PolyLC is the unchallenged leader in columns for peptide and protein analytics. We help you to become successful!
Dr. Andrew Alpert, owner of PolyLC is a world famous specialist for protein and peptide separation. He is also the inventor of the terms HILIC and ERLIC and associated methods. His products are very popular in the field of proteomics, protein engineering and metabolomics.
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Use: For cation-exchange separation of proteins
Synthesis: PolyCAT A is made by a unique process by 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. Operating conditions are similar to those used with other weak cation-exchange (WCX-) materials .
When to use PolyCAT A:
For larger proteins such as haemoglobins and immunoglobulins selectivity is optimal with PolyCAT A with 1000-angstrom pores.
Tips on Ion-exchange chromatography:
Volatile mobile phases
PolyCat A ion-exchange columns can be used with volatile mobile phases. Peptides are adsorbed from dilute solutions of ammonium acetate and eluted with a step or linear gradient to dilute (at 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.
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.
Asked for specific application help
For HPLC separation of complex carbohydrates
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:
Sialylated and asialo-glycans can be resolved using the same running conditions.
It is not necessary to operate at pH 7.3 or above with PA-derivatives or other reductively-aminated derivatives of carbohydrates.
For Hydrophilic Interaction Chromatography (HILIC) and Size Exclusion Chromatography (SEC)
PolyHYDROXYETHYL for use in HILIC process mode
What is HILIC? (click here)
PolyHYDROXY-ETHYL Aspartamide (or PolyHYDROXYETHYL A is an extreemly polar stationery phase that enables 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). It retains solutes almost solely on the basis of hydrophilic interaction. Volatile mobile phases can be used.
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.
PolyHYDROXYETHYL works well for:
When to use PoyHYDROXYETHYL in HILIC process mode:
For proteins and peptides, use 200- or 300-┼ material. For polar small solutes, try our premium 3-Ám, 100-┼ material.
PolyHXDROXYETHYL for use in Size Exclusion Chromatography (SEC)
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. Non-specific 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 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 PolyHYDROXYETHYL columns in SEC :
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).
PolyHYDROXYETHYL A phases SEC fractionation ranges (daltons)
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 PolyPROPYL A:
When to use PolyETHYL A
When to use PolyMETHYL
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:
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.
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%.
Use material with 1000- or 1500-├ pores for proteins > 20 KDa. Capacity is comparable to ion-exchange. Unless a protein is known to be unusually hydrophobic, use PolyPROPYL
For cation-exchange of peptides
PolySULFOETHYL A is the standard material for the SCX-RPC sequence for multidimensional LC of complex tryptic digests in Proteomics.
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 SCX and RPC, used in sequence, will yield sequenceable peptides from most crude mixtures.
PolySULFOETHYL A is unusually hydrophilic. This minimizes hydrophobic interactions with peptides, with high recovery and less peak tailing. Capacity is also high, permitting better retention and fractionation of the weakly basic peptides from tryptic digests. The capacity of an ion-exchange material like this is 4x that of a comparable RPC column. Therefore, ion-exchange should be the initial step of a multi-step purification.
When to use PolySULFOETHYL A:
Mapping of peptide digests (tryptic, V8, CNBr etc) and isoforms.
Multidimensional HPLC of peptide mixtures, such as tryptic digests in proteomics analyses (including iTRAQ® and ICAT®* reaction products)
QC and purification of synthetic peptides.
Isolation of natural peptides from crude natural extracts
Selective isolation of disulfide-linked peptides, phosphopeptides and C-terminal fragments from tryptic digests
Assay of N- and C-terminal variant peptides and peptides with blocked termini.
Quality control assays requiring a method orthogonal to RPC.
Most other other Strong Cation Exchange (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.
Proteins can be run on PolySULFOETHYL A columns too; at pH 3, retention is all but guaranteed. An example is the analysis of Lung Surfactant Protein on a PolySULFOETHYL A column operated in the Hydrophilic Interaction (HILIC) mode.
Standard material for peptide applications is 300-┼, in either 3- or 5-Ám. The 200-┼ material has about 25% greater capacity and is preferred for phosphopeptide isolation and fractionation of iTRAQ® reaction mixtures. For proteins, use 1000-┼ material or the 3-Ám, 1500-┼ material.
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 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 process mode if organic solvents are included in the mobile phase. This technique has been used for some membrane proteins.
PolyWax LP can be used to separate amino acides, peptides and proteins by ERLIC. ERLIC is a new mode of chromatography that permits some separations to be done isocratically that normally would require a gradient. (more about ERLIC click here
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 able to separate proteins differing by a single phosphate group
Anion-exchange is the method of choice for resolution of oligonucleotides and their analogs > 15 bases. It is also much faster and convenient than PAGE gels for purification of the double-stranded DNA products from PCR reactions.
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.
Use PolyWAX LP for:
Isocratic separation of amino acids, peptides, and proteins.
Selective isolation and separation of phosphopeptidesPurification of acidic proteins and polypeptides from natural product
Analysis and purification of oligonucleotides and their analogs as well as amplified PCR products
In proteomics, predigest fractionation of intact proteins via mixed-bed ion-exchange.
Anion-exchange of small, acidic solutes.
For small solutes, use our 100-┼ material. For peptides, use 300 ┼. For proteins > 20 KDa, we recommend pore diameters of at least 1000 ┼ for optimal selectivity and efficiency. Our 3-Ám, 1500-┼ material affords superior separations of closely-related protein variants.
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Javelin guard columns provide convenient, easy-to-use protection for your analytical HPLC column. The patented Javelin design1 has a 1/16" CPI tip that attaches directly to the inlet of your HPLC column without the need for tubing or wrenches.
To replace, simply remove and discard the entire guard column for fast, easy change over.
Javelin guard columns come in all popular PolyLC phases and are available in 10 mm lengths in 4.0, 2.1, 3.0 and 1.0 mm IDs. The 4.0 mm ID Javelin guard is designed for use with both 4.6 mm and 4.0 mm ID columns.
Javelin guard columns are popular also as analytical columns although they are provided without QC test results.1 US Patent 5,582,723
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