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Column Selection for Biomolecule Analysis by IEX

Most column manufacturers now offer a range of columns specifically designed for the analysis of biomolecules, such as monoclonal antibodies, peptides and proteins, oligonucleotides, and polynucleotides.  These columns are often constructed from PEEK instead of stainless steel. 

Biocolumns are specifically designed with larger pore sizes which are compatible with the larger molecule sizes found with biomolecules.  The packing material is designed to minimize non-specific binding of analytes which will result in better recoveries, for example, by the use of hydrophilic coatings.  It is also recommended that a metal free system is used, as halide-salt based mobile phases can cause corrosion of metal components.  Metal leaching from the system will lead to decreased column performance due to metal contamination. 

Therefore, most manufacturers now offer bio-inert HPLC and UHPLC systems which comprise a metal free sample flow path.

Impact of Stationary Phase

The selection of stationary phase and mobile phase pH strongly depends on the species being analyzed.  The conditions have to be selected such that the protein and the stationary phase are in their charged forms (Figure 1).

IEX stationary phase selection

Figure 1: IEX stationary phase selection.


Phase Chemistries

Ion exchange sorbents contain a variety of functional moieties.  Strong ion exchange materials contain functional groups which are charged over a wide pH range, whereas weak ion exchange materials comprise functional groups which lose their charge as the pH changes. 
Weak anion exchangers function poorly above pH 9 and weak cation exchangers begin to lose their ionization below pH 6.  When working with weak ion exchange resins it is important to work within the supplier-provided working pH range.  The number of charges on a strong ion exchanger remains constant regardless of the buffer pH.  These phases retain their selectivity and capacity over a wide pH range. 
Strong ion exchangers are often preferred for many applications because their performance is unaffected by pH.  Method development always begins using strong ion exchange columns, allowing a wide range of pH values to be assessed; however, weak ion exchangers can then be incorporated to provide different selectivity.

As with other HPLC columns, parameters such as maximum temperature, pressure, and pH range should be taken into account (Table 1).

 
Packed
Column
Name
Chemistry Particle Size/ Macropore Size Max Temperature (°C) pH Range Max Pressure (bar)
Protein-Pak Hi Res Q (Waters) Quaternary ammonium 5 60 3-10 150
Bio-Pro QA (YMC) Quaternary ammonium 4 60 2-12 30
Bio-Pro QA-F (YMC) Quaternary ammonium 4 60 2-12 120
 
Monolith
Column
Name
Chemistry Particle Size/ Macropore Size Max Temperature (°C) pH Range Max Pressure (bar)
Proswift SAX-1S (Thermo) Quaternary amine Info not available 70 2-12 70
TSKgel Q-STAT (Tosoh) Quaternary ammonium 7
10
60 3-10 50
Packed
Column
Name
Chemistry Particle Size/ Macropore Size Max Temperature (°C) pH Range Max Pressure (bar)
Poly WAX LP (PolyLC) Polyethyleneimine 5 Ambient Info not available Info not available
 
Monolith
Column
Name
Chemistry Particle Size/ Macropore Size Max Temperature (°C) pH Range Max Pressure (bar)
Proswift WAX-1S (Thermo) Tertiary amine Info not available 60 2-12 70
Packed
Column
Name
Chemistry Particle Size/ Macropore Size Max Temperature (°C) pH Range Max Pressure (bar)
Protein-Pak Hi Res SP (Waters) Sulfopropyl 7 60 3-10 100
MAbPac SCX-10 QA (Thermo) Sulfonic acid 3
5
10
60 2-12 480
480
200
Bio-Pro SP (YMC) Sulfopropyl 4 60 2-12 30
Bio-Pro SP-F (YMC) Sulfopropyl 4 60 2-12 120
 
Monolith
Column
Name
Chemistry Particle Size/ Macropore Size Max Temperature (°C) pH Range Max Pressure (bar)
Proswift SCX-1S (Thermo) Polymethacrylate Info not available 60 2-12 70
Proswift SCX (Thermo) Sulfonic acid 5 45 2-14 70
TSKgel SuperQ-5PW (Tosoh) Trimethylamine 10 45 2-12 50
TSKgel SP-STAT (Tosoh) Sulfopropyl 7
10
60 3-10 50
Packed
Column
Name
Chemistry Particle Size/ Macropore Size Max Temperature (°C) pH Range Max Pressure (bar)
Bio Mab (Agilent) Carboxylate 1.7
3
5
10
80 2-12 270
410
550
680
Antibodix
(Supelco, Sepax)
Carboxylate 1.7
3
5
10
80 2-12 270
410
550
680
Protein-Pak Hi Res CM (Waters) Carboxymethyl 7 60 3-10 100
Poly CAT A (PolyLC) Polyaspartic acid 5 Ambient Info not available Info not available
 
Monolith
Column
Name
Chemistry Particle Size/ Macropore Size Max Temperature (°C) pH Range Max Pressure (bar)
Proswift WCX-1S (Thermo) Carboxylic acid Info not available 60 2-12 70

Table 1: Commercially available IEX columns. 
SAX = strong anion exchange   |   WAX = weak anion exchange   |   WCX = weak cation exchange   |   SCX = strong cation exchange


Solid Support

Commercially available ion exchange columns are based on silica or polymer particles.   Polymers employed are normally poly(styrene divinylbenzene) (PS/DVB), which often have a hydrophilic coating to suppress secondary interactions.8  The use of polymeric particles provides the columns with greater pH stability (2 ≤ pH ≤ 12), unlike the much more restricted pH range with silica materials. Particles are available in both porous and non-porous formats; however, for the analysis of large biomolecules non-porous particles are preferred as this provides better mass transfer which in return can reduce band broadening.

Particle Size

Columns come in a range of particle sizes, most commonly, 3, 5, and 10 μm, with the recent introduction of sub-2 μm particles.  Decreasing particle size offers greater efficiency and improved resolution, while also reducing analysis times.  As with any HPLC separation, reducing particle size comes at the cost of increased system pressure and the possibility of frictional heating in the column.  When analyzing biomolecules, increases in temperature and pressure may cause unwanted on-column degradation or aggregation which will impact negatively on analytical results.
An alternative to using small particles to increase sample throughput involves using shorter columns at higher flow rates (Figure 2).

Protein separation

Figure 2: Protein separation on, WCX 3 μm column at 1 mL/min. flow rate (left), WCX 1.7 μm column at 1 mL/min. flow rate (middle), and WCX 1.7 μm column at 1.7 mL/min. flow rate (right).  Increased flow rate and smaller particle size results in a reduced analysis time of 3 minutes without loss of peak shape and resolution.


Capillary IEX

Reducing column internal diameter can increase analytical sensitivity.  However, changes to equipment may be required to minimize the system volume in order to avoid unwanted extra-column band broadening effects.

Monolithic columns

Monolithic phases contain continuous channels made up of non-porous materials, this helps to support mass transfer of slow diffusing biomolecules.  This reduced diffusion and the absence of pores and void volume allows for rapid transport of the molecules between the mobile and stationary phases.  These types of column can be applied to provide improved chromatographic performance - faster separations (from the use of higher linear velocities) and improved resolution (due to fast mass transfer).  Monolithic columns are available as strong and weak cation exchange materials.

Impact of Temperature

For analysis of biomolecules using ion exchange chromatography temperature affects peak capacity and in turn resolution, however, it does not have a great impact on selectivity.3,6  In comparison, selectivity in reversed phase separations can be tuned using temperature.  However, temperature should always be considered when analyzing biomolecules as it may cause denaturing which may impact negatively on chromatographic results.

 
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