PHD
  1. Inorganic chemistry  1.1. Coordination chemistry  1.1.1. Ligand field theory  1.1.2. Crystal field theory  1.1.3. Molecular Orbital Theory for Coordination Compounds  1.1.4. Stability of Coordination Compounds  1.1.5. Electronic spectra of coordination compounds  1.1.6. Isomerism in Coordination Compounds  1.1.7. Kinetics and mechanism of coordination reactions  1.2. Organometallic Chemistry  1.2.1. Metal Carbonyls  1.2.2. Metal alkyl and aryl  1.2.3. Fischer and Schrock carbonaceans  1.2.4. Oxidative Addition and Reductive Elimination  1.2.5. Metal Hydrides  1.2.6. Catalysis by organometallic compounds  1.2.7. Metallocenes and Sandwich Complexes  1.2.8. CH activation  1.3. Solid state chemistry  1.3.1. Crystal structures and lattices  1.3.2. Band theory and electrical properties  1.3.3. Defects in the crystal  1.3.4. Synthesis of solid state materials  1.3.5. Magnetic and optical properties of solids  1.3.6. Superconductivity  1.3.7. Solid state ionics  1.4. Bio-inorganic chemistry  1.4.1. Metalloproteins and enzymes  1.4.2. Metal ion transport and storage  1.4.3. The role of metals in medicine  1.4.4. Bioinspired catalysis  1.4.5. Metal–DNA interactions  1.4.6. Metal Complexes in Medical Science  1.5. Lanthanides and Actinides  1.5.1. Electronic configuration and oxidation states in lanthanides and actinides  1.5.2. Spectral and Magnetic Properties in Lanthanides and Actinides  1.5.3. Separation and Extraction of Lanthanides and Actinides  1.5.4. Coordination chemistry of f-block elements  1.6. Main Group Chemistry  1.6.1. Chemistry of boron compounds  1.6.2. Chemistry of Silicon and Germanium Compounds  1.6.3. Chemistry of Phosphorus and Sulfur Compounds  1.6.4. Organosilicon chemistry  1.6.5. Noble Gas Chemistry
  2. Organic chemistry  2.1. Reaction mechanism  2.1.1. Nucleophilic substitution reactions  2.1.2. Electrophilic addition and substitution reactions  2.1.3. Elimination reactions  2.1.4. Rearrangement reactions  2.1.5. Radical reactions  2.1.6. Pericyclic Reactions  2.1.7. Photochemical reactions  2.2. Stereoscopic  2.2.1. Chirality and optical activity  2.2.2. Structural analysis  2.2.3. Stereoelectronic effects  2.2.4. Asymmetric synthesis  2.2.5. Dynamic Stereochemistry  2.3. Organometallic Chemistry in Organic Synthesis  2.3.1. Organolithium and organomagnesium reagents  2.3.2. Transition metal catalyzed coupling reactions  2.3.3. Palladium-catalyzed reactions  2.3.4. Olefin Metathesis  2.3.5. Gold and silver catalysis  2.4. Natural products chemistry  2.4.1. Alkaloids and terpenoids  2.4.2. Steroids and Flavonoids  2.4.3. Total synthesis of natural products  2.4.4. Biosynthesis of natural products  2.5. Supramolecular Chemistry  2.5.1. Host-Guest Chemistry  2.5.2. Self-assembly and molecular recognition  2.5.3. Supramolecular Catalysis  2.5.4. Molecular Machines
  3. Physical Chemistry  3.1. Thermodynamics  3.1.1. Laws of Thermodynamics  3.1.2. Chemical Potential and Equilibrium  3.1.3. Statistical thermodynamics  3.1.4. Non-equilibrium thermodynamics  3.2. Quantum Chemistry  3.2.1. Schrödinger equation and its applications  3.2.2. Molecular orbital theory  3.2.3. Density functional theory  3.2.4. Post-Hartree–Fock methods  3.3. Chemical kinetics  3.3.1. Reaction rate theory  3.3.2. Mechanism and rate laws  3.3.3. Catalysis and enzyme kinetics  3.3.4. Reaction dynamics  3.4. Spectroscopy and molecular structure  3.4.1. Infrared Spectroscopy  3.4.2. UV-visible spectroscopy  3.4.3. Nuclear Magnetic Resonance Spectroscopy  3.4.4. Mass Spectrometry  3.4.5. Raman Spectroscopy  3.5. Surface and Colloid Chemistry  3.5.1. Absorption and Catalysis  3.5.2. Surfactants and micelles  3.5.3. Colloidal Stability  3.5.4. Nanomaterials and Interfaces
  4. Analytical chemistry  4.1. Chromatography  4.1.1. Gas Chromatography  4.1.2. High Performance Liquid Chromatography  4.1.3. Thin Layer Chromatography  4.1.4. Ion Chromatography  4.2. Electroanalytical techniques  4.2.1. Voltammetry and Polarography  4.2.2. Conductometry and potentiometry  4.2.3. Colometry  4.3. Spectroscopic Methods  4.3.1. Atomic absorption spectroscopy  4.3.2. Fluorescence Spectroscopy  4.3.3. X-ray diffraction  4.3.4. Electron paramagnetic resonance spectroscopy  4.4. Chemometrics  4.4.1. Data processing and statistical methods  4.4.2. Multidisciplinary analysis  4.4.3. Machine Learning in Analytical Chemistry
  5. Theoretical and Computational Chemistry  5.1. Molecular dynamics simulation  5.2. Quantum chemistry methods  5.3. Computational drug design  5.4. Machine Learning in Chemistry  5.5. Ab initio molecular dynamics
  6. Biophysics and Medicinal Chemistry  6.1. Drug discovery and design  6.2. Enzyme kinetics and inhibition  6.3. Chemical biology approach  6.4. Protein-ligand interactions  6.5. Bioorthogonal chemistry
  7. Materials chemistry  7.1. Polymer chemistry  7.1.1. Polymerization mechanism  7.1.2. Functional Polymers  7.1.3. Biodegradable Polymer  7.1.4. Conducting and semiconducting polymers  7.2. Nano Chemistry  7.2.1. Nanoparticles and nanostructures  7.2.2. Carbon-based nanomaterials  7.2.3. Quantum dots  7.3. Energy and Environmental Chemistry  7.3.1. Battery and Fuel Cell Chemistry  7.3.2. Green chemistry and sustainable materials  7.3.3. Photocatalysis

