GE SP HP, Q HP User Manual

GE Healthcare

Instructions 71-7149-00 AN HiTrap ion exchange columns

HiTrap SP HP, 1 ml and 5 ml
HiTrap Q HP, 1 ml and 5 ml

HiTrap™ SP HP and HiTrap Q HP are prepacked, ready to use cation and anion
exchange columns for method scouting, group separations, sample concentration
and sample clean-up of charged biomolecules. HiTrap SP HP and HiTrap Q HP provide
fast, reproducible, and easy separations in a convenient format.

The columns can be operated with a syringe, peristaltic pump or liquid
chromatography system such as ÄKTAdesign

™

or FPLC™ System.

Code No. Designation No. supplied

17-1151-01 HiTrap SP HP 5 x 1 ml
17-1152-01 HiTrap SP HP 5 x 5 ml
17-1153-01 HiTrap Q HP 5 x 1 ml
17-1154-01 HiTrap Q HP 5 x 5 ml

Connectorkit

Connectors supplied Usage No. supplied

1/16” male/luer female Connection of syringe to top of
HiTrap column 1

Tubing connector Connection of tubing (e.g. Peristaltic
flangeless/M6 female Pump P1) to bottom of HiTrap column* 1

Tubing connector Connection of tubing (e.g. Peristaltic
flangeless/M6 male Pump P1) to top of HiTrap column** 1

Union 1/16” female/ Connection to original FPLC Sys tem
M6 male through bottom of HiTrap column 1
Union M6 female/ Connection to original FPLC Sys tem
1/16” male through top of HiTrap column 1

Stop plug female, 1/16” Sealing bottom of HiTrap column 2, 5 or 7

* Union 1/16” fe male/M6 male i s also needed .
** Union M6 fema le/1/16” male is als o needed.

Table of contents

1. Description 3

2. Selection of ion exchanger and conditions 5

3. Operation 9

4. Optimization 12

5. Choice of gradient type 13

6. Determination of binding capacity 15

7. Scaling up 17

8. Storage 17

9. Ordering Information 17

p. 2

1. Description

Media properties

SP Sepharose™ High Per formance and Q Sepharose High Performance
are strong cation and strong anion exchangers respectively. Both remain
charged and maintain high capacity over broad pH ranges. The functional
groups are coupled to the matrix via chemically stable ether linkages.
Characteristics of HiTrap SP HP and HiTrap Q HP, 1 and 5 ml columns are
listed in Table 1.

Column

HiTrap Q HP and HiTrap SP HP are 1 ml and 5 ml columns made of
polypropylene, which is biocompatible and non-interactive with
biomolecules. The top and bottom frits are manufactured from porous
polyethylene. It is delivered with a stopper on the inlet and a snap-of f end
on the outlet.

The separation can be easily achieved using a syringe together with the
supplied luer adaptor, a peristaltic pump, or in a chromatography system
such as ÄKTA

Note: To prevent leakage it is essential to ensure that the adaptor is tight.
The column cannot be opened or refilled.

™

or FPLC.

p. 3

Table 1. HiTrap SP HP and HiTrap Q HP columns characteristics

Column volumes 1 ml or 5 ml
Column dimensions 0.7 × 2.5 cm (1 ml) and 1.6 × 2.5 cm (5 ml)
Total ionic capacity 0.14–0.20 mmol (Cl

0.15–0.20 mmol (H

–

)/ml medium (Q)

+

)/ml medium (SP)
Dynamic binding capacity SP: approx. 55 mg ribonuclease/ml medium
(0.1 M sodium acetate, pH 6.0 at 1 ml/min)
Q: approx. 50 mg HSA/ml medium (20 mM Tris-HCl,
pH 8.2 at 1 ml/min)
Mean particle size 34 μm
Bead structure 6% highly cross-linked spherical agarose
Maximum flow rates HiTrap 1 ml: 4 ml/min, HiTrap 5 ml: 20 ml/min
Recommended f low rates HiTrap 1 ml: 1 ml/min, HiTrap 5 ml: 5 ml/min
Maximum backpressure 0.3 MPa, 3 bar, 42 psi
Chemical stability All commonly used buffers
Charged group SP: – CH
Q: – CH2N+(CH3)
pH stability*

