GE Mono Q 5/50 GL, Mono S 5/50 GL Cleaning, Ordering information

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GE Healthcare Europe GmbH

Munzinger Strasse 5, D-79111 Freiburg, Germany

GE Healthcare UK Ltd

Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UK

GE Healthcare Bio-Sciences Corp

800 Centennial Avenue, P.O. Box 1327, Piscataway, NJ 08855-1327, USA

GE Healthcare Bio-Sciences KK

Sanken Bldg, 3-25-1, Hyakunincho, Shinjuku-ku, Tokyo 169-0073, Japan

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www.gehealthcare.com/protein-puriﬁ cation

www.gehealthcare.com

GE Healthcare Bio-Sciences AB

Björkgatan 30

751 84 Uppsala

Sweden

71-5017-88 AC 03/2006

Tricorn, Mono Q, Mono S, HiTrap, ÄKTA and Drop Design are trademarks of GE Healthcare

companies. GE and GE Monogram are trademarks of General Electric Company.

The Tricorn column and components are protected by US design patents USD500856, USD506261,

USD500555, USD495060 and their equivalents in other countries.

All goods and services are sold subject to the terms and conditions of sale of the company within

GE Healthcare which supplies them. General Electric Company reserves the right, subject to any

regulatory and contractual approval, if required, to make changes in speciﬁ cations and features

shown herein, or discontinue the product described at any time without notice or obligation.

GE Healthcare Bio-Sciences AB, a General Electric Company.

Optimization

Perform a first run as described in the section “Try these conditions first”. If the

results obtained are unsatisfactory, consider the following:

Action Eﬀ ect

Change pH/buffer salt (see Changes selectivity, gives

Figure 1 and Figure 2 for buffers) weaker/stronger binding.

Change salt, counter ions Changes selectivity.

and/or co-ions

Decrease the sample load Improves resolution.

Decrease the flow rate Improves resolution.

Change gradient slope Shallower gradients improve selectivity but

broaden peaks (decrease efficiency).

A steeper gradient will sharpen peaks, but

move them closer together.

For more information, please refer to the handbook “Ion exchange

chromatography, Principles & Methods”, which can be ordered from GE Healthcare

or downloaded from our web site.

Cleaning

It is recommended to reverse the direction of flow during column cleaning so that

contaminants do not need to pass through the entire length of the column.

Regular cleaning

Flow: 0.5 ml/min at room temperature

1. Wash with 2 column volumes (CV) of 2 M NaCl.

2. Wash with 4 CV of 1 M NaOH

3. Wash with at least 2 CV of 2 M NaCl

4. Rinse with at least 2CV of distilled water until the UV-baseline and the eluent

pH are stable.

4. Wash with at least 4 CV of start buffer or storage buffer until pH and

conductivity values have reached the required values.

More rigorous cleaning

Remove strongly hydrophobically bound proteins, lipoproteins and lipids by

washing with 4 column volumes (CV) of 30% isopropanol or 70% ethanol at

0.25 ml/min. Remove precipitated proteins with 1 CV of 1 mg/ml pepsin in

0.5 M NaCl, 0.1 M acetic acid (leave overnight) or wash with 2 CV of 6 M Guanidine

hydrochloride at 0.25 ml/min.

Depending on the nature of contaminant cleaning solution in the section ”Buffers

and solvent resistance” may be appropriate. After cleaning the column wash

with at least 2 CV of distilled water and 4 CV of start buffer or storage buffer. For

more information on how to clean your column, please refer to the handbook ”Ion

exchange chromatography & Chromatofocusing, Principles & Methods”.

As an alternative to more rigorous cleaning or if column performance still not

restored change the filter at the top of the column. (Since contaminants are

introduced with the liquid flow, many of them are caught by the filter.) Instructions

for changing the filter are supplied with the Filter Kit. Clean the column after filter

change according to regular cleaning.

Troubleshooting

Symptom Remedy

Increased back-pressure Reverse the flow direction and pump 5 ml elution

over the column buffer at a flow rate of 0.5 ml/min through the

column. Return to normal flow direction and run for

5 minutes at 1 ml/min through the column. If high

backpressure persists, clean the column.

Loss of resolution and/or Clean the column according to the procedure

decreased sample recovery described in the section “More rigorous cleaning”.

Air in the column Reverse the flow direction and pump 10 ml well de-

gassed start buffer through the column at a flow

rate of 0.5 ml/min.

Column performance control

Check the performance of the column when new by running the separation

described in Figures 4 and 5. Figures 4 and 5 shows a typical chromatogram run

on an optimized system. Since the system can profoundly affect the resolution,

it is meaningful to compare runs done on the same system. Check the column at

regular intervals and whenever you suspect a problem.

Function test of Mono Q 5/50 GL

Sample: 1. Conalbumin (3 mg/ml)

2. α-lactalbumin, bovine milk (4 mg/ml)

3. Soybean trypsin inhibitor (6 mg/ml)

Sample volume: 200 μl

Gradient: 0–100% elution buffer in 20 CV

Start buffer: 20 mM Tris-HCl, pH 7.0

Elution buffer: 20 mM Tris-HCl + 0.25 M NaCl, pH 7.0

Flow rate: 1.0 ml/min (room temperature)

Function test of Mono S 5/50 GL

Sample: 1. Ribonuclease A (1.5 mg/ml)

2. Cytochrome C (0.4 mg/ml)

3. Lysozyme (0.4 mg/ml)

Sample volume: 100 μl

Gradient: 0–100% elution buffer in 20 CV

Start buffer: 20 mM sodium phosphate, pH 6.8

Elution buffer: 20 mM sodium phosphate + 0.4 M NaCl, pH 6.8

Flow rate: 1.0 ml/min (room temperature)

mAU (280 nm) % Elution Buffer

200

100

150

100

0.0 5.0 10.0 15.0 20.0

Fig 4. Typical chromatograms from a function test of Mono Q 5/50 GL.

Fig 5. Typical chromatograms from a function test of Mono S 5/50 GL.

mAU (280 nm) % Elution Buffer

200

100

150

100

0.0 5.0 10.0 15.0 20.0

Ordering information

Product No. per pack Code No.

Mono Q 5/50 GL 1 17-5166-01

Mono S 5/50 GL 1 17-5168-01

Related products

Product No. per pack Code No.

Mono Q 10/100 GL 1 17-5167-01

Mono Q 4.6/100 PE 1 17-5179-01

Mono S 10/100 GL 1 17-5169-01

Mono S 4.6/100 PE 1 17-5180-01

HiTrapTM Desalting 5 × 5 ml 17-1408-01

Accessories

Product No. per pack Code No.

Tubing connectors:

Fingertight connector 1/16” male 10 18-1112-55

Tricorn 5 filter kit* 1 18-1153-02

Filter Tool 1 18-1153-20

Union M6 female/1/16” male 8 18-1112-58

On-line filter (1/16”) 1 18-1118-01

Handbook:

Ion Exchange Chromatography & Chromatofocusing,

Principles and Methods 1 11-0004-21

* includes top and bottom filters and O-rings, 5 of each.

Elanders Östervåla 2006