Titratable acidity was determined by following the method described by Bradely et al. [19 ]. The contents of lactose, lactic acid, acetic acid and ethyl alcohol were determined by using high performance liquid chromatography (HPLC). In order to make samples for HPLC analysis, the airag samples were acidified to pH 2.0–2.3 by adding 1 M sulfuric acid and centrifuged to 1,000xg for 30 min. The supernatants were passed through a column containing 0.2 g of Chelex 100 resin (Bio-Rad, USA) and filtered through a membrane filter with pore size of 0.45 μm. The filtrates were used as samples for the HPLC analysis. The HPLC system (Varian, USA) consisted of a solvent delivery system (9012Q), a refractive index detector (Star 9040), and a column oven (101). The temperature of Rezex ROA column with the size of 150x7.7 mm (Phenomenex, USA) was 60 °C. The eluant was 4 mM sulfuric acid and the flow rate was 0.6 mL/min.
Hplc system
Manufactured by Agilent Technologies
Sourced in United States, Germany, Belgium, Japan, United Kingdom, France, China
The HPLC system is a high-performance liquid chromatography instrument used for the separation, identification, and quantification of complex mixtures of chemical compounds. It consists of a solvent delivery system, an injection port, a chromatographic column, and a detector. The system operates by pumping a liquid mobile phase through the column, which contains a stationary phase material. The sample is injected, and the different components in the mixture are separated based on their interactions with the stationary phase and the mobile phase. The separated components are then detected and quantified by the detector.
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Lab products found in correlation
Chromasolv, by Merck Group (5 mentions)
6210 lc tof spectrometer, by Agilent Technologies (5 mentions)
Masshunter workstation software, by Agilent Technologies (5 mentions)
6495 triple quadrupole mass spectrometer, by Agilent Technologies (4 mentions)
Api 4000 triple quadrupole mass spectrometer, by AB Sciex (4 mentions)
Zorbax 300sb c8, by Agilent Technologies (4 mentions)
Eclipse xdb c18 column, by Agilent Technologies (3 mentions)
P680 pump, by Agilent Technologies (3 mentions)
C18 ods 2 hypersil analytical column, by Thermo Fisher Scientific (3 mentions)
C18 guard column, by Thermo Fisher Scientific (3 mentions)
Eclipse xdb c18, by Agilent Technologies (3 mentions)
Minidawn treos multi angle light scattering, by Wyatt Technology (3 mentions)
Zorbax sb c18 column, by Agilent Technologies (3 mentions)
Zorbax sb c18, by Agilent Technologies (3 mentions)
Rezex roa column, by Phenomenex (2 mentions)
Star 9040, by Agilent Technologies (2 mentions)
C4 column, by Phenomenex (2 mentions)
C18 column, by Agilent Technologies (2 mentions)
Rezex roa organic acid analysis column, by Phenomenex (2 mentions)
Xterra c18 column, by Agilent Technologies (2 mentions)
280 protocols using hplc system
1
Analytical Methods for Airag Characterization
2
Synthesis and Purification of miRNA Mimics
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The unmodified RNA oligomers were synthesized in Division of Bioorganic Chemistry of CMMS PAS Łódź by dr Anna Maciaszek using Gene World automated DNA/RNA synthesizer at a 0.2 μmol scale, with commercially available LCA–CPG supports (Biosearch Technologies, Inc., Petaluma, CA) and the standard phosphoramidite monomers (Glen Research, Sterling, VA, USA). The phosphoramidites were dissolved in anhydrous CH3CN at concentration of 0.07 M, and a 0.25 M solution of 5-Benzylmercapto-1H-tetrazole in anhydrous CH3CN was used as an activator. Oligonucleotides were deprotected and isolated using a binary Varian HPLC system, consisting of two PrepStar 218 pumps and a ProStar 325 UV/VIS detector set at 260 nm. A reverse phase HPLC column (PRP-1, C18, 7 μm, 305 × 7 mm, Hamilton, Reno, NV, USA) was eluted with a 1% min − 1 gradient of CH3CN in 0.1 M TEAB (pH 7.3) at a flow rate 2.5 ml min − 1. The miR494 and miR146a mimics sequences: miR494-5p 5ʹ-AGGUUGUCCGUGUUGUCUUCUC-3ʹ; miR494-3p 5ʹ-UGAAACAUACACGGGAAACCUC-3ʹ; miR146a-5p 5ʹ-UGAGAACUGAAUUCCAUGGGUU-3ʹ; miR146a-3p 5ʹ-CCUCUGAAAUUCAGUUCUUCAG-3’.
