Agilent Technologies BioHPLC series User manual

REVERSED-PHASE
COLUMN USER GUIDE
for Agilent BioHPLC
and AdvanceBio Columns
Guide d’utilisation decolonnes pour les colonnes BioHPLC
et AdvanceBio d’Agilent
BenutzeRINFORMATION zu Agilent BioHPLC- und
AdvanceBio-Säulen
Guida ALL’USO per colonne Agilent BioHPLC e AdvanceBio
Guía de usuario para columnas Agilent BioHPLC y
AdvanceBio
Руководство пользователя для колонок Agilent BioHPLC
и AdvanceBio
Guia do usuário de colunas para colunas Agilent
BioHPLC e AdvanceBio
カラムユーザーガイド Agilent BioHPLC および
AdvanceBio カラム
Agilent BioHPLC 和AdvanceBio 色谱柱
用户指南

This booklet provides general information for the
Agilent BioHPLC and AdvanceBio reversed-phase
columns. For additional detailed information about
your specific phase or family, see:
www.agilent.com/chem/biocolumnchoices
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 efficiency and assures a more consistent
product. An optimized LC system will generate similar results to the
chromatogram on your QC Performance Report.
If you have specific questions, contact the Technical Support team at
www.agilent.com/chem/columnsupport

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Installation
• The direction of flow is marked on the column.
• 1.8 µm columns (ZORBAX RRHD) can only be operated in the
direction of flow marked on the column.
• Agilent recommends using Polyketone fittings (p/n 5042-8957)
for columns up to 600 bar, and 1200 bar removable fittings
(p/n 5067-4733) for columns that will be operated at
UHPLC pressures.
Polyketone fitting,
p/n 5042-8957
Agilent 1200 bar removable fitting,
p/n 5067-4733
Using Your Column
Column Conditioning
Every column is tested before shipment. So, for first use, the shipping
solvent must be replaced with eluent, taking care that all components are
miscible and soluble. 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. 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.

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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.
Agilent does not recommend removing the column end fittings and
exposing the media. Columns should only be opened by trained
personnel in a well-ventilated area.
• Please adhere to operating pressure limits noted for each column
(see Table 1). Exceeding these limits will compromise
chromatographic performance and column lifetime, and could
be unsafe.
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”).
• It is recommended that the flow rate is started at a reduced rate
and then gently increased to the desired operating flow rate.
• 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.
• An inline filter or guard column may be used to protect your column
and increase its lifetime.
• If the column is used outside of recommended pH ranges for column
phase (see Table 2), a reduced lifetime will result.
(continued)

Other Operating Tips (continued)
• Columns should not be maintained at elevated pH or elevated
temperature when not in use (see Table 2).
• New columns may contain a mixture of organic solvents and water,
which may contain buffer salts. See Table 3 for details of the shipping
solvents. Initially, care should be taken not to pass any mobile phase
through the column that may cause a precipitate to form or may not
be fully miscible.
• Agilent BioHPLC and AdvanceBio columns are compatible with all
common eluents used for analysis of biomolecules.
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Column Particle size Pressure limit
AdvanceBio Peptide Mapping
2.7 µm 600 bar
AdvanceBio Peptide Plus
AdvanceBio Oligonucleotide
AdvanceBio Amino Acid Analysis
AdvanceBio RP-mAb 3.5 µm
ZORBAX 300SB 3.5 µm, 5 µm, 7 µm 400 bar
Poroshell 300 5 µm
ZORBAX RRHD 1.8 µm 1200 bar
PLRP-S
3 µm 275 bar
5 µm, 8 µm, 10 µm 207 bar
10-15 µm, 15-20 µm, 30 µm 103 bar
Table 1. Maximum operating parameters – columns
up to 4.6 mm

