The peptide-based traditional Maleimidocaproyl(Mc)-Val-Cit-PABC-PNP linker or the rebridging linker were reacted with monomethyl auristatin E (MMAE). In the construction of traditional linker-MMAE payload, 18.20 μmol potent molecule MMAE, 16.38 μmol Mc-Val-Cit-PABC-PNP, and 3.64 μmol hydroxybenzotriazole were dissolved and mixed in 500 μL dimethylformamide. Then 18.20 μmol pyridine was added to the mixture after 2 min, and 20 μmol trifluoroacetic acid (TFA) was added after 24 h. In the construction of rebridging linker-MMAE payload, 13.55 μmol N,N'-diisopropylcarbodiimide, 13.55 μmol N,N-diisopropylethylamine, and 33.85 μmol synthesized rebridging linker were mixed in 0.25 mL dichloromethane, followed by frequent mixing for 1 h at room temperature. Then 13.55 μmol MMAE was added and frequently mixed for additional 16 h. After linker-MMAE conjugates were synthesized, the solvents were removed by vacuum pump and the conjugates were purified by a Waters HPLC system equipped with 600 Controller/Pump and 996 PDA detector (Waters, Milford, MA). A reversed-phase C18 column with 5 μm C18(2) 100 Å and 250 x 10 mm (Phenomenex Luna; Torrance, CA) was used with gradient elution buffer of Phase A (water+0.1% TFA) and Phase B (acetonitrile). The purified products were confirmed by Agilent 6500 Series Accurate-Mass Q-TOF LC/MS (Agilent Technologies, Santa Clara, CA).
996 pda detector
Manufactured by Waters Corporation
Sourced in United States
The 996 PDA detector is a photodiode array detector used in high-performance liquid chromatography (HPLC) systems. It provides simultaneous multi-wavelength detection, enabling the analysis of a wide range of compounds. The 996 PDA detector collects full spectral data, allowing for the identification and quantification of analytes.
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Lab products found in correlation
Empower software, by Waters Corporation (2 mentions)
600 controller pump, by Waters Corporation (1 mentions)
Waters hplc system, by Waters Corporation (1 mentions)
Waters 2695 chromatography system, by Waters Corporation (1 mentions)
Empower 2 chromatographic manager system, by Waters Corporation (1 mentions)
2414 refractometer, by Waters Corporation (1 mentions)
Fast acid analysis hplc column, by Bio-Rad (1 mentions)
Pack pro c18 column, by YMC (1 mentions)
Alliance 2695 separation module, by Waters Corporation (1 mentions)
Zq mass detector, by Waters Corporation (1 mentions)
Silica gel 60, by Merck Group (1 mentions)
717 autosampler, by Waters Corporation (1 mentions)
Alliance 2690 hplc, by Waters Corporation (1 mentions)
Zorbax 300sb c18, by Agilent Technologies (1 mentions)
Waters 600 system, by Waters Corporation (1 mentions)
Xterra c18 column, by Waters Corporation (1 mentions)
Cary 60, by Agilent Technologies (1 mentions)
1525 binary pump, by Waters Corporation (1 mentions)
Luna 5 micron c18 column, by Phenomenex (1 mentions)
1525 binary hplc pump, by Waters Corporation (1 mentions)
28 protocols using 996 pda detector
1
Cytotoxic Payload Conjugation via Linkers
2
HPLC Analysis of Rosmarinic Acid and Carvacrol
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High-performance liquid chromatography (HPLC) analysis was carried out using Waters 2695 chromatography system (Waters, Milford, MA, USA) equipped with Waters 996 PDA detector. Data were collected and analyzed using a PC and the Empower 2 chromatographic manager system (Waters Corporation, Milford, USA). For determination of rosmarinic acid and carvacrol, an ACE 5 C18 250 × 4.6 mm column (Advanced Chromatography Technologies, Aberdeen, Scotland) was used.
3
Aromatic Substrate Analysis by HPLC
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Aromatic substrate utilization was analyzed using a Waters 600E multisolvent delivery (Waters Corporation) high performance liquid chromatography (HPLC) system with a BioRad Fast Acid Analysis HPLC column with 10% v/v acetonitrile and 0.01 N H2SO4 in a 1:1 mixture as the eluent, a flow rate 0.6 mL/min, temperature of 65 °C, and with a Waters 996 PDA detector. Phenol was detected at 270 nm, resorcinol at 274 nm, and pHBA at 254 nm. Glucose and xylose were measured using the same column at 85 °C, 5 mM H2SO4 eluent, and a Waters 2414 refractometer. Concentrations were calculated from standard curves created for each carbon source in the appropriate medium.
