PolySULFOETHYL Aspartamide™ Strong Cation Exchange Columns Technical Sheet

PolySULFOETHYL Aspartamide™ Strong Cation Exchange Columns Technical Sheet PolySULFOETHYL Aspartamide™ Strong Cation Exchange Columns

General operating conditions for SCX

(ALKYL) Aspartamide™ cation exchange chemistries are some of the best materials available for the HPLC separation of peptides. With proper care, your column will last for hundreds of hours of operation, and give superior results. These are wide-pore (300Å) silica packings with a bonded coating of hydrophilic, poly(2-sulfoethyl aspartamide) anionic polymer.

With the PolySULFOETHYL Aspartamide SCX column you can use mobile phase modifiers to help improve peptide solubility or to mediate the interaction between peptide and stationary phase. By varying the pH, ionic strength or organic solvent concentration in the mobile phase, chromatographic selectivity can be significantly enhanced. For more strongly hydrophobic peptides, a non-ionic surfactant (at a concentration below its CMC) and/or acetonitrile or n-propanol as mobile phase modifiers, can substantially improve resolution and recovery over conventional reverse phase methods. You may obtain additional selectivity by simply changing the slope of the KCl or (NH₄)2SO₄ gradient.

Using this column at pH 3 is better for retention of neutral to slightly acidic peptides. Use of a higher pH may be considered for basic hydrophobic peptides. The addition of MeCN or propanol to the A and B solvents changes the mechanism of separation (Ref: S1) and results in a separation based not only on positive charge, but also on hydrophobicity.

These columns are quite useful for 2-D LC MS/MS proteomics applications or for neuropeptides, growth factors, CNBr peptide fragments, and synthetic peptides as a complement to RPC, or to remove organic reagents from peptide samples which would cause smearing on a RPC column.

The operating conditions for these applications for an analytical column are:
Buffer A: 5 mM K-PO₄, pH 3.0 + 25% MeCN

Buffer B: 5 mM K-PO₄, pH 3.0 + 25% MeCN + 300-800 mM KCl

Linear gradient, 30 min at 1 ml/min.

Volatile buffers for LC MS/MS proteomics applications:
Buffer A: 25 mM NH₄-formate, pH 2.7 - 3.0 + 5-25% MeCN

Buffer B: 25 mM NH₄-formate, pH 2.7 - 3.0 + 5-25% MeCN + 300-800 mM NH₄-formate

If you want all tryptic peptides to stick to this SCX column, then use a pH low enough for the acidic residues (Asp or Glu) to be uncharged, i.e., pH 2.7 - 3.0. To make this buffer start with an aqueous solution of formic acid or acetic acid and add ammonium hydroxide until you get to this pH. Acetate (pKa=4.7) doesn't buffer well in this range, while Formate (pKa=3.7) does and is to be preferred. It has two additional advantages over acetate:

At pH 3, formate will be present as a salt, whereas acetate would be present almost completely as the unbuffered acid. If you use formate, then you do not need to use H3PO4. To elute use a higher concentration of ammonium formate.

The peptides are retained on the column by the positive charge of at least the terminus amino and elute by total charge, charge distribution and hydrophobicity. If your peptide does not stick to the column, be sure it is in a small amount of buffer, or decrease the concentration of organic in the A & B solvents to 5 or 10%. Organic solvent concentration is empirically determined and n-propanol can be substituted for MeCN for more hydrophobic species.

Since the total binding capacity of these columns is on the order of 100 mg/gm of packing (for nonresolved materials) there will be a considerable Donan effect present. It will be necessary to have your sample in 5-15 mM of salt or buffer to prevent exclusion from the column. Additionally, the gradient at the outlet of the column will be much more concave than that observed on the chart paper. Consequently, if you have had no prior experience using this column, we recommend following a standard methods development protocol to be sure that your protein is eluting properly. The Nest Group recommends an upper load limit of 1 milligram for an analytical column. For a guard column used as a methods development column, we recommend a load limit of one-tenth of a milligram.

