3.3. Packing Pilot-scale (>5 cm I.D.) or Manufacturing Columns
7. Thermal Stability and Storage
Please contact your local Pall representative or office.
4. Working Conditions and Basic Protocol
Temperature of use
2 to 30 °C
Storage temperature
2 to 8 °C
Storage solution between runs
Neutral buffer containing 1 M NaCl with
20% (v/v) ethanol
Instructions For Use
Caution
Product must never be frozen
Blue Trisacryl® M
Note
Product is shipped at ambient temperature
Dye-Affinity Chromatography Sorbent
4.1. Equilibration
1. After column packing, wash with 1.5 CV of a buffer of the same
composition as that chosen for equilibration, i.e., PBS.
2. Continue to equilibrate the column until the ionic strength and
pH of the buffer at both the outlet and the inlet of the column
are identical.
8. Ordering Information
Blue Trisacryl M Sorbent
4.2. Sample Application
1. Ensure the absence of bubbles in the sample.
Pack Size
Part Number
2. Inject the sample into the column through a pump and then
connect the pump to a buffer reservoir or to a gradient system.
5 mL
25896-051
25 mL
25896-045
3. Start the pump, the recorder and the elution gradient maker
simultaneously.
100 mL
25896-010
1L
25896-028
5L
25896-044
10 L
25896-036
4.3. Working Flow Rate
Dynamic binding capacity may slightly vary as a function of
residence time. For a 10 cm bed height column, use a flow rate
of 100 to 150 cm/hr, 6 to 4 minutes residence time.
Working flow rate should not be above packing flow rate
4.4. Choice of Elution Gradient and its Slope
Blue Trisacryl M AcroSep Column
Description
Part Number
Blue Trisacryl M, 1 mL, Dark Blue 5/pkg
25896-C001
For preliminary studies, make an ionic strength linear gradient using
sodium chloride up to 3 M in phosphate buffer.
After running, optimize the elution conditions, particularly the final
NaCl concentration. The elution can also be performed more
specifically by cofactor gradients (NAD, NADP) or with more eluents
such as ethylene glycol, urea, potassium isothiocyanate.
5. Regeneration and Cleaning
Regenerate the column with 2 to 4 CV of 3 M NaCl, followed by a
cleaning-in-place (CIP) with 5 CV of 0.1 M HCl or 10 mM NaOH at
room temperature. In some cases, 6 M urea or 50% ethylene glycol
can also be used.
After regeneration treatment, re-equilibrate Blue Trisacryl M sorbent
in the starting buffer.
6. Autoclaving
Equilibrate Blue Trisacryl M sorbent in a neutral buffer without any
phosphate and any other products unstable at high temperature.
Autoclave the sorbent at 121 °C for 20 minutes.
After sanitization, the column must be re-equilibrated in the normal
sterile pyrogen-free buffer.
For more information, please contact our technical services.
5
USD 2889a
Corporate Headquarters
Port Washington, NY, USA
+1 800 717 7255 toll free (USA)
+1 516 484 5400 phone
biopharm@pall.com e-mail
European Headquarters
Fribourg, Switzerland
+41 (0)26 350 53 00 phone
LifeSciences.EU@pall.com e-mail
Asia-Pacific Headquarters
Singapore
+65 6389 6500 phone
sgcustomerservice@pall.com e-mail
Visit us on the Web at
www.pall.com/biopharm
E-mail us at biopharm@pall.com
International Offices
Pall Corporation has offices and plants throughout the
world in locations such as: Argentina, Australia, Austria,
Belgium, Brazil, Canada, China, France, Germany, India,
Indonesia, Ireland, Italy, Japan, Korea, Malaysia, Mexico,
the Netherlands, New Zealand, Norway, Poland, Puerto
Rico, Russia, Singapore, South Africa, Spain, Sweden,
Switzerland, Taiwan, Thailand, the United Kingdom, the
United States, and Venezuela. Distributors in all major
industrial areas of the world. To locate the Pall office or
distributor nearest you, visit www.pall.com/contact.
The information provided in this literature was reviewed
for accuracy at the time of publication. Product data may
be subject to change without notice. For current information consult your local Pall distributor or contact Pall
directly.
© 2013, Pall Corporation. Pall,
, AcroSep
and Trisacryl are trademarks of Pall Corporation.
® indicates a trademark registered in the USA.
Filtration.Separation.Solution. is a service mark
of Pall Corporation.
5/13, GN13.8674
USD 2889a
USD 2889a
1. Product Description
Blue Trisacryl M sorbent is a dye-affinity chromatographic sorbent
used for the purification of a wide variety of enzymes and proteins
such as kinases, albumin, interferons, and some coagulation factors.
The basic matrix is the macroporous non-ionic Trisacryl GF2000 support
on which Cibacron blue 3GA has been covalently immobilized.
Blue Trisacryl M sorbent is available in a variety of package sizes, and
also in 1 mL AcroSep™ column for fast screening laboratory applications.
2. Properties
Particle size
Cibacron blue 3GA
107 Da
Exclusion limit
Capacity for human albumin
1
≥10 mg/mL
Thermal stability
2 to 121 °C
Working pH
4 to 10
Cleaning pH
1 to 12 (short term)
Working pressure at 100 cm/hr
Up to 3 bar (45 psi)
5. When the top of the bed stabilizes, stop the pump and
unscrew the O-ring seal. Position the adjustable piston
at the top of the packed sorbent by turning the screw-lock
mechanism, leaving no visible space between the frit and
packed sorbent at any point around the circumference.
