GE Compact Excavator Mono Q 5 50 GL User Manual

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 rst  
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  
Buers 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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