The hydrophobicity parameter was expressed as RP-HPLC mobile phase acetonitrile content (%ACN) at a retention time of a particular peptide. All peptides were analyzed using Waters Alliance e2695 system with a Waters 2998 PDA Detector (software–Empower®3). All HPLC analyses were conducted in triplicate on a Waters XBridge™ Shield RP-18 column (4.6 × 150 mm, 3.5-μm particle size, 130-Å pore size) using a linear 30–60% ACN (B) gradient in deionized water (A) over 30 min (1% ACN/min) at 25.0 ± 0.1 °C, at a flow rate of 0.5 mL/min and UV detection at 214 nm (A–0.1% aqueous TFA and B–0.1% TFA in ACN).
Xbridge shield rp18 column
Manufactured by Waters Corporation
Sourced in Japan
The XBridge Shield RP18 column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of compounds. It features a stable, endcapped C18 stationary phase that provides excellent retention and selectivity for a variety of analytes. The column's robust construction and consistent performance make it a reliable choice for various analytical applications.
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Lab products found in correlation
E2695 system, by Waters Corporation (6 mentions)
2998 pda detector, by Waters Corporation (5 mentions)
Alliance e2695 separation module, by Waters Corporation (2 mentions)
Waters 2998 pda detector, by Waters Corporation (2 mentions)
2695 system, by Waters Corporation (1 mentions)
Gemini nx c18 column, by Phenomenex (1 mentions)
C18 cartridge, by Phenomenex (1 mentions)
Prominence hplc instrument, by Shimadzu (1 mentions)
Lc solution 1.11 sp1, by Shimadzu (1 mentions)
Dgu 20a5 degasser, by Shimadzu (1 mentions)
E2695 separation module, by Waters Corporation (1 mentions)
E2695 series, by Waters Corporation (1 mentions)
Acquity sq detector, by Waters Corporation (1 mentions)
Nicolet ir200, by Thermo Fisher Scientific (1 mentions)
Automatic tlc sampler 4, by CAMAG (1 mentions)
Moe 2015, by Chemical Computing Group (1 mentions)
21 protocols using xbridge shield rp18 column
1
Hydrophobicity Profiling of Peptides
2
HPLC Analysis of Triphala Extract
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The HPLC analysis was performed on a Waters Alliance 2695 system (Waters Corp, Milford, USA) equipped with a Waters 2489 UV detector. The dried triphala extract was dissolved in water, injected into a Waters XBridge Shield RP18 column (250 mm × 4.6 mm × 5 μm) and eluted with a linear gradient from 15 to 80% aqueous acetonitrile solution. The absorbance was detected at 254 nm.
3
Plasma Quantification by HPLC
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The collected blood samples were centrifuged for 10 min at 4 °C and 3220 g. The generated plasma was removed and mixed in a ratio of 1:3 with ice-cold acetonitrile. The mixture was vortexed for 10 s and centrifuged for 25 min at 4 °C and 11,600 g. The plasma concentrations were quantified by HPLC using an Alliance e2695 separation module and the 2998 PDA detector (Waters, Eschborn, Germany). A Waters XBridge® Shield RP18 column (3.5 µm, 2.1 × 100 mm, 130 A) was used at 30 °C. Data were analyzed by Empower 3 software and quantified using an external reference standard. A solvent gradient was used comprising mobile phase A (acetonitrile/water 5/95 with 5 mM ammonium acetate and 40 µL acetic acid per liter) and mobile phase B (acetonitrile/water 95/5 with 5 mM ammonium acetate and 40 µL acetic acid per liter) with a gradient from 70%A/30%B to 20%A/80%B, stepwise within 30 min and a flow rate of 0.3 mL/min. A sample volume of 5 µL was injected and quantified at a wavelength of 300 nm.
4
HPLC Quantification of SER.DMP and DMP
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Isocratic separation was carried out on Waters X-bridge Shield RP18 column (150 × 3.9 × 5 μm particle size) at column temperature 25 °C, using mobile phase consisting of a mixture of 5 mM phosphate buffer (pH 5.5): acetonitrile (40:60 v/v)]. The mobile phase was degassed by a degasser before being pumped at flow rate 1 mL/min and detection at 220 nm. Aliquots of standard SER.DMP solution (1 mg/mL) equivalent to 2.5—25 μg were transferred into a series of 10-mL volumetric flasks and adjusted to volume with the mobile phase. The same was carried out for DMP. A 20 µL from each solution was injected into HPLC column and eluted with the mobile phase under the previously described chromatographic conditions. Calibration curves representing the relationship between peak area and the corresponding concentration μg/mL of SER.DMP and DMP were plotted, and the regression equations were computerized.
