Data Sheet
Agilent PLRP-S for Biomolecules
The PLRP-S media has an optimized pore size and structure for the analysis and
purification of biological macromolecules. PLRP-S media is available in prepacked
columns from 2.1 to 100 mm id or as bulk media up to 1 kg. 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.
For specific questions, contact the Technical Support team at
agilent.com/chem/columnsupport for more information.
Basic characteristics
Parameter
Value
Column Phase
Reversed-phase
Packing
Spherical, polymeric media
Particle Size
3, 5, 8, 10, 10 to 15, 15 to 20, 30, and 50 µm
Pore Structure
Totally porous, 100 Å, 300 Å, 1,000 Å, and 4,000 Å
pH Stability
1 to 14
Operating Temperature Limit
200 °C
Operating Pressure Limit
3 µm (275 bar)
5, 8, 10 (207 bar)
10 to 15, 15 to 20, 30, 50 µm (103 bar)
Mobile Phase Compatibility
Aqueous organic solvents including N,N-dimethylformamide and dimethyl
sulfoxide. 100% aqueous is not recommended, as it will reduce column
performance and lifetime.
Linear Flow Rate
180 to 360 cm/hr
Agilent PLRP-S for Biomolecules
Columns and Media
2
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 of 275 bar for 3 µm, 207 bar for 5 to 10 µm, and
103 bar for 15 to 50 µm particles. Exceeding these limits
will compromise chromatographic performance and
column lifetime and could be unsafe.
Installation
Ensure that your LC instrument is configured correctly to
minimize extracolumn band broadening and to ensure
that there are no pressure restrictions that could lead
to excessive operating pressure. Agilent recommends
choosing capillary tubing of the appropriate internal diameter
(id); 1/16 in stainless steel tubing is recommended for
column connections.
Table 1. Recommended capillary inner diameter.
1.0 to
2.0 mL/min
4.0 to
8.0 mL/min
15 to
40 mL/min
40 to
80 mL/min
80 to
200 mL/min
0.17 mm id
0.3 mm id
0.5 mm id
0.6 mm id
0.94 mm id
For more information on capillaries, visit
HPLC Capillaries | Agilent
Before connecting your column, use a barrel connector and
determine the backpressure from your LC system. Identify
any causes of high backpressure and rectify any problems
before installing your column.
Note: Agilent 50 and 100 mm id PLRP-S preparative columns
are suitable for use with 1/8 in outer diameter (od) tubing
using Valco 1/8 in nuts (PL1310-0038, 5/pk) and ferrules
(PL1310-0038, 5/pk). Alternatively, 1/8 to 1/16 in reducers are
supplied with each column to enable 1/16 in od tubing to be
used where appropriate. The direction of flow is marked on
your column.
Shipping eluent
PLRP-S columns are supplied containing 7:1
acetonitrile:water. Columns are securely sealed with endcaps
is disconnected from the system to prevent columns from
drying out.
Column compatibility
PLRP-S is compatible with aqueous organic solvents
including n,n-dimethylformamide and dimethyl sulfoxide.
100% aqueous is not recommended as it will reduce column
performance and lifetime.
Column conditioning
Every column is tested before shipment. Before the 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. Check that the column has been properly
equilibrated before use. This will ensure reproducibility and
help prevent retention time drifting.
When using formic acid as a mobile phase additive, condition
the column as recommended in Table 2.
Table 2. Recommended formic acid mobile phase additive conditions.
Column
id
Mobile Phase
Flow Rate
(mL/min)
Column
Temp.
(°C)
Time
(hrs)
After
Conditioning
2.1 mm
95/5 H
2O/CH3CN +
0.1% formic acid
0.1
60
4
Flush and
store in 100%
CH
3CN
4.6 mm
95/5 H
2O/CH3CN +
0.1% formic acid
0.4
60
4
Flush and
store in 100%
CH
3CN
Mobile phases
The PLRP-S media, being polymeric and macroporous,
is stable in most aqueous organic solvents including
N,N-dimethylformamide and dimethyl sulfoxide. PLRP-S 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.
The excellent chemical resistance of both the base polymer
enables the use of buffers in the pH range 1 to 14 without
accelerated column degradation or loss of capacity.
3
Operating tips
– Reverse flow will not usually harm the column, but should
be avoided except when removing a clogged frit.
– Start the flow rate at a reduced rate, and gently increase it
to the desired operating flow rate.
– Always use high purity reagents and chromatography
grade solvents to prepare your mobile phase. Degas and
filter all mobile phase before use.
– Use an inline filter to protect your column and increase
its lifetime.
– Avoid using 100% aqueous eluents with PLRP-S columns
as they will significantly reduce the column lifetime
and may result in a rapid deterioration in peak width
and symmetry.
Flow rate/pressure
The maximum operating pressure for the PLRP-S stainless
steel HPLC column is 275 bar for 3 µm, 207 bar for 5 to
10 µm particles, and 103 bar for 15 to 50 µm particles. With
low-viscosity mobile phases, linear flow rates of 180 to
360 cm/hr can be used.
Column id (mm)
Volumetric Flow Rate (mL/min)
4.6
0.5 to 1
7.5
1.3 to 2.7
25
14.7 to 29.5
50
58.8 to 117.8
100
235 to 471.7
If column pressures are high, due to mobile phase viscosity,
or to improve sample solubility or resolution, elevated
temperatures up can be used.
Sample preparation
The samples should be free from fat, which would otherwise
contaminate the column, and be filtered (<0.5 µm). If turbid
sample solutions are injected, even after being filtered, the
lifetime of the column may be significantly reduced. All
eluents should be freshly prepared and filtered before use.
If dealing with difficult samples prone to aggregation, inline
filters are recommended to prevent column clogging.
Column cleanup
An increase in column backpressure is likely to occur over
time. Absorption of protein to the packing material or on the
inlet frit will cause this increase in pressure and will decrease
column performance. Cleaning the column may decrease the
backpressure and improve performance. When using a guard
column or precolumn filter, replace the guard or filter and
remove the main column.
When performing cleaning, the column can be run in the
reverse direction. Start with a stronger (less polar) solvent.
1. Disconnect the column from the detector and the run
wash solvents into a beaker.
2. Start with the mobile phase without buffer salts
(water/organic). Run 10 to 20 column volumes through.
3. Next, use 100% organic solvent (methanol or acetonitrile).
4. Check the pressure to see if it has returned to normal. If
not, then:
5. Discard the column or consider stronger conditions, such
as 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 before
use and before returning to your mobile phase as it is not
miscible with aqueous).
With PLRP-S columns, additional aggressive cleanup cycles
using 80% 1 M NaOH or 1 M HCI with 20% organic, can
be used.
Storage recommendations
When removing the column from the system, end-fittings
should be tightly capped with end-plugs to prevent packing
from drying out. Columns may be safely stored for up to
several days in most mobile phases.
Where possible, flush the column and return it to the same
storage solution in which the column was originally shipped.
Do not leave columns stored in solutions that contain high
concentrations of buffers or salts. If alternative storage
solutions are required, ensure that there is no risk of bacterial
growth occurring. Some storage solution combinations (such
as aqueous alcohol) can be very viscous. To avoid pressure
damage to the column, only use these storage solutions at a
reduced flow rate.
DE44737408
This information is subject to change without notice.
© Agilent Technologies, Inc. 2022
Printed in the USA, May 19, 2022
5994-4896EN
