The concentration of sugars (glucose and galactose), metabolic intermediates (succinate and lactate), as well as the main short-chain fatty acids (SCFAs) acetate, propionate, butyrate and isovalerate, were identified and measured by means of high-performance liquid chromatography (HPLC). The HPLC unit (Agilent Technologies Inc., Santa Clara, CA, USA) was equipped with an Aminex HPXH-87H ion exclusion column (Bio-Rad Laboratories, Hercules, CA, USA) and a refractive index detector G1362A (Agilent Technologies Inc., Santa Clara, CA, USA). Separation was performed with 0.0005 mol L−1 H2SO4, 0.45 mL min−1 flow rate and 20 to 100 µL injection volume. Before injection, the samples were centrifuged (Hermel-Z233 M-2, Hermle Labortechnik GmbH, Wehingen, Germany; 6000× g, +20 °C, 30 min), and the supernatant was used after an additional filtration (0.22 µm). The metabolites were identified and quantified using external standards (Sigma Aldrich, Saint Louis, MO, USA) and the Agilent ChemStation Instrument 1 offline software (Agilent Technologies Inc., Santa Clara, CA, USA).
Hplc unit
Manufactured by Agilent Technologies
Sourced in United States
The HPLC unit is a high-performance liquid chromatography system designed for the separation, identification, and quantification of chemical compounds. It functions by using a high-pressure liquid solvent to pass a sample mixture through a column filled with a stationary phase material, which interacts with the sample components, causing them to separate based on their unique physical and chemical properties.
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Lab products found in correlation
Hermel z233 m 2, by Hermle (1 mentions)
Agilent chemstation instrument 1 offline, by Agilent Technologies (1 mentions)
Ultrahydrogel 250, by Waters Corporation (1 mentions)
Shodex ri 101, by Resonac (1 mentions)
Chromafil a 45 25, by Macherey-Nagel (1 mentions)
Empower software, by Waters Corporation (1 mentions)
Poroshell 120 ec c18 reverse phase column, by Agilent Technologies (1 mentions)
C18 column, by Dikma Technologies (1 mentions)
Eclipse plus c18, by Agilent Technologies (1 mentions)
Zorbax eclipse plus c18, by Agilent Technologies (1 mentions)
7 protocols using hplc unit
1
HPLC Analysis of Metabolic Compounds
2
Pectin Molar Mass Distribution Analysis
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High-performance size-exclusion chromatography (HPSEC) (HPLC unit, Agilent technologies, Diegem, Belgium) equipped with multi-angle laser light scattering (MALLS) (PN3621, Postnova analytics, Landsberg, Germany) and refractive index (RI) detection (Shodex RI-101, Showa Denko K.K., Kawazaki, Japan) was used to measure the molar mass distribution of the pectin samples following the procedure described by Shpigelman et al. [44 (link)], with minor modifications. Briefly, pectin samples (0.2% w/v) were dissolved overnight in a 0.1 M acetic acetate buffer (pH 4.4) containing 0.1 M NaNO3. The pectin samples were subsequently filtered (Chromafil® A-45/25, 0.45 mm pore size, Macherey-Nagel Gmbh, Duren, Germany), injected (100 μL), and separated using a series of Waters columns: Ultrahydrogel 250, 1000, and 2000, with exclusion limits of 8 × 104, 4 × 106, and 1 × 107 g/mol, respectively (Waters, Milford, MA, USA). Elution of the pectin polymers was accomplished with a 0.1 M acetate buffer (pH 4.4) containing 0.1 M NaNO3 at 35 °C and a flow rate of 0.5 mL/min. The dn/dc value used to calculate the concentration was 0.146 mL/g. Analysis of the samples was performed in duplicate. Calculation of the average molecular weight was achieved by applying the Debye fitting method (2nd order) of the operating software (Nova Mals, version 1.0.0.18, Postnova analytics, Lansberg, Germany).
3
Analytical HPLC Characterization of 11C-Glutamine
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The HPLC analysis of the final product was performed on an Agilent HPLC unit and the data was collected and analyzed using Waters Empower software. Astec CHIROBIOTIC™ T HPLC column 25 cm × 4.6 mm, 5 μm was used for analytical purpose. Final product was sterile filtered using 0.22 μm GS filter. The pH of the final product was approximately 6.0. The final product was obtained with the radiochemical purity of > 90% with less than 5% of [11C]D-glutamine (Fig. 2 ). The other radiochemical impurity was predicted to be the unhydrolyzed, deprotected intermediate and/or [11C] glutamic acid. The molar activity was greater than 7000 mCi/μmol at the EOB.![]()
HPLC chromatograms of C-11 glutamin
4
Preparative Column Chromatography Protocol
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Commercial grade reagents and solvents were used without further purification. Thin layer chromatography (TLC) was performed using precoated silica gel 60 F254 plates and visualized using anisaldehyde solution, heat, and UV light (254 nm). Flash column chromatography was undertaken on silica gel (400–630 mesh). 1H NMR was recorded at 400 MHz and chemical shifts are quoted in parts per million (ppm) versus an appropriate solvent peak or 2.50 ppm for DMSO-d6. The following abbreviations were used to describe peak splitting patterns: br = broad, s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublets, td = triplet of doublets, ddd = doublet of doublets of doublets. Coupling constants, J, are reported in hertz (Hz). HPLC was conducted using an Agilent HPLC unit equipped with an Agilent Poroshell 120 EC-C18 reverse phase column (4.6 × 50 mm, 2.7 Micron) and mass spectroscopy was performed using a quadrupole LC/MS unit.
