Advanced Practical HPLC Course - 5 days

Course duration:

Location:

(see yellow tab for course dates)

Laserchrom - Rochester

"Probably the most comprehensive HPLC Course anywhere in the world,

and definitely the best value"

Course content:

Day 1 (Monday)

Day 2 (Tuesday)

Column Technology

Column Packing Material

 - Silica, alumina, polymers, zirconia, carbon, monolithic silica

 - Particle Size

 - Bonded phase

Column Sizes and Shapes

Column Loading Capacity

Scale down to microbore & narrowbore

Normal Phase and Reversed Phase

Polymer-based Reversed phase

Chiral HPLC

GPC/GFC/SEC

Ion Chromatography

Ion Exchange Chromatography

Hydrophobic Interaction Chromatography

Protein Columns

Column Protection

 - Use of precolumns & guard columns

 - Mobile Phase & Sample Filtration

 - In-Line Filters

Column Regeneration

 - Eluent Sequences

 - Removing Air

 - Filling Voids

Scale up to Prep and semi-prep scale

 - Calculating column size required

 - Calculating new flow rate

 - System changes - pump heads, injection valve, tubing, autosampler, flow cell etc

 - Method development

 - Solvent recycling

 - Peak collection

Autosamplers

 - Fixed and variable volume

 - Heating, chilling

 - Preparative

 - Programming

 - Vial choice

 - Troubleshooting

Injection valves

 - Practical - Strip and rebuild a Rheodyne valve

Column Switching

Column Heaters

 - Ovens and Block heaters

 - The importance of temperature control

Sample Preparation

 - Solvent extraction

 - Filtration

 - Solid Phase Extraction

Day 3 (Wednesday)

Day 4 (Thursday)

Detectors

General detection parameters

Noise

Drift

Sensitivity

Linearity

Dynamic Range

UV / Vis

 - Eluent choice

 - Optical Layout

 - Lamps

 - Wavelenght Control

 - Wavelength selection

 - Flow cells

 - Lamp and cell replacement

 - Wavelength programming

 - Dual Wavelength detection

 - Spectral Scanning

Diode Array

 - Number of Diodes

 - Advantages of Diode Array

 - Time slice spectra

 - Peak Identification

 - Peak Purity assessment

Fluorescence

 - Introduction to fluorescence

 - Sensitivity

 - Excitation and Emission Wavelength determination

 - Selectivity

 - Derivatisation - post column and pre-column

Refractive Index

 - Preparing for use

 - Eluent choice

 - Importance of Temperature control

 - Mixing isocratic eluents with a gradient pump

 - Minimising the effect of Negative peaks

 - Importance of degassing

Evaporative Light Scattering

 - Operating Principles

 - Gas supply requirements

 - Nebulisation

 - Light source

 - Optimising detector sensitivity

 - Eluents which are and are not compatible

 - Maintenance

 - Troubleshooting

Electrochemical

 - Operating principles

 - Analogy with UV detection

 - Electrode materials

 - Coulometric and Amperometric detection

 - Pulsed Amperometric detection

 - Selection of applied potential

 - Eluent selection

 - Use with gradients

 - Pump requirements

 - Selectivity and applications

Conductivity

 - Chemical and electronic suppression

 - Effect of changing temperature

 - Detection limits

 - Optimising the eluent

 - Sensitivity

 - Limitations with certain eluents

Mass Spec

 - Principles of Mass Spec

 - Peak identification

 - Characterisation of unknowns

 - Interfaces and ionisation Sources

 - Mass Analysers

 - Fragmentation Patterns

 - Isotopes

 - Molecular ions

 - Single Ion Monitoring

 - Quantitation

Practical: The use of fluorescence detection for the analysis of Biogenic Amines using Pre-column and then Post-column derivatisation

Data Handling

Sampling frequency

Input Voltages

Integration Parameters

Peak Width

Peak Threshold

Minimum Area

Negative Peaks

Fused Peaks - perpendiculars, skims, valley-valley

Understanding noise

Reducing Noise

Limit of detection

Limit of Quantitation

Integration by height or area

Peak Table set up

Peak Identification

Peak time windows

Reference peaks

Internal Standards

Calibration

External Standard Calibration

-single point

-multi point

Internal Standard calibration

Choice of internal standard

Area%

Calibration range

Best fit curve to data

How to get a straight line calibration curve

Making up standards

Errors analysis

Accuracy and Precision

Systematic and Random Errors

Reporting

Assessment

Discussion of assessment

Presentation of course certificates

Course Dates in 2010

1. For those attending from Muslim countries, no courses are run during Ramadan, and lunch is timed to allow for time for prayer.
2. Vegetarian food is available on all courses.
3. If you do not have experience of HPLC , we recommend booking the Introduction and Intermediate courses. A combined offer is sometimes available.