PHD → Analytical chemistry → Chromatography ↓


High Performance Liquid Chromatography


High-performance liquid chromatography, often abbreviated as HPLC, is a powerful and widely used technique in analytical chemistry. This method is used to separate, identify, and quantify each component in a mixture. This technique is important in a variety of fields, including biochemistry, pharmaceuticals, and environmental testing. HPLC offers high precision and is able to analyze complex mixtures with great efficiency.

Principles of HPLC

HPLC works on the same basic principles as conventional chromatography, but it is much more sophisticated. In short, chromatography is a method for separating dissolved substances based on their different interactions with two media: a mobile phase and a stationary phase.

Here is a simple description of the HPLC process:

  1. Sample injection: The sample is inserted into the system using an injector.
  2. Transport through the column: The sample is transported through the column by a liquid (the mobile phase). The column contains a stationary phase.
  3. Separation: Different components of the sample pass through the column at different rates due to interaction with the stationary phase.
  4. Detection: As components leave the column, they are detected and quantified by a detector.

The chemistry behind HPLC

The separation process in HPLC basically depends on the interaction between the sample components, the stationary phase, and the mobile phase. The stationary phase is often a column filled with tiny silica particles, and the mobile phase is a solvent that flows through the column. The components of the mixture move at different velocities, causing them to separate over time.

S + R ⇌ SR

In this equation:

  • S represents the sample component.
  • R represents the stationary phase.
  • SR indicates the relationship of the sample with the stationary phase.

The degree of interaction between the sample and the stationary phase affects the retention time, which is the time it takes for a component to reach the detector from the column inlet.

