Department of Chemistry, Anurag University, Hyderabad, India.
Gas chromatography (GC) is a powerful analytical technique used for the separation, identification, and quantification of volatile and semi-volatile compounds. This review explores the fundamental principles of GC, focusing on its working mechanism, stationary phases, column selection, operational conditions, sample introduction techniques, detection devices, and detection processes. Additionally, it covers qualitative and quantitative methods, applications in drug substance development, sample preparation techniques, derivatization methods, and chiral gas chromatographic applications. The article concludes with a discussion on recent advancements and future prospects of GC in pharmaceutical analysis.
Gas chromatography (GC) is a vital analytical tool, particularly in the pharmaceutical industry, due to its ability to detect and quantify volatile and semi-volatile compounds with high sensitivity. It plays a crucial role in impurity profiling, quality control, and regulatory compliance by identifying trace levels of genotoxic and nitrosamine contaminants in drug substances. GC operates on the principles of separation science, utilizing an inert carrier gas to transport analytes through a column for effective isolation and detection [1]. Since its introduction in the mid-20th century, GC has undergone continuous advancements, improving its precision, efficiency, and applicability across various industries, including pharmaceuticals, environmental analysis, and food safety. Key technological developments, such as highly sensitive detectors, capillary columns, and GC-mass spectrometry (GC-MS) integration, have significantly enhanced its resolution and detection capabilities. Additionally, automation and advanced sample introduction techniques such as headspace sampling and solid-phase microextraction (SPME) have improved reproducibility and efficiency. Despite its many advantages, GC is primarily suited for volatile and semi-volatile compounds. High molecular weight or thermally unstable analytes often require derivatization to enhance volatility and detectability. Nonetheless, GC remains an indispensable technique in pharmaceutical research, ensuring drug safety and regulatory compliance. This review explores its fundamental principles, instrumentation, methodologies, and applications in pharmaceutical analysis while highlighting recent advancements and future prospects [2-3].
2. Physical Components of Gas Chromatography (GC)
Gas chromatography consists of Carrier Gas System, Sample Injection System, Heated Injection Port, Chromatographic Column and Oven, Detector System and Data Processing System. The block diagram of Gas chromatograph depicted in Figure1.
Figure1. Block Diagram Of Gas Chromatograph.
3. Working Principle of Gas Chromatography
Principles of Operation
GC operates by first vaporizing the sample in an injection port, typically maintained at a temperature higher than the boiling points of the analytes to ensure complete volatilization. The carrier gas commonly helium, nitrogen, or hydrogen acts as the mobile phase, sweeping the analytes into the column, where separation occurs based on differential partitioning [4].
Separation Mechanism
The chromatographic column, often a capillary column with an inner coating of a stationary phase, provides the medium for analyte separation. The interaction of analytes with the stationary phase is influenced by van der Waals forces, dipole interactions, hydrogen bonding, and solubility effects. Components with stronger interactions with the stationary phase elute later, while those with weaker interactions elute earlier. Factors such as temperature programming and column dimensions (length, inner diameter, and film thickness) play a crucial role in resolution and efficiency. The separation process of Gas chromatograph is shown in Figure2 [5].
Figure2. Separation process Diagram of Gas chromatograph
3. Stationary Phases in Gas Chromatography
The stationary phase in gas chromatography (GC) is a critical factor influencing the resolution, selectivity, and efficiency of analyte separation. It is typically a liquid or polymeric coating immobilized on the inner surface of the column, allowing differential interactions with the analytes as they migrate through the system. The choice of stationary phase is based on the physicochemical properties of the analytes, including their polarity, volatility, and functional groups [6].
Types of Stationary Phases
Dimethylpolysiloxane (100% PDMS): Non-polar and widely used for hydrocarbons, non-polar solvents, and lipophilic compounds.
Phenyl-Modified Polysiloxanes: Increased polarity for separating aromatic and moderately polar compounds.
Cyanopropyl-Modified Phases: Provide enhanced selectivity for polar compounds such as nitriles, esters, and ketones.
The some common commercially available GC columns tabulated in Table1.
