SepaFlash HILIC ARG cartridge and Its Application in the Purification of Oligosaccharides

Background of Applied Technology Research Center introduces that oligosaccharide is a kind of glycopolymer, which is made of a small number of (usually 3-10 [1-3]) monosaccharide molecules by glycosidic bond polymerization Oligosaccharides are ubiquitous in the cell membrane of animal cells and have many functions including cell recognition and cell binding [4] For example, glycolipid molecules play an important role in the immune response process [5] In nature, oligosaccharides are often part of glycoproteins and glycolipids Oligosaccharides bind to lipid or appropriate intramolecular amino acid side chains via n-or O-glycoside bonds Oligosaccharides linked by N-glycoside bonds are always pentasaccharides, which are linked to asparagine in the side chain of amino acids by β bonds [6] O-glycoside linked oligosaccharides are usually linked to threonine or serine on the alcohol group in the amino acid side chain According to nutrition experts and many medical researches, oligosaccharides have similar functions as water-soluble dietary fiber, which can promote intestinal peristalsis, improve constipation, diarrhea and other problems Since the human small intestine can only consume oligosaccharides incompletely, the indigestible part of oligosaccharides will let the intestinal colony use [7, 8] Therefore, it can change the intestinal ecology, normalize the flora ecology of the human digestive tract, increase the number of beneficial bacteria, help improve the normal digestion and movement of the intestinal tract, reduce the absorption of toxins, prevent the incidence of colorectal cancer and enteritis, and improve the blood lipid level Therefore, more and more researchers pay attention to the application of oligosaccharides in medicine, nutrition, immunology and so on In this application case, the sample is a synthetic oligosaccharide with strong polarity, which is very weak on the common C18 reversed-phase column In addition, its UV absorption is very weak, so it is not suitable to use UV detector to detect it According to the specific properties of the samples, the application engineers of Santai technology use sepaflash ® HILIC Arg column to prepare the chromatographic system sepabean ® machine with fast liquid phase It was successfully purified and prepared with external ELSD detector, and the target product meeting the preparation requirements was obtained, which provided a feasible scheme for the purification and preparation of oligosaccharide samples with strong polarity Experiment part the sample to be purified in this case is from a university laboratory engaged in oligosaccharide application research In order to synthesize oligosaccharide compounds, the sample contains other by-products with different degree of polymerization, which are very polar and soluble in water See Figure 1 for the schematic diagram of its molecular structure Figure 1 Schematic diagram of molecular structure formula of oligosaccharide sample due to the large polarity of sample molecule, easy to dissolve in water and low solubility in organic solvent, so the positive phase separation mode is excluded first In the reversed phase separation mode, C18 reversed phase separation column is a common separation column, which is suitable for the separation of all kinds of polar or non-polar samples Therefore, we first consider using a C18 reversed phase separation column to try to separate and purify the samples Take a small amount of sample and dissolve it in water, use syringe to sample on flash separation column, and the flash separation and purification experimental parameters of the sample are shown in Table 1 Table 1 Flash preparation and purification experiment parameters setting instrument SepaBean machine T column 12 g SepaFlash C18 reversed phase separation column (spherical silica gel, 20-45 μ m, 100 μ m, order No.: SW-5222-012-SP) 12 g SepaFlash HILIC ARG column (spherical silica gel, 20-45 μ m, 100 μ m, order No.: sw-5622-012-sp) Detection wavelength 254 nm, 280 nm, ELSD mobile phase solvent A: water solvent B: acetonitrile flow rate 30 ml / min injection volume 30 mg elution gradient time (CV) solvent B (%) time (CV) solvent B (%) 0 100 95 15.0 90 8.0 80 // 15.0 80 23.0 53 25.0 53 32.0 25 37.0 25 results and the flash preparation map of the sample on the common C18 reversed-phase column are shown in Figure 2 It can be seen from Fig 2 that, due to the large