Column Part No.:
Column Serial No.:
Stationary Phase:
Particle Size (µm):
Lot No.:
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PERFORMANCE SUMMARY
Column Test
Conditions
General HPLC Column Care and Use Instructions
Column
Type
Silica RP* Columns:
Polymer RP Columns: CN (Reverse Phase): CN (Normal Phase): NH2, Diol, Silica Ion Exchange Shipping
Solvent MeCN: H20
MeCN: H20 MeCN 3:97 IPA:Heptane 3:97 IPA:Heptane 3:97 IPA:Heptane StoragepH
Solvent Range
MeCN MeCN MeCN IPA:Heptane
IPA:Heptane
IPA:Heptane 2-7(1.5-10)**
1.5–13
2-7
2-7
Column Storage
Column storage conditions have a profound effect on
column lifetime and performance. If the column has been
used with eluents containing buffers, ion-pair reagents, or
inorganic solutes, the column should be flushed completely
clean of these components before extended storage by
flushing with 20-50 column-volumes of water. The column
should be flushed with 2 to 5 volumes the storage solvent
(100% MeCN or MeOH for most reverse-phase columns).
*RP columns include reverse phases Pursuit (XRs), Inertsil , Microsorb, OmniSpher,
Kromasil , MetaSil , Polaris , Pursuit , Nucleosil , and most other silica-based columns
provided by Agilent Technologies, Inc. See individual QC Test Data Sheet to confirm
the actual shipping solvent in a particular column.
Flow rates and pressure
The table below lists typical flow rates for various column
configurations. The back pressure of the column will vary directly
with flow rate and is greatly influenced by eluent composition and
viscosity. Columns should not be used over 3000 psi for extended
periods of time. 5000 psi should be considered the absolute
maximum pressure applied to an HPLC column. Wide-pore silicas
(e.g. >200Å pore size) should not be used over 2000 psi.
Typical
Column
Flow
ID
(ml/min)
1.0
0.05
2.0
0.2
3.0
0.5
3.2
0.5
3.9
1.0
4.0
9.6
10.0
12.2
1.0
1.0
5.0
10-25
Samples should be filtered prior to injection.
Only HPLC grade, pre-filtered, degassed solvents should be used. (see www.agilent.com for information on automated degassing.)
Stay within the pH limitations of the column.
All mobile phases should be completely miscible with solvents contained in the column.
(I.D. Column 2)2
S = Scale Factor = (I.D. Column 1)2
** Inertsil columns have a pH range of 2-9. Polaris and Pursuit(XRs) have a pH range
of 1.5-10. Note: Diphenyl columns have a lower pH range of 1.5-7.5. pH ranges are
specified at ambient temperature (±25 °C).
BEFORE flushing the column, be certain that the solvent to be introduced into the column is completely miscible with the solvent
contained in the column. If not, use an intermediary solvent which
is soluble in both solvents. IPA (isopropanol) is an acceptable
intermediary solvent for most reverse phase eluents. If uncertain
as to the solvent contained in the column during shipping, please
contact Agilent Technologies, Inc., before using the column.
Flow Rate Adjustments
When scaling up or scaling down
from one column dimension to
another, it is often desirable to
maintain constant retention values.
The flow rate and load capacities
must be adjusted as a function of
the change in the column’s cross
sectional area. Use the equation
below to determine the scale factor,
S, by which flow rates and loading
should be multiplied:
New Flow Rate = (Old Flow Rate) *S
Column Regeneration
Many samples and mobile phases contain components which
can adsorb to the column, eventually promoting a change in the
separation characteristics. Such columns can sometimes be restored
to near original condition by “regeneration” or removal of adsorbed
contaminants through washing. Follow the procedures outlined
below for regeneration of the indicated column types. In all cases,
reverse the column (e.g., attach the outlet end of the column to the
pump, and pump the eluent directly into a waste reservoir) and flush
the column with 10 column volumes of the indicated solvents in the
indicated sequences:
Reverse Phase (C18, C8, Phenyl, CN*): 5% MeOH in H20
(up to 55 °C) > MeOH > MeCN > THF > MeOH > Mobile Phase
(*Skip H20 rinse on CN)
Normal Phase (Silica, NH2, CN, Diol): Hexane and Chloroform >
Methylene Chloride > Isopropanol > Mobile Phase
Ion Exchange (SAX, SCX, DEAE, NH2, CM) : 5% MeOH in H20
(up to 55 °C) > MeOH > MeCN > Methylene Chloride > MeOH >
Mobile Phase*
*If mobile phase contains a buffer, flush column with mobile phase MINUS
the buffer first, to avoid precipitation of the buffer in the pure MeOH
remaining in the column.
Protein Removal (C18, C4, C8, Phenyl): 5% MeOH in H20 > 0.1%
TFA > Isopropanol > MeCN > 5% MeCN in H20 > Mobile Phase
This information is subject to change without notice.
© Agilent Technologies, Inc. 2010
Printed in the USA December 20, 2010