Dionex HPLC Manual

The Dionex HPLC (High Performance Liquid Chromatography) system is a sophisticated analytical instrument designed for precise separation, identification, and quantification of chemical compounds. This manual covers the UltiMate 3000 series with quaternary pump, autosampler, column compartment, and various detectors including DAD, FLD, and CAD. Below are key sections for safety, features, installation, setup, operation, maintenance, and troubleshooting.

Features at a Glance

Key components: Quaternary pump, autosampler, column compartment, detector array, Chromeleon software interface.

Feature	Description
Quaternary Pump	Four-solvent capability with high-pressure mixing
Autosampler	Precise sample injection with temperature control
Column Compartment	Temperature-controlled column environment
DAD Detector	Diode Array Detector for multi-wavelength detection
FLD Detector	Fluorescence Detector for sensitive analysis
CAD Detector	Charged Aerosol Detector for universal detection
Chromeleon Software	Comprehensive data acquisition and analysis
Degasser	Online solvent degassing system
Column Heater	Precise temperature control up to 80°C
Sample Tray	Capacity for 100+ vials with barcode reading

Installation

Unpack and inspect all components. Place on stable, level surface.

1. Connect solvent lines: Attach solvent reservoirs to degasser inlet ports.
2. Install column: Connect column between pump and detector, ensuring proper orientation.
3. Connect waste line: Route waste tubing to appropriate container.
4. Power connections: Connect all modules to power source and network.
5. Computer connection: Install Chromeleon software and connect to instrument.

WARNING! Handle columns with care; avoid dropping or shocking. Ensure all fittings are properly tightened.

First-Time Setup

Power on system; initialize Chromeleon software.

1. System priming: Prime pump with mobile phase to remove air bubbles.
2. Column conditioning: Condition new column according to manufacturer specifications.
3. Detector calibration: Perform wavelength calibration for DAD detector.
4. Method setup: Create initial method with flow rate, gradient, and detection parameters.
5. System suitability: Run test mixture to verify performance.

CAUTION! Use HPLC-grade solvents only. Wear appropriate personal protective equipment.

Specifications

• Pump Type: Quaternary gradient pump
• Flow Rate Range: 0.001 to 10.0 mL/min
• Pressure Limit: 6000 psi (413 bar)
• Autosampler: 100-vial capacity with temperature control 4-40°C
• Column Compartment: Temperature range 5-80°C
• DAD Wavelength: 190-800 nm
• FLD: Excitation 200-700 nm, Emission 280-900 nm
• Data System: Chromeleon Chromatography Data System
• Power Requirements: 100-240 VAC, 50/60 Hz
• Operating Environment: 15-30°C, 20-80% relative humidity

Controls and Settings

Access system controls via Chromeleon software interface or front panel displays.

Software Controls: Method editor, sequence setup, real-time monitoring, data analysis.

Pump Settings: Flow rate, pressure limits, gradient program, solvent composition. Autosampler: Injection volume, wash cycles, needle position, temperature. Detector: Wavelength selection, bandwidth, acquisition rate, reference wavelength. Column Oven: Temperature setpoint, equilibration time, temperature ramp.

Connecting Components

Proper connection sequence: Solvent reservoirs → Degasser → Pump → Autosampler → Column → Detector → Waste.

1. Ensure system is powered off before making connections.
2. Use appropriate HPLC tubing and fittings for all connections.
3. Verify flow direction: Pump to injector to column to detector.
4. Check for leaks at all connection points after startup.

Tip: Use finger-tight fittings; avoid over-tightening which can damage threads.

Method Development

Create and optimize HPLC methods for specific applications.

1. Column selection: Choose appropriate stationary phase for analytes.
2. Mobile phase: Select solvents and pH based on analyte properties.
3. Gradient optimization: Develop time program for solvent composition.
4. Flow rate: Optimize for resolution and analysis time.
5. Detection parameters: Set wavelengths, acquisition rate, and filter settings.
6. Method validation: Test for precision, accuracy, and robustness.

WARNING! Always equilibrate column with starting mobile phase before analysis.

Data Analysis

Using Chromeleon software for chromatographic data processing.

1. Peak integration: Automatic and manual integration methods.
2. Calibration curves: Create and validate using standards.
3. Quantitation: Calculate concentrations using internal or external standards.
4. System suitability: Monitor retention time, resolution, and peak symmetry.
5. Report generation: Customizable reports with chromatograms and results.

Maintenance & Cleaning

Regular maintenance schedule for optimal performance.

1. Daily: Check for leaks, monitor pressure, clean autosampler needle.
2. Weekly: Replace solvent filters, purge pump seals, clean detector flow cell.
3. Monthly: Replace pump seals, clean autosampler, validate detector performance.
4. As needed: Replace columns, tubing, and other consumables.

CAUTION! Flush system with appropriate solvents before storage. Use only HPLC-grade solvents for cleaning.

Regulatory Information

CE marked: Complies with European Union safety standards. FDA 21 CFR Part 11: Software complies with electronic records requirements. RoHS compliant: Meets Restriction of Hazardous Substances directive. For technical support, contact Thermo Fisher Scientific at 1-800-532-4752 or visit www.thermofisher.com.

Troubleshooting

Symptom	Possible Cause	Corrective Action
High pressure	Clogged column/filter	Replace inline filter; flush or replace column.
Peak tailing	Column degradation	Replace column; check mobile phase pH.
Retention time shift	Mobile phase/temperature	Prepare fresh mobile phase; check column temperature.
No peaks	Detector lamp/settings	Check detector lamp life; verify wavelength settings.
Baseline noise	Contamination/bubbles	Degas mobile phase; clean system; replace lamp.
Leaks	Loose fittings/seals	Tighten fittings; replace pump seals or tubing.
Poor resolution	Method/column issues	Optimize gradient; replace column; adjust flow rate.

Reset: Power cycle system; reinitialize Chromeleon software if needed.

Service: 1-800-532-4752; www.thermofisher.com