polarity of the sample, the sample is basically not retained on the common C18 reversed-phase column As the mobile phase is directly eluted from the separation column, the effective purification cannot be obtained In addition, it can be seen from the flash preparation map that there is no absorption signal in the UV band of the sample, so it is necessary to use the external ELSD detector to detect the signal of the sample Fig 2 Flash preparation map of sample on C18 reversed-phase column is based on the experimental results of common C18 reversed-phase column without reservation for the sample Next, we consider the separation and purification of the sample by using the hydrophilic interaction chromatography (HILIC) mode The HILIC separation mode adopts a highly hydrophilic polar stationary phase, such as sepaflash ® HILIC Arg separation column launched by Santai technology, whose packing surface is bonded with a highly polar Arg group (see Figure 3), which can produce enough retention effect on the hydrophilic compound sample Under the HILIC separation mode, the proportion of water phase in the mobile phase gradually increases during the elution process, so it is suitable for the separation and purification of samples with larger polarity We used a sepaflash ® HILIC Arg column to separate and purify the sample The flash preparation map is shown in Figure 4 It can be seen from Figure 4 that the sample has been effectively retained on the HILIC Arg separation column, and the impurities such as target product and by-product have a good separation TLC spot plate confirmation was carried out for the collected components Concentrated sulfuric acid / methanol (V: v = 1:5) was used as the color developing agent After baking the TLC plate for 15 min with a hot air gun, the color developing results of the sample spot were as shown in Figure 5 It was confirmed that the target product had been effectively separated from impurities such as by-products and could be used in the next research and development Fig 3 Schematic diagram of bonding phase on the packing surface of sepaflash HILIC Arg separation column Fig 4 Flash preparation map of sample on HILIC Arg separation column Fig 5 TLC spot plate test results of crude products and collected components Therefore, for purification preparation of oligosaccharide samples with great polarity and no UV absorption, sepaflash ® HILIC The Arg column with the rapid liquid preparation chromatographic system sepabean ® machine equipped with ELSD detector provides a feasible scheme for the rapid preparation and purification of such samples Sepaflash? HILIC Arg separation column series products launched by Santai technology have a variety of specifications (see Table 2) Table 2 Sepaflash ® HILIC Arg separation column parameters (packing: high efficiency spherical Arg, 20 – 45 μ m, 100 Ω) item number column size sample size max.pressure (PSI / bar) sw-5 6 22-004-sp 5.4 g 5.4 mg – 108 mg 400 / 27.5 sw-5 6 22-012-sp 20 g 20 mg – 0.40 g 400/27.5 SW-5 6 22-025-SP 33 g 33g –  0.66 g 400/27.5 SW-5 6 22-040-SP 48 g 48 mg –  0.96 g 400/27.5 SW-5 6 22-080-SP 105 g 105 mg –  2.1 g 350/24.0 SW-5 6 22-120-SP 155 g 155 mg –  3.1 g 300/20.7 SW-5 6 22-220-sp 300 g 300 mg – 6.0 g 300 / 20.7 sw-5 6 22-330-sp 420 g 420 mg – 8.4 g 250 / 17.2 to learn more about the detailed specifications of sepabean ® machine, or the ordering information of the flash purification column used together, please visit CBG online store: https://store.chembango.com/ References 1 Oligosacharides at the US National Library of medical subject headings (mesh) 2 Walstra P, Wouters JT, gears TJ (2008) Daily science and Technology (Second ed.) CRC, Taylor & Francis 3 Whitney e, rolfesr (2008) Understanding nutrition (eleven ed.) Thomson Wadsworth 4 Varki a, ( 1993 ) Biological roles of oligosaccharides: all of the theories are correct Glycobiology 3 (2): 97-130 5. Mattner J, DeBord KL, Ismail N, et al ( 2005 ) Exogenous and endogenous glycolipid antigens activate NKT cells during microbial infections Nature 434: 525-529 6. Voet D, Pratt C ( 2013 ) Fundamentals of Biochemistry: Life at the Molecular Level (4th ed.) Hoboken, NJ: John Wiley & Sons, Inc ISBN 978-0470-54784-7 7. Bode L ( 2009 ) Human milk oligosaccharides: prebiotics and beyond Nutrition Reviews 67 (2): S183–91 8. De Filippo C, Cavalieri D, Di Paola M, et al ( 2010 ) Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa Proceedings of the National Academy of Sciences 107 (33): 14691–14696.