Simple sugars and the major oligosaccharides, 3′-sialyllactose (3′SL), 6′-sialyllactose (6′SL) and 6′-sialyllactosamine (6′SLN), were quantified by high-performance anion-exchange chromatography with pulsed ampero-metric detection (Thermo Scientific, HPAE-PAD ICS-5000, Sunnyvale, CA). Whey permeate samples were diluted 10-fold for oligosaccharide analysis and 1000-fold for simple sugar analysis, and filtered through a 0.2-μm membrane (Pall, Port Washington, NY). For mono- and disaccharide analysis, a 25-μL aliquot was injected into a CarboPac PA10 analytical column (3 × 250 mm, Dionex, Sunnyvale, CA) and guard column (3 × 50 mm, Dionex) at a 1.2 mL/min flow rate with an isocratic eluent of 10 mM NaOH for 12 min and a gradient that increased to 100 mM sodium hydroxide over the next 13 min. For oligosaccharide analysis, a CarboPac PA200 column (3 × 250 mm, Dionex) and guard column (3 × 50 mm, Dionex) were used. The isocratic eluent was 10 mM sodium acetate in 100 mM sodium hydroxide at 0.5 mL/min. More details were described in a previous publication.29 (link)
Guard column
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
A guard column is a short, inexpensive column placed in front of the analytical column in a high-performance liquid chromatography (HPLC) or gas chromatography (GC) system. The primary function of a guard column is to protect the analytical column from contamination and wear, prolonging its lifetime and performance.
Automatically generated - may contain errors
Lab products found in correlation
Hypersil gold c18 column, by Thermo Fisher Scientific (3 mentions)
Carbopac pa200 column, by Thermo Fisher Scientific (2 mentions)
Ultimate 3000, by Thermo Fisher Scientific (2 mentions)
Carbopac pa1 column, by Thermo Fisher Scientific (1 mentions)
Carbopac pa 10 analytical column, by Thermo Fisher Scientific (1 mentions)
Hpae pad ics 5000, by Thermo Fisher Scientific (1 mentions)
Tsq vantage, by Thermo Fisher Scientific (1 mentions)
Propac pa1 column, by Thermo Fisher Scientific (1 mentions)
Agilent 1290 infinity series uhplc system, by Agilent Technologies (1 mentions)
C18 column, by Thermo Fisher Scientific (1 mentions)
Hpaec pad, by Thermo Fisher Scientific (1 mentions)
Carbopac ma 1 column, by Thermo Fisher Scientific (1 mentions)
Hypercarb column, by Thermo Fisher Scientific (1 mentions)
Agilent 1200 hplc system, by Agilent Technologies (1 mentions)
11 protocols using guard column
1
Quantification of Oligosaccharides and Sugars
2
Quantifying Chondroitin Sulfate Synthesis
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Sulfation of endogenous and [3H]‐glucosamine‐labelled CS was monitored in papain‐digested cartilage explants, as described previously.30 , 41 Briefly, papain‐digested tissue samples were centrifuged to remove tissue debris, and GAGs were pelleted, washed, dried, resuspended and digested with chondroitinases ABC and ACII (Seikagaku Corp., Tokyo, Japan). Enzymatically digested CS, containing the unsaturated disaccharides of the internal CS chains, and the nonreducing terminal mono‐ and disaccharides were concentrated using the MicroCon® 3 filters, reduced with NaBH4 and injected onto a CarboPac PA1 column that was connected to a guard column (Dionex GmbH, Idstein, Germany). Specimens were eluted by a step gradient and scanned on a HPAEC‐PAD system (model DX 500; Dionex GmbH, Idstein, Germany) to quantify the unlabelled, unsaturated disaccharides ΔDi6S, ΔDi4S and ΔDi0S, obtained from the digested interior part of the CS chains. To quantify [3H]‐radiolabelled mono‐ and disaccharides, 1‐ml fractions of the eluant were collected and the radioactivity was quantified by liquid scintillation counting. We calculated the mean length of the newly synthesized CS chains (ie the number of internal Δdisaccharides per chain). For this purpose, the ratio of the radioactively labelled internal unsaturated disaccharides to the terminal saccharides was computed.41 , 42
3
Quantifying Plant-Derived Alkaloid Compounds
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The PAs in the samples were measured using a LC-MS/MS system consisting of an UHPLC (Ultimate 3000, Thermo Scientific, San Jose, CA, USA) and a Triple Stage Quadrupole mass spectrometer (TSQ Vantage, Thermo Scientific, San Jose, CA, USA), as described previously with minor modifications [21 (link)]. Briefly, chromatographic separation was achieved on 150 × 2.1 mm, 1.9 μm particle sizes, C18 Hypersil Gold column fitted with a guard column (Thermo Scientific, Dreieich, Germany). Eluent A was 100% water with 0.1% formic acid and 5 mM of ammonium formate. Eluent B was 95% methanol and 5% water with 0.1% formic acid and 5 mM of ammonium formate. A stepwise gradient elution was conducted as follows: 0–0.5 min for 95% A/5% B, 7.0 min for 50% A/50% B, 7.5 min for 20% A/80% B, 7.6–9.0 min for 100% B and 9.1–15 min for 95% A/ 5% B. A flow rate of 300 μL/minute was applied and 10 μL of each sample was injected. The column temperature was maintained at 40 °C. Details of the mass parameters are listed in supporting data S1, Table S1 .
