Dr. Pramod Kr. Pandey is a distinguished Analytical Research Expert with over three decades of extensive experience in the pharmaceutical industry. He has contributed his expertise to both leading Indian and global pharmaceutical companies, consistently driving innovation and excellence in analytical research
Learn the fundamentals of gas chromatography (GC) in pharmaceutical development, including its principles, method development, column selection, applications, and more. Gain insights into its advantages, disadvantages, and real-world case studies
Gas Chromatography (GC) in Drug Development: Techniques, Case Studies, and Expert Tips
Gas chromatography (GC) is a vital tool in drug development, renowned for its reliability, sensitivity, precision, rapid results, and cost-effectiveness. It plays a critical role in the analysis of residual solvents and organic volatile compounds – tasks that would be nearly impossible without this technique. Given its significance, I have decided to share my expertise on the subject, providing a detailed guide to help you navigate the complexities of GC.
In this article, we will explore the fundamental principles of gas chromatography, various approaches for method development, the process of column selection, and its wide-ranging applications in the pharmaceutical industry. I will also discuss the advantages and disadvantages of GC, present insightful case studies, and address some frequently asked questions. By the end of this post, you’ll have a comprehensive understanding of GC and how to leverage its full potential in pharmaceutical development.
Gas Chromatography(GC)
Gas Chromatography (GC) is an analytical technique used for separating volatile components in a sample or sample mixture. In GC, the sample components are distributed between two phases: a stationary phase and a mobile phase. The mobile phase, known as the carrier gas, is typically helium or nitrogen, which flows through the stationary phase.
As the carrier gas moves through the column, it facilitates the separation of the sample components. This separation occurs through a series of sorption and desorption events, where the components interact with the stationary phase at different rates. The differences in how strongly each component is retained by the stationary phase, based on their respective distribution coefficients, are what lead to the separation of the individual components.
The following are the two types of Gas chromatography:
Gas-solid chromatography or GSC: When a solid stationary phase is used in GC, then it is called Gas-solid chromatography. Most commonly used stationary phases are molecular sieves, silica and alumina. GSC is restricted to the analysis of gases and low molecular weight hydrocarbons. Nowadays, GSC is not used in the industries due to its low efficiency.
Gas-liquid chromatography or GLC: When a liquid stationary phase is used in GC, then it is called Gas-liquid chromatography.
Now in the industries, only GLC is used for its high efficiency, selectivity and reliability of result and hence in this article, focus will be given on GLC
GC Column Types
Two types of Gas Chromatography column
The following are types of GC columns
Packed columns and
Capillary columns
Packed columns
The packed column is a tube packed with the support material coated with a liquid stationary phase. The most widely used and readily available support material is diatomaceous earth (siliceous material).
The column is made of glass or stainless steel. Its length is 2 to 6 meters, and its internal diameter is 1/4 inch to 1/8 inch.
The efficiency of this column is low (theoretical plate 1000 to 2000).
Capillary columns
These columns are made of fused silica, and their inner wall is coated with the liquid stationary phase. These columns are hollow from the inside
The length of the capillary column is 10 meters to 150 meters, and its internal diameter is 0.1mm to 0.7mm.
These columns have extremely high column efficiency (theoretical plate 10000 to 100000)
GC Instrument Flow Diagram
GC Components and Their Function
Carrier gas: Helium and Nitrogen are used as carrier gases. The carrier gas should be :
Inert to avoid interaction with the sample or solvent
Readily available
Purity >99.9% &
Inexpensive
Flow control valve: The flow control valve controls the flow rate of the carrier gas
Sample injection port: The injector injects the sample into the sample injection port. This is at a high temperature, and it converts the liquid sample into the vapour phase. Then this vapour goes into the column. Gas-tight syringes 1 microlitre to 10 microlitre are used for liquid samples, and 2 ml to 10 ml syringes are used for gases.. The injector port should be hot enough to vaporise the sample rapidly but not hot so as to decompose it. The temperature of the injector port is always controlled.