5
HPLC Analysis of Analyte Quantification
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Chromatographic analyses were carried out using an analytical Agilent HPLC unit equipped with a G1310A isopump, a 7725iManu sampler, a GA1314A UV detector and an AT-130 column heater.
The separation was performed on a C18 column (Dikma, Lake Forest, CA, USA, 150 ×4.6 mm i.d.) in the column heater set to 40°C. The mobile phase consisted of methanol:water (85:15, v:v) at a flow rate of 1.0 mL/min. The UV detector was set to 263 nm. The method had a good linearity (r2 = 0.9995) in the range of 4–1000 ng/mL with a low limit of quantification of 4 ng/mL and low limit of detection of 1.5 ng/mL.
The separation was performed on a C18 column (Dikma, Lake Forest, CA, USA, 150 ×4.6 mm i.d.) in the column heater set to 40°C. The mobile phase consisted of methanol:water (85:15, v:v) at a flow rate of 1.0 mL/min. The UV detector was set to 263 nm. The method had a good linearity (r2 = 0.9995) in the range of 4–1000 ng/mL with a low limit of quantification of 4 ng/mL and low limit of detection of 1.5 ng/mL.
6
HPLC Determination of Vitamin C in Honey
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Vitamin C content was determined as previously reported (Alvarez-Suarez, González-Paramás, Santos-Buelga, & Battino, 2010). Samples (1 g) were diluted in 5 mL of metaphosphoric acid (5 g 100 mL -1 ), and 20 μL was injected onto an HPLC system consisting of a modular 1260 Agilent Technologies HPLC unit (CA, USA) equipped with a quaternary pump and DAD detector set at 245 nm. The stationary phase comprised an Agilent Eclipse Plus C18 (4.6 × 150 mm, 5 μm) (CA, USA) column.
Elution was performed in an isocratic gradient with potassium diacid phosphate at a flow rate of 1 mL min -1 for 10 min. For quantitative analysis, a calibration curve was obtained using an external standard of ascorbic acid at a range of 5-50 mg L -1 (y = 370.65x + 153.23, R 2 = 0.998, LOD: 0.032 μg g -1 and LOQ: 0.107 μg g -1 ), and the results are expressed as μg of Vit C per gram honey (μg Vit C g -1 of honey).
Elution was performed in an isocratic gradient with potassium diacid phosphate at a flow rate of 1 mL min -1 for 10 min. For quantitative analysis, a calibration curve was obtained using an external standard of ascorbic acid at a range of 5-50 mg L -1 (y = 370.65x + 153.23, R 2 = 0.998, LOD: 0.032 μg g -1 and LOQ: 0.107 μg g -1 ), and the results are expressed as μg of Vit C per gram honey (μg Vit C g -1 of honey).
7
HPLC Analysis of Organic Acids in Honey
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Organic acids were determined using an HPLC system (Nour, Trandafir, & Ionica, 2010) (link). Samples (1 g) were diluted in 25 mL of distilled water, and 10 μL of this aqueous fraction was injected onto the HPLC system. This system consisted of a modular 1260 Agilent Technologies HPLC unit (CA, USA) with a quaternary pump and DAD detector set at 210 nm. The stationary phase involved a Zorbax Eclipse Plus C18 (4.6 × 250 mm, 5 μm) (Agilent, CA, USA) column. Elution was performed with potassium diacid phosphate (Merck, Germany) in an isocratic gradient at a flow rate of 0.7 mL min -1 for 15 min. For the quantitative analysis, a calibration curve was obtained using external standards for oxalic acid at a range of 5-75 mg L -1 (y = 8E-06x-0.0021, R 2 = 0.9994, LOD: 0.027 mg 100 g -1 and LOQ: 0.091 mg 100 g -1 ), lactic acid at a range of 1-60 mg L -1 (y = 5429.4x+ 8.3139, R 2 = 0.9992, LOD: 0.008 mg 100 g -1 and LOQ: 0.026 mg 100 g -1 ), acetic acid at a range of 1-60 mg L -1 (y = 4190.2x + 5.4518, R 2 = 0.9978, LOD: 0.007 mg 100 g -1 and LOQ: 0.024 mg 100 g -1 ) and citric acid at a range of 1-60 mg L -1 (y = 14892x-26.278, R 2 = 0.9883, LOD: 0.004 mg 100 g -1 and LOQ: 0.014 mg 100 g -1 ). The results are expressed as mg 100 g -1 honey.
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