Left ventricular apices were homogenized on ice with 0.4 mmol/L perchloric acid and precipitated with KOH. The samples were centrifuged for 10 min (3000× g) and the supernatants were injected twice onto a pre-equilibrated RP18 column (LiChroCART, Merck, Darmstadt, Germany) as previously published [31 (link)]. For ATP detection a HPLC-apparatus from Knauer (Berlin, Germany) and an UV-detector (PDA Detector 2800, Knauer, Berlin, Germany) were used. Peaks were measured at 259 nm. Standard curves were generated with 4 concentrations of ATP, (25-12.5-6.25-3.125 µg/mL). External standards and ventricular samples were measured together in one HPLC run.
Pda detector 2800
Manufactured by Knauer
Sourced in Germany
The PDA Detector 2800 is a photodiode array (PDA) detector designed for high-performance liquid chromatography (HPLC) applications. It provides simultaneous multi-wavelength detection and analysis of samples.
Automatically generated - may contain errors
5 protocols using pda detector 2800
1
HPLC-based ATP Quantification in LV
2
Purification and Characterization of Compounds
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Reversed-phase preparative high pressure liquid chromatography (prep-HPLC) analyses were carried out on a HPLC system (Knauer, Germany), equipped with a Knauer PDA detector 2800 (Heraeus, Germany), using a reversed-phase Reprosil 100 C18 column (10 μm particle size, 250 × 20 mm i.d.) (Dr. Maisch, Germany).
The NMR spectroscopic analyses (one- dimensional, 13C spectra) were obtained on a Bruker 200 NMR spectrometer (Bruker, Germany) (200 MHz for 1H, and 50 MHz for 13C). Chemical shifts (δ, ppm) are reported relative to transcranial magnetic stimulation (TMS) as an internal standard. Sephadex LH20 (Amersham Biosciences, Sweden) used for column chromatography and Sep-pak cartridge (10 g, Waters, Ireland) used for fractionating of methanol (MeOH) extract. Thin layer chromatography (TLC) was performed on silica gel GF-254 plates (Merck, Germany) and spots were detected by ultraviolet (UV) illumination.
The NMR spectroscopic analyses (one- dimensional, 13C spectra) were obtained on a Bruker 200 NMR spectrometer (Bruker, Germany) (200 MHz for 1H, and 50 MHz for 13C). Chemical shifts (δ, ppm) are reported relative to transcranial magnetic stimulation (TMS) as an internal standard. Sephadex LH20 (Amersham Biosciences, Sweden) used for column chromatography and Sep-pak cartridge (10 g, Waters, Ireland) used for fractionating of methanol (MeOH) extract. Thin layer chromatography (TLC) was performed on silica gel GF-254 plates (Merck, Germany) and spots were detected by ultraviolet (UV) illumination.
3
Quantifying Myocardial ATP Levels
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Left ventricular specimen were homogenized on ice with 0.4 mmol/L perchloric acid and precipitated with KOH. The samples were centrifuged and supernatants were injected twice onto a pre-equilibrated RP18 column (LiChroCART, Merck, Darmstadt, Germany) as previously published (Salameh et al., 2015 (link)). For ATP detection a HPLC-apparatus from Knauer (Berlin, Germany) and an UV-detector (PDA Detector 2800, Knauer, Berlin, Germany) were used. Peaks were measured at 259 nm. Standard curves were generated with 4 concentrations of ATP (25-12.5-6.25-3.125μg/ml) and measured together with the ventricular samples.
4
Cardiac Adenine Nucleotide Quantification
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Left ventricular specimen were homogenized at 4°C with 0.4 mmol/L perchloric acid and precipitated with KOH. Thereafter, the samples were centrifuged for 10 min and 20 μl of the supernatant was injected twice onto a pre-equilibrated RP18 column (LiChroCART, Merck, Darmstadt, Germany) as previously published (Salameh et al., 2015a (link)). For ATP, ADP AMP, and adenosine detection a HPLC-apparatus from Knauer (Berlin, Germany) and an UV-detector (PDA Detector 2800, Knauer, Berlin, Germany) were used. Peaks were measured at 259 nm, standard curves were generated with 4 concentrations of ATP, ADP, AMP, and adenosine respectively (25–12.5–6.25–3.125 μg/ml) and measured together with the ventricular samples.
5
Quantifying Residual Anthracene in Enzymatic Reactions
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Residual concentration of anthracene in the enzymatic reaction mixtures was determined by an HPLC system equipped with a Smartline Pump 1000, a PDA Detector 2800 (set at 254 nm), a Degasser 5000, and Lichrospher 100 RP & C18 reverse phase column (C18, 250 × 4.6 mm), all from Knauer (Berlin, Germany). Each sample (20 µL) was injected using a Smartline Autosampler 3950 with a sample loop of 100 µL. The mobile phase was acetonitrile/methanol at a ratio of 70:30. The retention time of anthracene (at flow rate of 1 mL.min1) was 18 min.
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