GC Column: The column separates each component of the sample mixture vapour phase, which is received from the injector. Each separated component goes one by one into the detector
Column oven: The column oven maintains the column temperature as per the method requirement (as per the temperature program)
Detector: The detector converts the eluted analyte into a signal and sends the signal to the data processor
Data processor: The Data processor converts each analyte signal (obtained from the detector) into a peak one by one. Hence, a GC chromatogram is obtained
Outlet
Note: If analysis is performed on a flame ionisation detector, in that case, apart from carrier gas, hydrogen and air gases are also used.
Type of GC Detectors
The following detectors are used in the GC:
Flame ionisation detector (FID)
GCMS Detector (MS)
Thermal conductivity detector (TCD)
Electron capture detector (ECD)
Nitrogen Phosphorus detector (NPD)
1. Flame ionisation detector (FID)
FID is widely used in the pharmaceutical industry since most of the pharmaceuticals contain carbon and hydrogen atoms. The molecules must have carbon and hydrogen atoms to be analysed in the FID detector. In this detector, nitrogen or helium is used as a carrier gas. Hydrogen gas and air are used to produce the flame. Most organic of organic compounds are readily pyrolysed when introduced into a hydrogen-oxygen flame and produce ions in the process. These ions are collected at the charged electrode, and the resulting current passes into the data processor/integrator, and a peak (signal is obtained. The resulting current is directly proportional to the concentration of the sample. This detector is highly sensitive and has a very good linearity range. Its minimum detectable limit is 1ng/ µl.
2. GCMS Detector (MS)
GCMS Detector is a universal detectorAmong the above detectors, flame ionisation detector and mass detectors are widely used in the industries, but it is mainly used for the identification of unknown impurities, structure elucidation and quantification at very low levels.
3.0 Thermal conductivity detector (TCD)
Thermal conductivity detector (TCD) is used for analysis of gases, light hydrocarbons and and those compounds that can not be analysed in the FID detector.
4.0 Electron capture detector (ECD)
This detector is mainly used for the analysis of volatile halo organic compounds like carbon tetrachloride and dichloromethane
5.0 Nitrogen Phosphorus detector:
Nitrogen Phosphorus detector is used for the analysis of volatile organic compounds containing Nitrogen and Phosphorus
Among the above detectors, flame ionisation detector and mass detectors are widely used in the pharmaceutical industry.
How to operate a GC (Gas chromatograph)?
Operating a Gas Chromatograph (GC) requires understanding both the instrument itself and the sample analysis process. The following is a step-by-step procedure to operate the GC:
1. Prepare the Instrument
Check the GC System: Ensure the GC is properly set up and connected to the necessary power, gas supply, and data system.
Column Installation: Make sure the column is correctly installed. The column should be attached to both the injector and the detector.
Carrier Gas: Check that the carrier gas (typically helium, nitrogen, or hydrogen) is at the correct pressure and flow rate. Ensure the gas cylinder is sufficiently full.
Detector: Ensure that the detector (FID, TCD, ECD, etc.) is correctly installed, calibrated, and ready for operation.
System Leak Test: Before starting, it’s good practice to check for leaks in the system, especially at connections, the injector, and detector.
2. Prepare the Sample
Sample Preparation: Prepare the sample by dissolving it in an appropriate solvent. The sample should be volatile and within the concentration range suitable for GC analysis.
Injection Method: Decide whether you will be injecting the sample manually or using an autosampler. In the case of manual injection, a syringe is used to inject the sample directly into the GC inlet.
3. Set Up the GC Parameters
Set the following GC parameters as per the method/STP/Momograph:
Injector Temperature
Column Temperature
Carrier Gas Flow Rate
Detector Settings
Program the Oven Temperature
4. Inject the Sample
Manual Injection: Inject the prepared sample into the injector port of the GC using a syringe. The sample should be injected quickly to minimise vaporisation loss.