4
HPLC Quantification of Chlorogenic Acids
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All PJ samples were analyzed using an HPLC instrument (Waters Alliance 2695 Separation Module, Milford, MA, USA) with a photodiode array detector (Waters 996 PDA Detector, Milford, MA, USA). The setup also included pumps and autosamplers coupled with a YMC Pack Pro C18 column (4.6 × 250 mm, 5 μm). HPLC-grade solvents [water, acetonitrile (ACN), and methanol (MeOH)] were purchased from J. T. Baker (Phillipsburg, Pennsylvania). Ethanol (EtOH) and acetic acid were purchased from Samcheon Chemical (Pyeongtaek, Korea). Gallic acid, quercetin, 5-CQA (1 ), 3-CQA (2 ), and 4-CQA (3 ) were obtained from the Natural Product Institute of Science and Technology (www.nist.re.kr (accessed on 1 March 2022)), Anseong, Republic of Korea (Figure 5 ).
5
Purification and Characterization of Organic Compounds
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All
the solvents and reagents were obtained
commercially and used as received unless noted otherwise. Anhydrous
diethyl ether was prepared by refluxing with sodium; anhydrous THF
was prepared by refluxing with sodium and benzophenone. Flash chromatography
was performed with Merck silica gel 60 (230 × 400 mesh). NMR
spectra were recorded on either a Bruker AVIII 400 spectrometer or
Bruker DRX500. For7a –7e , 9a –9c and 15a ,b , HPLC/MS
analyses were recorded on a Waters Alliance LC/MS System, consisting
of a Waters ZQ mass detector, photodiode array detector, and an Alliance
HPLC system, equipped with an XTerra column (C-18, 2.1 mm × 5
mm). HPLC analysis was done using gradient of ACN/H2O/10%
HCl (aq). For all remaining final compounds, mass spectra were obtained
on a Waters micromass ZQ detector or a Micromass Q-TOF micro hybrid
quadrupole/orthogonal high resolution time of flight MS. HPLC analysis
was performed on a reverse phase XBridge C18 column (4.6 mm ×
75 mm) column, using 40% H2O/50% ACN/10% of 0.2% TFA(aq)
as the mobile phase, in gradient conditions at a flow rate of 1 mL/min.
Eluted peaks were monitored at 254 nm with a Waters 996 PDA detector.
All final compounds tested were confirmed to be of ≥95% purity
by the HPLC methods described above.
the solvents and reagents were obtained
commercially and used as received unless noted otherwise. Anhydrous
diethyl ether was prepared by refluxing with sodium; anhydrous THF
was prepared by refluxing with sodium and benzophenone. Flash chromatography
was performed with Merck silica gel 60 (230 × 400 mesh). NMR
spectra were recorded on either a Bruker AVIII 400 spectrometer or
Bruker DRX500. For
analyses were recorded on a Waters Alliance LC/MS System, consisting
of a Waters ZQ mass detector, photodiode array detector, and an Alliance
HPLC system, equipped with an XTerra column (C-18, 2.1 mm × 5
mm). HPLC analysis was done using gradient of ACN/H2O/10%
HCl (aq). For all remaining final compounds, mass spectra were obtained
on a Waters micromass ZQ detector or a Micromass Q-TOF micro hybrid
quadrupole/orthogonal high resolution time of flight MS. HPLC analysis
was performed on a reverse phase XBridge C18 column (4.6 mm ×
75 mm) column, using 40% H2O/50% ACN/10% of 0.2% TFA(aq)
as the mobile phase, in gradient conditions at a flow rate of 1 mL/min.
Eluted peaks were monitored at 254 nm with a Waters 996 PDA detector.
All final compounds tested were confirmed to be of ≥95% purity
by the HPLC methods described above.
