First-time use
GE Healthcare
Sample recommendations
Net charge of target molecule
Recommended initial sample load
Preparation
Equilibrate the column for first-time use or after long-term storage as follows:
negative (Mono Q), positive (Mono S)
a)
b)
c)
d)
5 column volumes (CV) distilled water at 1 ml/min at room temperature.
5 CV start buffer at 2 ml/min at room temperature.
≤ 45 mg
Instructions 71-5017-88 AC
Ion Exchange Columns
Dissolve the sample in start buffer,
filter through a 0.22 μm filter or
centrifuge at 10 000 × g for 10 min
5 CV elution buffer at 2 ml/min at room temperature.
5 CV start buffer at 2 ml/min at room temperature.
Mono Q 5/50 GL and
Mono S 5/50 GL
Try these conditions first
Start buffer (Mono Q)*: 20 mM Tris-HCl, pH 8.0
In-depth information
Delivery/storage
The column is delivered in degassed 20% ethanol sealed with two stop plugs to
prevent the column from drying out. For column storage, wash with 5 column
volumes of distilled water followed by 5 column volumes of 20% ethanol. Degas
the ethanol/water mixture thoroughly and apply at a low flow rate to avoid over-
pressuring the column. Store at room temperature or, for long periods, store at +4°
C to +8 °C. Ensure that the column is sealed well to avoid drying out. Do not freeze.
Elution buffer (Mono Q)*: 20 mM Tris-HCl + 1.0 M NaCl, pH 8.0
Start buffer (Mono S)*: 20 mM 2-[N-morpholino] ethanesulphonic acid (MES), pH 6.0
Elution buffer (Mono S)*: 20 mM MES + 1.0 M NaCl, pH 6.0
*
Users of ÄKTATM design system may select one of the buffer recipes recommended for anion
exchange chromatography at pH 8 or cation exchange chromatography at pH 6.
Separation by gradient elution
Flow: 2 ml/min at room temperature
Choice of eluent
To avoid local disturbances in pH caused by buffering ions participating in the ion
exchange process, select an eluent with buffering ions of the same charge as the
substituent groups on the ion exchanger.
1. Equilibrate column with 5–10 column volumes (CV) of start buffer or until
baseline, eluent pH and conductivity are stable.
Quick information
Mono Q™ 5/50 GL and Mono STM 5/50 GL are TricornTM high performance columns.
The columns are pre-packed glass columns for high performance ion exchange
chromatography of proteins, peptides, polynucleotides and other biomolecules.
2. Adjust the sample to the chosen starting pH and ionic strength and apply to
the column (see sample recommendations).
Choose the start buffer pH so that substances to be bound to the ion exchanger
are charged, e.g. at least 1 pH unit above the isoelectric point for anion exchangers
and at least 1 pH unit below the isoelectric point for cation exchangers. Figure 2
and Figure 3 list a selection of standard aqueous buffers.
3. Wash with 5–10 CV of start buffer or until the baseline, the eluent pH and the
conductivity are stable i.e. when all unbound material has washed through
the column.
The columns are supplied with two union M6 female/1/16” male for connection
to FPLCTM System, two fingertight connector 1/16” for connecting 1/16” tubing
to column and ÄKTA, two stop plugs 1/16” male to seal the column (attached to
column when delivered) and instruction.
4. Begin elution using a gradient volume of 10–20 CV and an increasing ionic
strengt up to 0.5 M NaCl (50% elution buffer).
pKa
pH
4
5
6
7
8
9
10
11
(25 ˚C)
Column data
5. Wash with 2–5 CV of 1 M NaCl (100% elution buffer) to elute any remaining
ionically-bound material.
N-methyl piperazine
Piperazine
4.75
5.33
6.48
Matrix
Polystyrene/divinyl benzene
Rigid, spherical, porous monodisperse
10 μm
bis-Tris
6. Requilibrate with at least 5–10 CV of start buff er or until eluent pH and
conductivity reach the required values.
Bead form
Particle size
6.65; 9.10
bis-Trispropane
Triethhanolamine
Tris
7.76
8.07
8.52
8.88
9.50
9.73
N-methyldiethanolamine
Read the section ”Optimization” for information about how to optimize a
separation.
Column dimensions
Bed volume
5 × 50 mm
1 ml
Propane-1,3-diamino
Ethanolamine
Piperazine
Propane-1,3-diamino 10.55
Average loading capacity
50 mg
Piperidine
11.12
Buffers and solvent resistance
(will vary depending on sample and loading conditions)
Recommended to have an on-line filter upstream of the injection valve. Buffers
and solvents with increased viscosity will affect the back-pressure and flow rate.
De-gas and filter all solutions through a 0.22 μm filter.
pH stability
regular use
cleaning
Fig 2. Recommended buffers for anion exchange chromatography.
2–12
1–14
▼
▼
▼
pKa
Temperature
operating
Daily use
pH 2.5
3
4
5
6
7
8
9
(25 °C)
4 to 40 ºC
All commonly used aqueous buffers, pH 2–12
Urea, up to 8 M
Guanidine hydrochloride, up to 6 M
Acetonitrile, up to 30% in aqueous buffers
Non-ionic detergents
Flow rate (water at room temperature)
recommended
maximum
Citric acid
3.13
3.86
4.21
4.75
5.76
6.27
7.20
7.56
8.33
0.5–3.0 ml/min
3 ml/min
Lactic acid
Butanedioic acid
Acetic acid
Pressure over column
maximum
Methyl Malonic acid
MES
4 MPa, 40 bar, 580 psi
Phosphate
Mono Q
Strong anion
-CH2-N+(CH3)3
Mono S
Strong cation
Cationic detergents (Mono Q)
Anionic detergents (Mono S)
HEPES
Type of exchanger
Charged group
Ionic capacity
BICINE
-
-CH2-SO3
Fig 3. Recommended buffers for cation exchange chromatography.
0.27–0.37 mmol
Cl-/ml medium
0.12–0.15 mmol
H+/ml medium
Cleaning
Acetonitrile, up to 100%
Sodium hydroxide, up to 2 M
Ethanol, up to 100%
Note: Before connecting the column to a chromatography system, start the pump and remove all air
Table 1 lists suggested volatile buffers that can be used in cases where the purified
substance has to be freeze-dried.
and debris in the system, particularly in the tubing and valves.
Methanol, up to 100%
Acetic acid, up to 75%
Isopropanol, up to 100%
Hydrochloric acid, up to 1 M
1% Trifluoroacetic acid
Table 1. Volatile buffer systems.
pH
Substance
3.3–4.3; 4.8–5.8
3.3–4.3; 9.3–10.3
4.3–5.8
Pyridine/formic acid
Trimethylamine/formic acid
Pyridine/acetic acid
Avoid:
3.3–4.3; 8.8–9.8
4.3–5.3; 8.8–9.8
5.9–6.9; 9.3–10.3
5.9–6.9; 8.8–9.8
4.3–5.3; 7.2–8.2
Ammonia/formic acid
Oxidizing agents
Anionic detergents (Mono Q)
Cationic detergents (Mono S)
Ammonia/acetic acid
Trimethylamine/carbonate
Ammonium carbonate/ammonia
N-ethylmorpholine/acetate
Fig 1. Illustration of how to lock the upper adapter. The locking ring (black) must be
in down position to prevent uncontrolled adjustment of the column’s bed height.
™
Tricorn
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