Autosampler Injection: If an autosampler is used, load the vial with the sample and program the autosampler to inject at the required time.
The injector port will vaporise the sample, which is then carried by the carrier gas into the column.
5. Run the Analysis
Start the Run: Once all parameters are set, initiate the analysis. The sample will travel through the column, where it will separate based on its interaction with the stationary phase.
Monitor the Chromatogram: As the sample components exit the column, they are detected by the detector, generating a chromatogram that shows peaks corresponding to each compound.
Observe Peak Retention Times: Each component in the sample will elute from the column at a specific retention time. These retention times will be compared against known standards for identification.
6. Data Collection and Analysis
Review Chromatogram: The data system will produce a chromatogram. Review the peaks and their retention times to identify the components in your sample.
Quantification: Quantitative analysis is done by measuring the area under each peak (integration). This can be compared to calibration standards for concentration determination.
Interpret Results: Compare the observed retention times and peak areas to your standard references or calibration curves to identify and quantify the components in your sample.
7. Post-Analysis
Clean the Instrument: After the run is complete, perform any necessary post-analysis tasks, like cleaning the injector or flushing the system with a solvent.
Shut Down: Turn off the carrier gas and detector gases, and shut down the GC system according to the manufacturer’s instructions.
Tips for Successful GC Operation:
Regular maintenance, such as replacing the septum, cleaning the injector, and checking gas flow, is essential to keep the GC system running smoothly. Here’s how to read and interpret a gas chromatogram:
How to read a Gas chromatogram?
Reading a gas chromatogram involves interpreting the data presented in the form of peaks on a graph. Each peak corresponds to a different component in the sample.
1. Understand the Chromatogram
A gas chromatogram is typically a graph with:
X-axis: Retention time (time elapsed from injection to detection), usually measured in minutes or seconds.
Y-axis: Signal intensity (peak height or area), which corresponds to the concentration or quantity of the detected component.
2. Identify the GC Peaks
Each peak represents a different compound in your sample. The time at which a peak appears on the x-axis is called the retention time, and this is key to identifying the compound.
The area or height of the peak indicates the relative amount or concentration of the compound in the sample. A larger peak generally means a higher concentration of that compound.
3. Key Components of the Chromatogram
Retention Time: Retention time (RT) is the time it takes for a particular compound to pass through the column and reach the detector. By comparing the retention time of peaks in your sample chromatogram with those of known standards, you can identify the compounds.
Peak Shape: TheGC peak may be sharp or broad. Try to get peak as per typical chromatogram given in the method.
Peak Height vs. Area: Either peak height or peak area can be used for calculation purposes. But, Area is usually more reliable for quantifying the concentration of a compound, as it accounts for both the height and width of the peak.
Baseline: The baseline is the flat line at the bottom of the chromatogram.
Separation and Resolution: Each peak must be separated from neighbouring peaks
Case Study: Example Interpretation
Let’s say you run a chromatogram for a pharmaceutical sample, and you see the following:
Peak 1 at 3.5 minutes: Retention time matches a standard compound (e.g., Acetone). The peak area is moderate, suggesting a low concentration.
Peak 2 at 7.2 minutes: Retention time matches another standard (e.g., Ethanol). The peak area is larger, indicating a higher concentration of ethanol in the sample.
Peak 3 at 11.1 minutes: No standard available, but this could be a contaminant or another compound. Further analysis (e.g., comparing retention times with databases or performing mass spectrometry) would be required to identify it.
