Waters BioSuite Columns User Manual
[ Care and Use ManUal ]
BIosuIte columns
I. IntroductIon
Thank you for choosing a Waters BioSuite™ column. Every column is individually tested to assure maximum product quality. Waters BioSuite high
performance chemistries are dedicated to the isolation, analysis, and characterization of proteins and peptides. For the latest list on ion-exchange, size
exclusion, hydrophobic interaction and reversed-phase go to www.waters.
com/lifesciences
contents
I. IntroductIon
a. Mobile Phase
b. Flow Direction
II. connectIng your column to the hPlc system
III. Waters BIosuIte Pc
and PPhenyl rPc columns
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IV. Waters BIosuIte Phenyl hIc columns
V. Waters BIosuIte ultra hIgh resolutIon (uhr), hIgh
resolutIon (hr), and sIze exclusIon (sec) columns
VI. Waters BIosuIte Q-PeeK and sP-PeeK columns
VII. Waters BIosuIte deae and sP nP columns
VIII. Waters BIosuIte Q, deae, sP, and cm columns
BioSuite Columns 1
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0.175 inches
Void
a. Mobile Phase
Prevent air bubbles from entering the column during its installation, use,
and storage since this may cause degradation of column performance
through the formation of channels in the packed bed. Mobile phases must be
thoroughly degassed before use. This can be accomplished by vacuum filtra-
tion, helium sparging or by in-line vacuum degassing. In addition to degas-
sing the solvent, vacuum filtration of the solvent will also prevent small
particles from plugging the column frit. You can use 0.20 or 0.45 µm mem-
brane to filter aqueous and aqueous/organic mobile phases. (Note: Consult
with filtration membrane manufacturer for details on solvent compatibility.)
Note: Use high quality reagents, HPLC grade water, and HPLC grade solvents
for preparing buffers to maximize column efficiency especially when using
Waters BioSuite Non-Porous (NP) columns.
The useful column lifetime is a function of numerous factors including: the
cleanliness and composition of the mobile phase and the sample; the flow
rate and pressure used; and the temperature. Refer to the section below
about “Cleaning” for information on extending column life.
Note: Cleaning, however, is not effective when the column is damaged by
irreversible sample adsorption, channeling, or exposure of the packing mate-
rial to excessive heat or shock.
tubing and the column that will result in undesired peak broadening (Figure
B). To correct this problem, Cut the tubing, place a new ferrule on it and re-
make the connection (i.e., 0.175” depth) to the Waters BioSuite column.
Figure A
Figure B
In a proper tubing/column connection, the tubing touches the bottom of the
column end fitting with no void between them. (Figure C)
b. Flow Direction
The recommended flow direction through the column is indicated by the
arrow on the tag. Operating the column with the flow in the reverse direc-
tion is only recommended as part of a cleaning procedure when removing
particulates from a clogged frit.
II. connectIng your column to the hPlc system
Due to the absence of an industry standard, various column manufacturers
have employed different styles of chromatographic column connectors. The
chromatographic performance of your separation can be negatively affected
if the style of column end fitting does not match the existing instrumenta-
tion tubing ferrule setting. Waters BioSuite columns require end fittings
that have a 0.175” depth between the ferrule and the end of the extending
stainless steel tubing (Figure A) that must perfectly seat to the end of the
BioSuite column. Connecting a non-BioSuite style connector to a Waters
BioSuite column will leave a gap between the end of the stainless steel
BioSuite Columns 2
Figure C
After manufacturing and quality control, the column has been flushed with
storage solvent and closed with caps to prevent solvent evaporation. When
installing the column to the Liquid Chromatograph, it is important to prevent
air from entering the column. The following steps can be used to minimize
this potential problem:
• Remove the cap from the column’s inlet side. Solvent should be visible at
the inlet fitting (if not, see below).
