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Mono Q 5/50 GL and
Mono S 5/50 GL
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Instructions 71-5017-88 AC Ion Exchange Columns
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GE Healthcare
Tricorn
™
Sample recommendations
Net charge of target molecule negative (Mono Q), positive (Mono S)
Recommended initial sample load ≤ 45 mg
Preparation Dissolve the sample in start buffer,
filter through a 0.22 μm filter or
centrifuge at 10 000 × g for 10 min
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.
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.
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.
Quick information
Mono Q™ 5/50 GL and Mono S
TM
5/50 GL are Tricorn
TM
high performance columns.
The columns are pre-packed glass columns for high performance ion exchange
chromatography of proteins, peptides, polynucleotides and other biomolecules.
The columns are supplied with two union M6 female/1/16” male for connection
to FPLC
TM
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.
Column data
Matrix Polystyrene/divinyl benzene
Bead form Rigid, spherical, porous monodisperse
Particle size 10 μm
Column dimensions 5 × 50 mm
Bed volume 1 ml
Average loading capacity 50 mg
(will vary depending on sample and loading conditions)
pH stability
regular use 2–12
cleaning 1–14
Temperature
operating 4 to 40 ºC
Flow rate (water at room temperature)
recommended 0.5–3.0 ml/min
maximum 3 ml/min
Pressure over column
maximum 4 MPa, 40 bar, 580 psi
Mono Q Mono S
Type of exchanger Strong anion Strong cation
Charged group -CH
2
-N
+
(CH
3
)
3
-CH
2
-SO
3
-
Ionic capacity 0.27–0.37 mmol 0.12–0.15 mmol
Cl
-
/ml medium H
+
/ml medium
Note: Before connecting the column to a chromatography system, start the pump and remove all air
and debris in the system, particularly in the tubing and valves.
First-time use
Equilibrate the column for first-time use or after long-term storage as follows:
a) 5 column volumes (CV) distilled water at 1 ml/min at room temperature.
b) 5 CV start buffer at 2 ml/min at room temperature.
c) 5 CV elution buffer at 2 ml/min at room temperature.
d) 5 CV start buffer at 2 ml/min at room temperature.
Try these conditions fi rst
Start buffer (Mono Q)*: 20 mM Tris-HCl, pH 8.0
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 ÄKTA
TM
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
1. Equilibrate column with 5–10 column volumes (CV) of start buffer or until
baseline, eluent pH and conductivity are stable.
2. Adjust the sample to the chosen starting pH and ionic strength and apply to
the column (see sample recommendations).
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.
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).
5. Wash with 2–5 CV of 1 M NaCl (100% elution buffer) to elute any remaining
ionically-bound material.
6. Requilibrate with at least 5–10 CV of start buff er or until eluent pH and
conductivity reach the required values.
Read the section ”Optimization” for information about how to optimize a
separation.
Buff ers and solvent resistance
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.
Daily use
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
Cationic detergents (Mono Q)
Anionic detergents (Mono S)
Cleaning
Acetonitrile, up to 100%
Sodium hydroxide, up to 2 M
Ethanol, up to 100%
Methanol, up to 100%
Acetic acid, up to 75%
Isopropanol, up to 100%
Hydrochloric acid, up to 1 M
1% Trifluoroacetic acid
Avoid:
Oxidizing agents
Anionic detergents (Mono Q)
Cationic detergents (Mono S)
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.
Table 1 lists suggested volatile buffers that can be used in cases where the purified
substance has to be freeze-dried.
Table 1. Volatile buffer systems.
pH Substance
3.3–4.3; 4.8–5.8 Pyridine/formic acid
3.3–4.3; 9.3–10.3 Trimethylamine/formic acid
4.3–5.8 Pyridine/acetic acid
3.3–4.3; 8.8–9.8 Ammonia/formic acid
4.3–5.3; 8.8–9.8 Ammonia/acetic acid
5.9–6.9; 9.3–10.3 Trimethylamine/carbonate
5.9–6.9; 8.8–9.8 Ammonium carbonate/ammonia
4.3–5.3; 7.2–8.2 N-ethylmorpholine/acetate
Fig 2. Recommended buffers for anion exchange chromatography.
Fig 3. Recommended buffers for cation exchange chromatography.
5467891011pH
4.75
5.33
6.48
6.65; 9.10
7.76
8.07
8.52
8.88
9.50
9.73
10.55
11.12
Piperazine
bis-Trispropane
Triethhanolamine
Tris
N-methyldiethanolamine
Propane-1,3-diamino
Ethanolamine
Piperazine
Propane-1,3-diamino
Piperidine
pKa
(25 ˚C)
N-methyl piperazine
bis-Tris
3.13
3.86
4.21
4.75
5.76
6.27
7.20
7.56
8.33
pH 2.5 3 4 5 6 7 8 9
pKa
(25°C)
Citric acid
Lactic acid
Butanedioic acid
Acetic acid
Methyl Malonic acid
MES
Phosphate
HEPES
BICINE