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General Chemistry Principles
0/4
Atomic Structure and Bonding
11:44
Acid-Base Chemistry
09:00
Organic Chemistry Fundamentals
17:10
End of Topic Quiz
00:20:00
Drug Chemistry
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Functional Groups
09:17
Stereochemistry
14:35
Drug Design
07:30
Drug Activity
06:20
Drug Stability
18:43
Drug Degradation
End of Topic Quiz
00:24:00
Analytical Techniques
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Chromatography (HPLC, TLC)
09:16
Spectroscopy (UV, IR)
06:38
Mass Spectrometry
10:02
End of Topic Quiz
00:20:00
Principles of Drug Chemistry and Analysis

Types of Chromatography

1. High-Performance Liquid Chromatography (HPLC)

  • Definition: Utilises high pressure to force a liquid mobile phase through a column containing a packed stationary phase.

2. Thin Layer Chromatography (TLC)

  • Definition: A simpler technique where the stationary phase is a thin layer of adsorbent material coated on a plate. The mobile phase moves up the plate via capillary action.

Basic Principles

Both HPLC and TLC rely on the principle that compounds in a mixture will interact differently with the stationary and mobile phases, resulting in different rates of migration and separation.

Key Terms

  • Analyte: Target compound(s) of interest for detection.
  • Mobile phase: The moving phase, typically a solvent or mixture of solvents, that carries the sample through the system.
  • Stationary phase: The immobile phase where the actual separation of analytes occurs.
  • Retention time (tR): Time taken for an analyte to travel from the point of injection to the detector.
  • Void time (tM): Time taken for an unretained compound (no interaction with the stationary phase) to travel through the system.
  • Retention Factor (Rf): A ratio calculated in TLC representing the distance travelled by a component divided by the total distance travelled by the solvent.
    • Rf=distance travelled by component/distance travelled by solvent/text{Rf} = frac{text{distance travelled by component}}{text{distance travelled by solvent}}
  • Distribution Constant (Kc): Describes the equilibrium between the mobile and stationary phases for a given analyte.
  • Retention Factor (k): A quantitative measure of an analyte’s affinity for the stationary phase.
  • Selectivity (α): Ability of the chromatographic system to distinguish between two analytes.
  • Resolution (Rs): A measure of how well two peaks are separated in a chromatogram.

High-Performance Liquid Chromatography (HPLC)

Instrumentation:

  1. Solvent Reservoir: Contains the mobile phase, typically a mixture of solvents.
  2. Pump: Delivers the mobile phase through the system at a constant flow rate.
  3. Injector: Introduces the sample into the flowing mobile phase stream.
  4. Column: Contains the stationary phase where separation occurs.
  5. Detector: Detects the separated components as they elute from the column.
  6. Data System: Records and processes the signal from the detector to produce a chromatogram.

Types of HPLC:

  • Normal Phase HPLC: Stationary phase is polar, mobile phase is non-polar.
  • Reverse Phase HPLC (RPLC): Stationary phase is non-polar, mobile phase is polar. RPLC is the most common type of HPLC.
  • Isocratic Elution: Mobile phase composition remains constant throughout the separation.
  • Gradient Elution: Mobile phase composition changes over time, offering better separation for complex samples.

Applications of HPLC:

  • Pharmaceutical Industry: Quality control of drugs, identification of impurities.
  • Biopharmaceutical Analysis: Characterisation of proteins, peptides, and antibodies.
  • Food and Beverage Industry: Analysis of food additives, contaminants, and quality control.
  • Environmental Monitoring: Detection and quantification of pollutants in water and soil samples.

Thin Layer Chromatography (TLC)

Procedure:

  1. Plate Preparation: A thin layer of stationary phase is coated onto a plate, typically glass, aluminium, or plastic.
  2. Sample Application: Small spots of sample are applied to the plate using a capillary tube.
  3. Development: The plate is placed in a developing chamber containing a shallow layer of mobile phase. The mobile phase travels up the plate by capillary action, carrying the sample components with it.
  4. Visualisation: Separated components are visualised using UV light or chemical stains.

Advantages of TLC:

  • Simplicity: Easy to perform and requires minimal equipment.
  • Cost-Effective: Relatively inexpensive compared to other chromatographic techniques.
  • Versatility: Can be used to analyse a wide range of substances.
  • High Throughput: Multiple samples can be run simultaneously on the same plate.

Disadvantages of TLC:

  • Lower Resolution: Limited separation power compared to HPLC.
  • Qualitative Analysis: Primarily used for qualitative analysis, although semi-quantitative analysis is possible.
  • Open System: Susceptible to environmental variations like humidity and temperature.

Applications of TLC:

  • Qualitative Analysis: Identification of unknown compounds by comparing Rf values to standards.
  • Monitoring Reactions: Following the progress of chemical reactions and identifying intermediates.
  • Purity Checks: Assessing the purity of compounds.
  • Sample Preparation: A preliminary step for other analytical techniques like HPLC.

References

  1. Thermo Fisher Scientific. (n.d.). HPLC Basics. Retrieved from [Thermo Fisher](https://www.thermofisher.com/uk/en/home/industrial/chromatography/chromatography-learning-center/liquid-chromatography-information/hplc-basics.html#:~:text=High%2Dperformance%20liquid%20chromatography%20(HPLC,packed%20with%20a%20stationary%20phase)
  2. LibreTexts. (n.d.). High Performance Liquid Chromatography. In Instrumentation and Analysis. Retrieved from LibreTexts
  3. Sigma-Aldrich. (n.d.). Thin Layer Chromatography. Retrieved from Sigma-Aldrich
  4. LibreTexts. (n.d.). Thin Layer Chromatography. In General Lab Techniques. Retrieved from LibreTexts
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