The allantoin content was measured with a method described previously [41 (link)] with some further modification [24 (link)]. An Agilent 7100 Capillary Electrohoresis set coupled with a diode-array detector (UV-Vis/DAD, 190–600 nm; Agilent Technologies, Santa Clara, CA, USA) was used for separation and determination of allantoin. The analysis was carried out in fused silica 50 µm i.d., capillaries with a total length of 64.5 cm. The background electrolyte with pH 9.2 consisted of a 50 mM borate solution. The quantitative analysis of allantoin was performed at λ = 192 nm. Allantoin was identified based on comparison of the retention time and absorption spectrum similarity in a 190–400 nm range with allantoin standards purchased in Sigma-Aldrich (St. Louis, MO, USA).
Uv vis dad
Manufactured by Agilent Technologies
Sourced in United States
The UV-Vis/DAD is a high-performance spectrophotometer designed for a wide range of analytical applications. It provides accurate measurements of ultraviolet, visible, and diode array detection (DAD) spectra. The instrument is capable of scanning a broad wavelength range and has high-resolution optics for precise analysis.
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4 protocols using uv vis dad
1
Quantitative Analysis of Allantoin
2
Quantification of Allantoin by Capillary Electrophoresis
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The allantoin content was determined using an Agilent 7100 capillary electrophoresis set coupled with a diode array detector (UV-Vis/DAD, 190–600 nm; Agilent Technologies, Santa Clara, CA, USA). The modified method developed by Dresler et al. [45 (link)] was applied. The separation was carried out in a fused silica capillary (64.5 cm × 50 µm i.d.) at a temperature of 27 °C. The background electrolyte consisted of a 50-mM borate solution, pH 9.2. A quantitative analysis was performed at λ = 192 nm. Allantoin was identified based on a comparison of retention times and absorption spectrum similarity in a 190–400-nm range with a standard purchased from Sigma-Aldrich (St. Louis, MO, USA).
3
Separation and Determination of ALLA and LMWOAs
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An Agilent 7100 Capillary Electrohoresis set coupled with a diode-array detector (UV-Vis/DAD, 190–600 nm; Agilent Technologies) was used for the separation and determination of ALLA and LMWOAs.
The analysis of ALLA was conducted according to Dresler et al. [90 (link)]. The analysis of ALLA was carried out in a 50 µm i.d. silica capillary with a total length of 64.5 cm, filled with electrolytes consisting of a 50 mM borate solution, pH 9.2. ALLA was identified at λ = 192 nm based on comparison of the retention time and absorption spectrum similarity in a 190–400 nm range with allantoin standards purchased in Sigma-Aldrich (St. Louis, MO, USA).
The LMWOAs were prepared and measured as described in Hanaka et al. [6 (link)].
The analysis of ALLA was conducted according to Dresler et al. [90 (link)]. The analysis of ALLA was carried out in a 50 µm i.d. silica capillary with a total length of 64.5 cm, filled with electrolytes consisting of a 50 mM borate solution, pH 9.2. ALLA was identified at λ = 192 nm based on comparison of the retention time and absorption spectrum similarity in a 190–400 nm range with allantoin standards purchased in Sigma-Aldrich (St. Louis, MO, USA).
The LMWOAs were prepared and measured as described in Hanaka et al. [6 (link)].
4
HPLC Method for Quantification
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The experiments were performed using an Agilent 1100 LC instrument (Agilent Technologies, Waldbronn, Germany), equipped with a quaternary pump, degasser membrane, autosampler, oven column compartment, UV-Vis DAD, FSFD and the Chemstation software package (Agilent Technologies, Waldbronn, Germany) to control the instrument, the data acquisition and the data analysis. The procedure separation was carried out on a 3.5 μm Zorbax Eclipse XDBC18 analytical column (4.6×50 mm) (Agilent Technologies, Waldbronn, Germany).
All the chromatographic experiments were performed in gradient mode, setting the column temperature at 40 °C and the flow rate at 0.65 mL min -1 . The initial composition of the mobile phase consisted of MeOH:ACN:phosphate buffer 10 mmol L -1 pH = 3.50 (2.5:2.5:95). Then, the gradient elution was performed as follow: 0-
All the chromatographic experiments were performed in gradient mode, setting the column temperature at 40 °C and the flow rate at 0.65 mL min -1 . The initial composition of the mobile phase consisted of MeOH:ACN:phosphate buffer 10 mmol L -1 pH = 3.50 (2.5:2.5:95). Then, the gradient elution was performed as follow: 0-
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