LudgerSep N Buffer
For preparation of LS-N Buffer (50 mM ammonium formate buffer, pH 4.4) used in amide or HILIC (hydrophilic interaction liquid chromatography) HPLC analysis of LudgerTag fluorophore and UV-chromophore labeled glycans.
Part Number: LS-N-BUFFX40
The LudgerSep LS-N buffer was developed for the preparation of the normal phase buffer (50 mM ammonium formate buffer, pH 4.4) used in amide or HILIC (hydrophilic interaction liquid chromatography) HPLC analysis of 2-AB and 2-AA labeled glycans.
Includes 50ml of x40 LS-N buffer (2.0M ammonium formate buffer/solution) in a square HDPE bottle with leak-proof cap.
Dilute with de-gassed HPLC grade water (use 1 part of x40 buffer to 39 parts of water) to make the buffer (50 mM ammonium formate, pH 4.4). The 50 ml of x40 buffer will make 2 litres of LS-N buffer.
Use the buffer for glycan analysis with any of the following HPLC columns:
• LudgerSep N series columns
• Tosoh Amide-80 columns
• Waters ACQUITY UPLC BEH glycan columns
• GlycoSep N and N-Plus columns<
• Other HILIC HPLC columns for glycan analysis
HPLC Column System Suitability Application Note
• How well is your column performing?
• Are you obtaining the best results from your chromatography?
• How often do you check peak resolution and shape?
Storage Store unopened bottles of N buffer below 25 ˚C. As with any HPLC solvent we recommend preparation of the solvent immediately before use. Take care that the undiluted/diluted solvent is not exposed to excessive heat or sunlight as it contains volatile components. Stability times of the prepared solvent will vary according to your laboratory conditions. Longterm storage of the prepared solvent, ie longer than 1 month, at room temperature, may result in evaporation of some of the volatile components in the solvent leading to a change in retention times.
LudgerSep LS-N Buffer Instruction Protcol
1. Prepare De-gassed HPLC Grade Water (Optional)
Prepare de-gassed HPLC grade water if de-gassed solvents are required for your HPLC system.
De-gassing of solvents (e.g. by vacuum, sonication or helium sparging) is required for optimal performance of some HPLC systems. Note that the ammonium formate buffer is semi-volatile so water should be de-gassed before adding the buffer concentrate.
2. Dilute the Ammonium Formate x40 Buffer and Store
Different volumes of LS-N buffer can be prepared (see table below for details). In each case, transfer x40 buffer to an appropriately sized glass volumetric flask, add de-gassed water to make up to the mark (use 1 part of x40 buffer to 39 parts of water). Stopper the flask and invert to mix. Store the prepared 50mM LS-N buffer at 4˚C until use.
Avoid introducing air into the LS-N buffer. Also, note that ammonium formate is semi-volatile so vigorous degassing of the made up LS-N buffer may cause changes in pH and buffering capacity.
3. Use the 50mM Ammonium Formate, pH 4.4 LS-N Buffer
Use the 50mM LS-N buffer for glycan HPLC as per the relevant LudgerSep N column guide. We recommend the use of solvent line filters for all HPLC solvents.
The LudgerSep N2 column guide gives example gradients for amide HPLC of fluorescently labeled glycans. These conditions can also be used with the other glycan analysis HPLC columns listed in the specifications section.