SepaFlash Gamma C18AQ Cartridge and Its Application in the Purification of Glutamine Derivatives

The background of Applied Technology Research Center introduces glutamine, 2-amino-4-carbamoyl butyric acid Its molecular formula is C5H10N2O3, and its molecular weight is 146.16 g / mol Glutamine is a polar and uncharged derivative of acid amino acid glutamic acid It has formamide group It is neutral at physiological pH and can be converted into carboxylic acid by hydrolysis to form glutamic acid The formamide group in glutamine can form hydrogen bond Glutamine is a kind of nonessential amino acid, which means that glutamine can be synthesized by human body itself, and does not need to be obtained from external sources [1] Glutamine is one of the most abundant amino acids in human body It can directly pass through the blood-brain barrier through blood circulation In biochemistry, glutamine has many functions In addition to participating in protein synthesis, it can also help maintain a neutral pH in the liver by balancing acid and alkali levels [3] Similar to glucose, glutamine can provide energy for cell bodies [4] It provides nitrogen to cells through anabolic reactions [5], and carbon in the citric acid cycle [6] In the gastrointestinal system, glutamine is also an important source of energy for small intestine activities [7] In the research of anticancer drugs, some cancer cells show "addictive" effect on glutamine, which has become a potential target of new cancer therapy [8] However, because glutamine is essential for many basic physiological processes in the human body, such as synaptic communication in the brain, simple removal of glutamine from the human body is not a feasible treatment and is very dangerous Therefore, researchers focused on protein targets related to glutamine metabolism pathway [9,10] In clinical nutrition research, glutamine and its derivatives have multiple proven efficacy in the treatment of trauma, infection, critical patients, bone marrow transplantation, small intestine transplantation and other diseases Its functions include maintaining glutamine concentration in skeletal muscle, improving nitrogen balance, promoting protein synthesis, avoiding intestinal atrophy caused by trauma, reducing intestinal mucositis caused by chemotherapy, and enhancing immunity [11] Therefore, glutamine derivatives have been paid more and more attention in clinical nutrition research In this application case, the sample is a highly polar glutamine derivative, which is not easily soluble in normal mobile phases such as n-hexane and ethyl acetate, but hardly retained on the common C18 reversed-phase column According to the specific properties of the samples, the application engineers of Santai technology use the hydrophilic sepaflash ® c18aq column with the rapid liquid phase preparation chromatographic system sepabean ® machine to successfully purify the samples and obtain the target products to meet the preparation requirements, which provides a feasible scheme for the separation and purification of highly polar glutamine samples In the experimental part, the samples to be purified in this paper are from a pharmaceutical company, which are glutamine derivatives with great polarity See Figure 1 for the schematic diagram of their molecular structure and Figure 2 for the purity of the crude product (see Figure 2 for the HPLC analysis map) Fig 1 Schematic diagram of molecular structure of glutamine derivatives Fig 2 HPLC analysis of crude product Dissolve 1.5g sample in 1mldmso, and make it completely dissolved into clear and transparent solution by ultrasound Inject the sample onto the flash separation column with syringe, and the flash separation and purification experimental parameters of the sample are shown in Table 1 Table 1 Sepabean ® machine t chromatographic column 12 g sepaflash ® C18 reversed phase separation column (spherical silica gel, 20-45 μ m, 100 Å, order No.: sw-5222-012-sp) 120 g sepaflash ® c18aq reversed phase separation column (spherical silica gel, 20-45 μ m, 100 Å, order No.: sw-5222-120-sp (AQ)) Detection wavelength 220 nm, 254 nm mobile phase solvent A: water; solvent B: acetonitrile flow rate 25 ml / min 40 ml / min injection volume 300 mg 1.2 g elution gradient time (min) solvent B (%) time (min) solvent B (%) 0 000 15 20 Results and discussion of 10.00 / / 12.02.0 / / 16.02.0 / / 17.595 / / 30.095 First, we used a common C18 reversed-phase column to purify a small number of samples The flash preparation map of the sample on the common C18 reversed-phase column is shown in Figure 3 Fig 3 Analysis of flash separation spectrum of sample on common C18 column Fig 3 shows that the sample is almost not retained on common C18 reversed-phase column Along with sample solvent DMSO, it is directly eluted from flash column by mobile phase, so the target product cannot be effectively separated from impurities in the sample For this result, we think the reason can be attributed to the hydrophobic collapse effect of the stationary phase In reversed-phase chromatography, the commonly used eluting solvents can be sorted as follows: water < methanol < acetonitrile < ethanol < tetrahydrofuran < isopropanol according to their elution strength For the samples with high polarity or strong hydrophilicity, in order to ensure that such samples and the stationary phase of the separation column can produce enough retention effect, it is usually necessary to