RP-HPLC Fundamentals

Overview

This lecture covers the fundamentals and practical aspects of Reverse Phase High Performance Liquid Chromatography (RP-HPLC), focusing on separation principles, suitable compounds, mobile phases, optimization strategies, and column selection.

Reverse Phase HPLC Basics

• Reverse phase HPLC uses a non-polar (hydrophobic) stationary phase and a polar mobile phase.
• The most common stationary phase is a C18 (octadecyl) silica column.
• The polar mobile phase often starts with water, combined with polar organic solvents like methanol, acetonitrile, or THF.

Separation Principles and Compound Suitability

• Hydrophilic (polar) compounds have low retention times and elute quickly.
• Hydrophobic (non-polar) compounds have high retention times and elute slowly.
• RP-HPLC is suitable for compounds under 2,000 Daltons (small to medium molecules).
• Effective for separating acids, bases, small peptides, and some proteins.
• Not suitable for highly hydrophobic, highly hydrophilic compounds, inorganic ions, or stereoisomers.

Hydrophobicity, Log P, and Retention Times

• Retention time increases with hydrocarbon content and decreases with branching or unsaturation.
• Log P (partition coefficient): log([solute]_octanol/[solute]_water) quantifies hydrophobicity.
• Log P > 1: hydrophobic; Log P = 0: intermediate; Log P < 0: hydrophilic.
• Retention time trends: higher log P = longer retention; lower log P = shorter retention.

Mobile Phases and Solvent Selection

• Common solvents (in order of increasing elution strength): water < methanol < acetonitrile < THF.
• Solvents must be water-miscible, low in UV absorbance, low in viscosity, and non-reactive.
• Use HPLC-grade solvents to avoid contamination and ensure data quality.
• Solvent shelf life: water/buffers (48–72 hours), <20% organics (1 month), >20% organics (3 months), pure organics (3 months).

Optimizing Separation

• Poor resolution can be improved by adjusting the ratio of water to organic solvent.
• If peaks elute too quickly, decrease organic fraction; if too slowly, increase organic fraction.
• Try changing or adding new solvents if ratio adjustments are insufficient.

Column Selection

• Start with a C18 column; alternatives: C8, C4, cyano, phenyl, or amino columns.
• Moving from C18 to amino increases polarity and decreases hydrophobicity of the stationary phase.

Key Terms & Definitions

• Reverse Phase HPLC — Chromatography using a non-polar stationary phase and a polar mobile phase.
• C18 Column — Silica column bonded with an 18-carbon hydrophobic chain.
• Retention Time — How long a compound is held by the column before elution.
• Log P — Logarithmic partition coefficient measuring compound hydrophobicity.
• Elution Strength — Ability of a solvent to remove analytes from the stationary phase.

Action Items / Next Steps

• Review previous HPLC lectures for foundational column chemistry and pH effects.
• Make water/buffer mobile phases fresh every 48–72 hours.
• Use only HPLC-grade solvents in experiments.
• Prepare for next lecture covering pH effects or ion exchange chromatography.