Applications of Gas Chromatography
Gas Chromatography is used in various industries such as pharmaceutical industries, food industries, petrochemicals and wine industries. The following are the various applications of GC:
Purity test: GC is widely used for purity testing of volatile pharmaceuticals and other volatile chemicals
Chiral purity test: GC with a chiral column is used for the optical purity of the volatile pharmaceuticals or other volatile organic compounds
Structural identification of unknown volatile pharmaceuticals/compounds: GC with mass (GC-MS) detector is used for structural identification of the unknown compounds
Assay test: Gas Chromatography is used for assay testing of volatile pharmaceuticals/compounds. In GC, the assay is generally performed using the external standard method
Identification test: Identification is performed by comparing the retention time of an unknown peak with a known standard peak
Content test: It is used for Genotoxic impurities content in pharmaceuticals and pesticide contents in food products
Performance-enhancing drugs in an athlete’s urine sample
Forensic science: This is used for the detection of explosives and the analysis of arson accelerants
Clinical analysis
Identification of different alcohols from the sample mixture using a standard solution
Specified vs. Unspecified Impurities
Advantages and Disadvantages
Advantages
The following are the advantages of Gas Chromatography: Fast analysis: Analysis can be performed in a few minutes, and that is why this technique is widely used in pharmaceutical development
Cost-effective analysis: This is a low-cost (cost-effective) analysis. Even a direct sample can be analysed using GC
Reliable, precise and accurate results with high sensitivity: Gas Chromatography gives reliable, precise and accurate results with high sensitivity.
Sharp and selective peaks: Reliable: Gas Chromatography gives sharp and selective peaks since capillary columns are used. Even a 0.2-minute difference in retention time can give base-to-base separation between the close-eluting peaks
Small sample consumption
Disadvantages
The following are the disadvantages of Gas Chromatography:
Non-volatile pharmaceuticals/compounds can not be analysed or in other words, only volatile pharmaceuticals/compounds can be analysed by Gas Chromatography
Compounds to be analysed should be stable in GC-operating conditions
Gas Chromatographic Method: How to Design?
A GC method contains the following components:
Chemicals and reagents: This section contains all the chemicals and reagents and their grade, and the parts numbers used in the analysis
Instrument details: This section contains instrument and detector details required for analysis
GC column details: This section contains the column name, its part number, make and dimension or equivalent column details. For example: DB 624, (30m x 0.53mm), 3.0 µm film thickness, make: xxxx (as applicable)
Injector temperature:
Detector temperature
Oven temperature program: It contains details of the GC oven temperature
Carrier gas: Nitrogen or Helium as required by the method
Flow rate: It contains the carrier gas required by the method
Injection volume: It contains injection details like 1µl, 2µl or 3µl (as required by the method)
Run time: It contains the analysis run time (in minutes) required by the method
Diluent: It contains the solvent details in which the sample will be prepared
Sample, standard, sensitivity solution and system suitability preparation procedure: It contains solution preparation details with weight and glassware (e.g volumetric flask details)
Retention time and Relative retention time table: It contains Retention time(RT) and Relative retention time (RRT), structures of different analyte components
System suitability acceptance criteria: It contains the System suitability test (SST) acceptance criteria required by the method. For example Resolution should not be less than 2, the theoretical plate should not be less than 10000, etc.
Procedure: It contains blank, standard, sample and SST injection order and calculation procedure
Typical chromatogram: This section contains Blank, SST, sensitivity, standard and sample typical chromatogram
Case studies
Separate Acetic acid and Ethanol from a sample mixture: Both acetic acid and ethanol are polar, volatile molecules. The polarity of acetic acid is greater than that of Ethanol. Therefore, these compounds can be separated using capillary columns containing polar stationary phases like Polyethylene Glycol (Carbowax)
Separate Benzene acid and Toluene from a sample mixture: Both benzene and Toluene are nonpolar the volatile molecules. Hence, these molecules can be separated using a capillary column containing nonpolar stationary phase like Dimethylpolysiloxane and 5% Diphenyl 95% dimethyl polysiloxane
Conclusion
Gas chromatography plays a crucial role in ensuring the quality of pharmaceuticals, whether it’s for reaction monitoring, in-process control, or final material release. I hope this post has helped clarify your understanding of GC and its application in pharmaceutical development, enabling you to use this technique confidently and independently.
If you have any questions or suggestions regarding this article, feel free to leave a comment below. Also, don’t forget to explore the following insightful articles on my blog, such as:
