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Our materials have pressure limits in the range of 500-550 bar.  Please keep in mind that running at the pressure maximum will degrade the column quicker than running at a more conventional pressure such as 100 bar.

Our stationary phases are based on silica, so the upper limit of pH is around 8.  The lower limit is around pH 2.5.

We recommend using ambient temperature.  In any case, do not exceed 40°C.  The higher the temperature, the shorter will be the useful lifetime of the stationary phase.

There are two ways to determine this.  We prefer using an unretained peak and calculate the void volume using retention time.  For example, using a 35 x 2.1mm PolyHYDROXYETHYL A™ column;

Alternatively, one can mathematically determine this by using the equation V = πr²L. Calculating the void volume this way requires an assumption of the ratio of the packed bed versus total volume. It is generally accepted that the packed bed can be estimated as accounting for 1/3 of the total volume. In our experience the unretained peak method is most accurate.

Loading capacities are determined by the ID of the column. Use the following table as a guide.

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.

Yes, 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.

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).

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 ensures that your solution will be reproducible.

There could be a number of reasons for this:

1. Your sample solvent or starting mobile phase might contain TFA or HFBA. Try 5-10 mM K₂HPO₄, 0.1% formic acid, or 0.1% acetic acid instead.
2. If the salt concentration is > 30 mM. Try diluting or dialyze your sample.
3. Perhaps 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.
4. If you performed an iTRAQ® derivatization its possible you didn’t adjust the pH to 2.7-3.0 and desalt adequately.
5. The column load capacity was greatly over loaded.

This is generally a bad idea with the materials that PolyLC synthesizes.

1. The sample contains less organic solvent than does the mobile phase.
2. 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.
3. 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.