3
Analytical Characterization of Compounds
4
Recombinant Porcine Amelogenin Protein Expression
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Wild type (rP172) and variant recombinant porcine amelogenin
proteins were expressed in E. coli strain BL21(DE3)pLysS
(Stratagene, CA), and precipitated by 20% ammonium sulfate following the method
described previously [19] , [20] . The ammonium sulfate precipitate was dissolved in
water containing 0.1% TFA and purified using a high performance liquid
chromatography (HPLC) system (Varian, CA) equipped with a Phenomenex C4 column
(10×250 mm, 5 µm). The purified proteins
were lyophilized, kept at −20 °C, and dissolved in water before
use. Our group designed three double-variant strains of amelogenin; rP172(W45Y,
W161Y) will be referred to as rP172(W25), rP172(W25Y, W161Y) will be referred to
as rP172(W45), and rP172(W25Y, W45Y) will be referred to as
rP172(W161) [21] (link). The
positions of tryptophan residues of rP172 and its variants are illustrated in
Table
1 .Table 1.
proteins were expressed in E. coli strain BL21(DE3)pLysS
(Stratagene, CA), and precipitated by 20% ammonium sulfate following the method
described previously [19] , [20] . The ammonium sulfate precipitate was dissolved in
water containing 0.1% TFA and purified using a high performance liquid
chromatography (HPLC) system (Varian, CA) equipped with a Phenomenex C4 column
(10×250 mm, 5 µm). The purified proteins
were lyophilized, kept at −20 °C, and dissolved in water before
use. Our group designed three double-variant strains of amelogenin; rP172(W45Y,
W161Y) will be referred to as rP172(W25), rP172(W25Y, W161Y) will be referred to
as rP172(W45), and rP172(W25Y, W45Y) will be referred to as
rP172(W161) [21] (link). The
positions of tryptophan residues of rP172 and its variants are illustrated in
1
The tryptophan residues of wild type amelogenin and its
variants.
| Name | Location of Tryptophan residues | ||
|---|---|---|---|
| rP172 | W25 | W45 | W161 |
| rP172(W25) | W25 | ||
| rP172(W45) | W45 | ||
| rp172(W161) | W161 | ||
5
Fluorometric HPLC Quantification of KYNA
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Eluted KYNA was subjected to the HPLC and quantified fluorimetrically (Varian HPLC system; ESA catecholamine HR-80.3 μm, C18 reverse-phase column), as previously described [26 (link)]. The mobile phase (pH 6.2) contained 250 mM zinc acetate, 50 mM sodium acetate, and 4% acetonitrile. Each chromatographic assay was preceded by the measurements of standardized concentrations of KYNA (0.2, 0.4, 0.6, 0.8, and 1.0 pmol), in order to obtain calibration curve.
