Waters SunFire Columns User Manual
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sunFire Columns
Contents
I. GETTING STARTED
a. Column Installation
b. Column Equilibration
c. Initial Column Efficiency Determination
II. COLUMN USE
a. Guard Columns
b. Sample Preparation
c. pH Range
d. Solvents
e. Pressure
f. Temperature
III. SCALING UP/DOWN ISOCRATIC METHODS
IV. TROUBLESHOOTING
V. COLUMN CLEANING, REGENERATING AND STORAGE
a. Cleaning and Regenerating
b. Storage
VI. CONNECTING THE COLUMN TO THE HPLC SYSTEM
a. Column Connectors and System Tubing Considerations
b. Bandspreading Minimization
c. Measuring System Bandspreading Volume & System Variance
d. Measuring System Volume
Thank you for choosing a SunFire
™
column. The SunFire packing
materials were designed to provide excellent peak shape, minimal
column bleed, and high mass loading. The SunFire packing materials
are manufactured in an ISO 9000 certified plant using ultra pure
reagents. Each batch of SunFire material is tested chromatographically with acidic, basic and neutral analytes and the results are held
to narrow specification ranges to assure excellent, reproducible
performance. Every column is individually tested and a Performance
Chromatogram is provided with each column along with the Certificate
of Batch Analysis.
SunFire Column Physical Characteristics
Chemistry C
USP Class No. L1 L7 L3
Particle Shape Spherical Spherical Spherical
Particle Sizes
Pore Size 100Å 100Å 100Å
Surface Area 340 m
Carbon Load 16% 12% N/A
Endcapped Yes Yes N /A
ph Range 2-8 2-8 N/A
Temperature
Limits
18
2.5, 3.5,
5, 10 µm
2
/g 340 m2/g 340 m2/g
Low pH = 50 ˚C
High pH = 40 ˚C
C
8
2.5, 3.5,
5, 10 µm
Low pH = 40 ˚C
High pH = 40 ˚C
Silica
5, 10 µm
N/A
VII. ADDITIONAL INFORMATION
a. Use of Narrow-Bore (≤3.0 mm i.d.) Columns
b. Impact of Bandspreading Volume on 2.1 mm i.d.
Column Performance
c. Non-Optimized vs. Optimized LC/MS/MS System:
System Modification Recommendations
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I. GETTING STARTED
Each SunFire column comes with a Performance Test Chromatogram.
This Performance Test Chromatogram is specific to each individual
column and contains the following information: gel batch number,
column serial number, USP plate count, USP tailing factor, capacity
factor, and chromatographic conditions. The performance test chromatogram should be stored for future reference.
a. Column Installation
Note: Flow rates given in the procedure below are for a typical 4.6
mm i.d. column. Scale the flow rate up or down accordingly based
upon the column i.d., length, particle size, and backpressure of the
SunFire column being installed. See “Scaling Up/Down Isocratic
Separations” for calculating flow rates when changing column i.d.
and/or length. See “Connecting the Column to the HPLC System” for a
more detailed discussion on HPLC connections.
Reversed-phase Columns (SunFire C
1. Purge the pumping system of any buffer-containing mobile
phases and connect the inlet end of the column to the
injector outlet.
and SunFire C8)
18
4. Connect the column and equilibrate it with the mobile phase.
Note: Equilibration with the mobile phase may require a larger amount
of solvent than in reversed-phase chromatography.
b. Column Equilibration
SunFire columns are shipped in 100% acetonitrile. It is important
to ensure mobile phase compatibility before changing to a different
mobile-phase system. Equilibrate the column with a minimum of 10
column volumes of the mobile phase to be used (refer to Table 1 for
a listing of empty column volumes).
Reversed-phase (SunFire C
To avoid precipitating out mobile-phase buffers on your column or in
your system, flush the column with five column volumes of a water/
organic solvent mixture, using the same or lower solvent content as in
the desired buffered mobile phase. (For example, flush the column and
HPLC system with 60% methanol in water prior to introducing 60%
methanol/40% buffer mobile phase.)
Note: If mobile-phase additives are present in low concentrations (e.g.,
ion-pairing reagents), 100 to 200 column volumes may be required
for complete equilibration.
or SunFire C8) Columns
18
2. Flush column with 100% organic mobile phase (methanol or
acetonitrile) by setting the pump flow rate to 0.1 mL/min and
increase the flow rate to 1 mL/min over 5 minutes.
3. When the mobile phase is flowing freely from the column outlet,
stop the flow and attach the column outlet to the detector. This
prevents entry of air into the detection system and gives more
rapid baseline equilibration.
4. Gradually increase the flow rate as described in step 2.
5. Once a steady backpressure and baseline have been achieved,
proceed to the next section.
