Frequently Asked Questions
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In order to achieve consistent flow rates when running multiple tubes simultaneously it is advisable to use a vacuum pump which can generate a vacuum equivalent to 20" of mercury.
Temperature typically only has minor impacts on GPC separations, but it should be considered nevertheless. Elevating the column temperature can help improve solubility of some analytes which may improve the resolution of poorly soluble analytes. Further, increased column temperature can also reduce mobile phase/diluent viscosity mismatches which can improve the performance of a separation. One caution one must consider with elevated temperature relates to the flash point or boiling point of your mobile phase solvent. Although it is sometime tragically overlooked, one does not want to do a separation at a temperature anywhere close the flash point of a mobile phase.
The main factor on solvent use is solubility. What solvent is your analyte soluble in? That will dictate the mobile phase used for your separation because your analytes must be soluble in the mobile phase to be separated by the column. Sometimes poor resolution or low recovery of an analyte can be caused by poor solubility of the analyte in the selected mobile phase.
Rezex H+ separates analytes isocratically based on three different separation modes: GFC for sugar residues, ion exchange interactions for organic acids, and reversed phase for organic compounds (glycerol and ethanol). The size exclusion mechanism is most influenced by flow rate. The ion exchange mechanism can be influenced by pH and temperature. The reversed phase interactions are influenced by primarily by temperature.
The Glycans are typically released from the proteins by PNGase F in buffer solution and then labelled using 2-AA or 2-AB. The samples can then be cleaned up using a HILIC based SPE sorbent such as the bioZen N-Glycan Clean-up plate under typical HILIC conditions to remove excess labeling reagent, salts and surfactants.
Kinetex phenylhexyl is best suited to the separation of aromatic compounds which are relatively hydrophobic. It can also be used as an alternate selectivity to a C8 or C18, retaining compounds to a similar extend compared to a C8, but with the additional pi-pi interations allowing for aromatic positional isomers to be separated. Kinetex biphenyl is more applicable to polar aromatic compounds, and will also offer a good degree of shape selectivity. Kinetex F5 is the most polar phenyl phase, due to it's highly polar nature and the interactions it can undergo with analytes it is the best option for analyzing highly polar analytes, and those compounds which differ in terms of pi electron structure.
Pressure is released at 3.0 +/- 0.5 Bar (1 Bar = approximately 14.6 PSI)
The pressure release will split and vent to prevent the seal and vial from breaking. This prevents instrument down time and provides added lab safety
Only use pressure release for high temperature work
For the highest quality vial and cap products, we recommend Verex. Our vials and caps are manufactured to provide high quality, state-of-the-art solutions for your most challenging, sensitive applications.
PES (polyethersulfone) syringe filters show low non-specific binding for ionic species, this combined with their hydrophilic properties make them a good choice when the filtration of ionic species is required.
The alkylamine used directly affects retention time, with longer chains leading to increased retention, e.g. hexylamine will show longer retention times than triethylamine. There is also a difference in solubility so generally gradients need to be adjusted to allow for this. Generally, the effect of ion-pair is oligo-specific; for example, Gapmers longer amines offer better resolution whereas for siRNA shorter amines offer an improvement in resolution.
Ordinarily, the use of retentive SPE to clean up and preconcentrate analytes prior to analysis is desirable. In the case of saccharides, however, it isn’t possible to retain the analytes from aqueous solution, we therefore recommend the use of a non-retentive cartridge which will preferentially retain the interfering ionic species - Strata-ABW is suitable for this purpose.
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