Amide 80 column

Manufactured by Tosoh

Sourced in Japan

The Amide-80 column is a high-performance liquid chromatography (HPLC) column designed for the separation and purification of a wide range of organic compounds. The column utilizes an amide-based stationary phase to provide efficient separation and high resolution. The core function of the Amide-80 column is to facilitate the separation and analysis of various chemical species in complex mixtures.

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Lab products found in correlation

Lc 20ad pump, by Shimadzu (1 mentions) Analytical hplc system, by Shimadzu (1 mentions) Sil 20ac autosampler, by Shimadzu (1 mentions) Hplc system, by Shimadzu (1 mentions) Hypersil ods column, by Agilent Technologies (1 mentions) Rf 10axl, by Shimadzu (1 mentions) Xcalibur program, by Thermo Fisher Scientific (1 mentions) Superdex peptide column, by GE Healthcare (1 mentions) Ltq orbitrap velos, by Thermo Fisher Scientific (1 mentions) Ods 100v column, by Tosoh (1 mentions) Hplc system, by Tosoh (1 mentions) 2 aminobenzamide, by Merck Group (1 mentions) Dionex ultimate 3000, by Thermo Fisher Scientific (1 mentions) Orbitrap fusion tribrid mass spectrometer, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

TSKgel Amide-80 column (43 citations) TSK gel amide-80 HILIC column (5 citations) TSK Amide-80 column (3 citations) TSKgel Amide-80 5-μm particle column (3 citations) TSK-gel Amide-80 (amide-silica) column (2 citations)

11 protocols using amide 80 column

Quantitative Analysis of Human Milk Oligosaccharides

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High-performance liquid chromatography (HPLC) was used to characterize HMOs in breast milk as previously described (23 (link)). Briefly, 20 uL of human milk were spiked with the non-HMO raffinose as an internal standard to allow for absolute quantification. Oligosaccharides were extracted by high-throughput solid phase extraction over C18 and Carbograph microcolumns and fluorescently labeled with 2-aminobenzamide (2AB). Labeled oligosaccharides were analyzed by HPLC on an amide-80 column (4.6 mm inner diameter 3 25 cm, 5 mm; Tosoh Bioscience) with a 50-mmol/L ammonium formate–acetonitrile buffer system. Separation was performed at 25C and monitored with a fluorescence detector at 360 nm excitation and 425 nm emission. Peak annotation was based on standard retention times and mass spectrometric analysis on a Thermo LCQ Duo Ion trap mass spectrometer equipped with a Nano-ESI-source. The total concentration of HMOs was calculated as the sum of the most common oligosaccharides. The proportion of each HMO per total HMO concentration was calculated.

Bender J.M., Li F., Martelly S., Byrt E., Rouzier V., Leo M., Tobin N., Pannaraj P.S., Adisetiyo H., Rollie A., Santiskulvong C., Wang S., Autran C., Bode L., Fitzgerald D., Kuhn L, & Aldrovandi G.M. (2016). Maternal HIV Infection Influences the Microbiome of HIV Uninfected Infants. Science translational medicine, 8(349), 349ra100.

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Glycoconjugate Synthesis via Reductive Amination

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In a 1.5 mL Eppendorf tube, linker 12 (1.4 mg to 17.2 mg, 10 equiv., 1 M) in dioxane/ammonium acetate buffer 1:1 (v:v) pH 4.6 was added to the dried free reducing glycan (0.2 mg to 2.8 mg, 1 equiv., 100 mM). The mixture was stirred and incubated at 40 °C in a heating block for 72 h. The crude reaction was concentrated under vacuum (SpeedVac) and cleaned up using NH₂-SPE cartridges. An isocratic elution with 85% MeCN was used for branched ABH blood group trisaccharides and a gradient elution from 85% to 50% MeCN for sialylated glycoconjugates. Fractions containing the glycoconjugates were lyophilized, purified by NP-HPLC on a Tosoh Amide-80 column with flow rate 1 mL/min and a linear gradient from 75 to 40% B in A over 40 min (A= 10 mM ammonium formate, pH 7; B= 95% MeCN) and characterized by MALDI-TOF MS; 10 pmol of conjugate is estimated to result in a signal of 80 mV.

