6.3 High-performance liquid chromatography (HPLC) and applications
4 min read•august 14, 2024
() is a powerful separation technique used in analytical chemistry. It uses high pressure to push a liquid through a column packed with a , 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 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 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
- 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 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, , 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 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
- 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
Key Terms to Review (21)
Adsorption: Adsorption is the process by which atoms, ions, or molecules from a gas, liquid, or dissolved solid adhere to a surface. This phenomenon is crucial in various analytical techniques as it influences separation mechanisms and the efficiency of different materials and processes used in chemical analysis.
C18 Column: A C18 column is a type of chromatographic column used in high-performance liquid chromatography (HPLC), packed with octadecylsilane (C18) bonded phases. This type of column is designed for the separation of non-polar to moderately polar compounds and is widely utilized for its efficiency in retaining analytes during the separation process. C18 columns are favored in various applications, including pharmaceutical analysis and environmental testing, due to their ability to provide high resolution and reproducibility.
Environmental Testing: Environmental testing refers to the analysis and assessment of environmental samples to evaluate the presence and concentration of pollutants, contaminants, and other chemical substances in air, water, soil, and biota. This process is critical for ensuring compliance with environmental regulations, protecting public health, and maintaining ecological integrity.
Filtration: Filtration is a physical separation process that involves the removal of solid particles from a fluid (liquid or gas) by passing it through a porous medium that retains the solids while allowing the fluid to flow through. This method is essential in various analytical techniques, as it helps isolate components for further analysis and improves the purity of samples.
Fluorescence Detector: A fluorescence detector is an analytical instrument used in high-performance liquid chromatography (HPLC) to measure the fluorescence emitted by a sample after it has been excited by a light source. This type of detector is highly sensitive and can detect low concentrations of fluorescent compounds, making it valuable for various applications in chemistry, biochemistry, and environmental analysis.
Food analysis: Food analysis is the scientific process of examining food products to determine their composition, quality, and safety. This process involves various techniques and methodologies, including chemical, microbiological, and physical assessments, to evaluate nutritional content, contaminants, and overall quality. Understanding food analysis is crucial for ensuring that food products meet regulatory standards and consumer expectations.
Gradient elution: Gradient elution is a technique used in chromatography, particularly in high-performance liquid chromatography (HPLC), where the composition of the mobile phase changes over time during the separation process. This approach allows for improved resolution and efficiency by enabling the separation of compounds with varying polarities or affinities for the stationary phase, leading to enhanced peak shapes and reduced analysis time.
High-performance liquid chromatography: High-performance liquid chromatography (HPLC) is an advanced technique used to separate, identify, and quantify components in a mixture by passing a liquid sample through a column packed with solid adsorbent material under high pressure. This method allows for rapid and efficient analysis of various substances, making it essential in fields like pharmaceuticals, environmental monitoring, and food safety.
HPLC: High-performance liquid chromatography (HPLC) is an advanced technique used for separating, identifying, and quantifying components in a mixture. This method uses high pressure to push a solvent through a column packed with stationary phase material, allowing for improved resolution and speed compared to traditional liquid chromatography. HPLC is widely utilized in various fields, including pharmaceuticals, environmental monitoring, and food analysis due to its precision and reliability.
Ion-exchange column: An ion-exchange column is a type of chromatography device used to separate ions and polar molecules based on their charge. It contains a stationary phase that is charged, allowing for the selective exchange of ions from a liquid sample as it passes through. This method is essential in high-performance liquid chromatography (HPLC) for separating and analyzing various ionic compounds in complex mixtures.
Method Validation: Method validation is the process of confirming that an analytical method is reliable, reproducible, and suitable for its intended purpose. This involves assessing various parameters such as accuracy, precision, specificity, and robustness, which ensures that the method produces results that are consistent and can be trusted in various applications.
Mobile phase: The mobile phase is a solvent or gas that carries the analytes through the stationary phase in chromatography. This phase plays a crucial role in separating components based on their interactions with both the mobile and stationary phases, influencing the resolution and efficiency of the separation process.
Normal-phase HPLC: Normal-phase high-performance liquid chromatography (HPLC) is a chromatographic technique where the stationary phase is polar and the mobile phase is non-polar. In this setup, polar analytes interact more strongly with the stationary phase, allowing for the separation of compounds based on their polarity. This method is particularly useful for separating non-polar to moderately polar compounds in various applications, including pharmaceuticals and environmental analysis.
Partitioning: Partitioning refers to the distribution of compounds between two immiscible phases, typically a stationary phase and a mobile phase, during separation processes. This principle is fundamental to various chromatographic techniques, where the different affinities of compounds for these phases lead to their separation. The efficiency of partitioning is critical in determining the resolution and effectiveness of the separation achieved.
Pharmaceutical Analysis: Pharmaceutical analysis is the science of identifying, quantifying, and characterizing the chemical components of pharmaceutical substances. This discipline plays a crucial role in ensuring the quality, safety, and efficacy of drugs, linking it directly to various analytical methods and their applications, which are essential for regulatory compliance and research.
Resolution: Resolution refers to the ability of a technique to distinguish between two closely spaced components in a mixture. In analytical chemistry, resolution is critical because it determines how well different substances can be separated and identified during analysis, impacting the quality and reliability of results.
Retention time: Retention time refers to the duration that a compound spends in the chromatographic system from the moment it enters the column until it is detected at the end of the column. This time is crucial for identifying substances because it is influenced by the interactions between the compounds and the stationary phase, allowing for separation based on their unique properties. Retention time can vary depending on the type of chromatography being used, including gas chromatography and high-performance liquid chromatography, as well as different methods of ion exchange and size exclusion.
Reversed-phase HPLC: Reversed-phase high-performance liquid chromatography (HPLC) is a technique used to separate compounds based on their hydrophobic interactions with a non-polar stationary phase while using a polar mobile phase. This method is highly effective for analyzing a wide range of organic compounds, including pharmaceuticals, environmental samples, and biomolecules. Reversed-phase HPLC is particularly valuable in analytical chemistry for its ability to provide high resolution and sensitivity in the separation of complex mixtures.
Solid-phase extraction: Solid-phase extraction (SPE) is a sample preparation technique used to isolate and concentrate analytes from a liquid sample by passing it through a solid adsorbent material. This method enhances the purity and concentration of the target compounds, making it especially useful in various analytical applications, including liquid chromatography and environmental analysis.
Stationary phase: The stationary phase is a key component in chromatography, where it refers to the phase that remains fixed in place within the column or separation medium. This phase interacts with the sample components as they move through the system, helping to separate them based on their different affinities for the stationary material. The effectiveness of the stationary phase is critical in determining the resolution and efficiency of the separation process in techniques such as gas chromatography and high-performance liquid chromatography.
Uv-vis detector: A uv-vis detector is an analytical instrument used in chromatography to measure the absorbance of ultraviolet and visible light by a sample. This type of detector is vital in high-performance liquid chromatography (HPLC) as it provides real-time analysis of the sample components based on their light absorption characteristics, allowing for precise quantification and identification of compounds.