6
Quantification of GABA Hydrolysis Kinetics
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Spontaneous
hydrolysis of DNI-GABA·TFA derivatives (1–3 ) was analyzed by the direct measurement of GABA concentration by
the previously published standard method by the o-phthalaldehyde mercaptopropionic acid derivatization method.60 (link) The cage compound was dissolved in distilled
water or in 12 mL of ACSF (a final concentration 2.5 mM). The analytical
setup consisted of a Waters 996 PDA detector and a Waters 474 detector,
a Waters 616 controller quaternary pump, and a Waters 717 autosampler,
operating with the Millennium Software. The analytical column was
a Thermo Hypersil GOLD 20 cm × 4.6 mm, 5 μm with a guard
column. Detections were performed simultaneously on PDA (190 and 400
nm) and Fl detectors (λEx/λEm =
337/454 nm). Eluent system: A: 0.05 M sodium acetate of pH 7.20 ±
0.05; B: 0.1 M sodium acetate–acetonitrile–methanol
(46/44/10) at pH 7.20 ± 0.05. Elutions were performed in the
gradient mode (at 40 °C): starting with 1% B for 1 min with a
1.3 mL/min flow rate, reaching 100% B, and a 2.0 mL/min flow rate
within 7 min; afterward, 3 min isocratic elution with 100% B, and
finally returning to the initial concentration (1% B) in 1 min and
equilibrating for 4 min with this B content (Figure S9 ).
hydrolysis of DNI-GABA·TFA derivatives (
the previously published standard method by the o-phthalaldehyde mercaptopropionic acid derivatization method.60 (link) The cage compound was dissolved in distilled
water or in 12 mL of ACSF (a final concentration 2.5 mM). The analytical
setup consisted of a Waters 996 PDA detector and a Waters 474 detector,
a Waters 616 controller quaternary pump, and a Waters 717 autosampler,
operating with the Millennium Software. The analytical column was
a Thermo Hypersil GOLD 20 cm × 4.6 mm, 5 μm with a guard
column. Detections were performed simultaneously on PDA (190 and 400
nm) and Fl detectors (λEx/λEm =
337/454 nm). Eluent system: A: 0.05 M sodium acetate of pH 7.20 ±
0.05; B: 0.1 M sodium acetate–acetonitrile–methanol
(46/44/10) at pH 7.20 ± 0.05. Elutions were performed in the
gradient mode (at 40 °C): starting with 1% B for 1 min with a
1.3 mL/min flow rate, reaching 100% B, and a 2.0 mL/min flow rate
within 7 min; afterward, 3 min isocratic elution with 100% B, and
finally returning to the initial concentration (1% B) in 1 min and
equilibrating for 4 min with this B content (
7
HPLC-DAD Analysis of Polyphenolic Compounds
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In order to obtain UV spectra for compounds of interest, an HPLC Alliance 2690 (Waters) device coupled with a Waters 996 PDA detector was used. A Zorbax 300 sb-C18 (3.5 μm, 4.6 × 150 mm) from Agilent was mounted on the chromatographic module and the temperature was set to 25°C.
A binary mobile phase was used, consisting of a first solvent (A), distilled water with acetic acid (0.5%) and a second one (B), acetonitrile with acetic acid (0.5%). The total flow rate was 1 mL/min and the injection volume was 15 μL. The gradient began with 100% A. This proportion was held for 5 minutes, after which the gradient decreased to 85% A at 10 minutes, then steadily to 65% A at 30 minutes, and then to 50% at 35 minutes, and finally, cut off to 0% at 36 minutes. This ratio was held until 40 minutes. After that, the proportion of A increased to 100% at 41 minutes and held there until 46 minutes.
By scanning from 200 to 400 nm, calibration curves for gallic acid, caffeic acid, p-coumaric acid, trans-ferulic acid, quercetin, kaempferol, and chrysin prepared in a water–methanol mixture (20:80 v/v) were constructed. After identification, this technique was also used for quantification at 280 and 320 nm with external calibration. Parameters of the HPLC-DAD method are summarized inSupplementary Table 1 .
A binary mobile phase was used, consisting of a first solvent (A), distilled water with acetic acid (0.5%) and a second one (B), acetonitrile with acetic acid (0.5%). The total flow rate was 1 mL/min and the injection volume was 15 μL. The gradient began with 100% A. This proportion was held for 5 minutes, after which the gradient decreased to 85% A at 10 minutes, then steadily to 65% A at 30 minutes, and then to 50% at 35 minutes, and finally, cut off to 0% at 36 minutes. This ratio was held until 40 minutes. After that, the proportion of A increased to 100% at 41 minutes and held there until 46 minutes.
By scanning from 200 to 400 nm, calibration curves for gallic acid, caffeic acid, p-coumaric acid, trans-ferulic acid, quercetin, kaempferol, and chrysin prepared in a water–methanol mixture (20:80 v/v) were constructed. After identification, this technique was also used for quantification at 280 and 320 nm with external calibration. Parameters of the HPLC-DAD method are summarized in
8
Determination of Polyphenolic Content in Black Tea
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2.5% infusion of Black tea was prepared in boiling water and cooled. It was lyophilized in a SCANVAC lyophilizer. The lyophilized product was weighed, reconstituted in water and administered to the mice by gavage (at an interval of 8 h, cumulated dose being 0.33 mg/gm body weight per day) [7 (link)]. polyphenolic content of tea was determined by HPLC, which was performed according to the laboratory protocol [11 (link)].