Jiménez-Castells C., Stanton R., Yan S., Kosma P, & Wilson I.B. (2016). Development of a multifunctional aminoxy-based fluorescent linker for glycan immobilization and analysis. Glycobiology, 26(12), 1297-1307.

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N-Glycan Derivatization and Purification

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The dried N-glycans were dissolved in 14 μl of Dimethyl sulfoxide (DMSO) and 6 μl of acetic acid, and 1 mg 2-aminobenzoic acid (2-AA) and 2.7 mg 2-picoline borane (2-PB) were quickly added for 2-AA derivatization^{55 (link)}. The mixture was incubated at 65 °C for 2 h, diluted with 0.5 ml of 1-butanol/ethanol/H₂O (4:1:1, v/v/v) and purified with a microcrystalline cellulose (MCC) column (Sigma-Aldrich)^{56 (link)}. The MCC column was equilibrated with 3 ml of 1-butanol/ethanol/H₂O (4:1:1, v/v/v), and the diluted N-glycans were loaded and washed with 3 ml of the equilibrium buffer, followed by elution with 1 ml of ethanol/H₂O (1:1, v/v). The eluate was dried under vacuum and analyzed with an HPLC analytical system (Shimadzu, Nakagyo-ku, Kyoto, Japan) consisted of two LC-20AD pumps, a SIL-20AC auto sampler, and a RF10AXL fluorescence detector. The 2-AA-derivatized N-glycans were resolved in Solvent A (50 mM ammonium formate) and analyzed on an Amide 80 column (Tosoh, Tokyo, Japan; 4.6 mm I.D., 250 mm) set at 40 °C. After sample injection, the column was eluted with the mixture of Solvent A and B containing 68% Solvent B (100% ACN) for 5 min at a flow rate of 1.0 ml/min, and then a 60-min linear decreasing gradient of 68–43% Solvent B. The excitation/emission wavelengths of the fluorometric detection were λ_ex = 360 nm and λ_em = 419 nm for 2-AA derivatives.

Zhang L., Wang P., Wang C., Wu Y., Feng X., Huang H., Ren L., Liu B.F., Gao S, & Liu X. (2019). Bacteriophage T4 capsid as a nanocarrier for Peptide-N-Glycosidase F immobilization through self-assembly. Scientific Reports, 9, 4865.

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HPLC-FL Separation of 2AB-Labeled HMOs

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Isolated, dried HMOs from intestinal samples were fluorescently labeled with 2AB and cleaned with silica spin columns as previously described. The 2AB-glycans were separated by HPLC-FL on an amide-80 column (4.6 mm ID × 25 cm, 5 mm, Tosoh Bioscience, Tokyo) with a linear gradient of a 50 mM ammonium formate/acetonitrile buffer system. The separation was performed at 25 °C and monitored by a fluorescence detector at 360 nm excitation and 425 nm emission.

Pruss K.M., Marcobal A., Southwick A.M., Dahan D., Smits S.A., Ferreyra J.A., Higginbottom S.K., Sonnenburg E.D., Kashyap P.C., Choudhury B., Bode L, & Sonnenburg J.L. (2020). Mucin-derived O-glycans supplemented to diet mitigate diverse microbiota perturbations. The ISME Journal, 15(2), 577-591.

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Glycan Separation and Analysis via HPLC

