Bio-Rad Nuvia™ Q Media - Manual (Page 7)

Section 3 : Preparation
Nuvia ™ media are supplied fully hydrated in 20% ethanol + 1 M NaCI as
a 50% (v/v) slurry. For column packing, removal of the shipping buffer
is recommended. Small volumes of Nuvia media are easily washed in a
Büchner funnel with 4–5 volumes of packing buffer. For large volumes,
cycling through 3–4 settling and decanting steps using the packing buffer
in the shipping container is recommended.
Removal of fines from Nuvia media is not required due to the narrow
particle size range. If fines have been generated during handling,
resuspend the sediment and remove the opaque supernatant before
sedimentation is complete. Repeat several times.
Section 4 : Column Packing
Nuvia ™ media can be packed using pressure, volumetric flow, or
vacuum packing methods. To pack columns for highly efficient
operation, it is recommended that a 20–50% slurry volume be used.
Packing Small Columns
This slurry packing method was designed to pack 25 ml of Nuvia
media in a conventional column with an internal diameter of 5–15 mm.
All buffers should be degassed. Because a relatively large volume of
slurry is required, it is recommended that a packing reservoir be used.
1. Prepare degassed 1.0 M NaCl, 20–50 mM buffer salt (see Table 2)
referred to herein as the packing buffer.
2. Nuvia media are shipped as a 50% slurry. Measure 50 ml of
suspended slurry into a 100 ml graduated cylinder. Allow the resin bed
to settle. Decant the shipping solution away from the resin bed.
3. Add 50 ml degassed packing buffer to the resin.
4. Seal the cylinder and rotate it to suspend the resin. Caution: Do not
mix with a magnetic stir bar as damage may occur. Larger amounts
of slurry may be mixed with a low-shear marine impeller at low to
moderate speed.
5. Add 10 ml packing buffer to the column. Pour in 75 ml resin slurry.
6. Insert the column flow adaptor and flow pack at a linear velocity of
300–600 cm/hr with packing buffer for at least 10 min. Note the
compressed bed height, stop the flow, and adjust the flow adaptor to
compress the bed 0.1–1.0 cm.
7. Attach the column to your chromatography system, and purge the
column with starting buffer at linear velocities up to 600 cm/hr. If the
bed compresses,repeat steps 6 and 7.
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Packing Process-Scale Columns
After removing the storage buffer (Section 3), prepare a 20–50% slurry
(v/v) with packing buffer (see Table 2). Follow the column manufacturer’s
recommendations with one major exception: do not recirculate the Nuvia
slurry through the packing pump. Use a low-shear impeller for automatic
mixing or a plastic paddle for manual mixing. The best overall performance
of Nuvia S and Nuvia Q will be obtained with a compression ratio of 1.15–
1.20 and 1.10 –1.15 respectively defined as settled bed height /packed bed
height. After the desired compression is achieved, it is recommended to
flow condition the column with fresh packing or equilibration buffer for
3 column volumes (CV) in upflow followed by 3 CV in downflow at the
process flow rate. After flow conditioning, it is recommended to evaluate
column efficiency using your standard operating procedures or the
procedure described in Section 5.
Section 5 : Column Packing Evaluation
When column packing is complete, equilibrate the column with up to
5 CV equilibration buffer. To test the effectiveness of column packing,
inject a sample of a low molecular weight, unretained compound (for
example, acetone or 1 M NaCl). If acetone is used as the test marker (use
a UV absorbance monitor set at 280 nm), the equilibration buffer must
have a salt concentration <100 mM. If 1 M NaCl is the test marker (use
a conductivity monitor), then the equilibration buffer salt concentration
should be 100–200 mM. The sample volume should be 1–3% of the total
column volume. Column testing should be operated using the same linear
velocity used to load and/or elute the sample.
To obtain comparable height equivalent to a theoretical plate (HETP)
values among columns, the same conditions must be applied. Minimum
theoretical plate values should be 1,000–3,000 plates/m for linear
velocities of 50–500 cm/hr.
HETP = L/N
N = 5.54(Ve/W
½h
) 2
L = Bed height (cm)
N = Number of theoretical plates
Ve = Peak elution volume or time
W
½h
= Peak width at peak half height in volume or time
Ve and W
½h
should always be in the same units
Peaks should be symmetrical and the asymmetry factor as close as
possible to 1. Values of 0.8–1.8 are acceptable.
Peak asymmetry factor calculation:
As = b/a
a = Front section of peak width at 10% of peak height bisected by line denoting Ve
b = Latter section of peak width at 10% of peak height bisected by line denoting Ve
As = 0.8–1.8 is acceptable.
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