Flow rates of 0.7 to 1.0 ml/min with a 4.6mmID column, and a 30-minutes gradient should be used for the analytical column. If using the 4.6 x 20 mm guard column as a methods development column, gradient times should be shortened to 8-10 min at the same flow rate since the void volume is only 0.3 ml. The semiprep columns, 9.4 mm ID, require flow rates and equilibration volumes 4x that of the analytical columns.

Methods development

For the first run, equilibrate the analytical column in the high salt (or final pH) solution (at least 25 ml, or for a guard column used as a methods development column use 8 ml, or on the semiprep column use 100 ml), and inject your sample under these isocratic conditions to observe the elution profile. The protein should elute at the void volume. Then equilibrate the column in low salt (or low pH if doing a pH gradient) conditions and run the gradient to the final conditions. Comparison of the chromatograms will assure that the proteins will elute in a predictable fashion. To decrease elution times increase the salt concentration (in a convex or step manner), increase the pH, or shorten the equilibration times between gradient runs. Exposure to a pH above 7 should be avoided since this will affect the silica support and will shorten column life, as will temperatures above 45C. For buffer gradients, phosphate or bis-tris are good buffers to use since they allow monitoring in the low UV range. For salt gradients, acetate salts are frequently used, although formate is a better buffer at low pH. It may be necessary to use sulfate or chloride if the buffering capacity of acetate or formate is undesirable or if the absorbance is to be monitored below 235 nm. When chloride has been used for salt gradient elution, flush the column with at least 30 ml of deionized water at the end of the day to prevent corrosion. If a denaturant such as 4M urea is used in the mobile phase to increase the accessibility of the ionizable groups, be sure to have a silica saturator column in line in front of the injector, to minimize attack of the silica on the ion exchange column.

Conditioning new columns before use

Columns are filled with methanol when shipped so the (analytical) column should be flushed with at least 30 ml water before elution with salt solution to prevent precipitation. The hydrophilic coating imbibes a layer of water. The resultant swelling of the coating leads to a slight and irreversible increase in the column back pressure. Some additional swelling occurs with extended use of the column. Since the swelling increases the surface area of the coating, the capacity of the column for proteins increases as well. Thus, retention times may increase by up to 10%. This process should be hastened by eluting the column with a strong buffer for at least one hour prior to its initial use. A convenient solution to use is 0.2 M monosodium phosphate + 0.3 M sodium acetate.

The conditioning process is reversed by exposing the column to pure organic solvents. Accordingly, to minimize the time to start the column after a 1-2 day storage, the column should be flushed with at least 30 ml of deionized water (not methanol), and the ends should be plugged. For extended storage it is recommended that a 100% methanol storage be used to prevent bacterial growth and contamination. Exercise care when using organic solvents to prevent precipitation of salts.

It is recommended that a new column be conditioned with two injections of an inexpensive protein (e.g. BSA) before it is used to analyze very dilute or expensive samples since new HPLC columns sometimes absorb small quantities of proteins in a nonspecific manner. The sintered metal frits have been implicated in this process. Fortunately these sites are quickly saturated. Mobile phases should be filtered before use, as should samples. Failure to do so may cause the inlet frit to plug. A guard column (PJGCSE0503, or other configurations) will prevent damage to the analytical or preparative columns. Use of 0.1% TFA or high concentrations of formic acid in the mobile phase is not recommended.

Using the PolySULFOETHYL A™ Column

Initial Use (Mfg. Instruction Sheet):
PolySULFOETHYL A is a silica-based material with a bonded coating of a hydrophilic, anionic polymer: poly(2-sulfoethyl aspartamide). Thus, it is a strong cation-exchange (SCX) material. Columns of this material are shipped in methanol. Flush new columns with at least 15 column volumes of water (30 ml for 4.6mm x 200 column), then condition with a salt solution for at least 1 hour prior to initial use. A convenient solution to use is 0.2 M NaH₂PO₄ + 0.3 M sodium acetate. The column should then be flushed with an additional 15 column columns of water. New HPLC columns sometimes adsorb small quantities of proteins in a nonspecific manner. The sintered metal frits have been implicated in this. The problem is generally overcome by conditioning a new column with two injections of a solution of an inexpensive protein before its initial use, particularly if dilute or expensive samples are to be resolved.