• StartsolventflowthroughtheliquidchromatographBEFOREconnecting
the Waters BioSuite column to the system. Turn off the pump after a
steady flow of solvent from the system is observed. You can now connect
the waters BioSuite column to the liquid chromatograph and slowly begin
delivering solvent to the column (Note: If the Waters BioSuite column
inlet fitting appears dry, we recommend that you first disconnect the
bottom cap and hook up the column exit to the injector. Then slowly start
[ Care and Use ManUal ]
the flow in this reversed flow direction until a few drops of mobile phase
exit from the column. Turn off the flow, let the pressure go to zero, and
disconnect the column from the system. Turn the column around and hook
it up so that the flow is now in the direction of the arrow. Start the flow
at a low setting and stop it as soon as the mobile phase exits from the
bottom fitting. Now you can hook up the column to the detector inlet and
increase the flow to the desired setting.)
• Besuretosettheflowwithintherecommendedrangeshowninthe
following tables.
Sample
If possible, always dissolve your sample in mobile phase or the starting
mobile phase when operating under gradient conditions. Alternatively, try
to match the pH, salt concentration and organic solvent of the sample with
those of the mobile phase and run a test to ensure that no precipitate, sus-
pension or flocculate is formed. Finally, before making an injection, filter the
sample through a 0.20-0.45 µm porosity membrane.
III. Waters BIosuIte Pc18 and PPhenyl rPc columns
This section contains recommended operating conditions and specifications
for the Waters polymer-based, C
and phenyl reversed-phase columns.
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Operating Conditions
1. Shipping Solvent: 70% Methanol - 30% Water
Note: When using a high viscosity buffer and ambient temperature, the maximum flow rate may have to be reduced to avoid exceeding the maximum
recommended operating pressure for the column. When changing solvents,
use 50% of the maximum flow rate (20% for the 21.5 mm i.d. preparative
column).
3. Recommended pH Range: 2 - 12 (Avoid extended operation at pH >
10 to maximize column life.)
4. Recommended Organic Concentration Range: 0 - 100% (Note:
Reduced organic concentrations may be required if buffers are present
in the eluent in order to avoid salt precipitation.)
5. Recommended Temperature Range: 5 - 50 °C (Note: Reduce flow rate
when operating at low temperatures (e.g.. 5 C) to avoid excessive
column pressure.)
6. Recommended Cleaning Solvents/Procedures:
A) Acetonitrile or Methanol, or
B) Aqueous buffer in organic solvent, or
C) 0.1 - 0.2M NaOH, or
D) 20 - 40% acetic acid in HPLC grade water.
Clean the column regularly with any of the listed solvents. Cleaning
solvents (C) and (D) can be injected onto the column in 250 µl incre-
ments for a total of one column volume. Use proportionally larger
volumes for the 21.5 mm i.d. prep column. Following use of cleaning
solvent, rinse the column with 3 to 5 column volumes of HPLC grade
water before storage.
2. Recommended Operating Flow Rates/Max Pressure:
Description Matrix i.d.
(mm)L (mm)
BioSuite pC
NP RPC
BioSuite pC
7 µm RPC
BioSuite pC
7 µm RPC
BioSuite pC
13 µm RPC
BioSuite pPhenyl, 1000,
10 µm RPC
BioSuite pPhenyl, 1000,
10 µm RPC
BioSuite pPhenyl, 1000,
13 µm RPC
BioSuite Columns 3
, 2.5 µm
18
, 500,
18
, 500,
18
, 500,
18
polymer 4.6 35 1.0 - 1.6 mL/min /
polymer 4.6 150 0.08 - 0.22 mL/min
polymer 4.6 150 0.5 - 1.2 mL/min /
polymer 21.5 150 3.0 - 8.0 mL/min /
polymer 2.0 75 0.05 – 0.12 mL/min
polymer 4.6 75 0.5 - 1.2 mL/min /
polymer 21.5 150 6.0 - 8.0 mL/min /
Flow Rates / Max
Pressure
3000 PSI
/ 1500 PSI
1750 PSI
500 PSI
/ 150 PSI
450 PSI
450 PSI
7. Recommended Storage: For overnight storage, continuously flush the
column with the mobile phase at 10 - 20% of the maximum recom-
mended flow rate. Store the column in the shipping solvent when the
column will not be used within 48 hours.
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