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Scaling-up sample volumes using
HiPrep 26/10 Desalting
A. Heijbel
Amersham Biosciences, Uppsala, Sweden
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HiPrep 26/10 Desalting column is packed with
Sephadex™ G-25 Fine for rapid, reproducible and easy
separations. The cross-linked dextran beads that comprise this medium allow for excellent resolution and high
flow rates. Globular proteins with molecular weights
between 1 000–5 000 fall within the fractionation range
of the matrix. The Mr 5 000 exclusion limit ensures that
proteins/peptides larger than Mr 5 000 can be separated
from molecules, such as salts, whose molecular weights
are less than Mr 1 000. Other characteristics of the
HiPrep 26/10 Desalting column are listed in Table 1.
HiPrep™ 26/10 Desalting is an excellent alternative to
dialysis, especially when larger sample volumes are used
or when sample degradation may occur with time. By
coupling four columns in series, 60 ml of sample can be
processed in 20–30 min, with high flow rates and
without encountering back pressure problems.
Buffer exchange and desalting are common operations
in any laboratory engaged in sample purification and
analysis. It is often necessary to change the buffer
composition of a sample between chromatography
steps or to satisfy special requirements of an assay.
Desalting of a sample is a prerequisite for mass spectroscopy analysis, lyophilization, and after certain
procedures such as ion exchange chromatography.
Table 1. Characteristics of HiPrep 26/10 Desalting
Although both procedures can be accomplished by
dialysis, this is a time-consuming process, and samples
sensitive to degradation may be at risk. Because of its
high speed and high volume capacity, HiPrep 26/10
Desalting is an excellent alternative to dialysis, especially
when larger sample volumes are used or when samples
need to be processed rapidly to avoid degradation.
Samples of 15 ml (30% of the total bed volume) or less
can be applied to a single column, and by coupling up
to four columns in series (Fig 1), a maximum sample
volume of 60 ml can be used. Even with four columns
in series, high flow rates can be maintained without back
pressure problems, resulting in fast separations. In fact,
in 20–30 min, up to 60 ml of sample can be desalted or
buffer-exchanged.
matrix
Sephadex G-25 Fine,
cross-linked dextran
particle mean diameter
90 µm
exclusion limit for
proteins/peptides (Mr)
5 000
bed volume
53 ml
column dimensions (i.d. × h)
2.6 × 10 cm
recommended sample volume
≤ 15 ml
maximum flow rate*
40 ml/min (450 cm/h)
recommended flow rate*
9-31 ml/min (100–350 cm/h)
maximum pressure drop, ∆p
1.5 bar (22 psi, 0.15 MPa)
chemical stability
all common aqueous buffers
pH stability
2–13 (long- and short-term)
storage
20% ethanol
avoid
oxidizing agents
*Water at room temperature
Fig 1. Four HiPrep 26/10
Desalting columns
connected in series.
This configuration enables
60 ml of sample to be
processed using high flow
rates without encountering
high back pressures.
Table 2 shows how sample volumes can be scaled up
using HiPrep 26/10 Desalting columns in series.
Samples consisted of either 30 or 60 ml of a fungal
culture supernatant, containing a secreted recombinant protein. Samples were run on two (30 ml sample)
or four (60 ml sample) columns connected in series.
In addition to HiPrep 26/10 Desalting, Amersham
Biosciences offers a range of columns for
desalting and buffer exchange that can accommodate
variations in sample size and yield acceptable levels of
dilution. These are summarized in Table 3.
Life Science News 2, 1999 Amersham Biosciences
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Table 2. Run data for buffer exchange of 30 ml and 60 ml samples on
HiPrep 26/10 Desalting columns connected in series
columns
sample
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Table 3. Summary of desalting/buffer exchange columns
two HiPrep 26/10 Desalting in
series (VT = 106 ml) for 30 ml
sample or 4 × HiPrep 26/10 Desalting
in series (VT = 212 ml) for 60 ml sample
applied
volume
(ml)
column
HiPrep 26/10
Desalting
collected
volume
(ml)
dilution
factor
operation
10
15 (max)
10–15
15–20
1–1.5
1–1.3
pump
pump
30 ml or 60 ml Pichia pastoris culture
supernatant containing a secreted
recombinant protein
2 × HiPrep 26/10
Desalting
30 (max)
30–40
1–1.3
pump
3 × HiPrep 26/10
Desalting
45 (max)
45–55
1–1.2
pump
sample preparation
filter through 0.45 µm filter
sample loop
Superloop™ 150 ml
buffer
0.1 M Tris, 0.15 M NaCl,
0.05% Tween 20, pH 7.6
flow rates (30 ml)
sample loading
elution
12 ml/min
17 ml/min
flow rates (60 ml)
sample loading
elution
8 ml/min
11 ml/min
back pressure
4.8 bar (70 psi, 0.48 MPa)
fractions
5 ml
instrument
ÄKTA™explorer 100
eluted sample
30 ml
60 ml
35 ml (dilution factor, 1.2)
70 ml (dilution factor, 1.2)
U
4 × HiPrep 26/10
Desalting
60 (max)
60–70
1–1.2
pump
HiTrap™ Desalting
0.25
0.5
1.0
1.5 (max)
1.0
1.5
2.0
2.0
4
3
2
1.3
syringe/pump
syringe/pump
syringe/pump
syringe/pump
2 × HiTrap Desalting
3.0 (max)
4–5
1.3–1.7
syringe/pump
3 × HiTrap Desalting
4.5 (max)
6–7
1.3–1.7
syringe/pump
NAP™-5
0.1
0.25
0.5 (max)
1.0
1.0
1.0
10
4
2
gravity
gravity
gravity
NAP-10
0.75
1.0 (max)
1.5
1.5
2
1.5
gravity
gravity
PD-10
1.5
2.0
2.5 (max)
3.5
3.5
3.5
2.3
1.8
1.4
gravity
gravity
gravity
0.5
1–2
2–4
pump
Fast Desalting
HR 10/10
ORDERING INFORMATION
HiPrep 26/10 Desalting
1
17-5087-01
HiTrap Desalting
5 × 5 ml
17-1408-01
NAP-5 Columns
20
50
17-0853-01
17-0853-02
NAP-10 Columns
20
50
17-0854-01
17-0854-02
PD-10 Columns
30
17-0851-01
Fast Desalting Column HR 10/10
1
17-0591-01
Superloop (150 ml)
1
18-1023-85
Life Science News 2, 1999 Amersham Biosciences
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