5
Quantifying Adenylate Levels in Stressed Embryos
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Dechorinated embryos were weighted before hypoxia or cyanide treatments and were snap-frozen immediately in liquid nitrogen at the end of the treatments. After adding 250 µl of 6% perchloric acid per 20–40 mg embryos, embryos were homogenized and incubated on the ice for 10 min. After 10 min of spin-down at 12,000 g at 4°C, supernatant was neutralized to pH 7 by adding 44 µl 2M K2CO3, incubated for 30 min on ice, and centrifuged at 12,000 g for 10 min at 4°C. Supernatant was filtered using a 0.45-µm spin filter by 12,000 g for 5 min at 4°C and stored at −80°C until HPLC injection. Concentrations of ATP, ADP, and AMP were measured by HPLC according to established methods (Celotto et al., 2011 (link)). HPLC separation of adenylate nucleotides was performed on 20 µl of extract using a gradient-mobile phase on a Waters XBridge Shield RP18 column and the following conditions: flow rate of 0.8 µl/min, detection wavelength of 257 nm, and column temperature of 30°C. The gradient consisted of 100% Buffer A from 0 to 6.5 min, 100% Buffer B from 6.5 to 12.5 min, and 100% Buffer A from 12.5 to 25 min. Buffer A was 50 mM NH4H2PO4, pH 5.7; Buffer B was 60:40 acetonitrile/H2O. Adenylate standards were linear through the range of 100–250 µM. Retention times were ∼4.2, 4.7, and 6.2 min for ATP, ADP, and AMP, respectively.
6
Peptide Purification and Characterization
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Purifications were carried out on a Phenomenex Gemini-NX C18 column (21.20 × 100 mm, 5.0 µm particle size, 110 Å pore size). UV detection at 214 nm was used, and the crude peptides were eluted with a linear 10–70% acetonitrile gradient in deionized water over 90 min at room temperature. The mobile phase flow rate was 10.0 mL/min. Acetonitrile and water, both containing 0.1% of TFA, were used as a mobile phase. The purity and identity of the peptide was confirmed with LC-MS analysis. The RP-HPLC system was used—Waters Alliance e2695 system with Waters 2998 PDA and Acquity QDA detectors (software—Empower®3). All analyses were carried out on a Waters XBridge™ Shield RP-18 column (4.6 × 150 mm, 3.5 µm particle size, 130 Å pore size). Samples (10 µL) were analyzed with a linear 10–90% acetonitrile gradient in deionized water over 15 min at 25.0 ± 0.1 °C. The mobile phase flow rate was 0.5 mL/min. Both eluents contained 0.1% (v/v) of formic acid. Mass analysis and UV detection at 214 nm were used. Pure fractions (>95%, by HPLC analysis) were collected and lyophilized.
7
HPLC-DAD Quantification of CorA in Plasma
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CorA plasma concentrations were quantified by HPLC-DAD with an Alliance e2695 separation module and a 2998 PDA detector (Waters, Eschborn, Germany). For the analysis, blood samples were centrifuged for 10 min at 4 °C and 3220× g. The generated plasma (20 µL) was mixed 1:3 with ice-cold acetonitrile, vortexed for 10 s and centrifuged for 25 min at 4 °C and 11,000× g. The supernatant was transferred into a HPLC vial and a sample volume of 5 µL was injected and quantified at a wavelength of 300 nm. A Waters XBridge® Shield RP18 column (3.5 μm, 2.1 × 100 mm, 130 A) was used at 30 °C. Two solvent gradients comprising mobile phase A (acetonitrile/water 5/95 with 5 mM ammonium acetate and 40 μL acetic acid per liter) and mobile phase B (acetonitrile/water 95/5 with 5 mM ammonium acetate and 40 μL acetic acid per liter) with a gradient from 70%A/30%B to 20%A/80%B, stepwise within 30 min and a flow rate of 0.3 mL/min were used. Data were analyzed by Empower 3 software and quantified using an external reference standard. No major matrix effects were seen when mixing ACN containing CorA with plasma [22 (link)].
