A one-day short course focused on revealing the important factors involved in reversed-phase chromatographic separations, how each affects retention and resolution and how to most efficiently and effectively develop reversed-phase HPLC methods.

Reversed-phase method development is looked upon as a mystery by many chromatographers. Resolution seems to be a hidden treasure which either requires too much time to find or entirely eludes the earnest seeker. The class is presented as a search for the treasure of resolution. A map is clearly laid out guiding the chromatographer through each step of the pathway that leads to resolution. Those attending the class will learn the important principles which affect resolution and will be presented with a systematic, efficient approach to developing reversed-phase HPLC methods.

Course Content

• Introduction to Resolution. The concepts of efficiency, retention factor and selectivity are discussed in detail as they relate to chromatographic resolution. The way to optimize each parameter and its role in reversed-phase resolution is explained.
• Reversed-Phase HPLC Columns. The characteristics of reversed-phase columns are explained as they contribute to retention and resolution.
• The matrix and the nature of the stationary phase
• How hydrophobic, polar and ionic interactions contribute to and affect reversed-phase
• retention and selectivity.
• The type of stationary phases available and how each affects selectivity.
• How to evaluate and compare columns in order to select the best column for a given separation.
• Reversed-Phase HPLC Mobile Phases. The characteristics of mobile phases used in reversed-phase HPLC are explained.
• How to find the best solvent concentration for a method
• How solvent type and concentration affects selectivity and why
• How to best perform gradient elution
• How gradient elution affects selectivity.
• How the pH of the mobile phase is used to optimize the separation of basic and acidic compounds.
• Temperature. The affect of column temperature on retention and selectivity is described.
• Method development. The principles of reversed-phase method development are brought together in a systematic approach to developing effective, robust reversed-phase HPLC methods in the most efficient manner.

Course Outline

Morning

• An introduction to the principles of chromatographic resolution.
• Reversed-phase HPLC columns

Afternoon

• The mobile phase: Solvents used and their characteristics.
• How the solvent affects retention and selectivity. The affect of solvent strength and type.
• Gradient elution and how it affects retention and selectivity
• The use of pH to control the retention of acids and bases.
• The affect of temperature on reversed-phase separations
• A systematic approach to developing reversed-phase HPLC methods

Who Should Attend:

Chromatographers wishing to improve their knowledge of reversed-phase method development.

Ordering Information:

Schedule and Registration:

Contact us for current schedule.

Questions:

Please send all training inquiries to us.training.analyze@thermofisher.com.