Overview
This lecture covers the fundamentals of normal phase HPLC, including its definition, column and solvent choices, mechanisms, appropriate applications, and a summary of its advantages and disadvantages compared to other HPLC methods.
What is Normal Phase HPLC?
- Normal phase HPLC is the original form of HPLC, using a polar stationary phase and nonpolar mobile phase.
- It is called "normal" because it predates reverse phase HPLC, which uses reversed polarity conditions.
- Reverse phase is now more commonly used in research and industry.
Columns and Solvents in Normal Phase HPLC
- Columns: Typically use bare silica (non-modified), cyano (nitrile), diol, or amino modifications for varying polarity.
- Most common column is bare silica due to its high polarity.
- Mobile phases are mainly nonpolar solvents like hexane, often mixed with ethyl acetate, methylene chloride, or chloroform for increased polarity.
- Ethers and acetonitrile may be used for mid-range polarity but are less common.
- Water should typically be avoided to prevent unpredictable results and strong adherence.
Mechanism of Separation
- Polar analytes interact strongly (localization) with the polar stationary phase, resulting in longer retention times.
- Nonpolar analytes elute faster as they interact less with the column.
- Intermolecular forces (IMF) drive the separation based on analyte and column polarity.
When to Use Normal Phase HPLC
- Suitable for separating large, primarily hydrocarbon compounds with poor solubility in polar solvents (e.g., fat-soluble vitamins, hormones).
- Used when reverse phase HPLC fails to provide effective separation.
- Effective for separating geometrical isomers due to selective localization.
Pros and Cons of Normal Phase HPLC
Advantages
- Solubilizes and separates a wide range of hydrocarbon-rich compounds.
- Nonpolar solvents have low viscosity, allowing faster flow rates and shorter run times.
- Wide variety of nonpolar solvents enables high selectivity and control over retention.
- Enables separation of geometrical isomers, which can be difficult by other methods.
- Solvents used often have low boiling points, making them easier to recover.
Disadvantages
- Not effective for highly polar or ionic compounds (poor solvent elution and excessive localization).
- Water must be strictly excluded, which can be difficult in practice.
- Results can become unpredictable if water contaminates the system.
- Solvents such as hexane are costly and have a higher environmental impact than water- or alcohol-based mobile phases.
Key Terms & Definitions
- Normal Phase HPLC — Chromatography using a polar stationary phase and nonpolar mobile phase.
- Stationary Phase — The column material (e.g., silica) that retains analytes based on polarity.
- Mobile Phase — The liquid solvent(s) used to move analytes through the column.
- Localization — The adherence of analytes to the stationary phase due to intermolecular forces.
- Retention Time — Time an analyte spends interacting with the stationary phase before eluting.
Action Items / Next Steps
- Review earlier HPLC lectures, especially on basic column concepts.
- Avoid water contamination when running normal phase HPLC.
- Practice identifying when to use normal vs. reverse phase techniques.
- Check for the instructor’s guide and playlist on ChemComplete.com for further study.