N1 4.6mm Guard Column
Dimensions: 4.6 x 10 mm
Part Number: LS-N1-4.6×10
Application: Analysis and purification by HPLC of LudgerTag™ fluorophore and UV-chromophore labeled glycans.
Description: LudgerSep™ N1 HPLC columns contain particles with a polymeric amide coating optimized for high resolution chromatography of complex glycan mixtures.
Particles: 5 µm particle size with 80 angstrom pores and polymeric amide coating.
Cat # Diameter x Length Dimensions Volume
LS-N1-4.6×10 LudgerSep™ N1 Guard Column 4.6 x 10 mm 0.17 ml
LS-N1-4.6×250 LudgerSep™ N1 HPLC Column 4.6 x 250 mm 4.2 ml
Column Tube: Stainless steel
Flow Rates: Typical flow rates = 0.4 – 1.0 ml/min. Maximum flow rate = 1.2 ml/min
Column Pressure: Maximum pressure = 2250 psi (150 kg/cm2)
PH Range: 2.0 – 7.5
Temperature:Typical operating temperature = 30oC. Maximum temperature range = 10 – 80oC.
Solvents: Typical solvent systems for glycan analysis include gradients of acetonitrile (aq) and buffers containing ammonium formate, pH 4.4. This is available from Ludger and can be diluted for use. Cat No. LS-N-BUFFX40
Shipping Solvent: 75% acetonitrile – 25% water
Water [to remove very polar solutes from the bonded phase]
45% acetonitrile (aq) [to desorb hydrophobic compounds]
0.1% triethylamine in 80% acetonitrile [to remove desorbed basic compounds]
50 mM ammonium formate pH 4.4 / acetonitrile (1:1 v/v) [to remove ionic compounds
Storage: Before long-term storage flush the column with at least 5 column volumes of 75% acetonitrile (aq).
Column Protection: Filter all solvents to 0.2 µm and degas using either helium sparging or vacuum degassing. Filter all samples using a 0.2 µm filter membrane before loading onto the column. Install a good quality in-line filter between the sample injector and the column. Please call us for advice on the most suitable sample and in-line filters to use.
Amout Of Sample: The maximum amount of glycan sample that can be loaded on the column depends on the number and type of glycan components as well as the nature of any non-glycan material. The typical range for successful analytical runs is 1pmol – 1 nmol per sample peak and up to 200 nmol of total glycans.
Suitable Sample: Suitable samples include glycans labeled with the following LudgerTag™ labels: 2-AA (2-aminobenzoic acid), 2-AB (2-aminobenzamide), AMAC (2-aminoacridone), 2-AP (2-aminopyridine), AMC (7-Amino-4-methylcoumarin), ABEE (4-Aminobenzoic ethyl ester).
Filter samples to 0.2 µm then dry using a centrifugal evaporator.
Re-dissolve in 5 – 50 µl of the starting buffer (i.e. the solvent mixture used at the very start of the HPLC gradient) then inject.
If possible, inject the sample soon after dissolution to minimise problems with sample precipitation in high organic solvent conditions.
Sample Detection: Either fluorescence or UV-absorbance depending on the dye used (see the appropriate LudgerTag instruction guide).
Handling: Ensure that any glass, plastic ware or solvents used are free of glycosidases and environmental carbohydrates. Use powder-free gloves for all sample handling procedures and avoid contamination with environmental carbohydrate.
Safety:Please read the Safety Data Sheets (SDS’s) for all chemicals used. All processes involving labeling reagents should be performed using appropriate personal safety protection – eyeglasses, chemically resistant gloves (e.g. nitrile), etc. – and where appropriate in a laboratory fume cupboard
For Research Use Only. Not For Human Or Drug Use
HPLC System Requirements
LudgerSep N1 columns can be used with an HPLC system capable of delivering accurate gradients at a flow rate of 0.3 to 1.0 ml/min. In general, systems which mix eluants at high pressure (after the pump head) have lower dead volumes and supply more accurate gradients that are appropriate at the flow rate needed for LudgerSep columns.
For the 4.6mm column inject the sample in up to 100 µl of the starting buffer (i.e. the solvent mixture used at the very start of the HPLC gradient).
A fluorescence detector is required with the following detection wavelengths:
2-AB [2-aminobenzamide] – λex 330 nm / λem 420 nm
2-AA [2-aminobenzoic acid] – λex 330 nm / λem 420 nm
AA-Ac [3-(acetylamino)-6-aminoacridine] – λex 442 nm / λem 525 nm
For optimal detection, use wide slit widths (e.g. 10 – 20 nm). Sub-picomole levels of 2-AB or 2-AA labelled glycans can be detected with good signal-to-noise (depending on the sensitivity of the detector used).
To improve repeatability and intermediate precisions for glycan analyses use a column temperature controller. Good results can be obtained with a column temperature of 35˚C.