Components of HPLC

HPLC consists of several important components:

  1. Solvent reservoir: Stores the mobile phase. It is important that this solvent be degassed to prevent air bubbles in the system.
  2. Pump: Provides a specific flow rate of the mobile phase through the system.
  3. Injector: Introduces the sample into the chromatograph stream.
  4. Column: This contains the stationary phase. This is where the actual separation of the components takes place.
  5. Detector: Identifies the components separated from the column. Common detectors include UV-VIS, refractive index, and mass spectrometric detectors.
  6. Data system: Provides data processing and display. Often the computer runs software that controls the entire system, processes the data, and displays the results.

Types of HPLC

1. Normal phase HPLC

The stationary phase is polar, and the mobile phase is non-polar. Substances that are more polar have longer retention times. This type is used less often nowadays as reverse phase HPLC is gaining popularity.

Polar stationary phase: Silica (SiO2) Non-polar mobile phase: Hexane

2. Reverse phase HPLC

The most popular form of HPLC. The stationary phase is non-polar, and the mobile phase is more polar. Molecules that are less polar will have a shorter retention time.

Non-polar stationary phase: C18 Polar mobile phase: Water/acetonitrile mixture

3. Size-exclusion HPLC

Also known as gel filtration, this type separates components based on size. Larger molecules pass through quickly because they cannot penetrate the pores in the packing material. Smaller molecules enter the pores and take longer to pass through.

4. Ion-exchange HPLC

This type separates ions and polar molecules based on their affinity for the ion exchanger. The stationary phase contains charged groups, and the substances can be either cation or anion exchangers.

Cation Exchange: Stationary phase contains negatively charged groups. Anion Exchange: Stationary phase contains positively charged groups.

Applications of HPLC

HPLC is employed in a variety of fields due to its versatility.

1. Pharmaceutical industry

The role of HPLC in the pharmaceutical industry is undeniable. It is used for:

  • To check the purity of a drug compound.
  • Determining the quantity of active ingredients in pharmaceutical formulations.
  • To identify any impurities or decomposition products.

2. Biochemistry

In biochemistry, HPLC allows the separation of biomolecules such as proteins and nucleic acids. Common uses include:

  • Proteomic studies to separate and identify different proteins.
  • Determination of enzyme activities or hormone levels.

3. Environmental science

Scientists use HPLC to analyze water, soil, and air samples to detect contaminants. HPLC can quantify the following:

  • Pesticides in water bodies.
  • Pollutants in air samples.
  • Heavy metals in soil samples.

4. Food and beverage industry

Quality control is important in this industry. HPLC is used to:

  • To analyse the concentration of vitamins and organic acids in foods.
  • Detection of food additives and preservatives.
  • Checking for contaminants or toxins in beverages.

Experimental procedure of HPLC

The typical experimental procedure of HPLC involves several steps:

  1. Sample preparation: Ensuring the sample is well dissolved in the appropriate solvent for addition to the system.
  2. Selection of mobile and stationary phases: Selecting the ideal phases based on the properties of the sample. For example, using a water/methanol mixture in reverse phase HPLC.
  3. Setting operational parameters: defining column temperature, mobile phase flow rate and other relevant parameters.
  4. Sample injection: Carefully pouring the sample to avoid sample loss or errors.
  5. Leaching operation: separation of components within the column, monitored via a detector.
  6. Data collection and analysis: collecting data from the detector, analyzing the retention times, and comparing them with known standards.

Advantages and limitations of HPLC

Benefits

  • Sensitivity: Highly sensitive and capable of detecting small quantities.
  • Versatility: Suitable for a wide range of samples, including large biomolecules and small organic compounds.
  • High resolution: can efficiently separate closely related compounds.
  • Automation: Many HPLC systems are automated, increasing repeatability and precision.

Boundaries

  • Cost: Initial installation and operating costs can be high.
  • Complexity: expertise is required to manage the systems and interpret the results.
  • Limited samples: Some samples may require derivatization to make them detectable.

Conclusion

High-performance liquid chromatography is an indispensable tool in modern analytical chemistry. Its ability to separate, identify, and quantify components efficiently and effectively makes it invaluable in many scientific fields. Although HPLC systems can come with their own challenges, their role is vital in ensuring product quality, environmental safety, and scientific research.

As technologies advance, HPLC systems continue to be developed and refined, potentially providing even greater speed, sensitivity, and applications in the future.


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High Performance Liquid Chromatography

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