6.3 High-performance liquid chromatography (HPLC) and applications

High-performance liquid chromatography (HPLC) is a powerful separation technique used in analytical chemistry. It uses high pressure to push a liquid mobile phase through a column packed with a stationary phase, separating mixture components based on their interactions.

HPLC has two main types: normal-phase and reversed-phase. The choice depends on the compounds being separated. HPLC is widely used in pharmaceuticals, environmental science, and food analysis for both qualitative and quantitative analysis of complex mixtures.

Principles of HPLC

HPLC Fundamentals

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• HPLC is a form of column chromatography that utilizes a liquid mobile phase to separate components of a mixture based on their interactions with a stationary phase
• As the sample components interact with the stationary phase, they are separated based on their relative affinities for the mobile and stationary phases
• The detector generates a signal proportional to the concentration of each analyte as it elutes from the column, allowing for quantitative analysis

HPLC Instrumentation

• The main components of an HPLC system include a high-pressure pump, an injector, a column packed with a stationary phase, and a detector (UV-Vis, fluorescence, or mass spectrometer)
• The high-pressure pump delivers the mobile phase through the system at a constant flow rate, typically ranging from 0.1 to 10 mL/min
• The injector introduces the sample into the mobile phase stream, which then carries the sample through the column
• The column is typically made of stainless steel and packed with a stationary phase, such as silica or polymer beads with specific surface modifications

Normal-Phase vs Reversed-Phase HPLC

Normal-Phase HPLC (NP-HPLC)

• NP-HPLC employs a polar stationary phase (silica or alumina) and a non-polar mobile phase (hexane or chloroform)
• In NP-HPLC, polar analytes are more strongly retained on the stationary phase and elute later than non-polar analytes
• NP-HPLC is suitable for separating polar compounds, such as amino acids, carbohydrates, and certain drug molecules
• Example: Separation of lipids using a silica column with a hexane/isopropanol mobile phase

Reversed-Phase HPLC (RP-HPLC)

• RP-HPLC uses a non-polar stationary phase (C18 or C8-modified silica) and a polar mobile phase (water, acetonitrile, or methanol)
• In RP-HPLC, non-polar analytes are more strongly retained on the stationary phase and elute later than polar analytes
• RP-HPLC is the most widely used mode of HPLC and is suitable for separating a broad range of compounds, including proteins, peptides, and small organic molecules
• Example: Separation of peptides using a C18 column with a water/acetonitrile gradient containing 0.1% trifluoroacetic acid

Mobile Phase in HPLC Separations

Mobile Phase Composition

• The mobile phase composition plays a crucial role in HPLC separations by influencing the interactions between the analytes and the stationary phase
• In isocratic elution, the mobile phase composition remains constant throughout the separation, which is suitable for simple mixtures or when analytes have similar properties
• The choice of mobile phase components depends on the nature of the analytes and the stationary phase, with factors such as solvent polarity, pH, and ionic strength influencing the separation
• Example: Separation of organic acids using an isocratic mobile phase of phosphate buffer and methanol

Gradient Elution

• Gradient elution involves changing the mobile phase composition during the separation, typically by increasing the proportion of the organic solvent (acetonitrile or methanol) relative to the aqueous component
• Gradient elution is useful for separating complex mixtures or analytes with a wide range of polarities
• By gradually increasing the elution strength of the mobile phase, gradient elution can improve peak resolution and reduce analysis time compared to isocratic elution
• Example: Separation of a protein mixture using a gradient from 5% to 95% acetonitrile in water over 30 minutes

HPLC Applications for Analysis

Sample Preparation and Calibration

• HPLC is widely used for the analysis and quantification of compounds in various fields, such as pharmaceutical, environmental, and food analysis
• Sample preparation is a critical step in HPLC analysis, which may involve extraction, filtration, or derivatization to ensure compatibility with the HPLC system and improve detection
• Calibration curves are constructed using standards of known concentrations to relate the detector response to the analyte concentration
  • External standard calibration involves preparing separate solutions of the analyte at different concentrations and constructing a calibration curve based on the detector response
  • Internal standard calibration involves adding a known amount of a reference compound to both the sample and the calibration standards to account for variations in sample preparation and injection

Quantification and Method Validation

• Peak identification is based on the retention time of the analyte, which can be compared to that of a reference standard or confirmed using complementary techniques such as mass spectrometry
• Quantification is performed by integrating the peak area or height and comparing it to the calibration curve to determine the concentration of the analyte in the sample
• Method validation is essential to ensure the accuracy, precision, and robustness of the HPLC method, which includes assessing parameters such as linearity, limit of detection, limit of quantification, and recovery
• Example: Quantification of caffeine in energy drinks using RP-HPLC with UV detection and external standard calibration