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Column
Recommended
pH range
Maximum operating
temperature
AdvanceBio Peptide Mapping 2.0-8.0 60 ºC
AdvanceBio Peptide Plus
ZORBAX RRHD 1.0-8.0 80 ºC
ZORBAX 300SB-C8, C3, CN
ZORBAX 300SB-C18
1.0-8.0 90 ºCAdvanceBio RP-mAb
Poroshell 300
Poroshell 300 Extend-C18 2.0-11.0 60 °C below pH 8,
40 °C above pH 8
AdvanceBio Oligonucleotide 3.0-11.0 65 ºC
AdvanceBio Amino Acid Analysis
PLRP-S 1.0-14.0 200 ºC
Table 2. 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.
Column Shipping solvent Compatibility
AdvanceBio Peptide Mapping
Mixture of acetonitrile
and water
Water and all common organic
solvents.
AdvanceBio Peptide Plus
ZORBAX RRHD
ZORBAX 300SB
AdvanceBio RP-mAb Mixture of acetonitrile
and water Water and all organic solvents,
including N,N-dimethylformamide
and dimethylsulfoxide.
Poroshell 300 and 300 Extend Mixture of methanol
and water
AdvanceBio Oligonucleotide Mixture of acetonitrile
and water
Water and all common organic solvents.
Modifiers including HFIP and TEAA.
AdvanceBio Amino Acid Analysis
PLRP-S 7:1 acetonitrile/water Aqueous organic solvents
including N,N-dimethylformide and
dimethylsulfoxide. 100% aqueous is
not recommended as it will reduce
column performance and lifetime.
Table 3. Shipping solvents

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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 reduces the
retention time of the sample.
When the maximum temperature is used for prolonged periods this will
reduce column lifetime.
Using 100% aqueous eluents with PLRP-S columns will significantly
reduce the column lifetime and may result in a rapid deterioration in peak
width and symmetry.
Recommended Starting Gradients
Separations of biological molecules typically use gradient conditions –
increasing the amount of the organic component to achieve elution from
the column. Separations of peptides, polypeptides, and proteins, are most
commonly carried out using acidic eluents with trifluoroacetic acid (TFA)
or formic acid (FA) used as the modifier for pH control and/or as an
ion-pairing additive to achieve the desired retention and selectivity.
Organics, such as acetonitrile, methanol, and ethanol, are used for
elution, with acetonitrile being the most commonly used.
Additional information on peptide separations can be found in chapter 11, pages
497-508, Introduction to Modern Liquid Chromotography, Second Edition. L.R.
Snyder and J. J. Kirkland, (John Wiley & Sons, 1979).

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Column Care
Columns with a particle size of 1.8 µm have a 0.5 µm inlet frit. Particulates
will block the column inlet frits and should be removed before the sample
is analyzed. Where this is not possible, an inline filter or guard column
should be used to protect and increase the lifetime of the analytical
column. The AdvanceBio Peptide Mapping column has a 2 µm inlet frit.
Samples that contain bioparticulate matter larger than 2 μm will plug the
inlet frit. Guard columns are recommended for use with such samples. It
is recommended that samples are filtered before injecting them onto any
column and all eluents are freshly prepared and filtered prior to use.
See www.agilent.com/chem/guards for more information about
guard columns.
Cleaning Your Column/Extending Column Life
For columns that can be backflushed (particles >1.8 µm), clean the
column in the reverse direction. 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 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:
(continued)

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Storage Recommendations
In general, columns may be safely stored for short periods in most
mobile phases. 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.
5. Discard column or consider stronger conditions, e.g.
75% acetonitrile: 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 mobile phase as it is not miscible with aqueous).
Aggressive cleanup cycles using 80% 1 M NaOH or 1 M HCI with
20% organic can be used with the PLRP-S columns.
For columns with 1.8 µm particles, do not backflush the column –
can attempt the cleanup procedure as detailed above but maintain
direction of flow.
Cleaning Your Column/Extending Column Life
(continued)
(continued)

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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 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 (ZORBAX RRHD).
• Use sample filtration or other sample prep as appropriate for your
sample. Learn more at www.agilent.com/chem/sampleprep
• Use certified lamps in your LC instruments for best performance.
Storage Recommendations (continued)
To protect equipment, it is 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/H2O 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 possibility of corrosion from the salts is
eliminated.