Frequently Asked Questions
1) Why can't I use reversed-phase HPLC for proteins?
Retention of a solute reflects the number of sites at which it interacts with the stationary phase. Proteins contain a lot of hydrophobic residues. Therefore, many of them do not desorb readily. Peaks may be extremely broad if they elute at all.
2) Do I need to degas mobile phases?
Only if you plan to run a gradient of organic solvent in water. It is not necessary to do so in ion-exchange or hydrophobic interaction chromatography. Literature from instrument companies generally assumes that their machines will be used only for RPC.
3) How should I store columns?
If you plan to use it within a day or two, you can frequently leave it in the starting mobile phase if it doesn't contain > 20 mM salt. Just plug the ends. If it does contain > 20 mM salt, flush it with water before storage. If you plan to use it after several days but within two weeks, you should definitely flush it with water. If you aren't going to use it within two weeks, flush it with water, then a mixture of ACN:water (any ratio between 50:50 and 80:20) and store in the refrigerator with the ends plugged. When this column is subsequently taken out of storage, it should be conditioned again as if it were new (generally with a high-salt buffer).
4) What's the correct way to prepare a mostly organic mobile phase?
Adjust the pH and filter the aqueous salt portion prior to adding the organic solvent, then pour the aqueous solution into a volumetric flask. Add the organic solvent to within several ml of the calibration mark. Invert 8-9x and allow the solution to reach room temperature. In the case of alcohols, this means to cool. In the case of ACN, this means to warm up (mixing of ACN and water is endothermic). The warming can be accelerated by putting the vol. flask in a sonicator bath containing warm water. This also degasses the solution. Once the contents have reached room temperature, add more organic solvent to the calibration mark and invert 8-9x.
Liquids expand when heated and contract when cooled. The above procedure insures that your solution will be reproducible.
5) Why aren't my peptides retained on the PolySULFOETHYL A™ column?
a) Your sample solvent or starting mobile phase contains TFA or HFBA. Use 5-10 mM K2HPO4, 0.1% formic acid, or 0.1% acetic acid instead.
b) The salt concentration is > 30 mM. Dilute or dialyze your sample.
c) You performed a tryptic digestion at pH 8 and haven't adjusted the pH to 2.7-3.0. It's easier to do this if you use NH4HCO3 as the trypsinization buffer instead of Tris. NOTE: If using a SpeedVac®*, it is necessary to take a sample to dryness 3x in succession in order to get rid of excess NH4HCO3.
d) You performed an iTRAQ® derivatization and didn't adjust the pH to 2.7-3.0 and desalt adequately.
e) You overloaded the column by more than 6x the recommended maximum loading limit.
6) Can I use TFA or HFBA in the mobile phase?
This is generally a bad idea with the materials that PolyLC makes.
7) My peaks are badly skewed or split in HILIC. Why?
a) The sample contains less organic solvent than does the mobile phase.
b) If the solute has a high charge-to-mass ratio (e.g., arginine), then the sample solvent should supply the same counterion that the mobile phase does to pair with the charged groups in the solute.
c) If the solute has an extremely high charge-to-mass ratio (e.g., ATP; aminoglycoside antibiotics), then it may be necessary to use 125 mM salt in both mobile phases or to run an increasing salt gradient.
PolySULFOETHYL A™ , PolyCAT A™, PolyWAX LP™, and PolyHYDROXYETHYL A™ are trademarks of PolyLC Inc. All Rights Reserved
SpeedVac® is a trademark of Savant Corp.
*ICAT® and iTRAQ® are trademarks of Applied Biosystems Inc.,
a branch of Applera Corp.