Re-tighten the O-ring seal.
6. Operate the pump again and repeat the adjustment of
the piston until no visible space appears under flow
between the frit and the top bed.
3.2.4. Evaluating Column Performance
Column performance is evaluated by determining column
efficiency, expressed as either plates/meter (N/m), or HETP
(Height Equivalent to one Theoretical Plate). Additionally, the
asymmetry factor (AF) is calculated. Required formulas are
shown below.
Measurements are made as follows:
1. Equilibrate a column of 15 mm I.D. x 20 cm length such
as Pall LRC column 15/80-200, with packing buffer (PBS).
2. Inject 1% column volume (CV) of 5% acetone solution.
Apply a flow rate of 100 cm/hr. Record UV absorbance
at 280 nm.
To determine the packing performance, use the following
formulas:
3.2.2. Preparing Column and System for Packing
40 – 80 μm
Ligand
5. Gently agitate the slurry and let the sorbent settle. Remove
the supernatant and add 3 to 5 volumes of fresh packing
buffer.
6. Repeat step 4 a total of 3 times minimum.
7. Gently agitate the slurry, pour it into a measured cylinder
and allow the sorbent to settle.
8. Remove the supernatant and add a volume of packing
buffer equal to one-half the volume of settled sorbent.
A slurry of 50 % (v/v) – the concentration recommended
for packing – is obtained.
1
Determined in 25 mM phosphate sodium, 150 mM NaCl, pH 7.4 using
5 mg/mL protein, column dimensions: 16 mm I.D. x 6 cm bed height, flow
rate 25 cm/hr
3. Column Packing
3.1. General Considerations
Blue Trisacryl M sorbent is supplied as a slurry/suspension in 1 M
NaCl with 20% (v/v) ethanol as bacteriostatic.
1. Prime the solvent delivery system, lines and valves to
ensure that all air is displaced. Ensure that top and bottom
frits or nets are fully wetted and free of air. Fill the column
with packing buffer and operate the system over a range
of flow rate representative of values that will be used
during packing and anticipated chromatographic procedures. Record pressure associated with the empty column
and system (including detectors, etc.).
2. Determine pressure/flow characteristics for the empty
system, taking into account recommendations concerning
flow rate during packing and chromatographic operation.
3. Stop the pump, close the column outlet, and remove the
upper flow-adaptor or piston.
4. Open the outlet and drain buffer from the column, leaving
1 to 4 cm of buffer above the bottom frit.
Following completion of blank pressure/flow rate measurements, proceed with column packing as described in
Section 3.2.3.
l
l
3.2. Packing a Small Column (≤ 5 cm I.D.)
3.2.3. Packing the Column
3.2.1. Preparing the Sorbent Slurry
Do not use stainless steel paddles or magnetic stirrers
which may damage the beads and create fine particles
1. Wash with 5 column volumes (CV) of 1 M NaCl.
2. Gently agitate the container to fully suspend the sorbent.
3. Depending on the desired bed volume, transfer a suitable
volume of slurry to a graduated beaker including a
“practical” excess of sorbent (20 to 25% of the desired
bed volume are recommended).
4. Allow the sorbent to settle and remove the supernatant.
Add 3 to 5 volumes of packing buffer, e.g. PBS, pH 7.4.
2
USD 2889a
With: a = First half peak width at 10% peak height
b = Second half peak width at 10% peak height
1. Gently resuspend the slurry and pour it into the column in
one continuous motion against the wall of the glass tube
to minimize introduction of air bubbles (if the pouring
process is done in several motions, gently homogenize
the slurry in the column using a plastic rod).
2. Fill with packing buffer up to the top of the glass tube.
Allow the suspension to settle so that a layer of clear
supernatant ≤ 1cm is visible at the top of the column.
3. For column such as Pall LRC column, connect the upper
adjustable piston to the system and prime with packing
buffer to ensure that no air is trapped under the frit. Stop
the pump and insert the adjustable piston into the column.
First tighten the O-ring seal and then engage the screwlock mechanism.
4. Open the column outlet and operate the pump at a
selected linear velocity of 100 cm/hr (~1.31 mL/min for a
1 cm I.D. column) for efficient packing; in any case, flow
rate must not be lower than 150 cm/hr for guaranteeing
high packing performance.
3
Figure 1
Peak Trace in a Typical Test Evaluation of Column Performance
Absorbance / Conductivity
Blue Trisacryl M sorbent is compatible with traditional low or
medium pressure chromatography columns and equipment. For
preliminary laboratory studies, a column equipped with an adjustable
piston such as Pall LRC column 10/80-200 of 1 cm I.D. x 20 cm
length (see Pall brochure USD 2480) facilitates optimal packing.
Direct scale-up is accomplished by maintaining bed height constant
while diameter is increased.
2
N/m = 5.54 x 100 x (Ve / W½)
BH
With: N = Number of theoretical plates
Ve = Elution volume on the chromatogram (cm)
W½= Width of the acetone peak at half of the height (cm)
BH = Bed height (cm)
b
AF =
a
W½
100%
Injection
Ve
50%
a
b
10%
Volume / Time
Typical values for Blue Trisacryl M sorbent N/m at 100 cm/hr are
≥ 2,500 plates/m after packing in PBS, pH 7.4. Typical values for
AF at 100 cm/hr range from 0.8 and 1.8 in equilibration buffer.
These values are given as the average of experimental values.
More important than the values by itself, the reproducibility of
the values over the successive packing operations is critical.
USD 2889a
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