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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