PolyCAT A column for Cation-Exchange
1. Uniqueness of PolyCAT A
PolyCAT A™ is made by attaching covalently poly(aspartic acid) to silica. Proteins elute from this polypeptide coating in sharp peaks with little tailing. Moreover, recovery and binding capacity is high.
2. Overview of Applications of PolyCAT A column for Cation-Exchange
1) Protein variants involving deamidation, PEGylation, determining position of attachment, desialylation and many more. It is widely used by the biotechnology industry.
2) Hemoglobin variant analysis by clinical chemistry labs.
3) Specifically for Proteins with pI above 6.0 (5.0 in special cases).
3. Properties of PolyCAT A column for Cation-Exchange
PolyCAT A™ is a weak cation-exchange (WCX) material. It is used at pH values above 4. A gradient to unbuffered acetic acid will uncharged PolyCAT A™, permitting the elution of proteins in a volatile solvent. See our poster on this subject.
Peptides can be run on PolyCAT A™ if they contain at least two excess positive charges above pH 4. More weakly basic peptides, such as tryptic fragments, are not reliably retained. Instead use PolySULFOETHYL A™ at pH = 2.7 – 3.0
For proteins larger than 20 KDa, we recommend the use of pore diameters particles. Use at least 1000 Å Porediameter for optimal selectivity and efficiency. Our 3-µm material with 1000- or 1500-Å pores is the finest cation-exchanger available for protein separations.
4. How professionals work with PolyCAT A column for Cation-Exchange
PolyCAT A™ is a silica-based material with a bonded coating of polyaspartic acid. It is a weak cation-exchange (WCX) material. Columns are shipped in methanol. Flush new columns with at least 15 column volumes of water (30 ml for a 200 x 4.6-mm), then condition with a salt solution prior to initial use. A good conditioning solution is 40 mM EDTA.2Na (filtered, but pH not adjusted) at a low flow rate for 20-24 hours.
New HPLC columns sometimes absorb small quantities of proteins or phosphorylated peptides in a nonspecific manner. The sintered metal frits have been implicated in this. Eluting the column for 20-24 hr. at a low flow rate with 40mM EDTA.2Na usually solves the problem. This passivates all metal surfaces in the HPLC system, as well as the column [CAUTION: This treatment can affect the integrity of the frits in some cases, and should probably be avoided with columns packed with 3-µm material. In some cases this has also caused the collapse of 5-µm, 200-Å column packings]. Alternatively, after flushing with water, condition the column for 2 hours with 0.2 M NaH2PO4 + 0.3 M sodium acetate. This solution conditions the coating but does not passivate metal surfaces.
Proteins can be eluted from PolyCAT A™ columns with salt and/or pH gradients. The most useful range for cation- exchange of proteins is pH 6-7. Phosphate and Bis-tris are good buffers in this range. The higher the pH, the weaker the retention. Avoid prolonged exposure to a pH above 8. For weakly basic peptides or for cation-exchange below pH 4, use PolySULFOETHYL A™, our strong cation-exchange (SCX) material.
Use ambient temperature (20-25°C), as this polypeptide-based coating is more sensitive to elevated temperatures than are other materials. Filter mobile phases and samples before use. Failure to do so may cause the inlet frit to plug. This frit can be replaced. At the beginning of the day, flush the column with 15 column volumes of the high-salt buffer before equilibration with the low-salt buffer. At the end of the day, flush the column with 15 column volumes of water and plug the ends.
The loading capacity of a 4.6mm ID column is about 4 mg of protein/injection, depending on the strength of the protein’s binding to the support.
1) Overnight: 100% mobile phase A. 2) Several days: Store in water. 3) Longer periods: Store in water in the refrigerator, with the ends plugged. ACN can be added to the storage solvent (e.g., ACN:Water = 80:20) to retard microbial growth.
After every 250 runs, invert the column and run it backwards overnight, at a low flow rate, with 40 mM EDTA.2Na. Continue using the column in this inverted direction for the next 250 samples, then repeat this treatment. If possible, open the inlet and fill in any voids with bulk PolyCAT A™ after running 500 samples.
