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
Developing a Gas Chromatography (GC) method is a systematic process that requires a deep understanding of both the analytical technique and the sample’s characteristics. Here’s a clear, step-by-step approach to developing an effective GC method GC Method Development GC Method Development is the systematic process of selecting and optimising the appropriate chromatographic conditions, including the […]
GC Method Development: How to Get Mastery In 11 Minutes
Developing a Gas Chromatography (GC) method is a systematic process that requires a deep understanding of both the analytical technique and the sample’s characteristics. Here’s a clear, step-by-step approach to developing an effective GC method
GC Method Development
GC Method Development is the systematic process of selecting and optimising the appropriate chromatographic conditions, including the choice of column, detector, carrier gas, diluent, temperature program, and calculation procedure, to create a robust, reproducible, and validatable analytical method for the analysis of a volatile pharmaceutical. This process ensures that the method meets the required accuracy, precision, sensitivity, and resolution for the intended application while adhering to regulatory and quality standards.
Developing a Gas Chromatography (GC) method is a systematic process that requires a deep understanding of both the analytical technique and the sample’s characteristics.
The following steps play an important role in developing an effective GC Method:
Define the Method Objective
Knowledge about the sample and its impurities
Selection of diluent for sample preparation
Column selection
Liner selection
Detector selection
Carrier gas selection
Certified standards, samples and markers
Sample concentration and preparation procedure selection
Develop the column oven Temperature Program
Detector temperature selection
Develop Injection Parameters
GC parameters optimisation
SST (system suitability testing) selection
Develop a calculation method
Verification/validation of the method
Documentation and STP (Standard Test Procedure) preparation
1. Define the Method Objective
Determine the Goal: Understand what you are analysing (e.g., qualitative vs. quantitative analysis) and identify the target compounds.
Choose the Right Sensitivity: Decide whether you need trace analysis, routine analysis, or specific compound identification.
Route of Synthesis (ROS): Collect ROS of all purchasing materials like starting materials, key starting materials, reagents, chemicals and final API (Active Pharmaceutical Ingredients).
Possible impurities and degradants: Collect information about possible impurities and degradation products with the help of ROS and literature reports.
Note: Find out all the concerned impurities related to a method with the help of literature and write it down in tabular form. It is required to prove the selectivity and specificity of the HPLC method which you are going to develop.
Literature reported impurities: Collect all the published data and make the literature report. This report contains a piece of information related to impurities, chromatographic conditions, pH, pKa, stability, etc.
Stability of the sample and impurities (under certain pH, temperature, or light): Collect all the information related to the stability of the main analyte and impurities using ROS and literature report. It is very helpful during diluent selection
Structures of the main analyte and impurities: In this step, write down the structure, molecular weight and polarity nature of the molecule in tabular form. It is very helpful during column selection and mobile phase selection during method development.
Understand chemical properties: polarity (Hydrophilicity/hydrophobicity), boiling point, and solubility,
3.0 Selection of diluent for sample preparation
The diluent must dissolve the sample completely, be compatible with the GC system, and not interfere with the target analytes or the chromatographic process.
4.0 GC Column Selection
The column is the heart of gas chromatography, and it is only responsible for the separation. Column selection or stationary phase selection is critical in the GC, and it needs both knowledge and skills. No matter how sophisticated the instrument may be, it is the column which determines the success or failure of the separation.
Column selection is the integral approach of the structural analysis of the molecule, application of polarity, Trial and error, experience and literature survey
Selection of the liquid phase also depends upon the sample, and it becomes easier if pieces of information such as suspected components, boiling range, and structure are known about the sample.
For the separation of non-polar compounds, one should use a non-polar stationary phase
For the separation of polar compounds, one should use a polar stationary phase
Phase
Polarity
Temperature range
Dimethylpolysiloxane
Non-polar
-60o to 320o C
5% Diphenyl 95% dimethyl polysiloxane
Non-polar
0 to 300oC
50% Diphenyl 50% dimethyl polysiloxane
Intermediate polar
50o to 320o C
(Cyanopropyl)(phenyl)dimethylpolysiloxane
Intermediate polar
-20o to 280o C
Polyethylene Glycol (Carbowax)
Strongly polar
40 to 250oC
(Bis cyanopropyl)(cyanopropylphenyl)polysiloxane
Strongly polar
0 to 275oC
Stationary phases used in GC analysis
Use the following increasing functional group polarity order to decide the molecule’s polarity during method development:
The inlet liner (also called injector liner) plays a critical role in achieving good separation, sensitivity, and reproducibility in Gas Chromatography (GC). Though often overlooked, selecting the right liner is essential for efficient sample vaporisation, minimising active site interaction, and achieving accurate results, especially in complex or trace-level analyses.
