Agilent Technologies ZORBAX RRHT Series User manual

COLUMN USER GUIDE
for Agilent Reversed-Phase
Columns
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
Poroshell 120/300 / Pursuit / Polaris / HC-C18(2) /
TC-C18(2)

This booklet provides general
information for all ZORBAX, Poroshell,
Pursuit, and Polaris reversed-phase
columns. For additional detailed
information about your specic phase or
family, see:
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 modied 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
congured 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
specications. Always review the specications before
putting in place a nal method.

Using Your Column
Installation
• The direction of ow is marked on the column.
• 1.8 µm columns (ZORBAX RRHT, ZORBAX RRHD) can only
be operated in the direction ow marked on the column.
For removable, zero-dead volume column connections
Agilent recommends the use of the InnityLab Quick Connect
and Quick Turn family. Choices are:
Maximum
System
Pressure Recommended Fitting Part Numbers
Up to 400 bar InnityLab Quick Turn Fitting
(nger-tight)
Fitting: 5067-5966
Up to 800 bar InnityLab Quick Turn Fitting
(with Mounting tool)
Fitting: 5067-5966
Mounting Tool:
5043-0915
Up to 1300 bar InnityLab Quick Connect Fitting Fitting: 5067-5965
For more information and part numbers, please see to the
AgilentInnityLab Fitting Brochure (5991-5164EN).
ENGLISH
InnityLab Quick Connect
assembly, p/n 5067-5961
InnityLab Quick Turn
tting, p/n5067-5966
Learn more at: www.agilent.com/chem/innitylabttings

Column conditioning
Every column is tested before shipment and shipped
in the test eluent. So, for rst 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 ush 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 ammability 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 table). Exceeding these limits will
compromise chromatographic performance and could
beunsafe.
Other operating tips
• While generally not harmful to the column, reverse ow
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
lter 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 Poroshell 120 or ZORBAX RRHT or RRHD columns,
consider using a Fast Guard for UHPLC to protect your analytical
column. See agilent.com/chem/fastguards for more information.
ENGLISH

Column operating parameters: pH and temperature
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 signicant impact on column
lifetime.
Phase
Recommended
pH Range
Maximum Operating
Temperature
Poroshell HPH pH 3.0 to 11.0 60 °C
Poroshell 120 SB-C18,
StableBond C18,
ZORBAXRx-C8,
ZORBAX SB-Aq,
ZORBAX SB-Phenyl,
SB-C3, Poroshell 300,
300SB-C3
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)
70 °C for pH <5.0; 40°C
for pH 5.0 to 8.0 (Poroshell
300 and 300SB-C3)
ZORBAX Eclipse Plus C18
and C8, Eclipse XDB-C8,
XDB-C18, XDB-Phenyl,
Poroshell 120 EC-C18 and
EC-C8, ZORBAX Bonus-RP,
HC-C18(2) and TC-C18(2)
pH 2.0 to 9.0 60 °C
ZORBAX Extend C18 pH 2.0 to 11.5 60 °C (40 °C at high pH)
Poroshell 120 PFP,
Poroshell 120 Phenyl-Hexyl,
ZORBAX ODS,
ZORBAXPhenyl,
ZORBAXEclipse Plus
Phenyl-Hexyl,
ZORBAX XDB-CN,
ZORBAXEclipse PAH,
AllPursuit and Polaris
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

Maximum operating pressures—columns up to 9.4 mm id
Column type Particle size Pressure limit
Poroshell 120 1.9 µm
2.7 μm, 4 μm
1300 bar (19,500 psi)
600 bar (9000 psi)
Poroshell 300 5 μm 400 bar (6000 psi)
ZORBAX Rapid Resolution 3.5 µm 400 bar (6000 psi)
ZORBAX Rapid Resolution High
Throughput (RRHT)
1.8 μm 600 bar (9000 psi)
ZORBAX Rapid Resolution High
Denition (RRHD)
1.8 μm 1200 bar (17,000 psi)
All ZORBAX, Pursuit, and Polaris
columns, not noted as RRHT or
RRHD, and HC-C18(2), TC-C18(2)
3.0 μm, 3.5 μm,
5 μm
400 bar (6000 psi)
200 bar (3000 psi)
(Metaguards)
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.
ENGLISH

Recommended starting gradients
Stationary phase Usage notes
Most reversed-phase
columns
5% methanol or acetonitrile initially and 100%
methanol or acetonitrile as the nal solvent.
ZORBAX Eclipse PAH* 30 or 40% acetonitrile initially, to 100%
acetonitrile as nal solvent. It may be necessary
to cool column to 15 to 20 °C for improved
resolution.
ZORBAX Bonus-RP Lower concentration of organic mobile phase
modier is required for compound elution,
compared to traditional long-chain alkyl
stationary phases.
Large molecule
columns (for example,
Poroshell300 or
ZORBAX300SB
columns)
Separations of polypeptides, proteins, DNA
fragments, etc. often use triuoroacetic or
formic acid modier for pH control and/or as
an ion-pairing additive for desired retention
and selectivity. Organic buffers such as TRIS
are especially useful for intermediate pH
applications.
* Best column lifetime is achieved at <40 °C.

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. ZORBAX
and Fast Guard columns and hardware kits (as required) are
recommended for use with such samples, and are gener-ally
recommended for all column use.
See agilent.com/chem/fastguards for more information
about guard columns.
Cleaning Your Column/Extending Column Life
For columns that can be backushed (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, for
example, 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 ush 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 backush the
column—replace the column.
ENGLISH

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 rst 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-ttings
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 (for example 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.