Graduate
  1. Physical Chemistry  1.1. Thermodynamics  1.1.1. Laws of Thermodynamics  1.1.2. Enthalpy and heat capacity  1.1.3. Entropy and free energy  1.1.4. Phase Equilibrium  1.1.5. Statistical thermodynamics  1.1.6. Thermodynamic cycle  1.1.7. Fugacity and activity  1.2. Quantum Chemistry  1.2.1. Principles of quantum mechanics  1.2.2. Schrödinger Equation  1.2.3. Particle in a box  1.2.4. Operators and eigenvalues  1.2.5. Atomic orbitals  1.2.6. Molecular orbital theory  1.2.7. Perturbation theory  1.2.8. Valence bond theory  1.2.9. Hybridization and chemical bonding  1.3. Chemical kinetics  1.3.1. Rate laws and reaction mechanisms  1.3.2. Collision theory  1.3.3. Transition state theory  1.3.4. Enzyme Kinetics  1.3.5. Chain Reactions and Polymerization  1.3.6. Photochemical reactions  1.4. Statistical mechanics  1.4.1. Partitioning functions  1.4.2. Molecular distribution functions  1.4.3. Boltzmann Distribution  1.4.4. Bose–Einstein and Fermi–Dirac statistics  1.5. Spectroscopy  1.5.1. Rotational Spectroscopy  1.5.2. Vibrational Spectroscopy  1.5.3. Electronic Spectroscopy  1.5.4. Nuclear magnetic resonance spectroscopy  1.5.5. Mass Spectrometry  1.5.6. Raman Spectroscopy  1.5.7. Electron paramagnetic resonance spectroscopy  1.6. Surface and colloidal chemistry  1.6.1. Absorption isotherm  1.6.2. Surface tension and wetting  1.6.3. Colloidal Stability  1.6.4. Catalysis  1.6.5. Emulsions and micelles  1.7. Electrochemistry  1.7.1. Nernst equation  1.7.2. Electrochemical cells  1.7.3. Conductivity and mobility  1.7.4. War  1.7.5. Fuel cells  1.7.6. Electrolysis
  2. Organic chemistry  2.1. Reaction mechanism  2.1.1. Nucleophilic substitution reactions  2.1.2. Electrophilic addition reactions  2.1.3. Elimination reactions  2.1.4. Rearrangement reactions  2.1.5. Radical reactions  2.1.6. Pericyclic reactions  2.2. Spectroscopy and structural determination  2.2.1. UV-Vis Spectroscopy  2.2.2. IR Spectroscopy  2.2.3. NMR Spectroscopy  2.2.4. Mass Spectrometry  2.2.5. X-ray crystallography  2.3. Stereoscopic  2.3.1. Chirality and optical activity  2.3.2. Structural analysis  2.3.3. Geometrical isomerism  2.3.4. Dynamic Stereochemistry  2.4. Organometallic Chemistry  2.4.1. Organolithium and organomagnesium reagents  2.4.2. Palladium-catalyzed cross-coupling reactions  2.4.3. Transition Metal Complexes  2.4.4. Metal–carbon bond  2.5. Polymer chemistry  2.5.1. Polymerization mechanism  2.5.2. Polymer Characteristics  2.5.3. Biodegradable Polymer  2.5.4. Conducting polymer  2.5.5. Supramolecular polymers  2.6. Medicinal chemistry  2.6.1. Drug design and development  2.6.2. Pharmacokinetics and pharmacodynamics  2.6.3. Structure-activity relationships  2.6.4. Molecular docking and drug screening
