The Polyaspartic Acid WCX™ (Poly CAT A™) columns are particularly useful for separation of proteins, peptides larger than 50 amino acids or for very basic peptides. Many newer applications are for larger, glycosylated proteins like antibodies or hemoglobin variants or more common enzyme separations. Proteins will elute from these columns with increasing salt and/or pH gradients. The most useful range for cation-exchange of basic proteins is pH 6-7. However, the lower the pH, the stronger the binding and the longer the elution times. This is useful for weakly basic or neutral peptides. At a pH of 3.2, there is still some ionization from the electron donating derived from the secondary carboxyl. However, below this pH the capacity of these columns decreases.
Since the total binding capacity of this material 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 are to be used for the 4.6mmID analytical column, and gradient times of 30 min. 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. The semiprep columns, 9.4 mm ID, require flow rates and equilibration volumes 4x that of the analytical columns.
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. However, it may be necessary to use sulfate or chloride if the buffering capacity of acetate 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. You may consider using Na2SO4 in place of NaCl to prevent salt problems with your HPLC.
Columns are filled with methanol when shipped so the (analytical) column should be flushed with at least 40 ml water before elution with salt solution to prevent precipitation (Mfg. Instruction Sheet) . 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 40 ml of deionized water (not methanol), and the ends should be plugged. For extended storage it is recommended that a 20 mM phosphate buffer with 0.05% sodium azide or thimerosal 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 scintered 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 P410-2WCX 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.
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 PolyAspartic Acid (PolyCAT™ A) WCX for Part Numbers and Prices.
Last Updated: 08/20/21