Routine Use: Mobile phases and samples should be filtered before use. Failure to do so may cause the inlet frit to plug. This frit is replaceable. At the beginning of the day, flush the column with 10 ml of the high-salt buffer before conditioning with the low-salt buffer. At the end of the day, flush the column with 30-40 ml water or (preferably) a weakly acidic buffer containing 1mM EDTA but no chloride, and plug the ends. If the column is to be stored for more than a few days, it should be refrigerated (but do not freeze).

Cation-Exchange of Peptides: This material has been developed specifically for cation-exchange of peptides in the pH range 2.7-4.0. It will function as a cation-exchanger above pH 4, but in that range it has no particular advantage over weak cation-exchangers such as PolyCAT A™ for analytical applications. At pH 3, basic residues in peptides (His, Arg, Lys) are positively charged, as are the amino-termini. Acidic residue (Asp, Glu) are unchanged, and the carboxyl-termini are predominantly unchanged. Thus, most peptides with free amino-termini will have net charges of at least +1, and bind to PolySULFOETHYL A. They can then be eluted with a salt gradient. A good general-purpose buffer system is 5 mM KH₂PO₄, pH 3.0, with 25% (v/v) acetonitrile; a linear gradient is run to the same buffer but with 0.25 M KC1 (with higher concentrations required to elute the most basic peptides). Peptides elute in order of increasing net positive charge at pH 3. In many cases, mixed-mode effects permit the resolution of different peptides with the same net charge. Selectivity can be manipulated by increasing or decreasing the amount of organic solvent in the buffers, since this differentially affects the retention of different peptides. While peptide recovery is generally high or quantitative, it may be advisable to use guard cartridges with potentially troublesome mixtures such as cyanogen bromide cleavage digests or crude tissue extracts.

Peptide Purification and Sequencing

: PolySULFOETHYL A is a good alternative to reversed-phase chromatography (RPC) columns, since the selectivity is almost completely complementary. The two types of columns are excellent complements when used in sequence to purify peptides. Use RPC columns last, since they have several times less capacity than SCX columns and yield products in volatile mobile phases.

All chromatography packings bleed minute amounts of stationary phase when in use. In this case, tiny amounts of poly(2-sulfoethyl aspartamide) could be introduced into the mobile phase. While this is usually not a problem, it could conceivably result in artifactual elevation of measured aspartic acid and taurine levels in peptides which are being collected for sequencing. When PolySULFOETHYL A and RPC columns are used in sequence, use the RPC column second (as any stationary phase contaminants would elute in the void volume). Alternatively, use a reversed-phase sample cleanup cartridge for the purpose. This also permits changeover to a lyophilizable solvent if desired.

Specific Isolation of Disulfide-Linked Peptides

A typical tryptic peptide has a charge (at pH 3) of +2, due to the N-terminus at one end and the Lys- or Arg- at the other. Linking two such peptides with a disulfide bridge results in a peptide with a charge of +4. Such peptides are eluted later from a PolySULFOETHYL A column than the typical tryptic peptide, permitting their convenient identification and isolation.

Normal phase and HILIC polarity considerations

By adding even more organic solvent to the mobile phase, these columns offer enough flexibility so that they may be used in a normal or Hydrophilic Interaction (HILIC) mode. Here, more polar peptides having little or no retention under conventional reverse-phase or even ion-exchange conditions are retained, and very hydrophobic peptides may have enhanced solubility and thus chromatograph better. There are three approaches to this mode: 1) using isocratic HILIC conditions, 2) using a sodium perchlorate gradient, or 3) using a decreasing organic solvent gradient. The key to achieving HILIC conditions is to use greater than 70% organic solvent with the SCX column. Care should be taken to assure solubility of salts under these conditions.

Satisfaction with this product is guaranteed

If the performance of this column does not meet the specifications of the attached chromatogram upon initial use, or if within 45 days of normal use the column fails to maintain adequate performance, The Nest Group will replace this product with a new column. We will need to have operating conditions which led to the failure and the column identification number to process your replacement order.

See PolySULFOETHYL Aspartamide for Part Numbers and Prices.

For more information contact:
The Nest Group, Inc.™ 17 Hayward St., Ipswich, MA 01938-2041 USA
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Last Updated: 08/20/21