  3. Inorganic chemistry  3.1. Coordination chemistry  3.1.1. Crystal field theory  3.1.2. Ligand field theory  3.1.3. Spectrochemical Series  3.1.4. Chelation and stability  3.2. Organometallic Chemistry  3.2.1. Metal Carbonyls  3.2.2. Catalysis by organometallic complexes  3.2.3. Metallocenes  3.3. Bio-inorganic chemistry  3.3.1. Metalloproteins and enzymes  3.3.2. The role of metals in biological systems  3.3.3. Metal ion transport and storage  3.4. Solid state chemistry  3.4.1. Crystal Structures  3.4.2. Band theory of solids  3.4.3. Superconductors  3.4.4. Defects in the crystal  3.5. Lanthanides and Actinides  3.5.1. Electronic configuration in lanthanides and actinides  3.5.2. Coordination chemistry of the lanthanides  3.5.3. Magnetic Properties of Lanthanides
  4. Analytical chemistry  4.1. Chromatography  4.1.1. Gas Chromatography  4.1.2. High Performance Liquid Chromatography  4.1.3. Thin Layer Chromatography  4.2. Spectroscopic Techniques  4.2.1. Atomic absorption spectroscopy  4.2.2. X-ray diffraction  4.2.3. Inductively coupled plasma spectroscopy  4.3. Electroanalytical methods  4.3.1. Potentiometry  4.3.2. Voltammetry  4.3.3. Colometry  4.4. Mass Spectrometry  4.4.1. Ionization Technique  4.4.2. Fragmentation Pattern  4.5. Chemometrics  4.5.1. Multidisciplinary analysis  4.5.2. Machine Learning in Chemistry
  5. Theoretical and Computational Chemistry  5.1. Molecular dynamics simulation  5.1.1. Force fields and energy minimization  5.1.2. Monte Carlo simulation in molecular dynamics simulations  5.2. Quantum chemical methods  5.2.1. Hartree–Fock theory  5.2.2. Density functional theory  5.2.3. Semi-empirical methods  5.3. Computational drug design  5.3.1. Molecular Docking  5.3.2. QSAR Modeling  5.3.3. Virtual Screening
  6. Biochemistry  6.1. Enzyme Kinetics  6.1.1. Michaelis-Menten kinetics  6.1.2. Inhibition mechanism  6.2. Metabolism and Bioenergetics  6.2.1. Glycolysis  6.2.2. Citric acid cycle  6.2.3. Electron transport chain  6.3. molecular Biology  6.3.1. DNA replication and repair  6.3.2. Protein synthesis  6.3.3. Gene Regulation  6.4. Structural Biochemistry  6.4.1. Protein Folding  6.4.2. Membrane Biophysics
  7. Environmental Chemistry  7.1. Atmospheric Chemistry  7.1.1. Greenhouse gases  7.1.2. Ozone depletion  7.1.3. Air pollutants and smoke  7.2. Water Chemistry  7.2.1. pH and water hardness  7.2.2. Wastewater treatment  7.2.3. Aquatic Chemistry  7.3. Soil Chemistry  7.3.1. Heavy metal contamination  7.3.2. Soil pH and Buffering  7.3.3. Nutrient cycling  7.4. Toxicology and Chemical Safety  7.4.1. Toxicokinetics  7.4.2. Risk Assessment in Toxicology and Chemical Safety in Environmental Chemistry  7.4.3. Effects of pollutants on the environment

