HPLC Instrumentation Lecture Notes

HPLC Instrumentation Lecture Notes

• Lecture: 7, WS24
• Instructor: Prof. Dr. Rasha Hanafi
• Topic: HPLC Instrumentation

Competencies

• Students should be able to compare polarities of different mobile phases.
• Students should be able to identify the cut-off of a mobile phase.
• Students should be able to describe sample preparation prior to HPLC.
• Students should be able to use a nomograph to find alternative mobile phases.
• Students should be able to describe the function of pump, detector, and injector in HPLC.
• Students should be able to compare different detectors in HPLC.
• Students should be able to describe the setup of an HPLC system in detail.

HPLC Setup

• Components: HPLC instrument includes a sample syringe, pump, HPLC column, detector, and data acquisition system.
• Solvent reservoir: Contains the solvent mixture used as the mobile phase.
• Pump: Used to deliver solvent at a precise flow rate to the HPLC column.
• HPLC column: Contains the stationary phase, where separation of components takes place.
• Detector: Measures properties of each component and generates a signal.
• Data acquisition system: Records and analyzes the signal from the detector.
• Waste: Used to collect and discard waste.
• Components separated: Separation of components happens on the HPLC column.

When to Use HPLC

• Drug discovery: Locating new chemical entities for new drug development.
• Chemical development: Developing effective and efficient methods of synthesizing active pharmaceutical ingredients (APIs).
• Pharmaceutical development: Optimizing dosage forms for stability and delivery profiles.
• Drug metabolism/pharmacokinetics (DMPK): Evaluation of drug candidates in animal models and human clinical studies.
• Quality control (QC): Evaluating the final product quality against standards for release.

Filtering and Degassing

• Filtering and degassing solvents is essential for HPLC operation.
• Filters: Prevent particles from damaging pumping or injection systems or clogging the column.
• Degassers: Remove air bubbles that can cause noise in the baseline of the chromatogram, which leads to errors in peak area calculations.

HPLC vs. Classical LC

• High resolution: HPLC provides better separation of components compared to conventional liquid chromatography.
• Small diameter columns: Utilizing smaller diameter columns increases the separation efficiency.
• Small stationary phase particles: Using tiny stationary phase particles improves the separation quality of HPLC.
• High inlet pressures: HPLC uses higher inlet pressures for controlling the mobile phase flow.
• Continuous flow detectors: Detecting small flow rates and amounts is possible with continuous flow detectors in HPLC.
• Rapid analysis: HPLC processes analysis faster than conventional liquid chromatography.

Components of an HPLC

• Mobile phase reservoirs: Contain solvents used in the mobile phase.
• Pumps: Deliver solvents at a precise flow rate.
• Injection port: Used for introducing a sample into the HPLC system.
• Column: Separates the sample components.
• Detector: Measures the properties of each component, thereby producing corresponding signals.

Solvents for HPLC

• Solvents are classified based on their eluent strength and UV cut-off wavelength.
• Solvent type affects their separation and detection capabilities.
• Many solvents are used during HPLC methods, each with specific characteristics. The most common solvents include pentane, hexane, heptane, chloroform, dichloromethane, diethyl ether, etc.

Mobile Phases in HPLC

• Single solvents: Selected based on the desired elution strength, according to the eluotropic series. High purity is a crucial factor. Degassing solvents is mandatory before use. Water requires purification prior to use.
• Mixtures: HPLC frequently utilizes mixtures of solvents, including binary to higher mixtures, organic solvents, water, and buffers. A note in the slides indicates the volume percentage of a solvent mixture should be considered before the solvents are combined.

Pumps

• HPLC pumps produce stable and reproducible flow.
• Fluctuations in the flow will generate detector noise.
• Pulse-free output, flow rates between 0.1-10 ml/min, and consistent flow reproducibility (error ≤ 0.5%) are necessary in HPLC systems.
• Materials like stainless steel and Teflon are ideal for construction.
• Pumping systems are capable of pressures up to 6000 psi.

Reciprocating Pumps

• Reciprocating pumps are a type of pump used in HPLC, involving a reciprocating (repetitive back and forth) movement for liquid transfer.
• Diagram depicts the interior workings of a reciprocating pump. The system uses valves and seals.

The Column

• Analytical columns: Have lengths of 5-30 cm, internal diameters (ID) of 1-5 mm, constructed from steel or plastic.
• Porous titanium frits are frequently used in these columns. Columns can be damaged by dust or solvent particles.
• Preparative columns: Are used for larger-scale separations.
• Guard columns are strategically positioned to catch impurities and keep the main column from contamination.
• A guard column and the main column use the same type of stationary phase material.

Injection Port

• Injection valves: Most commonly used in HPLC systems are six-port or Rheodyne type valves.
• Syringes are used for loading fresh samples into sample loops under atmospheric pressure.
• Valves rotate a specific angle for introducing sample into the column.

Sample Preparation

• Generally, "dilute and shoot" approach is used for most drug substances.
• A common process is "grind → extract → dilute → filter."
• More complex dosage forms, like suppositories, might require extra sample clean-up and extraction procedures such as liquid-liquid extraction or solid-phase extraction.
• A vial containing the extracted sample is used for transferring into the HPLC system.

Detection

• Sensitivity: Detectors are required for detecting various analytes at low concentrations.
• Small volume: Avoiding peak broadening ensures accurate detection.
• Linearity: Linear response ensures a consistent signal across a broad range.
• Temperature/solvent insensitivity: Detectors should not be significantly affected by changes in temperature or solvent composition.

UV and Fluorescence Detectors

• UV detector: A photodiode array (PDA) records the absorbance spectrum. Useful for a wide variety of substances.
• Fluorescence detector: The eluate is excited by a laser for fluorometric detection. More specific to only certain substances.
• Flow cell: Typically z-shaped design, to minimize extra-column broadening and to prevent excessively small possible volumes.

Refractive Index Detector

• Responds to all analytes: Measures the change in refractive index for detecting a broad variety of analytes.
• Useless for gradient elution owing to sensitivity to temperature and pressure changes.
• Poorer detection limit compared to UV detectors.

References

• Provided a list of references, including specific chapters and web links.

This quiz covers the essential aspects of HPLC instrumentation, including the setup, components, and functions of each part within the system. Students will learn to compare mobile phases, understand sample preparation, and identify the roles of key components such as pumps and detectors. Enhance your knowledge of High-Performance Liquid Chromatography with this focused assessment.