Waters Symmetry Columns User Manual
[ Care and Use ManUal ]
symmetry columns
i. introduction
Thank you for choosing a Waters Symmetry® c o l u m n . S y m m e t r y
c o l u m n s c o n t i n u e t o s e t t h e s t a n d a r d o f p e r f o r m a n c e f o r
reproducibility. As today’s chemists establish new analytical methods
for the latest pharmaceutical and biopharmaceutical products, the
selection of a reproducible HPLC column is essential. The selected
column needs to provide the same chromatographic results over the
life of the new drug product. The excellent reproducibility of Symmetry, SymmetryShield™ and Symmetry300™ columns is a result
of our commitment to maintaining the tightest specifications in the
HPLC column industry.
Physical C haracteristics
Packing Chemistry Particle Size Particle
Shape
Symmetry C
SymmetryPrep C
SymmetryShield R P18
RP8
Symmetry300 C
C
C
C
18
8
18
8
18
4
3.5, 5 µm
3.5, 5 µm
7 µm
7 µm
3.5, 5, 7 µm
3.5, 5, 7 µm
3.5, 5 µm
3.5, 5 µm
Spherical
Spherical
Spherical
Spherical
Spherical
Spherical
Spherical
Spherical
Pore
Size
100 Å
100 Å
100 Å
100 Å
100 Å
100 Å
300Å
300Å
Carbon
Load
19%
12 %
19%
12 %
17%
15%
8.5%
2.8%
End-
capped
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
contents
i. introduction
ii. GettinG started
a. Column Installation
b. Column Equilibration
c. Initial Column Efficiency Determination
iii. column use
a. Guard Columns
b. Sample Preparation
c. pH Range
d. Solvents
e. Pressure
f. Temperature
iV. scalinG up/down isocratic methods
V. troubleshootinG
Vi. column cleaninG, reGeneratinG and storaGe
a. Cleaning and Regenerating
b. Storage
Symmetry Columns 1
Vii. connectinG the column to the hplc
a. Column Connectors and System Tubing Considerations
b. Band Spreading Minimization
c. Cartridge Installation
d. Measuring System Bandspreading Volume & System Variance
e. Measuring System Volume
Viii. 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
d. Waters Small Particle Size (3.5 μm) Columns – Fast Chromatography
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ii. GettinG started
E a c h S y m m e t r y c o l u m n c o m e s w i t h C e r t i f i c a t e o f A n a l y s i s a n d a
Performance Test Chromatogram. The Certificate of Analysis is
specific to each batch of packing material contained in the Symmetry
column and includes the gel batch number, analysis of unbonded
particles, analysis of bonded particles, and chromatographic results
and conditions. The Performance Test Chromatogram is specific
to each individual column and contains the information: gel batch
number, column serial number, USP plate count, USP tailing factor,
capacity factor, and chromatographic conditions. These data should
be stored for future reference.
a. Column Installation
Note: The 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
Symmetry 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” for a more
detailed discussion on HPLC connections.
1. Purge the pumping system of any buffer-containing mobile
phases and connect the inlet end of the column to the
injector outlet.
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.
b. Column Equilibration
Symmetry 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). 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
m o b i l e p h a s e . ( F o r e x a m p l e , f l u s h t h e c o l u m n a n d H P L C s y s t e m
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. In addition, mobile phases that
contain formate (e.g., ammonium formate, formic acid, etc.) may also
require longer initial column equilibration times.
c. Initial Column Efficiency Determination
1 . P e r f o r m a n e f f i c i e n c y t e s t o n t h e c o l u m n b e f o r e u s i n g i t .
Waters recommends using a suitable solute mixture, as found
in the “Performance Test Chromatogram”, to analyze the
column upon receipt.
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
o b t a i n e d o n t w o d i f f e r e n t H P L C s y s t e m s d u e t o t h e q u a l i t y o f
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 0.1 4 0.33 – – –
30 mm – 0 .1 0.2 0.5 – 8 –
50 mm 0 .1 0.2 0.3 0.8 2.4 14 35
100 mm 0 .1 0.4 0.7 1.7 5 28 70
150 mm 0 .1 0.5 1.0 2.5 7 42 –
250 mm – 0.9 1.8 4 – 70 –
1.0 2 .1 3.0 4.6 7. 8 19 30
Column internal diameter (mm)
Symmetry Columns 2
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iii. column use
To ensure the continued high performance of Symmetry columns, follow these guidelines:
a. Guard Columns
Use a Waters guard column of matching chemistry and particle size between the injector and main column. It is important to use a high performance 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 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
solid-phase extraction cartridges/columns or Sep-Pak
cartridges of the appropriate chemistry to clean up the sample
before analysis. Link to www. waters.com/sampleprep
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
i s d i s s o l v e d i n a s o l v e n t t h a t c o n t a i n s a n o r g a n i c m o d i f i e r
(e.g., acetonitrile, methanol, etc.) ensure that the membrane
material does not dissolve in the solvent. Contact the
2. It is preferable to prepare the sample in the operating mobile
phase or a mobile phase that is weaker (less organic modifier)
than the mobile phase for the best peak shape and sensitivity.
m e m b r a n e m a n u f a c t u r e r w i t h s o l v e n t c o m p a t i b i l i t y q u e s t i o n s .
Alternatively, centrifugation for 20 minutes at 8,000 rpm,
f o l l o w e d b y t h e t r a n s f e r o f t h e s u p e r n a t a n t l i q u i d t o a n
appropriate vial, could be considered.
c. pH Range
The recommended operating pH range for Symmetry columns is 2 to 8. A listing of commonly used buffers and additives is given in Table 2.
Additionally, the column lifetime will vary depending upon the operating temperature, the type and concentration of buffer used. For example,
the use of phosphate buffer at pH 8 in combination with elevated temperatures will lead to shorter column lifetimes.
Table 2: Buffer Recommendations for Using Symmetry Columns from pH 2 to 8
Additive or Buffer pK
a
Buffer range
(±1 pH unit)
Volatility Used for Mass Spec? Comments
Formic Acid 3.75 Volatile Yes Maximum buffering obtained when used with Ammonium Formate salt.
Used in 0.1-1.0% range.
Acetic Acid 4.76 Volatile Yes Maximum buffering obtained when used with Ammonium Acetate salt.
Used in 0.1-1.0% range.
Ammonium Formate
COOH)
(NH
4
Trifluoroacetic Acid (TFA) 0.3 Volatile Low conc. When used in LC/MS, due to signal suppression, it is generally recommended to
Ammonium Acetate
COOH)
(NH
4CH2
Phosphate 1 2.15 1.15 – 3.15 Non-volatile No Traditional low pH buffer, good UV transparency.
Phosphate 2 7.2 6.20 – 8.20 Non-volatile No Above pH 7, reduce temperature/concentration and use guard column to
Symmetry Columns 3
3.75 2.75 – 4.75 Volatile Yes Used in the 1-10 mM range.
Note: sodium or potassium salts are not volatile.
use TFA at concentrations < 0.1%
4.76 3.76 – 5.76 Volatile Yes Used in the 1-10 mM range.
Note: sodium or potassium salts are not volatile.
maximize lifetime.
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