Table1. Some examples of commercially available GC column with their properties
|
Column Name |
Type of Stationary Phase |
Basic Structure |
Density (g/mL) |
Viscosity |
Average Molecular Weight (g/mol) |
|
DB-1 (Agilent J&W) |
100% Dimethylpolysiloxane |
Non-polar Polysiloxane |
~0.97 |
High |
~10,000–100,000 |
|
DB-5 (Agilent J&W) |
5% Phenyl, 95% Dimethylpolysiloxane |
Moderately polar Polysiloxane |
~1.02 |
High |
~40,000–150,000 |
|
DB-624 (Agilent J&W) |
6% Cyanopropyl, 94% Dimethylpolysiloxane |
Slightly polar Polysiloxane |
~1.03 |
Medium-High |
~50,000–200,000 |
|
Rtx-1 (Restek) |
100% Dimethylpolysiloxane |
Non-polar Polysiloxane |
~0.97 |
High |
~10,000–100,000 |
|
Rtx-5 (Restek) |
5% Phenyl, 95% Dimethylpolysiloxane |
Moderately polar Polysiloxane |
~1.02 |
High |
~40,000–150,000 |
|
HP-5 (Agilent J&W) |
5% Phenyl, 95% Dimethylpolysiloxane |
Moderately polar Polysiloxane |
~1.02 |
High |
~40,000–150,000 |
|
BPX-5 (SGE) |
5% Phenyl, 95% Dimethylpolysiloxane |
Moderately polar Polysiloxane |
~1.02 |
High |
~40,000–150,000 |
|
HP-35 (Agilent J&W) |
35% Phenyl, 65% Dimethylpolysiloxane |
Moderately polar Polysiloxane |
~1.05 |
Medium-High |
~50,000–200,000 |
|
DB-WAX (Agilent J&W) |
Polyethylene Glycol (PEG) |
Highly polar PEG-based phase |
~1.13 |
Very High |
~50,000–200,000 |
|
Rtx-WAX (Restek) |
Polyethylene Glycol (PEG) |
Highly polar PEG-based phase |
~1.12 |
Very High |
~50,000–200,000 |
|
SP-2560 (Supelco) |
100% Cyanopropyl Polysiloxane |
Highly polar Polysiloxane |
~1.04 |
Medium |
~50,000–200,000 |
|
Rtx-200 (Restek) |
100% Trifluoropropyl Methylpolysiloxane |
Highly polar Fluorinated phase |
~1.08 |
Medium |
~50,000–200,000 |
4. Selection Considerations for Gas Chromatography Columns
The choice of a gas chromatography (GC) column is a crucial factor that influences separation efficiency, resolution, and analysis time. GC columns vary in length, internal diameter, and film thickness, with capillary columns being the preferred choice for most modern applications due to their superior separation performance compared to packed columns. Several key factors must be considered when selecting an appropriate column for a specific analytical application. The selection considerations for Capillary GC Columns with Examples has shown in Table2.
Table2. Selection considerations for Capillary GC Columns with Examples.
|
Selection Parameter |
Description |
Example Columns |
|
Column Length (m) |
- Affects resolution and analysis time. - Longer columns (30–75 m): Higher resolution but longer run times. - Shorter columns (10–15 m): Faster analysis with lower resolution. |
- DB-1 (60 m, Agilent J&W) for complex separations. - HP-5 (15 m, Agilent J&W) for rapid screening. |
|
Column Internal Diameter (ID, mm) |
- Influences sample capacity and resolution. - Narrow-bore (0.10–0.25 mm ID): High efficiency, better resolution, but lower sample capacity. - Standard-bore (0.32 mm ID): Good balance between resolution and sample load. - Wide-bore (0.45–0.53 mm ID): Higher sample capacity but lower resolution. |
- Rtx-5 (0.25 mm ID, Restek) for general-purpose separations. - DB-WAX (0.53 mm ID, Agilent J&W) for higher sample loading. |
|
Column Film Thickness (µm) |
- Affects retention time and sensitivity. - Thin films (0.1–0.25 µm): Fast elution, sharper peaks, suitable for high-boiling analytes. - Thick films (1–5 µm): Increased retention, suitable for highly volatile compounds. |
- DB-5 (0.25 µm, Agilent J&W) for standard separations. - SPB-624 (1.4 µm, Supelco) for volatile organic compounds (VOCs). |
By carefully selecting a GC column based on these factors, analysts can achieve optimal separation, peak resolution, and reproducibility for their specific applications [7].
