The HPLC-DAD equipment used was an Agilent Series 1290 infinity with vacuum degasser, quaternary pump, autosampler, thermostated column compartment and photodiode array detector (DAD). Data analysis was performed with Agilent HPLC EZChrom software. The chromatographic separation was performed at 35 °C on a Grace Alltima C18 column (4.6 mm × 250 mm, 5 μm) with a flow rate of 1 mL/min, and the injection volume was 20 μL. Mobile phase A consisted of ultrapure type 1 water (Simplicity Water Purification System, Millipore) adjusted to pH 2.5 with trifluoroacetic acid (TFA), and mobile phase B contained acetonitrile (ACN). The gradient was programmed as follows: 0–3 min, 5% B; 3–43 min, 5–30% B; 43–73 min, 30–85% B; 73–75 min, 85–5% B. Simultaneous monitoring was performed for determination at 254 and 350 nm. ACN and methanol (MeOH) were HPLC grade (JT Baker) and TFA HPLC grade (Sigma-Aldrich). Prior to injection, 10% MeOH was added to each sample to improve solubility https://dx.doi.org/10.17504/protocols.io.wy9ffz6
Alltima c18 column
Manufactured by Grace Bio-Labs
Sourced in United States
The Alltima C18 column is a reversed-phase high-performance liquid chromatography (HPLC) column. It is designed for the separation and analysis of a wide range of organic compounds. The column features a C18 stationary phase, which is a common choice for HPLC separations.
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Lab products found in correlation
Simplicity water purification system, by Merck Group (1 mentions)
Tfa hplc grade, by Merck Group (1 mentions)
Hplc grade, by Avantor (1 mentions)
2998 photodiode array detector, by Waters Corporation (1 mentions)
2695 separations module, by Waters Corporation (1 mentions)
Empower software version 3, by Waters Corporation (1 mentions)
5 protocols using alltima c18 column
1
HPLC-DAD Analysis of Organic Compounds
2
Quantification of BPA in Thermal Paper
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Approximately 100 mg of thermal paper were accurately weighed, cut into small pieces and dispersed in 40 ml of a methanol/water mixture (90:10). Extraction of BPA from thermal paper was performed by sonication for 15 min. The extract was then filtrated before analysis by high-performance liquid chromatography with fluorimetric detection (Perkin Elmer system). HPLC separation was achieved on an Alltima C18 column (150 × 3, 5 µm) from Grace. The mobile phase used was a mixture of acetonitrile/water (35:65) in isocratic mode. The flow rate of the mobile phase was 1 ml/min and the injection volume 10 µl. The fluorimetric detector operated at an excitation wavelength of 230 nm and at an emission wavelength of 315 nm. Quantification was performed by external calibration. The recovery rate of the extraction was about 98 % and the detection limit was 0.73 µg/L or 0.03 % (0.03 mg BPA/100 mg paper).
3
Purification of Target Saponins by Pre-HPLC
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After separation by macroporous resins, the individual target saponins were further separated and purified by one-step pre-HPLC. A Shimadzu LC-20AP pre-HPLC apparatus (Shimadzu, Tokyo, Japan) coupled with an SPD-M20A photodiode array detector (Waters, Milford, MA, USA) was used. Chromatographic separation was performed on a Grace Alltech Alltima C18 column (20 mm × 250 mm, 10μm, Grace, Milford, MA, USA). Preparative separation of the four target saponins was achieved by a two-phase solvent system composed of acetonitrile-water (33:67, v/v) with a flow-rate of 10 mL/min. The monitoring wavelength was set at 203 nm. The peaks corresponding to gypenoside XVII (6 ), notoginsenoside Fe (7 ), ginsenoside Rd2 (8 ), and notoginsenoside Fd (9 ) were collected by an automatic fraction collector. After that, the fractions were combined according to the results of HPLC analysis.
4
Quantification of L-Ascorbic Acid in Sea Buckthorn Flour
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A Shimadzu 2010 HPLC system equipped with PDA detector and Grace Alltima C18 column (100 × 3 mm, 3 µm) was used to determine L-ascorbic acid content in sea buckthorn berry flour. The elution gradient used 15 mM phosphate buffer at pH 2.7 (A) and methanol (B) as a mobile phase with a flow rate of 0.4 mL/min. The gradient was achieved as follows: min 0: 10% B, min 5: 20% B, min 10: 10% B. The column temperature was 30 °C and the volume injected, 20 μL. UV wavelength for L-ascorbic acid detection was 244 nm, and the retention time determined was 1.85 min. The results of the total vitamin C content, expressed in mg/100 g, were obtained using the calibration curve of L-ascorbic acid (0.3–1 mg/L, R2 = 0.999) [45 ].
5
HPLC Analysis of Compounds
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The samples were analyzed using a HPLC system (Waters 2695 Separations Module, Milford, USA) coupled to a PDA detector (Waters 2998 Photodiode Array Detector, Milford, USA). The analysis was performed on an Alltima C18 column (250 mm × 3 mm; 5 μm particle size) (Grace, Columbia, USA). The mobile phase consisted of a gradient with an ammonium formate buffer (0.020 M) pH = 3 and methanol. First, a ratio of 90 % buffer and 10 % methanol was held for 2 min. During the next 5 min, the ratio changed to 50 % buffer and 50 % methanol. This ratio was kept for 7 min. The next 6 min, the gradient altered to 10 % buffer and 90 % methanol, which was held for 5 min. During the last 5 min, the gradient returned to its starting condition, making a total run of 30 min for each sample. This gradient was run at a flow rate of 0.5 mL min -1 . Five microliters of each sample was injected at a temperature of 15 °C, while the column temperature was set at 30 °C. PDA signals were measured in the range of 210 to 400 nm. Data acquisition was achieved using the Empower software version 3 (Waters, Milford, USA).
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