Hewlett-Packard gradient System

Quantitation of 1. Quantitation was performed by high performance liquid chromatography using a Hewlett Packard 1050 system. One mg of root exudates were dissolved in I mL of acetonitrile. Components of the root exudates was separated on an Alltech EPS CIS column 100 A 3p (ISO mm length x 4.5 mm internal diameter). The mobile phase was eluted at a flow rate of 2 mL/min as follows (solvent A is 2.5% acetic acid in water, solvent B is acetonitrile) 0-15 min 45% A / 55% B isocratic 15-22 min linear gradient from 55% to 100% B 22-25 min 100%B 25-26 min 100% to 55% B 26-30 min 45% A / 55% B isocratic. The peaks were monitored at X280 nm, and the volume of injection was 20 pL. Quantitation was based on a calibration curve of an external standard of purified 1. 

Procedure Phenolic acids were detected between 210 and 360 nm using a Hewlett Packard diode array detector (HP 1100 HPLC system). The separation was achieved with a Nucleosil 100-5 C18 column 5 pm 4.0x250 mm (Agilent Technologies, USA) at a flow rate of 1.0 ml/min and injection volume of 5 mL. For the elution, a discontinuous acetonitrile-water gradient was used 15% acetonitrile (5 min), 30% acetonitrile (20 min), 40% acetonitrile (25 min), 60% acetonitrile (30 min), 60% acetonitrile (35 min) and... 

The HPLC system consist of an HP 1100 series binary gradient pump, a vacuum degasser, and a column temperature controller (all from Hewlett Packard), connected to a Gilson 231 XL autosampler (Gilson). 

Analysis of Phenolic Compounds. A Hewlett-Packard (Palo Alto, CA) Model 1090 HPLC System, was used to determine the levels of specific phenolic components. The HPLC system was equipped with a ternary solvent delivery system, a diode array UV-VIS detector, and HP ChemStation software for data collection and analysis. Full chromatographic traces were collected at 280, 520, 316, and 365 nm, and spectra were collected on peaks. The stationary phase was a Hewlett-Packard LiChrosphere C-18 coliram, 4mm X 250 mm, with 5 pM particle size packing. Operating conditions include an oven temperature of 40 C, injection volume of 25 pL, and flow rate of 0.5 mL/minute. The mefriod was based on a previously published method for phenolic components in wine (30) and used the modified solvent gradient shown in Table II. Solvent A was 50 mM dihydrogen ammonium phosphate, adjusted to pH 2.6 with orthophosphoric acid. Solvent... 

The [l Nlr-metHuLeptin isoforms were separated by applying 750 pg of a leptin preparation to a Vydac C-4 reverse-phase serai-preparative column (10x250 mm) using a Hewlett Packard HPLC (Model 1090) system equipped with a diode array detector. The column was initially equilibrated with 47% mobile phase A (0.1%TFA) and 53% mobile phase B (90% acetonitrile in 0.1% TFA). A linear gradient from 53 to 55% mobile phase B was run over a period of 35 minutes at a flow rate of 2.0 ml/min. 

RP-HPLC was performed using a Vydac C4 column (4.6 mm X 250 mm) connected to a Hewlett Packard 1090 HPLC system. The HPLC was equipped with a diode array detector and a PC based computer system for data processing. Solvent A was 0.1% TFA, while solvent B was composed of 0.1%, TFA in 90% acetonitrile. The column was initially equilibrated at 3% B using a flow rate of 0.7 ml/min, with the absorbance monitored at 230 nm. The elution gradient consisted of isocratic conditions at 3% B for 5 minutes, followed by linear gradients to 40% B over 10 minutes, to 50% B over 20 minutes, and to 80% B over 5 minutes, and finally isocratic conditions at 80% B for 5 minutes. 

The samples (50 (jul) were analysed on a Hewlett Packard 1050 DAD system upgraded with a 1090 DAD optical bench. The column was a Luna 5 j,m phenyl-hexyl, 250 mm x 4.6 mm maintained at 30 °C. The flow rate was 1 ml min-1 using a gradient elution of acetonitrile and water as follows ... 

Major manufacturers of HPLC instruments include Waters, Agilent (formerly Hewlett Packard), and Shimadzu, PerkinElmer, Thermo, Beckman, Varian, Hitachi, Jasco, Dionex, Gilson, Scientific Systems (SSI), and Isco. The Internet addresses of these companies can be found in the reference section. HPLC is a mature technology and most manufacturers have highly reliable products with sufficient performance and feature sets to be competitive in the market place. However, there can still be significant differences between the vendors on these performance characteristics on systems (dwell volume, dispersion), pumps (low flow, seal life), autosamplers (carryover, speed, sample capacity, minimum sample volume), and detectors (sensitivity, gradient baseline shift). 

The HPLC system used for the separation of nonpolar lipid classes consisted of a Hewlett Packard (Avondale, PA, USA) Model 1050 ternary gradient system (HPLC pump, autosampler, and UV/visible detector), and an Alltech-Varex Mark HI ELSD (Alltech Associates, Deerfield, IL). The column was a LiChrosorb DIOL, 5 jiun, (3 x 100 mm from Chrompack, Inc., Raritan, NJ), and the flow rate was 0.5 ml/min. The solvents were A, hexane/acetic acid, 1000/1, v/v and B, hexane/isopropanol, 100/1, v/v, (Both were mixed fresh daily to eliminate variability caused by evaporation and/or absorption of moisture). The linear gradient timetable was At 0 min, 100/0 at 8 min, 100/0 at 10 min, 75/25 at 40 min, 75/25 at 41 min, 100/0 at 60 min, 100/0 (%A/%B, respectively). 

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