Bio-Rad UNO Monolith Cation Exchange Columns User Manual

UNO

™

Q&S R Continuous

Bed Ion Exchange

Replacement Column

Instruction Manual

Catalog Numbers

720-0011, 720-0013, 720-0015,

720-0031, 720-0033 and 720-0035

For Technical Service Call Your Local Bio-Rad Office or in the U.S. Call 1-800-4BIORAD (1-800-424-6723)

Introduction

UNO prepacked ion-exchange columns are designed to meet the needs of the bio-chro­matographer for rapid and reproducible high-resolution separations of biomolecules includ­ing proteins, peptides and polynucleotides. The UNO Replacement columns provide an
economical alternative to purchasing a new complete UNO column. The UNO Replacement
column hardware utilizes the existing hardware from your worn-out UNO column.

Installing the Replacement Column

Preparing the Used UNO Column (refer to Figure 1)

1. Remove the tube end fittings (2) from the UNO column end caps (3).

2. Remove one end cap (3) from the column and remove glass tube assembly (10) from the

clear shield (4).

3. Remove both tan bed supports (5) and spacers (7), if they are still attached, from the glass

tube and set aside.

4. Discard the glass tube filled with the used continuous bed support material in accordance

to local regulations.

Preparing the New UNO Replacement Column

1. Unthread the end caps (11) and remove UNO Replacement Column glass tube (16) from

the clear shield (12).

2. Remove the solid white plug (14) from both ends of the glass tube.

Assembling the New UNO Replacement Column

1. Each of the tan bed supports (5) from the used UNO column should have a 4 mm wide

brown spacer (7) located between the o-ring (6) and the top of the bed support. If the bed

support does not have the spacer, insert a new spacer, from the UNO Replacement col-

umn parts kit, onto the bed support (5).

2. Insert each tan bed support (5) with spacer (7) into each end of the UNO Replacement

Column glass tube (6). Push the bed supports into the column until they touch the frits (9).

3. Insert the UNO replacement column glass tube into the clear shield (4) and attach the

end caps (3) from the used UNO column onto the shield (4). Tighten the end caps.

4. Attach the tube end fittings (2) to the column and chromatography system and follow

equilibration protocol listed in the next section.

1

2

Fig. 1. Drawing of UNO column and UNO replacement column.

The columns are supplied in a storage buffer of 0.1 M NaCl + 20% ethanol. The counter
ion for the Q column is Cl-and for the S column is Na+. Prior to initial use and after extend­ed storage periods, each column should be conditioned as described below (steps 1–4). Always
use HPLC grade reagents and filter and degas the buffers. During this operation do not exceed
the following flow rates:

S-1 or Q-1 1 ml/min
S-6 or Q-6 2 ml/min
S-12 or Q-12 3 ml/min

1. Wash with 5 column volumes of water. Elevated backpressures may occur when wash-

ing with deionized water. Do not exceed 700 psi.

2. Wash with 5 column volumes of low ionic strength start buffer [e.g. 20 mM Tris-HCl

(Q) or 20 mM Sodium Phosphate(S)].

3. Wash with 5 column volumes of high ionic strength elution buffer (e.g. starting buffer +

1.0 M NaCl).

4. Wash with 5 column volumes of low ionic strength equilibration buffer [e.g. 20 mM

Tris-HCl (Q) or 20 mM Sodium Phosphate(S)].

The column may now be further equilibrated in start buffer at the desired flow rate.

Tubing, .020 I.D. x 15 cm (1)

Cap (11)

Shipping tube (12)

O-ring (13)
Plug (14)

Centering ring (15)

Glass column (16)

Centering ring (15)

Plug (14)
O-ring (13)

Cap (11)

Tube end fitting, 1/4 x 28 (2)

End cap (3)

Shield (4)

Bed support (5)
O-ring (6)

Spacer (7)
Distribution screen (8)
Frit (9)

Glass tube (10)

Frit (9)
Distribution screen (8)

Spacer (7)
O-ring (6)

Bed support (5)

End cap (3)

Tube end fitting, 1/4 x 28 (2)

Tubing, .020 I.D. x 15 cm (1)