8
HPLC Analysis of Pharmaceuticals
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A prominence HPLC instrument (Shimadzu, Japan) was equipped with a DGU-20A5 degasser, a LC-20AD pump, a SIL-20AC autoinjector, a CTO-20AC column oven, a SPD-M20A DAD, and a CBM-20A communications bus module. The data were acquired and then processed using the LC Solution 1.11 SP1 software for chromatographic analysis (Shimadzu, Japan).
The chromatographic analyses were carried out on an XBridge Shield RP18 column (150 mm L. × 4.6 mm I.D., 3.5 μm) purchased from Waters Corporation (Massachusetts, USA) and protected by a SecurityGuard Guard system equipped with C18 cartridge (4.0 mm L. × 3.0 mm I.D.) from Phenomenex Inc. (CA, USA) under a linear gradient elution at 35°C. The mobile phases used were composed of ACN (A) and 0.30% TFA aqueous solution (B). Time program for the separation was as follows: 0 min, 15% A–85% B; 20 min, 20% A–80% B; 31 min, 30% A–70% B, followed by cleaning the column with 100% A for 5 min and then conditioning with 15% A for another 5 min. A constant flow rate was set at 1.0 mL/min throughout the entire analysis. Injection volume was 10.0 μL, the wavelength for monitoring the separation was set to 254 nm, and the contour view by DAD was plotted for an overall profile of the sample as well.
The chromatographic analyses were carried out on an XBridge Shield RP18 column (150 mm L. × 4.6 mm I.D., 3.5 μm) purchased from Waters Corporation (Massachusetts, USA) and protected by a SecurityGuard Guard system equipped with C18 cartridge (4.0 mm L. × 3.0 mm I.D.) from Phenomenex Inc. (CA, USA) under a linear gradient elution at 35°C. The mobile phases used were composed of ACN (A) and 0.30% TFA aqueous solution (B). Time program for the separation was as follows: 0 min, 15% A–85% B; 20 min, 20% A–80% B; 31 min, 30% A–70% B, followed by cleaning the column with 100% A for 5 min and then conditioning with 15% A for another 5 min. A constant flow rate was set at 1.0 mL/min throughout the entire analysis. Injection volume was 10.0 μL, the wavelength for monitoring the separation was set to 254 nm, and the contour view by DAD was plotted for an overall profile of the sample as well.
9
Analytical Determination of Peptide Hydrophobicity
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To determine peptide hydrophobicity, a Waters Alliance e2695 system with a Waters 2998 PDA Detector (software-Empower 3, Waters, Milford, MA, USA) was used. All analyses were carried out on a Waters X-Bridge Shield RP-18 column (3.0 × 100 mm, 3.5 μm particle size, 130 Å pore size). The Shield Technology column with embedded polar groups was used to minimize interactions of unreacted silanol groups with basic lipopeptides [68 ]. The peptides were dissolved in water (0.1% TFA, v/v) up to a concentration of 1 g/L. UV detection at 214 nm was used, and samples (10 μL) were eluted with a linear 20–65% acetonitrile gradient in deionized water over 30 min at 25.0 ± 0.1 °C. The mobile phase flow rate was 0.5 mL/min. Both eluents contained 0.1% (v/v) of TFA. Each peptide sample was analyzed in triplicate. Maximum standard deviation and coefficient of variation were 0.042 and 0.25%, respectively.
10
Peptide Hydrophobicity Determination by RP-HPLC
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To determine peptide hydrophobicity, the RP-HPLC system was used. The equipment used was Waters Alliance e2695 system with a Waters 2998 PDA Detector (software-Empower®3). All analyses were carried out on a Waters X-Bridge Shield RP-18 column (4.6 × 150 mm, 3.5 µm particle size, 130 Å pore size) and each peptide sample was analyzed in triplicate. UV detection at 214 nm was used, and samples (10 µL) were eluted with a linear 20–50% acetonitrile gradient in deionized water over 30 min at 25.0 ± 0.1 °C (gradient 1% of ACN/min). The mobile phase flow rate was 0.5 mL/min. Both eluents contained 0.1% (v/v) of TFA. The peptides were dissolved in water (0.1% TFA, v/v) to obtain a concentration of 1 mg/mL. The hydrophobicity was expressed as acetonitrile content in the mobile phase at a retention time of a peptide (tR). The dead time (t0) was determined by injecting water and measuring the elution time of negative peaks. To calculate hydrophobicity, Eq. (2 ) was used. where Y %ACN is the acetonitrile content [%] at the peak maximum; tR is the retention time [min]; t0 is the dead time [min]; 1% ACN/min is the organic phase gradient; 20% ACN is the content of organic solvent at the beginning of method.
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