GE Ion Exchange Column Mono Q 5/50 GL User's Manual

GE Compact Excavator User's Manual - Ion Exchange Column Mono Q 5/50 GL.
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Mono Q 5/50 GL and
Mono S 5/50 GL

Instructions 71-5017-88 AC 

Ion Exchange Columns

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 distil ed water fol owed 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 TricornTM 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 -CH2-SO3

-

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) distil ed 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 wil  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. Il ustration of how to lock the upper adapter. The locking ring (black) must be 
in down position to prevent uncontrol ed  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.

5

4

6

7

8

9

10

11

pH

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

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