HPLC was carried out using a 515 lb dual pump with a control (Waters, USA), a Rheodyne injector, a RP C-18 column by Nova-Pak which was attached to the guard column regulated at 30 °C and a 996 PDA detector (Waters, USA) adjusted to 278 nm. Catechins were separated using gradient of 5–25% acetonitrile in 0.025 M KH2PO4, pH 2.4. Isocratic mode was used to separate theaflavin, where the mobile phase consisted of acetic acid: acetone: water in the ratio of 1:60:39 and detected at 365 nm. Integration and calibration of mixture containing catechins and theaflavins were done using the software Millennium 32. The quantification of the content of catechins and theaflavins was done by plotting against the standard.
HPLC was carried out using a 515 lb dual pump with a control (Waters, USA), a Rheodyne injector, a RP C-18 column by Nova-Pak which was attached to the guard column regulated at 30 °C and a 996 PDA detector (Waters, USA) adjusted to 278 nm. Catechins were separated using gradient of 5–25% acetonitrile in 0.025 M KH2PO4, pH 2.4. Isocratic mode was used to separate theaflavin, where the mobile phase consisted of acetic acid: acetone: water in the ratio of 1:60:39 and detected at 365 nm. Integration and calibration of mixture containing catechins and theaflavins were done using the software Millennium 32. The quantification of the content of catechins and theaflavins was done by plotting against the standard.
9
Purification of Crude Peptides by RP-HPLC
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All crude peptides were purified by semi-preparative RP-HPLC using a Waters 600 system (Waters, Milford, MA, USA) equipped with a C18 column (MultoKrom 100–5 C18, 5 µm particle size, 100 Å pore size, 250 × 20 mm, CS Chromatographie Service, Langerwehe, Germany). The gradient elution system was 0.1% trifluoroacetic acid (TFA) in water (eluent A) and 0.1% TFA in acetonitrile (eluent B). The peptides were eluted with a gradient of 5–40% eluent B in 60 min and a flow rate of 8 mL/min. The peaks were detected at 220 nm. Collected fractions were combined, freeze-dried and stored at −28 °C. Purity was confirmed by analytical RP-HPLC on a Waters XC e2695 system (Waters, Milford, MA, USA) employing a Waters PDA 2998 diode array detector or a Waters 600 system employing a Waters 996 PDA detector. Both systems were equipped with a Multospher 120 RP 18 HP column (C18, 3.1 µm particle size, 120 Å pore size, 60 × 4.6 mm, CS Chromatographie Service, Langerwehe, Germany). The peptides were eluted with a gradient of 5–40% eluent B in 10 min at a flow rate of 2 mL/min. Chromatograms were extracted at 214 nm.
In order to avoid sample oxidation during HPLC sample preparation and measurements Ni(II)-peptide/buffer solution was acidified by diluting the sample 1:10 with aqueous TFA (1% or 0.1%, v-v), which effectively slows down the oxidation process allowing for reliable HPLC analysis.
In order to avoid sample oxidation during HPLC sample preparation and measurements Ni(II)-peptide/buffer solution was acidified by diluting the sample 1:10 with aqueous TFA (1% or 0.1%, v-v), which effectively slows down the oxidation process allowing for reliable HPLC analysis.
10
Chromatographic Separation of Analytes
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Chromatographic separation was done on a Waters LC 2695 system (Milford, MA, USA) with a quaternary, low-pressure mixing pump, inline vacuum degassing, and a Waters 996 PDA detector (200–600 nm). Data collection and analysis were done by Empower software (version 3, Waters Corporation, Milford, MA, USA). Mixture separation was achieved using an Xterra C18 column (4.6 × 100 mm, 5 µm, Waters, Milford, MA, USA). The column temperature was maintained at 25 ± 5 °C. A mobile phase composed of methanol (A) and phosphate buffer of pH 3 ± 0.1 (B) was used. The analytes were separated by the gradient elution of A/B (v/v) as follows: 50/50 for 5 min, 98/2 for 5 min, then 50/50 for 5 min; the total run time was 15 min. The injection volume was 10 µL and UV detection was done at 280 nm.
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