6
Recombinant Amelogenin Protein Expression
7
Characterization of Organic Compounds
8
Analytical Techniques for Chemical Separations
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Flash chromatography separations were performed using an automated Isco CombiFlash RF system over silica gel columns (Teledyne Isco, Lincoln, NE). High performance liquid chromatography (HPLC) separations were accomplished using a Varian HPLC system (ProStar 210 pumps, ProStar 710 fraction collector, ProStar 335 photodiode array detector) with Galaxie Chromatography Workstation software (version 1.9.3.2). An Acquity ultra-high performance liquid chromatography (UHPLC) system (Waters Corporation, Milford, MA) coupled to a LTQ Orbitrap XL Hybrid mass spectrometer (Thermo Fisher Scientific, Waltham, MA) was used for all LC-MS analyses. NMR spectra were acquired with a JNM-ECS 400 MHz NMR spectrometer (JOEL USA, Peabody, MA). Unless otherwise stated, all solvents used in chemical analyses were purchased from Thermo Fisher Scientific (Waltham, MA).
9
Analytical Techniques for Microbial Metabolites
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The biomass concentration was measured as absorbance at 600 nm. During the fermentation experiments, the value of the optical density was converted to cell dry weight by an appropriate calibration curve. A molecular weight of 25.73 g/mol was used to convert the cell dry weight from g/L to M. Glycerol, PDO, lactate, acetate, succinate, formate and ethanol were quantified with an HPLC system (Varian, Belgium) coupled with a Refractive Index Detector and a dual UV Detector (wavelength of 210 and 265 nm). The compounds were separated by using an Aminex 300 × 7.8 mm HPX-87H Organic Acid Analysis Column (Bio-Rad Laboratories, Belgium) and eluted at 600 µL/min isocratically in 5 mM H2SO4 at 65 °C. Glucose was analyzed using the YSI 2700 SELECT Biochemistry Analyzer (YSI Life Sciences, Ankersmid Scientific, Belgium). 3-hydroxypropionaldehyde (3-HPA) was determined by an HPLC system with a Rezex ROA Organic Acid Analysis column (Phenomenex, Belgium) using a dual Ultraviolet Detector with a wavelength of 210 and 265 nm. The metabolite was eluted at 500 µL/min isocratically in 10 mM H2SO4 at 40 °C.
10
Enzymatic Synthesis of Secoiridoid Compound
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3,4-DHPEA-EA (methyl-4-(2-(3,4-dihydroxyphenethoxy)-2-oxoethyl)-3-formyl-2-methyl-3,4-dihydro-2H-pyran-5-carboxylate) was obtained by enzymatic hydrolysis of oleuropein, as a molecular evolution consequence of the hemiacetal functionality of the aglycon 2 , formed by glycosidic bond cleavage (Figure 1 ). The lipidic/water interface promotes the rapid rearrangement of the intermediate oleuropeinenol 3 into the final stable biomolecule, the transposed secoiridoid 4 , within 5 min [18 (link)].
Thus, to a solution of 100 mg of oleuropein, previously extracted from olive leaves, endogenous β-glucosidase was added at 40°C for 6 h in 20 ml of a H2O/CHCl3 1:1 mixture, The mixture was evaporated and purified using Waters XTerra C18 column on a Varian HPLC system (H2O/MeCN H2O/MeCN gradient) with a flow rate of 2 ml/min.
Methyl-4-(2-(3,4-dihydroxyphenethoxy)-2-oxoethyl)-3-formyl-2-methyl-3,4-dihydro-2H-pyran -5-carboxylate was obtained as a yellow oil (20% yield). 1H and 13C NMR spectra were in agreement with literature data [10 (link)].
Thus, to a solution of 100 mg of oleuropein, previously extracted from olive leaves, endogenous β-glucosidase was added at 40°C for 6 h in 20 ml of a H2O/CHCl3 1:1 mixture, The mixture was evaporated and purified using Waters XTerra C18 column on a Varian HPLC system (H2O/MeCN H2O/MeCN gradient) with a flow rate of 2 ml/min.
Methyl-4-(2-(3,4-dihydroxyphenethoxy)-2-oxoethyl)-3-formyl-2-methyl-3,4-dihydro-2H-pyran -5-carboxylate was obtained as a yellow oil (20% yield). 1H and 13C NMR spectra were in agreement with literature data [10 (link)].
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