Graduate → Analytical chemistry → Chromatography ↓


High Performance Liquid Chromatography


High-performance liquid chromatography (HPLC) is an advanced technique in analytical chemistry used to identify, quantify, and purify individual components of a mixture. Since its development in the mid-20th century, it has been an indispensable tool for separating complex mixtures in the laboratory. Understanding HPLC involves exploring its components, operational principles, and applications in various scientific fields.

Fundamentals of HPLC

HPLC is a type of column chromatography. In this process, a liquid sample dissolved in a solvent is passed through a column filled with a solid adsorbent. Different components of the sample pass through the column at different rates due to differences in their interactions with the adsorbent and the solvent, leading to their separation.

Basic components of HPLC:
1. Solvent reservoir
2. Pump
3. Injector
4. Column
5. Detector
6. Data system

Components of HPLC

1. Solvent reservoir: This is where the mobile phase is stored. The mobile phase is a liquid that is used to carry the sample through the column. It can be a single solvent or a mixture of solvents. The choice of mobile phase affects the separation process.

Solvent Reservoir

2. Pump: The pump moves the solvent through the system. It delivers the solvent at high pressure, and this pressure is what allows for rapid movement of the sample through the column.

3. Injector: The sample is introduced into the HPLC system using the injector. This allows a precise volume of sample to be introduced into the mobile phase for analysis. Automatic injectors are often used for better accuracy and reproducibility.

4. Column: The main part of the HPLC system is the column. It is filled with small particles that provide a large surface area for interaction with the sample components. The column material (stationary phase) is selected based on the type of analytes being separated.

5. Detector: The detector identifies and quantifies the components separated from the column. Many types of detectors exist, each suited to different applications. Common detectors include ultraviolet-visible (UV-Vis) detectors, fluorescence detectors, and mass spectrometers.

Column

6. Data system: This component records the signals from the detector and processes it into usable data. Advanced software allows the generation of chromatograms and peak analysis, making quantitative and qualitative analysis of sample components possible.

Operating system

The operation of HPLC involves the following steps:

  • Prepare the mobile phase and fill the solvent reservoir.
  • Turn on the pump to establish the desired flow rate and pressure.
  • Inject the sample into the injector, which adds it to the mobile phase.
  • The sample passes through the column where the separation takes place.
  • The separated components move toward the detector, which sends a continuous signal to the data system.
  • The data system generates a chromatogram and converts the signals into quantitative data.

The key to effective HPLC is to optimize the column, mobile phase, and detection method according to the specific needs of the analysis, whether this involves maximizing separation effectiveness or improving detection sensitivity.

Visual example of component interaction

Solvent Reservoir Pump Injector Column Detectors

Applications of HPLC

HPLC is used for various applications in different fields:

1. Pharmaceuticals

In the pharmaceutical industry, HPLC is important for the following:

  • Analyzing pharmaceutical products for purity and potency.
  • To determine the stability and degradation of medicinal compounds.
  • Conducting pharmacokinetic studies to understand the absorption, distribution, metabolism, and excretion of drugs.

For example, HPLC is used to measure the concentration of C18H21NO3 in blood samples, helping to determine the proper dosage.

2. Environmental monitoring

Environmental scientists use HPLC to:

  • Detection and quantification of pollutants in water, soil and air samples.
  • Analyze pesticide residues in agricultural products.

An example of this is measuring the levels of atrazine (a common herbicide) in water bodies to assess environmental impact.

3. Food and drinks

In food science, HPLC helps in:

  • Ensuring quality control by detecting food additives and contaminants.
  • Analyzing the nutritional content of food products.

An example of this is using HPLC to determine the concentration of caffeine in coffee to ensure it meets labeling requirements.

4. Clinical and forensic analysis

In clinical laboratories and forensics, HPLC is used for the following:

  • Drug testing in biological samples such as blood or urine.
  • Toxicology analysis to identify exposure to chemicals or toxins.

For example, HPLC can separate and identify different metabolites of a drug present in a urine sample.

Advantages of HPLC

HPLC has several advantages, which explain its widespread use:

  • The high resolution and sensitivity allow accurate separation and detection of components.
  • Versatility to analyze non-volatile, thermally unstable and complex mixtures.
  • Speed of analysis, especially with modern rapid column technologies.
  • Quantitative and qualitative data provide comprehensive information about samples.

Challenges and limitations of HPLC

Despite its strengths, HPLC also faces challenges:

  • This equipment is expensive and requires significant investment for its installation and maintenance.
  • Complex samples can pose difficulties in achieving maximum resolution and quantification.
  • Some types of samples may require extensive preparation before analysis.

In addition, incorrect parameter settings may lead to poor separation and erroneous results.

Conclusion

High-performance liquid chromatography remains an important tool in the field of analytical chemistry. The ability to separate, identify, and quantify compounds with precision has established it as an essential technique in research, industry, and environmental monitoring. As the technology continues to advance, HPLC is likely to evolve, providing even more sophisticated capabilities and applications.

Understanding the fundamental components and principles of HPLC, as well as adapting methods to specific analytical challenges, helps scientists extract meaningful data efficiently and effectively, leading to further advancements in their field of study.


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High Performance Liquid Chromatography

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