5. Operational Conditions of Gas Chromatography Columns
The performance of a gas chromatography (GC) column is significantly influenced by operational parameters such as carrier gas flow rate, temperature programming, and column conditioning. Proper optimization of these factors ensures high resolution, reproducibility, and sensitivity in analytical separations.
Carrier Gas Flow Rate
The carrier gas serves as the mobile phase in GC, transporting analytes through the column. Common carrier gases include helium, nitrogen, and hydrogen.
Table3. Effect of Carrier Gas Flow Rate on Gas Chromatography Performance
|
Flow Rate |
Resolution |
Retention Time |
Peak Shape |
Analysis Time |
Example/Application |
|
Low Flow Rate (e.g., 0.5–1.0 mL/min) |
High (better separation) |
Long |
Narrow, well-resolved peaks |
Slow |
Complex mixtures requiring high resolution (e.g., impurity profiling in pharmaceuticals, enantiomeric separation) |
|
Moderate Flow Rate (e.g., 1.0–2.0 mL/min) |
Optimal balance |
Moderate |
Symmetrical peaks |
Reasonable |
Routine pharmaceutical quality control (e.g., residual solvent analysis, stability studies) |
|
High Flow Rate (e.g., 2.0–4.0 mL/min) |
Low (peak coelution risk) |
Short |
Broadened, overlapping peaks |
Fast |
High-throughput screening (e.g., environmental VOC analysis, rapid industrial process monitoring) |
A lower carrier gas flow rate in gas chromatography improves resolution by allowing analytes more time to interact with the stationary phase, leading to better peak separation. However, this comes at the cost of longer retention times, increasing overall analysis time. A moderate flow rate provides the best compromise between separation efficiency, speed, and sensitivity, ensuring well-resolved peaks while maintaining a reasonable analysis time. In contrast, a higher flow rate reduces retention time, allowing for faster separations, but it can negatively impact resolution by causing peak coelution and broadening (Table3) [8]. Additionally, sensitivity may decrease due to reduced analyte interaction time with the detector, leading to lower signal intensity. Therefore, selecting the optimal flow rate requires balancing speed, resolution, and sensitivity based on the analytical requirements.
Temperature Programming
Temperature plays a crucial role in analyte separation. Isothermal operation (constant temperature) is suitable for analyzing compounds with similar boiling points, while temperature programming is used for complex mixtures with a wide range of volatilities. A graphical presentation of gradient program depicted in Figure3. Impact of Temperature Programming on Resolution and Sensitivity is tabulated in Table4.
Figure3. Typical graph for a gradient temperature program in GC
Table4. Impact of Temperature Programming on Resolution and Sensitivity
|
Temperature Program |
Effect on Resolution |
Effect on Sensitivity |
Example/Application |
|
Slow Temperature Ramp (e.g., 2–5°C/min) |
High resolution, better separation of closely eluting peaks |
Increased sensitivity for low-volatility analytes due to better peak shape |
Pharmaceutical impurity profiling, chiral separations |
|
Moderate Ramp Rate (e.g., 5–10°C/min) |
Balanced resolution and faster analysis |
Good sensitivity for a broad range of compounds |
Essential oil analysis, fatty acid methyl esters (FAMEs) in food |
|
Fast Temperature Ramp (e.g., 10–30°C/min) |
Lower resolution, risk of peak coelution |
Decreased sensitivity for late-eluting compounds due to peak broadening |
High-throughput environmental VOC analysis, industrial process monitoring |
|
High Final Hold Time (e.g., 5–10 min at max temperature) |
Prevents carryover and ensures full elution of heavy compounds |
Ensures complete detection of high-boiling analytes |
Petroleum hydrocarbons, pesticide residue analysis |
Gradient temperature programs improve resolution by reducing retention of late-eluting compounds while maintaining sharp peak shapes. A typical temperature ramp increases the oven temperature at 5–20°C/min, optimizing separation without excessive peak tailing or broadening [9].
Column Conditioning
Before use, GC columns must be conditioned to remove residual contaminants and stabilize the stationary phase. This involves heating the column to its upper temperature limit under carrier gas flow for several hours. Proper conditioning minimizes baseline noise and prevents unwanted interactions that could affect analytical performance.
By carefully optimizing carrier gas flow rate, temperature programming, and column conditioning, analysts can achieve precise, reproducible, and high-resolution chromatographic separations, ensuring reliable analytical outcomes.