use a high proportion of water phase system as the mobile phase, sometimes even need to use 100% of water phase When pure water phase system (including pure water or pure salt solution) is used as mobile phase, the long carbon chains on the C18 reversed-phase column stationary phase tend to avoid pure water mobile phase due to hydrophobic effect and mix with each other, resulting in the instantaneous retention capacity of chromatographic column decreased or even no retention effect, which is called hydrophobic collapse of stationary phase (see the left part of Figure 4) Although this situation is reversible (it can be washed with methanol or acetonitrile and other organic solvents), it will cause certain damage to the chromatographic column, so it is necessary to avoid this situation as much as possible In addition, for the separation and purification of samples with large polarity or strong hydrophilicity, the common C18 reversed-phase column is a little powerless Figure 4 Schematic diagram of bonding phase on the surface of silica gel of common C18 column and c18aq column In view of the above situation, the manufacturer of chromatographic packing has made technical improvement Through some modifications on the surface of silica gel matrix, such as introducing hydrophilic cyano (see the right part of Figure 4), the hydrophilicity of silica gel surface is stronger, so as to prevent the occurrence of hydrophobic collapse The modified C18 column is named as c18aq column, i.e hydrophilic C18 column, which is specially designed for high water phase elution conditions and can withstand 100% water phase system C18aq column has been widely used in the separation and purification of organic acids, peptides, nucleosides and water-soluble vitamins We used a C18AQ column to separate and purify the sample The Flash preparation profile of the sample on the C18AQ column is shown in Fig 5 It can be seen from Figure 5 that glutamine derivative samples have sufficient retention on c18aq column, and other impurities in the crude products have been effectively separated After freeze-drying of the collected components, the purity of the purified product is more than 98% (see Figure 6) after HPLC analysis, which can be used for the next research and development Figure 5 Flash separation map of sample on c18aq column Figure 6 HPLC analysis map conclusion of target product after purification Due to the effect of hydrophobic collapse of stationary phase, the common C18 reversed-phase column is limited in the separation and purification of glutamine derivatives, while the improved c18aq column overcomes the above effect and is successfully applied in the preparation and purification of such samples in this case Therefore, for the separation and purification of large polar glutamine derivatives, sepaflash ® c18aq reversed-phase separation column combined with sepabean ® machine rapid liquid chromatography system provides a fast and efficient solution for the rapid separation and purification of such samples Sepaflash ® c18aq reversed phase separation column series products launched by Santai technology have a variety of specifications (see Table 2) Table 2 Sepaflash ® c18aq reversed phase separation column parameters (packing: high efficiency spherical C18 (AQ), 20 – 45 μ m, 100 μ m) item number column size flow rate (ml / min) max.pressure (PSI / bar) sw-5222-004-sp (AQ) 5.4G 5-15 400 / 27.5 sw-5222-012-sp (AQ) 20 g 10-25 400 / 27.5 SW-5222-025-SP(AQ) 33 g 10-25 400/27.5 SW-5222-040-SP(AQ) 48 g 15-30 400/27.5 SW-5222-080-SP(AQ) 105 g 25-50 350/24.0 SW-5222-120-SP(AQ) 155 g 30-60 300/20.7 SW-5222-220-SP(AQ) 300 g 40-80 300/20.7 Sw-5222-330-sp (AQ) 420 g 40-80 250 / 17.2 to learn more about the detailed specifications of sepabean ® machine, or the ordering information of flash purification column used together, please visit CBG online store: https://store.chembiango.com/ References 1 Lacey, J M.; Wilmore, D W (1990) Is glutamine a conditionally essential amino acid? Nutrition Reviews 48 (8): 297 – 309 2 Roth, e (2008) Nonnutritive effects of glutamine The Journal of nutrition 138 (10): 2025s-2031s 3 Hall, J E.; Guyton A C (2006) Textbook of medical physiology (11th ed.) St Louis, Mo: Elsevier Saunders p 393 4. Aledo, J C ( 2004 ) Glutamine breakdown in rapidly dividing cells: Waste or investment?  BioEssays 26 (7): 778–785 5. Brosnan, J T ( 2003 ) Interorgan amino acid transport and its regulation.  The Journal of Nutrition 133(6 Suppl 1): 2068S–2072S 6. Yuneva, M.; Zamboni, N.; Oefner, P.; et al ( 2007 ) Deficiency in glutamine but not glucose induces MYC-dependent apoptosis in human cells.  The Journal of Cell Biology  178 (1): 93–105 7. Yamamoto, T.; Shimoyama, T.; Kuriyama, M ( 2016 ) Dietary and enteral interventions for Crohn's disease.  Current Opinion in Biotechnology  44: 69–73 8. Wise, D R.; Thompson, C B ( 2010 ) Glutamine addiction: a new therapeutic target in cancer.  Trends in biochemical sciences , 35(8): 427–433 9. Smith, B.; Schafer, X L.; Ambeskovic A.; et al ( 2016 ) Addiction to coupling of the Warburg Effect with glutamine catabolism in cancer cells.  Cell Reports , 17: 821-836 10. Altman, B J.; Stine, Z E.; Dang, C V ( 2016 ) From Krebs to clinic, glutamine metabolism to cancer therapy.  Nature Reviews Cancer , 16: 619-634 11. Rajendram R.; Preedy, V R.; Patel V B ( 2015 )  Glutamine in Clinical Nutrition (2015th ed.) 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