Chromatographic techniques have become indispensable tools in pharmaceutical analysis, enabling precise separation, identification, and quantification of complex mixtures. Among these, Gas Chromatography (GC) and High-Performance Liquid Chromatography (HPLC) stand out as the most widely used instrumental methods in research laboratories, quality control units, and regulatory testing facilities. Unit 4 focuses on the theory, instrumentation, advantages, limitations, and applications of these powerful analytical techniques that form the backbone of modern drug analysis.
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Gas Chromatography (GC): Analysis of Volatile and Thermally Stable Compounds
Introduction to Gas Chromatography
Gas Chromatography is a separation technique in which the mobile phase is an inert gas and the stationary phase is either a liquid coated on a solid support or a solid adsorbent. GC is primarily used for the analysis of volatile, thermally stable, and low-molecular-weight compounds. Its high resolution and sensitivity make it a preferred method for analyzing solvents, impurities, essential oils, and drug degradation products.
Theory of Gas Chromatography
The separation in GC is based on differential partitioning of analytes between the stationary phase and the gaseous mobile phase. As the carrier gas moves through the column, components of the sample repeatedly partition between the gas phase and stationary phase. Compounds with lower boiling points or weaker interaction with the stationary phase elute faster, while strongly retained compounds elute later. Retention time serves as a key qualitative parameter for compound identification.
Instrumentation of Gas Chromatography
Carrier Gas and Flow System
Common carrier gases include helium, nitrogen, and hydrogen. The gas must be inert and of high purity to avoid interference and ensure reproducible results.
Sample Injection System
The injection port rapidly vaporizes the sample and introduces it into the column. Split and splitless injection modes are used depending on sample concentration.
Chromatographic Columns
GC columns may be packed columns or capillary (open tubular) columns. Capillary columns provide superior resolution and sensitivity and are widely used in pharmaceutical analysis.
Detectors
Common detectors include Flame Ionization Detector (FID), Thermal Conductivity Detector (TCD), and Electron Capture Detector (ECD). FID is particularly popular due to its sensitivity to organic compounds.
Derivatization in Gas Chromatography
Some compounds are non-volatile or thermally unstable. Derivatization chemically modifies such analytes to increase volatility, thermal stability, or detector response. Silylation, acylation, and alkylation are commonly used derivatization techniques in pharmaceutical GC analysis.
Temperature Programming
Temperature programming involves gradually increasing column temperature during analysis. This improves separation of compounds with a wide boiling point range, reduces analysis time, and enhances peak resolution.
Advantages, Limitations, and Applications of GC
GC offers excellent resolution, high sensitivity, and rapid analysis. However, it is limited to volatile and heat-stable compounds.
Applications include residual solvent analysis, impurity profiling, essential oil analysis, forensic testing, and environmental monitoring.
High-Performance Liquid Chromatography (HPLC): The Workhorse of Pharmaceutical Analysis
Introduction to HPLC
High-Performance Liquid Chromatography is a versatile separation technique where a liquid mobile phase is forced through a packed column under high pressure. Unlike GC, HPLC can analyze non-volatile, thermolabile, and high-molecular-weight compounds, making it indispensable in pharmaceutical sciences.
Theory of HPLC
Separation in HPLC occurs due to differences in polarity, adsorption, partition, ion exchange, or molecular size, depending on the chromatographic mode. The most widely used mode is reverse-phase HPLC, where a non-polar stationary phase and polar mobile phase are employed. Retention time and peak area are used for qualitative and quantitative analysis, respectively.
Instrumentation of HPLC
Solvent Delivery System
High-pressure pumps deliver the mobile phase at a constant and reproducible flow rate. Isocratic and gradient elution modes allow flexibility in separation.
Sample Injector
Manual or autosamplers introduce precise volumes of sample into the mobile phase without interrupting flow.
HPLC Columns
Columns are packed with small particle stationary phases such as C18 or C8 silica. Smaller particle size increases surface area and enhances separation efficiency.
Detectors
Common detectors include UV-Visible detectors, Photodiode Array (PDA) detectors, Fluorescence detectors, and Refractive Index detectors. UV detection is most widely used due to simplicity and reliability.
Advantages of HPLC
HPLC offers high accuracy, reproducibility, and sensitivity. It requires minimal sample preparation and can analyze a wide range of pharmaceutical compounds. Automation and compatibility with hyphenated techniques such as LC-MS further enhance its analytical power.
Applications of HPLC in Pharmaceutical Sciences
HPLC is extensively used for:
Assay of bulk drugs and formulations
Stability and degradation studies
Impurity profiling
Bioanalytical studies and pharmacokinetics
Quality control and regulatory compliance
Its versatility makes HPLC a regulatory-approved method across global pharmacopeias.