6. Sample Introduction into Gas Chromatography Inlet Systems
Sample introduction is a critical step in gas chromatography (GC) that influences sensitivity, reproducibility, and chromatographic performance. The choice of injection technique depends on the sample type, concentration, and volatility. Proper sample preparation and injection method selection ensure optimal analyte transfer into the column while minimizing matrix effects and degradation.
Types of Sample Introduction Techniques
Importance of Sample Preparation
Proper sample preparation enhances sensitivity, protects the column, and minimizes matrix interferences. Techniques such as solid-phase microextraction (SPME), derivatization, and filtration improve the reliability of GC analysis. By selecting the appropriate injection technique and optimizing sample preparation, analysts can achieve precise and reproducible results across a wide range of applications [10].
7. Detection Devices and Detection process in Gas Chromatography
Detection devices in gas chromatography (GC) are essential for identifying and quantifying analytes after their separation in the column. Each detector operates based on distinct principles, offering varying sensitivity, selectivity, and application suitability.
Some common GC Detectors are Flame Ionization Detector (FID), Thermal Conductivity Detector (TCD), Electron Capture Detector (ECD), Mass Spectrometry (MS) detector, Flame Photometric Detector (FPD), Nitrogen-Phosphorus Detector (NPD), Photoionization Detector (PID), Helium Ionization Detector (HID), Atomic Emission Detector (AED) making it useful for multi-elemental analysis in petrochemical and pharmaceutical applications. The comparison of detection devices with respect to common detectable compounds and its reported minimum detectable count is tabulated in Table5.
Detection Process
The detection process involves three key steps: ionization or signal generation, signal processing, and data interpretation.
Data Interpretation: The processed signals produce a chromatogram, where peak areas or heights correspond to analyte concentration. Retention time and peak shape help in compound identification and quantification.
Table5. Comparison of GC Detectors
|
Detector Type |
Example of Detectable Compound |
Example of Minimum Detectable Amount |
Detection Process |
|
Flame Ionization Detector (FID) |
Hydrocarbons, alcohols, esters, fatty acids |
~1 pg (picogram) |
Organic compounds are burned in a hydrogen-air flame, producing ions detected as an electrical signal. |
|
Thermal Conductivity Detector (TCD) |
Permanent gases (H?, He, N?, CO?), simple organic molecules |
~1 ng (nanogram) |
Measures changes in thermal conductivity of carrier gas due to the presence of analytes. |
|
Electron Capture Detector (ECD) |
Halogenated compounds, pesticides, nitrosamines |
~0.1 fg (femtogram) |
Uses a radioactive ?-emitter (Ni-63) to generate electrons; electronegative analytes capture electrons, reducing current. |
|
Mass Spectrometry (MS) |
Volatile and semi-volatile organics, drug metabolites |
~1 fg (femtogram) |
Ionizes analytes and separates fragments based on mass-to-charge (m/z) ratio for identification and quantification. |
|
Flame Photometric Detector (FPD) |
Sulfur and phosphorus compounds (e.g., organophosphates) |
~10 pg (Sulfur), ~1 pg (Phosphorus) |
Analytes are burned in a hydrogen-rich flame, and element-specific light emission is detected. |
|
Nitrogen-Phosphorus Detector (NPD) |
Nitrogen-containing drugs, explosives, pesticides |
~10 fg (femtogram) |
Similar to FID, but an alkali metal bead enhances ionization of nitrogen and phosphorus compounds. |
|
Photoionization Detector (PID) |
Volatile organic compounds (VOCs), aromatics, ketones |
~1 pg (picogram) |
Uses UV light to ionize analytes, and the resulting ions generate an electrical signal. |
|
Helium Ionization Detector (HID) |
Permanent gases, noble gases |
~500 pg (picogram) |
Analytes pass through a helium plasma, ionizing and altering electrical conductivity. |
|
Atomic Emission Detector (AED) |
Element-specific detection (C, S, N, P, Cl, etc.) |
~10 pg (picogram) |
Uses microwave-induced plasma (MIP) to break analytes into atoms, measuring their characteristic emission spectra. |
The choice of detector depends on the chemical nature of the analytes, detection limits, and required specificity. Sensitivity (ability to detect low concentrations) and selectivity (ability to distinguish specific analytes) are critical factors in selecting an appropriate detector for a given application [11-12].
