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PolyLC Switzerland, Europe, Germany, Austria | HPLC Columns Purification and Analysis of Proteins and Peptides

CATALOGUE

 

PolyGLYCOPLEX- for complex carbohydrates

PolyCAT A- for cation-exchange of proteins

Hydrophilic Interaction Chromatography (HILIC)

Size Exclusion Chromatography (SEC)

PolySULFOETHYL Aspartamide- for cation-exchange of peptides

PolyPROPYL-, PolyETHYL-, PolyMETHYL Aspartamide- for hydrophobic interaction chromatography (HIC) of proteins and peptides

PolyBUTYL-, PolyPENTYL-, PolyHEXYL-, PolyHEPTYL-, PolyOCTYL-, PolyNONYL-, PolyDECYL Aspartamide for HIC-MS in top-down proteomics

Getting rid of SDS

PolyWAX LP- for anion-exchange of proteins 

PolySAX LP- for anion-exchange of peptides, proteins and oligonucleotides

 


PolyGLYCOPLEX- for complex carbohydrates

Our new PolyGLYCOPLEX columns permit the separation of complex carbohydrates in volatile mobile phases; typically, acetonitrile and water. Direct flow to mass spectrometry is convenient. Compared to reversed-phase HPLC, selectivity is generally superior. Selectivity is quite good for both native glycans and derivatives such as those with the 2-aminopyridine (PA)-fluorophore. Oligosaccharide mixtures can often be resolved with isocratic elution, although gradients are recommended for especially varied samples. Columns are available from microbore to process-scale.

Some advantages over basic HPLC materials (eg. amino-silica) are:
1.  Sialylated and asialo-glycans can be resolved using the same running conditions.
2.  It is not necessary to operate at pH 7.3 or above with PA-derivatives or other reductively-aminated derivatives of carbohydrates.
                                                                                                      



PolyCAT A- for cation-exchange of proteins

PolyCAT A is made through a unique process for attaching poly(aspartic acid) covalently to silica. Proteins elute from this polypeptide coating in sharp peaks with little tailing. Binding capacity and recovery are high as well. Operating conditions are similar to those used with other weak cation-exchange (WCX-) materials (eg. CM-type).

When to use PolyCAT A:
1.  Hemoglobin analysis (Hb A1c and variants).
2.  Proteins with isoelectric points above 6.0.
3.  Polypeptides with more than 3 basic residues, such as hormones and growth factors (easier elution than with PolySULFOETHYL A).
4.  Purification of very basic solutes by ion-exchange with a volatile mobile phase.
5.  Quality control assay of isoforms, cf. sialylation variants of monoclonal antibodies.
6.  Histones (using mixed modes of ion-exchange and hydrophilic interaction).

With larger proteins such as hemoglobins and immunoglobulins, selectivity is optimal with PolyCAT A with 1000-angstrom pores.

Tips on Ion-exchange:
1.  Volatile mobile phases
It is possible to use our ion-exchange columns with volatile mobile phases. Peptides are adsorbed from dilute solutions of ammonium acetate and eluted with a step or linear gradient to dilute (@5-10%) acetic acid. With very basic peptides, use PolyCAT A; otherwise, use PolySULFOETHYL A. This method also works with other very basic solutes, such as aminoglycoside antibiotics.  Ask for our applications sheet about this.
2.  Organic solvents in the mobile phases
When the mobile phase contains over 40% organic solvent, then our cation-exchangers become sensitive to changes in polar as well as basic residues. Examples include the presence or absence of a Ser-residue or the methylation of a Lys-residue. This is proving to be particularly useful for resolution of minor forms of large recombinant or synthetic polypeptides.
                                                                                                        



Hydrophilic Interaction Chromatography (HILIC)

What is HILIC?
HILIC is a variant of normal-phase chromatography which can be performed with partially aqueous mobile phases. This permits normal-phase separation of peptides, carbohydrates, nucleic acids, and many proteins. The elution order is least to most polar, the opposite of that in reversed-phase HPLC (RPC). The stationary phase in HILIC must be extremely polar. PolyLC has developed a material specifically for this purpose: PolyHYDROXY- ETHYL Aspartamide (or PolyHYDROXYETHYL A). It retains solutes almost solely on the basis of hydrophilic interaction. Volatile mobile phases can be used.

How to use HILIC
Retention is proportional to the amount of organic solvent in the mobile phase (the opposite of RPC). Typical HILIC mobile phases contain 65-80% acetonitrile or propanol. Gradient elution may be performed either with a decreasing organic or increasing salt gradient. 10 mM salt is necessary with charged solutes such as peptides but is not necessary with uncharged solutes such as carbohydrates. Salts with good solubility in HILIC mobile phases include potassium methylphosphonate, triethylamine phosphate (TEAP), and sodium perchlorate. Ammonium formate and acetate are volatile but not transparent below 230 nm; they can be used for direct mass spec analysis.

For more details on the HILIC mode, see J. Chromatogr. (1990) 177-196.

HILIC works well with:
1.  Peptide mapping.
2.  Purification of synthetic and natural peptides.
3.  Glycopeptides and phosphopeptides.
4.  Complex carbohydrates.
5.  Oligonucleotides and their analogues.
6.  Membrane proteins.

When to use HILIC:
1.  When you need a volatile phase and RPC does not suffice.
2.  With solutes too weakly or too strongly retained in RPC.
3.  For HPLC of solutes which aggregate or aren't soluble in aqueous media (eg. amyloid peptides).
4.  To separate solutes differing in a hydrophilic residue (eg. Ser-).
5.  Purifications and quality control assays which require a complementary ("orthogonal") mode.
6.  Separating electroeluted proteins from SDS, Coomassie blue, and salts.
                                                                                                 



Size Exclusion Chromatography (SEC)

Our PolyHYDROXYETHYL Aspartamide columns can each be used in two different fractionation ranges, merely by changing the mobile phase. With conventional salt buffers, the fractionation range is determined by the pore diameter of the packing. Nonspecific interactions with polypeptides are generally lower than with other SEC columns. If the mobile phase contains a denaturing agent (eg. 50 mM formic acid or hexafluoro-2- propanol (HFIP), then sieving occurs between the polymer chains of the coating. This results in a dramatic shift of the fractionation range to lower values; solutes as small as formic acid can be separated by size! Moreover, these separations can be effected with volatile mobile phases.

With our new 60-angstrom pore column, the fractionation range is 20-600 daltons. This permits SEC of small solutes not possible heretofore. Examples include desalting a dipeptide, or separation of small solutes from a large excess of an even smaller derivatizing agent.

Use our SEC columns for:
1.  Routine SEC of enzymes and other proteins.
2.  SEC of polypeptides which exhibit nonspecific interaction or poor recovery from other SEC columns.
3.  Resolution of the smallest peptides and other solutes by size.
4.  SEC in a volatile mobile phase. This permits direct feed to a mass spectrometer.
5.  Analysis of residual monomer content of a polymer.
6.  Desalting of just about anything, including removal of derivatizing reagents present in great excess.
7.  Peptide mapping, either before or after reversed-phase HPLC (RPC).

For routine SEC applications, we recommend the 200 x 9.4 mm columns, which offer optimal separations at @ 0.5 ml/min. Smaller columns can be used if the HPLC system can deliver low flow rates accurately (eg. 0.12 ml/min for 200 x 4.6 mm columns).
                                                                                                         



PolySULFOETHYL Aspartamide- for cation-exchange of peptides

This strong cation-exchange (SCX) material was developed specifically for HPLC of peptides. At pH 2.7-3.0, peptides lose their (-) charges, and have net (+) charge. They can be retained by a SCX column such as PolySULFOETHYL A. With a salt gradient, peptides elute in order of increasing number of basic residues. Thus, the selectivity complements that of RPC. The capacity is several times greater than that of RPC. SCX and RPC, used in sequence, will yield sequenceable peptides from most crude mixtures.

When to use SCX:
1.  Mapping of peptide digests (tryptic, V8, CNBr etc) and isoforms.
2.  Purification of synthetic peptides.
3.  Isolation of natural peptides from crude extracts.
4.  Specific isolation of disulfide-linked peptides from digests.
5.  Specific isolation of C-terminal peptides.
6.  Assay of N- and C-terminal variant peptides and peptides with blocked termini.
7.  Quality control assays requiring a method orthogonal to RPC.

Most other SCX columns are based on sulfopropyl- (SP-) groups. Hydrophobic interactions are significant with such groups. This often results in poor recovery and efficiency with hydrophobic peptides. By contrast, PolySULFOETHYL A is based on sulfoethyl- groups, and recovery of peptides is generally high or quantitative.
                                                                                                            



PolyPROPYL-, PolyETHYL-, PolyMETHYL Aspartamide- for hydrophobic interaction chromatography (HIC) of proteins and peptides

These materials separate proteins on the basis of hydrophobic character, as does RPC. However, HIC uses totally aqueous buffers, maintaining tertiary structure and biological activity. Typically, a sample is eluted with a decreasing gradient of a salt such as sulfate or phosphate. Proteins elute in order of increasing surface hydrophobicity. Surfactants (eg. CHAPS, octylglucoside) can be added to the mobile phase if necessary. The relative hydrophobic character of PolyPROPYL A, PolyETHYL A, and PolyMETHYL A is 100, 60 and 15, resp.

When to use HIC:
1.  Characterization of antibodies (use PolyPROPYL A or PolyETHYL A with 1000-angstrom pores).
2.  Purification of polypeptides (eg. glycopeptides and venoms).
3.  Isolation of proteins from crude extracts (capacity is several times greater than in RPC).
4.  Quality control assay using a method complementary to ion-exchange and RPC.
5.  Isolation of integral membrane proteins and their complexes (use PolyMETHYL A or PolyETHYL A).
                                                                                                       


 

PolyBUTYL-, PolyPENTYL-, PolyHEXYL-, PolyHEPTYL-, PolyOCTYL-, PolyNONYL-, PolyDECYL Aspartamide for HIC-MS in top-down proteomics

Our new series of materials for HIC permit the retention of proteins using concentrations of ammonium acetate that are compatible with direct analysis via mass spectrometry. Proteins can be separated and eluted with their native structures intact. This is useful for top-down proteomics and permits the analysis of some proteins that are not compatible with the conditions of reversed-phase chromatography. 