4
UHPLC Analysis of Compounds
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All measurements were conducted on an UltiMate 3000 (Thermo Fisher Scientific, Waltham, USA) Ultra-High-Performance Liquid Chromatography (UHPLC) system. Chromatographic reversed-phase (RP) separation with 5 µL injection volume was performed on a C18 Hypersil Gold column (150 mm × 2.1 mm; 1.9 μm particle size) with guard column (Thermo Fisher Scientific, Waltham, USA) at a flow rate of 0.3 mL/min and with a column temperature of 40 °C. The binary mobile phase was composed of water as mobile phase A and methanol as mobile phase B, both containing 0.1% formic acid and 5 mM ammonium formate. The gradient conditions were as follows: 0–0.5 min A: 95% / B: 5%, 7.0 min A: 50%/B: 50%, 7.5 min A: 20% / B: 80%, 7.6 min A: 0% / B: 100%, 10.1–15 min A: 95% / B: 5%.
5
HPLC Analysis of CHG Receptor and Tape Samples
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HPLC analysis of both CHG receptor phase and tape strip samples used the method detailed previously (42 (link)). A Thermo Scientific guard column with replaceable guard cartridges was used to ensure HPLC pressure remained stable throughout the study. The limit of detection (LoD) and limit of quantification (LoQ) were calculated from this calibration graph according to the following Eqs. (43 (link)):
The mobile phase was used as the solvent for all CHG extractions and was validated on the Shimadzu system (r2 value 0.9992). The LoD was calculated to be 0.362 µg/mL and the LoQ was calculated to be 1.098 µg/mL.
The mobile phase was used as the solvent for all CHG extractions and was validated on the Shimadzu system (r2 value 0.9992). The LoD was calculated to be 0.362 µg/mL and the LoQ was calculated to be 1.098 µg/mL.
6
Disaccharide Quantification by UHPLC
7
UHPLC Analysis of Compounds Using C18 Column
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All measurements were conducted on an Agilent 1290 Infinity Series UHPLC system (Agilent Technologies, Santa Clara, USA). Chromatographic reversed-phase separation with 2 μL injection volume was performed on a C18 Hypersil Gold column (150 mm × 2.1 mm; 1.9 μm particle size) with guard column (Thermo Fisher Scientific, Waltham, USA) at a flow rate of 0.3 mL/min and with a column temperature of 40 °C. The binary mobile phase was composed of water as mobile phase A and methanol as mobile phase B, both containing 0.1% formic acid and 5 mmol ammonium formate. The gradient conditions were as follows: 0-0.5 min A: 95%/B: 5%; 7.0 min A: 50%/B: 50%; 7.5 min A: 20%/B: 80%; 7.6 min A: 0%/B: 100%; 10.1-15 min A: 95%/B: 5%.
8
Phytochemical Analysis of YMJ by HPLC
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Phytochemical analysis of YMJ was performed using high-performance liquid chromatography (HPLC) (Shimadzu, Kyoto, Japan) with a diode array detector (DAD), a C18 column with particle size of 5 μm and dimensions of 250 × 4.6 mm, and a guard column (Thermo Fisher Scientific, MA, USA). Temperature was maintained at 40 °C throughout the analysis. Two solutions were used for the mobile phase: Solution A, which consisted of phosphoric acid in ultra-purified water (0.1% v/v); and solution B, which consisted of acetonitrile (100%). The two solutions were delivered using a gradient system as follows: 0–60 min/95–5% (A), 60–70 min/5–95% (A), and 70–75 min/95% (A), at a flow rate of 0.6 mL/min. A volume of 40 μL of the analytical standards and samples was injected, and detection was performed by the DAD at a wavelength of 340 nm.
9
Oligosaccharide Enzymatic Hydrolysis Analysis
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Oligosaccharides (10 mg/mL) with a degree of polymerization (DP) of 2 (cellobiose, laminaribiose and xylobiose) and DP 6 (cellohexaose and xylohexaose) in 500 µl, 20 mM citrate phosphate buffer, pH 5.6 were incubated with 5–10 µg/mL enzyme in duplicate reactions in a ThermoMixer (HLC Biotech, Bovenden, Germany) at 60 °C, 600 rpm. Samples (30 µL) were withdrawn at 20 min intervals for 120 min, and diluted with 970 µL 0.5 mM NaOH before analysis on high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) (Thermo Fisher Scientific, Waltham, USA), using a CarboPac PA200 column (250 mm × 3 mm, 5.5 µm) and a guard column (50 mm × 3 mm) (Thermo Fisher Scientific, Waltham, USA) of the same material. A mobile phase of 100 mM NaOH at 0.5 mL/min and a linear gradient of sodium acetate (0–120 mM) was used for 25 min. Standards included glucose, cellooligosaccharides (DP 2 to 6), xylose and xylooligosaccharides (DP 2 to 6). An identical method was applied to analyse hydrolysis of acetylated chitooligosaccharides, with N-acetylated chitooligosaccharides (DP 2 to 5) and glucosamine as standards.
10
Carbohydrate and Alditol Quantification
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Carbohydrates and alditols were quantified on a Dionex ICS 5000 ion chromatography system (Dionex, Idstein, Germany) consisting of an ICS 5000 DP-5 dual pump, an ICS 5000 AS-AP autosampler, an ICS 5000 DC-5 detector/chromatography module with column oven, and an ICS 5000 electrochemical detector cell. Aliquots (25 μL) were analyzed on a Carbo Pac MA-1 column (250 mm × 4.0 mm, Thermo Fisher Scientific, Dreieich, Germany) equipped with a guard column (50 mm × 4.0 mm, Thermo Fisher Scientific, Dreieich, Germany) and detected by a pulsed amperometric detector equipped with an Ag/AgCl electrode operating with a standard quadrupole waveform. Analytical separation was conducted at 30 °C at a flow rate of 0.4 mL/min using water and aqueous NaOH (1 M, 90:10) for 140 min. By comparison of retention times and co-chromatography with reference compounds, the identified compounds were further quantified using an external standard calibration. Data were acquired using Chromeleon v7.2 (Dionex, Idstein, Germany).
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