2CH2CH2SO3

3

Sho rt term SP: 3–14, Q : 1–14
Working SP: 4–13, Q: 2–12
Long term SP: 4–13, Q: 2–12
Storage temperature +4° to +30 °C
Storage buffer SP: 20% ethanol, 0.2 M sodium acetate
Q: 20% ethanol
Avoid SP: Oxidizing agents, cationic detergents and
buffers
Q: Oxidizing agents, anionic detergents and
buffers

* Th e ranges given are estimates b ased on our knowledg e and experience.

Pleas e note the follow ing:

pH stability, long term refers to the pH i nterval where the medium is s table over

a long pe riod of time wit hout advers e eff ects on its subsequent chromatographic
performance.

pH stability, short term refers to the pH interval for regeneration, cleaning-in-place and

sanitization procedures.

p. 4

2. Selection of ion exchanger and conditions

Ion exchange chromatography is based on adsorption and reversible
binding of charged sample molecules to oppositely charged groups
attached to an insoluble matrix. The pH value at which a biomolecule carries
no net charge is called the isoelectric point (pI). When exposed to a pH below
its pI, the biomolecule will carry a positive charge and will bind to a cation
exchanger (SP). At pH’s above its pI the protein will carry a negative charge
and will bind to an anion exchanger ( Q). If the sample components are most
stable below their pI’s, a cation exchanger should be used. If they are most
stable above their pI´s, an anion exchanger is used. If stability is high over a
wide pH range on both sides of pI, either type of ion exchanger can be used
(Figure 1).

Selection of buff er pH and ionic strength

Buffer pH and ionic strength are critical for the binding and elution of
material (both target substances and contaminants) in ion exchange
chromatography. Selection of appropriate pH and ionic strength for
the start and elution buffers allows the use of three possible separation
strategies.

Fig 1. The net charge or a protein as a function of pH.

p. 5

Strategy 1. Binding and elution of all sample components

Binding is achieved by choosing a start buffer with a low pH for HiTrap SP
HP or a high pH for HiTrap Q HP. The ionic strenght should be kept as low as
possible to allow all components to bind to the ionic exchange (<5 mS/cm).
This results in a concentration of the target substance and a complete
picture of the whole sample. The drawback of this strategy is that the
binding capacity of the ion exchanger for the target substance is dependent
on the amount of contaminant in the sample. Strongly binding contaminants
can also displace bound target protein if a large volume of sample is loaded.

Note: Start conditions are subject to the stability of the sample

components.

Strategy 2. Enrichment of target protein

This is achieved by choosing a start buffer with a pH optimized to allow
maximal binding of target protein, and as high as possible ionic strength
to suppress binding of sample contaminants. This strategy results in a
concentration of the target substances.

Strategy 3. Binding of sample contaminants

This is achieved by choosing a start buffer with a pH and ionic strength
that promotes the binding of some or all contaminating substances but
allows the substance of interest to pass through the column. The drawback
of this approach is that the target substance is not concentrated and the
sample volume applied to the ion exchanger is dependent on the amount of
contaminants in the sample.

Start buff er

The concentration of buf fer required to give effective pH control varies
with the buffer system. A list of suitable buffers and suggested starting
concentrations is shown in Tables 2 and 3, Figs 2 and 3. In the majority of
cases a concentration of at least 10 mM is required to ensure adequate
buffering capacity. The ionic strength of the buf fer should be kept low
(< 5 mS/cm) so as not to interfere with sample binding. Salts also play a
role in stabilizing protein structures in solution and it is important the ionic
strength should not be so low that protein denaturation or precipitation
occurs.

p. 6