Normal-phase Columns (SunFire Silica)
Note: It is assumed that your system has been used for reversed-phase
chromatography. If this is not the case, you can start with step 3.
1. Purge the pumping system of any buffer-containing mobile phases.
2. Flush the system thoroughly with acetonitrile.
3. Switch the system over to the mobile phase that you are planning
to use in normal-phase chromatography.
Normal-phase Columns (SunFire Silica)
SunFire normal-phase (NP) columns are delivered in 96% heptane/
4% isopropyl alcohol. Care should be taken not to pass any mobile phase
through the column that might cause a precipitate (see above). SunFire
Silica normal-phase columns are compatible with water and all common
organic solvents, provided that solvent miscibility is accounted for.
Equilibrate normal-phase silica columns in the mobile phase. Very
small quantities of water in the mobile phase can dramatically affect
the activity of normal-phase packings. For good reproducibility,
ensure that the mobile phase always has the same water content.
It is difficult and usually unnecessary to completely eliminate the
water from the mobile phase. Dry mobile phases can take a very long
time to equilibrate the column. A water content of 50 percent of
saturation is recommended for most applications.
To equilibrate your column:
1. Starting at 0.0 mL/min, increase the flow rate in
0.1 mL/min increments to 1.0 min.
2
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2. Purge the column with the mobile phase until you obtain a stable
baseline.
3. Verify that retention times and peak areas for a standard are
stable by comparing 2-3 replicate, consecutive injections.
Before you perform the first analysis on your new column, perform an
efficiency test to confirm the performance of the column.
c. Initial Column Efficiency Determination
1. Perform an efficiency test on the column before using it. Waters
recommends using a suitable solute mixture, as found in the
“Performance Test Chromatogram”, to analyze the column upon
receipt. However, if the column is used only for a single routine
isocratic assay, it may be more convenient to test the column
under these assay conditions. Keep a record of the initial column
performance.
2. Determine the number of theoretical plates (N) and use this value
for periodic comparisons.
3. Repeat the test at predetermined intervals to track column
performance over time. Slight variations may be obtained on
two different HPLC systems due to the quality of the connections,
operating environment, system electronics, reagent quality,
column condition and operator technique.
Table 1. Empty Column Volumes in mL (multiply by 10 for flush
solvent volumes)
Column
Length
20 mm - 0.07 0.14 0.33 - - - - 30 mm - 0.1 0.2 0.5 - 2.4 8 - 50 mm 0.1 0.2 0.3 0.8 2.4 4 14 35 98
100 mm 0 .1 0.4 0.7 1.7 5 8 28 70 196
150 mm 0.1 0.5 1.0 2.5 7 12 42 106 294
250 mm - 0.9 1.8 4 - 20 70 176 490
1.0 2 .1 3.0 4.6 7.8 10 19 30 50
Column Internal Diameter (nm)
II. COLUMN USE
To ensure the continued high performance of SunFire columns, follow
these guidelines:
a. Guard Columns
Use a SunFire guard column of matching chemistry and particle size
between the injector and main column. It is important to use a matching
guard column to protect the main column while not compromising or
changing the analytical resolution. Guard columns need to be replaced
at regular intervals as determined by sample contamination. When
system backpressure steadily increases above a set pressure limit, it
is usually an indication that the guard column should be replaced. A
sudden appearance of split peaks or other changes in chromatographic
performance is also indicative of a need to replace the guard column.
b. Sample Preparation
1. Sample impurities often contribute to column contamination.
®
One option to avoid this is to use Oasis
®
cartridges/columns or Sep-Pak
cartridges of the appropriate
chemistry to clean up the sample before analysis. Link to
www.waters.com/sampleprep
2. It is preferable to prepare the sample in the operating mobile
phase or a mobile phase that is weaker (less organic modifier
in the case of reversed-phase chromatography, less polar
modifier in the case of normal-phase chromatography or
hydrophilic interaction chromatography, less salt in the case
of ion exchange) than the mobile phase for the best peak shape
and sensitivity.
3. If the sample is not dissolved in the mobile phase, ensure that the
sample, solvent and mobile phases are miscible in order to avoid
sample and/or buffer precipitation. Filter sample with 0.2 μm
membranes to remove particulates. If the sample is dissolved in a
solvent that contains an organic modifier (e.g., acetonitrile,
methanol, etc.) ensure that the membrane material does not
dissolve in the solvent. Contact the membrane manufacturer with
solvent compatibility questions. Alternatively, centrifugation for
20 minutes at 8,000 rpm, followed by the transfer of the
supernatant liquid to an appropriate vial, could be considered.
solid-phase extraction
3
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