Agilent Technologies InfinityLab Poroshell 120 Manuale utente

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

ENGLISH

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

ENGLISH

• 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.

ENGLISH

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.

ENGLISH

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.

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