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Separation of PA-labelled glycans was carried out on a Shimadzu HPLC system equipped with a fluorescence detector (RF 10 AXL). First the glycans were fractionated by NP-HPLC using a Tosoh Amide-80 column (4.6 × 250 mm); dried samples were taken up in 50 μl of a 25:75 mixture of buffer A (10 mM ammonium formate, pH 7) and buffer B (95% acetonitrile) prior to injection and the following gradient was applied: 0-5 mins, 75% B; 5-15 mins, 75-65% B; 15-40 mins, 65% B; 40-55 mins, 65-57% B; followed by a return to the starting conditions. Selected fractions were then subject to RP-HPLC on a Hypersil ODS column (5 μm, 4 × 250 mm; Agilent), whereby buffer C was 0.1 M ammonium acetate, pH 4, and buffer D was 30% (v/v) methanol. Gradients of increasing methanol (1% buffer D per minute) were applied. Fluorescence was recorded at 320 nm (excitation) and 400 nm (emission). The columns were calibrated daily in terms of glucose units (g.u.) with a pyridylaminated partial dextran hydrolysate. Various modifications of N-glycans have different effects on retention time as compared to the ‘parent’ structure: α1,3-fucose and α1,6-fucose resulting in either 2 g.u. earlier or 3 g.u. later RP-HPLC elution; bisecting galactose in 3 g.u. earlier elution on RP-HPLC; intersecting galactose in 1 g.u. earlier elution on RP-HPLC; methylation in 0.3 g.u. earlier on NP-HPLC, but later elution on RP-HPLC.

Yan S (.闫.石.)., Wang H (.王.彗.捷.)., Schachter H., Jin C (.金.春.生.)., Wilson I.B, & Paschinger K. (2018). Ablation of N-acetylglucosaminyltransferases in Caenorhabditis induces expression of unusual intersected and bisected N-glycans. Biochimica et biophysica acta. General subjects, 1862(10), 2191-2203.

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HPLC Purification and Labeling of HMOs

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HMOs were extracted from mouse ileum and feces, purified over C18 and Carbograph microcolumns, fluorescently labeled with 2-aminobenzamide (2AB) and separated by HPLC on an amide-80 column (4.6 mm ID × 25 cm, 5 μm, Tosoh Bioscience, Tokyo) with a 50 mM ammonium formate/acetonitrile buffer system. The separation was monitored by a fluorescence detector at 360 nm excitation and 425 nm emission. Peak annotation was based on standard retention times and mass spectrometric (MS) analysis on a Thermo LCQ Duo Ion trap mass spectrometer equipped with a Nano-ESI-source.

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Sodium Alginate Degradation Analysis

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The products resulting from the degradation of sodium alginate were examined by electrospray ionization mass spectrometry, with the resulting data analyzed by the Xcalibur program (Thermo Fisher Scientific, Waltham, MA, USA). The mass spectrometer was operated in the negative mode. To prepare the samples, the reaction mixtures were incubated at 30 °C for 48 h. After centrifugation, the samples were applied to a Superdex peptide column (GE Healthcare). The degradation products, rAlgSV1-PL7 and undigested sodium alginate, were separated based on the absorbance at 232 nm. The collected fractions were applied to an Amide-80 column (Tosoh, Tokyo, Japan) and the samples were eluted with a linear gradient of 95–70% acetonitrile.

Yagi H., Isobe N., Itabashi N., Fujise A, & Ohshiro T. (2017). Characterization of a Long-Lived Alginate Lyase Derived from Shewanella Species YH1. Marine Drugs, 16(1), 4.

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Glycan Profiling by LC-MS

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The LC-MS analysis were performed on a Thermo Dionex UltiMate HPLC system with a fluorescent detector, equipped with a Tosoh Amide 80 column (2.1 micron, 15 cm × 2.0 mm). The chromatographic conditions were as follows: solvent A, 100% acetonitrile; solvent B, ammonium formate, 50 mM, pH 4.4 in 100% water; flow rate, 350 μl min⁻¹ throughout the run; gradient, started at 80% A, decreased to 40% from 2 to 34 min, before decreased further to 10% than back to 80% for reconditioning. The fluorescence detector was set at 350 nm for excitation and 420 nm for emission. The HPLC was coupled with a Thermo LTQ Orbitrap Velos mass spectrometer, equipped with a heated electrospray ionization interface. The major parameters were as follows: ESI spray voltage, 3.5 kV; capillary temperature, 250 °C; sheath gas, 11 psi; Aux gas and sweep gas flow rates, 0; S-lens RF level, 66%; Multiple 00 offset, 2.5 V; Lens 0 voltage, 6.5 V; Multiple 0 offset, 7.0 V; Lens 1 voltage, 16 V; Multiple 1 offset, 6.5 V; Multiple RF Amplitude, 600; Front lens, 7.75 V. The fluorescence traces of glycans released were superimposed for display. The identity of the major fluorescent peaks was assigned by corresponding mass value at the specific retention time, according to the glycan compositions of common mammalian N-glycan structures.