Conventional materials like PolyBUTYL A are not hydrophobic enough to retain many proteins well with Am-OAc buffers. Our PolyPENTYL A and more hydrophobic new materials do retain them. However, sensitive proteins (eg. RNAse) may denature on PolyOCTYL A and more hydrophobic materials before they can elute. The new method is probably going to be implemented using PolyPENTYL A, PolyHEXYL A and PolyHEPTYL A. A particularly hydrophobic protein, such as an antibody-drug conjugate (ADC), might best be run using PolyPENTYL A or even PolyBUTYL A.

 

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Getting rid of SDS

SDS is sometimes used to solubilize proteins, and is often present in proteins and peptides electroeluted from SDS-PAGE gels. Unfortunately, the SDS in the samples often ruins subsequent runs by reversed-phase HPLC (RPC). Moreover, in automated peptide sequencers, SDS in samples can lead to bubble formation in the sample intake lines, which blocks further sample intake. SDS can also accumulate on PVDF membranes used for sequencing, washing off at an inconvenient time. All in all, it's a good idea to get it out of your samples. PolyLC offers two ways to accomplish this:

1.  Hydrophilic Interaction Chromatography (HILIC)
When our PolyHYDROXYETHYL Aspartamide columns are used in the HILIC mode, SDS and Coomassie blue elute immediately after the void volume. Peptides and proteins are retained and can be eluted with a decreasing gradient of acetonitrile (peptides) or propanol (proteins). Thus, one can eliminate the SDS and obtain a good peptide map at the same time. This method works with proteins as large as 100 kD (Anal. Biochem. 215 (1993) 292). Volatile mobile phases can be used. This method also gets rid of neutral surfactants such as Triton X-100 and Nonidet P-40.

2.  For reversed-phase HPLC: our 2SDS and 4SDS guard cartridges
These are wide-pore, 5-micron materials packed into 20 x 2.1 mm and 20 x 4.6 mm cartridges, respectively. Used as guard cartridges with reversed-phase columns, they selectively remove SDS from peptide mixtures. Conventional TFA/acetonitrile gradients can be used. Accumulated SDS is washed off the cartridges (and out of the RPC column) by levels of acetonitrile higher than 70%.
                                                                                                          



PolyWAX LP- for anion-exchange of proteins

Most proteins have isoelectric points below 7, and are best purified or analyzed by anion-exchange chromatography. PolyWAX LP is a weak anion-exchange (WAX) material developed by Poly LC for HPLC of enzymes and other proteins. Selectivity is excellent, with high or quantitative recovery of applied activity. PolyWAX LP can be used in the HILIC mode if organic solvents are included in the mobile phase. This technique has been used for some membrane proteins.

Most anion-exchange materials based on polyethyleneimine (PEI) are prepared with the conventional branched polymer. PolyWAX LP is prepared with linear PEI, which confers greater selectivity and recovery.

PolyWAX LP is also used for anion-exchange of acidic small molecules. Examples include analysis of fruit juice for Red Dye #2 and #40, as well as benzoate and sorbate.

 

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PolySAX LP- for anion-exchange of peptides, proteins and oligonucleotides

PolySAX LP is a silica-based material with an adsorbed, cross-linked coating. It is a strong anion-exchange (SAX) material. The capacity is high for both small and large solutes.

Anion-exchange of peptides: In contrast to proteins, acidic peptides can be run at lower pH down to the range where Asp- and Glu- residues start to lose charge (@ pH 4). Retention reaches a maximum around pH 4.5. Peak shape and selectivity can sometimes be improved by inclusion of 10% organic solvent (MeOH or ACN) in the mobile phase.

Anion-exchange of proteins: This is typically performed in the pH range 7.0-8.0, using either increasing salt or decreasing pH gradients. The higher the pH, the stronger the binding of proteins. Tris is a good buffer in this range. Since halide salts tend to corrode stainless steel, acetate salts are often used to generate the gradient. However, it may be necessary to use a chloride salt gradient if the absorbance is to be monitored below 235 nm. If so, flush with 15 column volumes of water at the end of the day to preclude corrosion. It is also helpful to passivate the column every 3-4 weeks with EDTA.2Na if chloride salts are used with regularity. For proteins < 15 KDa, a 300 angstrom material can be used. For larger proteins, a 1000 angstrom column is recommended.

Anion-exchange of oligonucleotides and oligonucleotide analogs: For oligos < 25 base pairs long, use 300 angstrom material. For oligos > 25 base pairs long and dsDNA, use of a 1000 angstrom column will afford elution without excessive amounts of salt. It is helpful to include 25-50% acetonitrile or propanol in both mobile phases. Large oligos may require over 1M salt for elution. It is advisable to use a chaotropic salt (eg. NaBr or NaClO4) for the gradient in cases where there could be a tendency for self-association like GC-rich sequences; otherwise, ammonium acetate can be used.