The homogeneity and molecular weights of the purified polysaccharide fractions were determined by size exclusion chromatography on a Superose 6 prepacked column (Amersham Biosciences) combined with the Äkta system (FPLC, Pharmacia Äkta, Amersham Pharmacia Biotech). Dextran polymers (Pharmacia) B512 (5.6 kDa), T8360 (19 kDa), T250 (233 kDa) and T500 (475 kDa) were used as calibration standards. Approximately 5 mg of the samples were dissolved in 2 mL of 10 mM NaCl buffer, the resulting solution was filtered through a Millipore filter (0.45 μm) and then applied to the column. The samples were eluted with 0.3 M sodium acetate at pH 5.2, with a speed of 0.5 mL/min, collecting 2 mL fractions. The eluent was monitored with a Shimadzu RI detector. The retention volume of the main peak of each sample was converted to molecular weight based on a standard curve prepared on the basis of the standards given above.
Ri detector
Manufactured by Shimadzu
Sourced in Japan, United States
The RI detector is a type of analytical instrument used in liquid chromatography to detect and quantify compounds based on their refractive index. It measures the change in the refractive index of the mobile phase as it passes through the detector cell, which is proportional to the concentration of the analyte. This information is then used to generate a signal that can be analyzed to identify and quantify the compounds present in the sample.
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Lab products found in correlation
5 protocols using ri detector
1
Polysaccharide Molecular Weight Analysis
2
Molecular Weight Characterization of Polymers
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All chemicals were purchased
from Sigma-Aldrich, TCI, and Oakwood Chemical and used as received
unless otherwise indicated. Gel-permeation chromatography (GPC) was
done using a Shimadzu pump coupled to a Shimadzu RI detector. Poly(styrene)
standards purchased from Agilent Technologies were used for column
calibration. Two GPCs with different eluents were used. One GPC ran
with a 0.03 M LiCl solution in N,N-dimethylformamide (DMF) as the eluent at a flow rate of 1
mL/min at 65 °C. A set of Polymer Standards columns (AM GPC gel,
10 μm, precolumn, 500 Å, and linear mixed bed) was used.
The second GPC ran with THF as the eluent at a flow rate of 1 mL/min
at ambient temperature. A set of Shodex GPC columns (KF-804 and KF-802.5)
was used. Mw, Mn, and Đ represent respectively the apparent
weight-average molecular weight, apparent number-average molecular
weight, and dispersity index. 1H NMR and 13C
NMR spectra were recorded at 25 °C on a Bruker AVIII400 MHz,
a Bruker AV 500 MHz, or a Bruker AVIII 600 MHz spectrometer. All chemical
shifts are reported in parts per million (ppm) with reference to solvent
residual peaks. Mass spectra of samples in methanol were acquired
with an Agilent 6224 Accurate-Mass TOF/LC/MS spectrometer. Circular
dichroism (CD) spectra and UV–vis spectra were obtained at
25 °C on a Jasco J-1500 circular dichroism spectrometer.
from Sigma-Aldrich, TCI, and Oakwood Chemical and used as received
unless otherwise indicated. Gel-permeation chromatography (GPC) was
done using a Shimadzu pump coupled to a Shimadzu RI detector. Poly(styrene)
standards purchased from Agilent Technologies were used for column
calibration. Two GPCs with different eluents were used. One GPC ran
with a 0.03 M LiCl solution in N,N-dimethylformamide (DMF) as the eluent at a flow rate of 1
mL/min at 65 °C. A set of Polymer Standards columns (AM GPC gel,
10 μm, precolumn, 500 Å, and linear mixed bed) was used.
The second GPC ran with THF as the eluent at a flow rate of 1 mL/min
at ambient temperature. A set of Shodex GPC columns (KF-804 and KF-802.5)
was used. Mw, Mn, and Đ represent respectively the apparent
weight-average molecular weight, apparent number-average molecular
weight, and dispersity index. 1H NMR and 13C
NMR spectra were recorded at 25 °C on a Bruker AVIII400 MHz,
a Bruker AV 500 MHz, or a Bruker AVIII 600 MHz spectrometer. All chemical
shifts are reported in parts per million (ppm) with reference to solvent
residual peaks. Mass spectra of samples in methanol were acquired
with an Agilent 6224 Accurate-Mass TOF/LC/MS spectrometer. Circular
dichroism (CD) spectra and UV–vis spectra were obtained at
25 °C on a Jasco J-1500 circular dichroism spectrometer.
3
Molar Mass Determination of Liquid Samples
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The molar mass determination of the liquid phase (without preparation) was performed with a HPSEC-MALLS system (miniDAWN, Wyatt Technology, Santa Barbara, CA) with RI-detector (Shimadzu Corp., Tokyo, Japan). A combination of two columns (2× Ultrahydrogel™ linear 7.8 × 300 mm, Waters, Milford, MA) was used in series. NaNO 3 (0.1 M) was used as eluent with a flowrate of 0.5 cm 3 min -1 . Residual solid particles which could be present in the samples were removed by using nylon syringe filters (25 mm, w/0.2 μm nylon membrane, VWR). All samples were concentrated to about 3 g/L and the injection volume of 200 μL was applied to the system. The dn/dc value was set to 0.150 cm 3 g -1 and the overall data analysis was performed by Astra software (Wyatt Technology) (Rissanen et al., 2014a; (link)Rissanen, Grénman, Willför, et al., 2014; Xu et al., 2008; (link)Xu et al., 2009) (link) The results obtained with HPLC were supported by data from UV adsorption and GC analysis.
4
Molecular Weight Determination of QZ-7
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The molecular weight of QZ-7 was determined using a TSK G4000 PWXL column operated at 40 °C via high-performance gel permeation chromatography (HPGPC) coupled to a refractive index (RI) detector (Shimadzu). The column was calibrated using dextran standards. The mobile phase was deionized-distilled (DDI) water at a flow rate of 0.6 mL/min. Before injection, the sample was filtered through a 0.45 µm filter [55 (link),60 (link)]. The following regression equation was obtained:
where mass (Da) and T (min) are the molecular mass and retention time of the samples, respectively, and K1 and K2 are constants.
where mass (Da) and T (min) are the molecular mass and retention time of the samples, respectively, and K1 and K2 are constants.
5
Polysaccharide Molecular Weight and Polydispersity Analysis
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The molecular weight (Mw) and the polydispersity index (PDI) of polysaccharide samples were determined by high-performance size exclusion chromatography coupled with multi-angle laser light scattering and refractive index detector (HPSEC-MALLS-RID). The chromatographic signals were collected by Multi-angle Light Scattering Detectors (MALLS, DAWN HELEOS, Wyatt Technology Co., Santa Barbara, CA, United States) and RI detector (Shimadzu Company, Japan) in series. Each sample (10 mg) was dissolved in the mobile phase (1 ml) and then filtered through a 0.45 µm membrane. Two size exclusion columns Shodex SB-806 (300 mm × 7.8 mm, i. d.), and Shodex SB-804 (300 mm × 7.5 mm, i. d.) were used. The mobile phase included a 0.1 mol/L NaCl aqueous solution applied at a flow rate of 0.5 ml/min. An injection volume of 100 µL was used. Each sample was run for 50 min, and the temperature of the column was maintained at 40°C.
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