Chen M., Shi X., Duke R.M., Ruse C.I., Dai N., Taron C.H, & Samuelson J.C. (2017). An engineered high affinity Fbs1 carbohydrate binding protein for selective capture of N-glycans and N-glycopeptides. Nature Communications, 8, 15487.

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Purification of Saccharide 1 from Super Ohtaka

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Super Ohtaka^® (1 kg, Brix: 59 %) was loaded onto a 4.5 × 35 cm carbon-Celite column consisting of 1:1 charcoal/Celite-535 (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and successively eluted in water (14 L) and 5 % ethanol (30 L). Almost all of glucose and fructose were eluted with water (0–4 L), while the fraction containing the target saccharide named saccharide 1 was eluted in 5 % ethanol fraction (1–3 L). Then, the fraction (2 L) containing the saccharide was concentrated to 10 mL. Subsequently, the concentrated fraction was repeatedly applied to preparative-HPLC (Tosoh Corporation, Tokyo, Japan) equipped with an Amide-80 column (7.8 mm × 30 cm, Tosoh Corporation) at 80 °C and eluted with 80 % acetonitrile at 2 mL/min using refractive index detection (Fig. 1). Furthermore, saccharide 1 was purified at room temperature using an HPLC system (Tosoh Corporation) equipped with an ODS-100V column (4.6 mm × 25 cm, Tosoh Corporation) and eluted with distilled water at 0.5 mL/min using refractive index detection. The purified saccharide 1 solution was freeze-dried to give a white powder (1.8 mg).

Yamamori A., Takata Y., Fukushi E., Kawabata J., Okada H., Kawazoe N., Ueno K., Onodera S, & Shiomi N. (2017). Structural Analysis of a Novel Oligosaccharide Isolated from Fermented Beverage of Plant Extracts. Journal of Applied Glycoscience, 64(4), 123-127.

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Quantitative Analysis of Human Milk Oligosaccharides

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Concentrations of HMOs (mg/mL) were measured, as previously described^{57 (link)}. Briefly, 20 µL of human milk were dried in a 96-well plate and oligosaccharides were fluorescently labeled with 2-aminobenzamide (2AB, Sigma) in a thermocycler heat block at 65 °C for exactly 2 h. The reaction was stopped abruptly by reducing the thermocycler temperature to 4 °C. The amount of 2AB was titrated to be in excess to account for the high and variable amount of lactose and other glycans in milk samples. Labeled oligosaccharides were analyzed by HPLC (Dionex Ultimate 3000, Dionex, now Thermo) on an amide-80 column (15 cm length, 2 mm inner diameter, 3 μm particle size, Tosoh Bioscience)) with a 50-mmol/L ammonium formate–acetonitrile buffer system. Separation was performed at 25 °C and monitored with a fluorescence detector at 360 nm excitation and 425 nm emission. Peak annotation was based on standard retention times of commercially available HMO standards and a synthetic HMO library and offline mass spectrometric analysis on a Thermo LCQ Duo Ion trap mass spectrometer equipped with a Nano-ESI-source. Concentrations were estimated by calculating area under the curve for each annotated HMO. Absolute quantification was not calculated because of fortifier interference. HMO concentrations between the groups were compared by Mann–Whitney test.

Bai-Tong S.S., Thoemmes M.S., Weldon K.C., Motazavi D., Kitsen J., Hansen S., Furst A., Geng B., Song S.J., Gilbert J.A., Bode L., Dorrestein P.C., Knight R., Leibel S.A, & Leibel S.L. (2022). The impact of maternal asthma on the preterm infants' gut metabolome and microbiome (MAP study). Scientific Reports, 12, 6437.

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