Minimize Iron in the System:
This coating chelates Fe+3, which ruins its performance. If chloride-containing mobile phases are used regularly, passivate the column and the HPLC system every 4 weeks with the 40 mM EDTA.2Na solution as described above. NOTE: If the HPLC system has not been used for several days (e.g., over a weekend), then Fe+3 ions tend to accumulate in the fluid in the lines. When restarting the system, flush this fluid to waste offline before diverting flow through the column.
Below pH 4 the coating loses its negative charge. Thus, peptides can be eluted by a gradient to dilute acetic acid.
1) Hemoglobins: These are well separated by PolyCAT A™! See the specific Application Note.
2) Growth Factors or Protein Variant separations: Try an ammonium acetate gradient in 40% ACN. For separation of Asp- vs, isoAsp- variants, try mobile phases at pH 4.2.
3) Antibodies: Human: try pH 6.4-7. Murine (= mouse): try pH 7.2-8.0.
4) Chloride vs. acetate: Unlike chloride ion, acetate does not corrode stainless steel. However, it is not transparent below 230 nm, and 10% more acetate is required to match the eluting power of chloride.
5) Mixed-mode effects: When the mobile phase contains over 60% organic solvent, then hydrophilic interactions will be superimposed on the electrostatic effects. PolyCAT A™ can then resolve many peptides that differ in polarity but not charge (e.g., methylation of a Lys- residue). It may be necessary to use a gradient salt with good solubility in org. solvents, such as sodium perchlorate or triethylamine phosphate (TEAP).
5. Advanced HPLC Strategies for PolyCAT A column using Volatile Mobile Phases
This method works with PolyCAT A™, our weak cation-exchange (WCX) material. A gradient to dilute acetic acid (HOAc) can uncharge the carboxyl- groups on the surface, leading to the elution of retained peptides. This gradient does not uncharge our strong cation-exchange (SCX) material, PolySULFOETHYL A™. Peptides are reliably retained on a WCX material only if they have three or more basic residues or two basic residues and a free N-terminus. Thus, most tryptic peptides are not well-retained. This method also works with nonpeptide basic solutes such as polyamines and aminoglycoside antibiotics. Elution is generally in order of least to most basic, but there is also some separation of sequence variants differing in nonbasic residues.
Apply the mixture to a PolyCAT A column equilibrated with 10 mM ammonium acetate, pH 5-5.5. Binding capacity is approx. 4 mg. peptide for a 4.6-mm i.d. column.
Run a linear gradient to 15% aq. HOAc. Some extremely basic peptides or polyamines have required as much as 30% HOAc for elution.
Absorbance detection below 240 nm is not possible with these mobile phases. Thus, absorbance detection is confined to peptides with aromatic residues; 254 nm (for Phe-), 270 nm (for Tyr-), or 280 nm (for Trp-). Suitable alternatives include mass spectroscopy or an evaporative light scattering detector (ELSD). Alternatively, just collect fractions for bioassay after lyophilization or drying in a SpeedVac.
1) Extremely basic solutes: If elution requires over 20% HOAc, try PolyCAT A™ with 1000-Å pore diameter instead of 300-Å. This can decrease by 3x the concentration of HOAc required.
2) Synthetic peptides: This is a convenient way to clean up a crude synthetic product. Basic peptides are retained while deprotection fragments are not. This eliminates the need for an ether precipitation step (and potential oxidation of labile side chains).
3) Unusually hydrophobic peptides: To promote solubility, @ 20% organic solvent can be included in both mobile phases. Use of >50% organic solvent will result in hydrophilic interactions being superimposed on the electrostatic effects.
4) Counterions: This method yields peptides with acetate counterions. This is compatible with bioassays, unlike the trifluoroacetate counterion frequently contributed by reversed-phase HPLC.
Acknowledgements to Mike Selsted @ U.C.-Irvine for initial use of this technique.