To Increase Retention:
To Decrease Retention:
Raise the temperature. Change from the ammonium to a sodium salt. Use a longer chain length (e.g.pentyl or phenyl) chemistry. Increase the salt concentration of the sample. Increase the salt concentration of the solvent. Use a better structure promoting salt (Citrate> Tartrate>Sulfate>Phosphate). See: The Hofmeister Effect and its implications for HIC. A discussion of the ranking of various ions toward their ability to retain (precipitate) a mixture of proteins.
Use a shorter chain length (ALKYL) Aspartamide (e.g. methyl). Decrease the salt concentration of the solvent. Use small amounts of sucrose, glycol, or propanol in solvent A&B (<5%) Use of Octyl Glucoside, CHAPS, and CHAPSO work well (below their CMC) although use of hydrophobic detergents is not recommended. Change the pH of the buffer to increase the ionic character of the peptide.
To best determine how changes in salt concentration will affect your separation, we recommend you start your methods development with the fastest elution conditions and incrementally increase the salt composition of the gradient run-by-run. Simultaneously, the salt concentration of the sample should be proportionately increased by dilution with the high salt solution (solvent A) (minimum 25% sample v/v.)
If using a guard column as a methods development column, gradient times should be shortened to 5-8 minutes; the flow rate should remain at the 1.0 ml/minute analytical level (This way you can accomplish 4-6 experiments in one hour (including flushing and equilibration), speeding up methods development, since a column void volume will take 10 seconds for a 4.6mmID x 10mm column). The semiprep columns, 9.4 mm ID, require flow rates and equilibration volumes 4x that of the analytical columns.
First, make an injection under the low salt strength conditions to assure that the protein will elute. If considerable delay in retention is seen, increase the rate of solubilization and release from the column by lowering the buffer to 10 mM, or use detergent or 1-5% organic solvent in both reservoirs.
If the protein elutes in the void volume, then use stepwise increases (i.e. 500 mM, 1M, 1.5M then 2M salt) of the high salt conditions (solvent A) until the binding profile doesn't change. This will assure sample binding, shorter operation times and minimize the risk of precipitation or irreversible conformational changes of the proteins.
Because proteins are sorted by surface hydrophobicity rather than total contact hydrophobicity, you can change the water of hydration by adding or deleting small amounts of salt to cause subtle changes in their surface hydrophobicity. Structure promoting salts in the Hofmeister series (citrate>tartrate>sulfate> phosphate>chloride) increase the ability of these columns to retain and discriminate between proteins or peptides by affecting the surface hydration and conformation (note that as few as 9 amino acids are necessary to form a beta-pleated sheet and 30 can form an alpha helix.) Using different alkyl chain length columns will then maximize these differences, depending upon the accessibility of the hydrophobic pockets of the proteins.
A dramatic example of this methods development technique working better than a single linear gradient from high salt, was in the isolation of anti-Factor VIII from ascites fluid. It was achieved by determining that the separation of anti-Factor VIII from other blood proteins on the PolyPROPYL Aspartamide column could be achieved if the gradient was started at 1.0 M salt and ran to 20mM buffer. If the gradient started at 1.3M salt they co-eluted at 800-700 mM salt.
If sulfate is not available or the baseline excursions from viscosity changes in the UV detector are unsatisfactory, initial operation with phosphate gradients has been shown to be a good alternative.
If the protein is sensitive to sulfate, use of 1.2 M potassium citrate is recommended, but then use 280 nm for UV detection. Alternatively, fluorescence detection (280ex, 330em ) will avoid all baseline changes from impurities or refractive index effects on the UV detector. Application of the sample should be in the smallest practical volume (<100µl for analytical columns) and in the most concentrated salt solution as possible without precipitating the proteins outside of the column. Make up a 0.1M K-PO4 solution to dissolve the sample and dilute it with the strong salt solution to the desired concentration for injection (minimum 25% sample v/v) to promote retention to the head of the column.
The capacity of the Alkyl Aspartamide packing is high (400 mg/gm for batch adsorption) and an analytical column should be able to handle 1-5 mg of protein in a high resolution separation of moderate complexity.
In the refrigerator, microbial growth is minimal even if stored in 100% water. Based on our experience, it is possible to store it in ACN/water=25/75. If you're going to use the column within a day or two, then there's no need to bother with anything but water. If organic is used, be sure to flush thoroughly with water to remove it prior to introducing any salt.
Generally, a column will fail from something other than microbial growth. However, if running ammonium acetate gradients at pH 5.5 without organic, then one has an excellent growth medium for microbes so we recommend flushing with water prior to storage.
If you flush the column with 100% ACN (or any other organic solvent) for storage, then condition the column with water prior to high salt when you take it out of storage, as if it were new.
Last Updated: 12/12/07