8. Qualitative and Quantitative GC Methods Gas chromatography (GC) is widely used for both qualitative and quantitative analysis of volatile and semi-volatile compounds. Qualitative analysis focuses on identifying compounds, while quantitative analysis determines their concentration in a sample. The accuracy and reliability of
these methods depend on calibration strategies and data interpretation techniques.
Qualitative GC Analysis
Qualitative analysis in GC is primarily based on retention times and, when coupled with spectroscopic detection (e.g., mass spectrometry), spectral data.
Quantitative GC Analysis
Quantification in GC involves measuring peak areas or peak heights to determine analyte concentrations. Several calibration techniques are used:
Qualitative GC methods rely on retention times, spectral data, and reference standards for compound identification, while quantitative GC methods use calibration techniques to measure analyte concentrations accurately. The choice of method depends on sample complexity, required accuracy, and the presence of potential interferences [13].
9. Applications in Drug Substance Development GC is widely used in pharmaceutical research for impurity profiling, stability testing, and quality control. The detection of volatile organic impurities and nitrosamine contaminants is crucial for regulatory compliance.
Gas chromatography (GC) plays a vital role in pharmaceutical research and development, ensuring drug safety, efficacy, and compliance with regulatory standards. It is particularly valuable for detecting and quantifying volatile organic compounds (VOCs), residual solvents, and genotoxic nitrosamine contaminants. GC is widely employed in impurity profiling, stability testing, and quality control of active pharmaceutical ingredients (APIs) and finished dosage forms.
1. Impurity Profiling
Regulatory agencies such as the ICH (International Council for Harmonisation), FDA, and EMA mandate impurity profiling in pharmaceutical substances to ensure patient safety. GC is a powerful tool for identifying and quantifying trace-level impurities, particularly volatile and semi-volatile organic compounds.
2. Stability Testing
Stability studies ensure that APIs and formulations retain their chemical integrity over time. GC is used to monitor changes in drug composition under different storage conditions.
3. Quality Control (QC) and Batch Release
GC is essential for routine quality control in pharmaceutical manufacturing. It ensures that raw materials, intermediates, and final drug products meet required specifications before batch release [17].
4. Detection of Nitrosamine Contaminants
Nitrosamines are highly potent genotoxic impurities that can form during drug synthesis, storage, or packaging. Regulatory agencies have set strict limits for nitrosamines, as they are classified as probable human carcinogens [18-23].
Gas chromatography is indispensable in pharmaceutical research for impurity profiling, stability testing, and regulatory compliance. Its ability to detect and quantify volatile organic impurities, residual solvents, and nitrosamines ensures drug safety and quality, making it a cornerstone of modern drug substance development. [24-30]
CONCLUSION
Gas chromatography (GC) has undergone significant advancements, solidifying its role as a powerful analytical technique in pharmaceutical research. Its ability to separate, identify, and quantify volatile and semi-volatile compounds with exceptional sensitivity makes it indispensable for impurity profiling, stability testing, and regulatory compliance. The integration of advanced detection systems, including GC-MS and selective detectors, has enhanced its capability to detect trace-level contaminants such as nitrosamines, ensuring drug safety and quality control. Recent developments in column technology, stationary phases, and sample introduction techniques have further improved GC’s efficiency, resolution, and applicability. Automation and hyphenated techniques continue to expand its scope, enabling more comprehensive analysis of complex pharmaceutical matrices. While GC remains primarily suited for volatile compounds, derivatization methods and alternative detection strategies have broadened its application range. As pharmaceutical regulations become more stringent and analytical challenges evolve, ongoing innovations in GC are expected to drive improvements in sensitivity, selectivity, and operational efficiency. Future research will likely focus on enhancing miniaturization, automation, and eco-friendly methodologies, ensuring that GC continues to play a crucial role in pharmaceutical analysis and quality assurance.
REFERENCES
Deshoju Srinu*, B. Jainendra Kumar, A Review on Gas Chromatography: Techniques, Methodologies, and Applications-Implications to Pharmaceutical Research, Int. J. of Pharm. Sci., 2025, Vol 3, Issue 2, 837-848. https://doi.org/10.5281/zenodo.14857270
10.5281/zenodo.14857270
