAX HILIC Plus
InfinityLab Poroshell 120
HILIC
Most common HILIC applications are for the analysis of
polar compounds that are unretained or poorly retained
on reversed-phase columns.
Best to equilibrate the column with 30-40% water in
acetonitrile before use.
Equilibration may require 20-50 column volumes.
A typical mobile phase is acetonitrile:water with an
acetate or formate buffer at 5-10 mM. Increasing buffer
concentration can improve peak shape and retention.
Decrease the aqueous/buffer component to increase
retention.
HILIC Method Development Notes
Operating Guidelines: HILIC
•Agilent HILIC columns are shipped containing acetonitrile:water or acetonitrile.
• Agilent HILIC columns are compatible with water and all common organic
solvents.
• All silica has some solubility in pH >6 aqueous mobile phases. Solubility of the
silica is also increased at elevated temperatures. Using the columns above pH 6
and 40 °C will reduce the column lifetime.
Column Name pH Range Maximum
Temperature
Suggested
Temperature
ZORBAX HILIC Plus
InfinityLab Poroshell 120
HILIC
pH 1.0 to 8.0 40 °C 30 °C
ZORBAX Carbohydrate pH 2.0 to 8.0 40 °C 30 °C
HILIC Column Operating Parameters
Operating at extreme ends of pH and temperature ranges will have a significant impact
on column lifetime.
As acetonitrile content of the mobile phase increases, the resolution of the sugars
may improve, with an accompanying increase in analysis time. The typical range
for the mobile phase is 60% to 75% acetonitrile. Resolution may be improved when
column temperature is above room temperature. Operation at 30 °C or 35 °C may
give better resolution of a carbohydrate mixture.

Cleaning Your Column/Extending Column Life:
HILIC Columns
Samples should be filtered before injecting on the column. This is particularly
important for the ZORBAX RRHD HILIC Plus column, as the column inlet frit is
nominally 0.5 µm and samples should be filtered through a 0.2 µm sample filter.
If solvent flow appears to be restricted (unusually high column backpressure),
check first to see that solvent flow is unobstructed up to the column inlet. If the
restriction is prior to the column, replace the appropriate piece of tubing or filter
that is plugged. If the column is plugged, InfinityLab Poroshell 120 HILIC can
be backflushed with 20-30 column volumes of mobile phase. The RRHD HILIC
column cannot be backflushed. To remove strongly-retained materials from the
column, flush the column with a strong solvent which in HILIC mode, is water.
Storage Recommendations: HILIC Columns
Acetonitrile:water (95:5) is recommended as the long-term storage solvent for
HILIC columns. It may be necessary to flush the column with acetonitrile:water
(60:40) to remove strongly-retained compounds prior to switching to the storage
solvent. Before storing the column, tightly cap the end fittings with the end plugs
to prevent the packing from drying out. Columns may be safely stored for short
periods in most HILIC mobile phases. However, to protect equipment, it is best
to remove salts from the instrument and column by purging the column with the
same mobile phase without the buffer (for example, using 90:10 ACN:H2O to
remove a 90:10 ACN:0.01M formate buffered mobile phase). Re-equilibration is
faster with the original mobile phase when using this approach, but several (3 to
6) injections should be made to verify column equilibration.