UNO Column

UNO Replacement Column

Table 1. UNO Q Column Characteristics

Q-1 Q-6 Q-12

Column volume (ml) 1.3 6 12
Recommended max. protein loading (mg) 20 80 160
Recommended flow-rates (ml/min) 0.5 to 5.0 0.5 to 8.0 0.5 to 10.0
Column Dimensions (mm) 7 x 35 12 x 53 15 x 68
Maximum operating pressure (psi/MPa/bar) 700/4.5/48 700/4.5/48 700/4.5/48

Table 2. UNO S Column Characteristics

S-1 S-6 S-12

Column volume (ml) 1.3 6 12
Recommended max. protein loading (mg) 20 80 160
Recommended flow-rates (ml/min) 0.5 to 6.0 0.5 to 8.0 0.5 to 10.0
Column Dimensions (mm) 7 x 35 12 x 53 15 x 68
Maximum operating pressure (psi/MPa/bar) 700/4.5/48 700/4.5/48 700/4.5/48

Column Hygiene

Careful preparation (especially filtration) of the sample and the buffers will maintain the
column performance and lifetime. Normally, washing with 1.0 M NaCl or KCl will remove
most bound components. However, if there is a significant decrease in column performance
(i.e. increasing backpressures or a significant drop in resolution) then a more extensive clean-
ing protocol as described below (steps 1–6) should be used. Always reverse the flow during
this procedure so tightly-bound substances at the top of the column are quickly removed.

This process should be performed within the following flow rates.

S-1 or Q-1 1 ml/min
S-6 or Q-6 2 ml/min
S-12 or Q-12 3 ml/min

1. Wash with 2 column volumes of 2.0 M NaCl or KCl.

2. Wash with 1 column volume of 2.0 M NaOH followed by 3 column volumes of 0.1 M

NaCl.

3. Wash with 1 column volume of 50% acetic acid followed by 3 column volumes of 0.1 M

NaCl. Wash with 1 column volume of deionized water.

4. If lipid contamination is a problem, wash with 1 column volume of MeOH followed by

1 column volume of deionized water and 3 column volumes of 0.1 M NaCl.

5. Wash with 2 column volumes of 2.0 M NaCl or KCl or the salt containing the desired

counter-ion.

6. Change the top frit and wash with 3 column volumes of start buffer.

References

1. Kopaciewicz, W., Rounds, M. A., Fausnaugh, J. and Regnier, F. E. Retention Model for High-per-

formance Ion-Exchange Chromatography, J. Chromatography, 266, 3-21 (1983).

2. A Guide to the Properties and Uses of Detergents in Biology and Biochemistry. J. Neugebauer

(Calbiochem Biochemicals).

3

Product Information

Catalog
Number Product Description

720-0001 UNO Q-1 Column
720-0003 UNO Q-6 Column
720-0005 UNO Q-12 Column

720-0009 UNO Q Polishing Column
720-0011 UNO Q-1 R Column, replacement column

720-0013 UNO Q-6 R Column, replacement column
720-0015 UNO Q-12 R Column, replacement column

720-0021 UNO S-1 Column
720-0023 UNO S-6 Column
720-0025 UNO S-12 Column

720-0029 UNO S Polishing Column
720-0031 UNO S-1 R Column, replacement column

720-0033 UNO S-6 R Column, replacement column
720-0035 UNO S-12 R Column, replacement column

751-0091 Bio-Scale 2 Replacement Part Kit, includes 5 frits, 5 distribution

screens, 2 o-rings, 1 frit remover. Use this kit for the UNO S-1 or
Q-1 Column.

751-0095 Bio-Scale 10 Replacement Part Kit, includes 5 frits, 5 distribution

screens, 2 o-rings, 1 frit remover. Use this kit for the UNO S-6 or
Q-6 Column.

751-0097 Bio-Scale 20 Replacement Part Kit, includes 5 frits, 5 distribution

screens, 2 o-rings, 1 frit remover. Use this kit for the UNO S-12 or
Q-12 Column.

751-0099 Bio-Scale Fittings Kit, includes 2 Super Flangeless Nuts (1/4 x 28

threads) and 6 ferrules, 2 Flangeless M6 Nuts, 4 ferrules and 2
caps, 2 Fingertight II fittings (10-32 threads). Use this kit for any

UNO Column.
125-0561 Anion Exchange Standards, 6 pack
125-0562 Cation Exchange Standards, 6 pack

4

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