Minimise Dead Volume: Liner should match injector geometry to avoid sample pooling and band broadening.
Use Deactivated Liners: For active or polar compounds, choose silanized/deactivated liners to reduce adsorption and peak tailing.
Avoid Overfilling: Match liner volume to your injection volume to prevent backflash and poor peak shape.
Routine Maintenance: Replace liners regularly, especially in matrix-rich or thermally dirty samples.
6.0 Detector Selection
Detector Selection: Choose a detector that is sensitive to your analytes (e.g., Flame Ionization Detector (FID), Mass Spectrometer (MS), Thermal Conductivity Detector (TCD), or Electron Capture Detector (ECD)).
Detection Limits: Determine the detection limits needed for your application.
Detector Sensitivity & Linearity: Make sure the detector provides sufficient sensitivity and a linear response over the expected concentration range.
7.0 Carrier Gas Selection
Carrier Gas Selection: The most commonly used carrier gases are Helium (He), Hydrogen (H2), and Nitrogen (N2). Choose based on cost, efficiency, and detector compatibility.
Flow Rate: The carrier gas flow rate impacts the analysis time and resolution. Optimise it for the best separation and speed, typically in the range of 1-2 mL/min for most applications.
Linear Velocity: In addition to flow rate, the linear velocity should be optimised for column efficiency.
8.0 Certified standards, samples and markers
Use only certified standards, samples and markers to confirm the different analytes during GC method development
9.0 Sample Concentration and Preparation Procedure Selection
Properly prepare the sample, whether by dilution, filtration, or derivatization, to avoid interference with the chromatography.
10. Develop the Column Oven Temperature Program
The oven temperature program is one of the most critical parameters in GC method development. It controls how analytes are separated as they travel through the column. The goal is to optimize separation, minimize analysis time, and achieve sharp, well-resolved peaks for all components. Use the following steps to finalise Column Oven Temperature Program:
Know Your Analytes
Boiling points: Determines starting and final temperatures.
Polarity & volatility: Affects elution order and speed.
Choose the Starting Temperature
Typically 30–50°C below the boiling point of the most volatile compound.
Must be above the solvent boiling point to ensure the solvent peak elutes quickly.
Note: Common starting points: 40–60°C for volatiles, 80–100°C for semi-volatiles.
Select the Ramp Rate
Typical range: 5–15°C/min.
Slower ramps (1–5°C/min): Better resolution but longer run time.
Faster ramps (15–30°C/min): Shorter run time, but may reduce separation quality.
Note: Start with 10°C/min for general use; adjust based on resolution and peak spacing.
Set Final Temperature
It should be 20–30°C higher than the boiling point of the heaviest analyte.
Final hold (isothermal step) helps elute late compounds and clean the column.
Note: Example: For semi-volatiles ending at ~250°C, use a final temp of 270–290°C.
Determine Hold Times
Initial hold: Helps resolve early-eluting peaks and stabilize the baseline.
Final hold: Ensures all analytes elute and the column is ready for the next injection.
Note: Initial: 1–3 min; Final: 5–10 min (or longer if heavy compounds present).
Caste study: Column Oven Temperature Program for a mixture of solvents and semi-volatiles
Initial Temp: 50°C, hold 2 min
Ramp: 10°C/min to 280°C
Final Hold: 10 min
11.0 Detector temperature selection
Setting the correct detector temperature is crucial for sensitivity, stability, and reproducibility in GC analysis. Each type of detector has different requirements, but the overall goal is to ensure analytes remain in the gas phase, prevent condensation, and avoid thermal degradation. Consider the following steps while selecting the detector temperature:
Match to Analyte Volatility
The temperature should be higher than the boiling point of your highest-boiling analyte.
Prevents condensation inside the detector or transfer line.
Match or Exceed Column Oven Final Temperature
Detector temp should be ≥ final oven temp to ensure analytes stay in the vapor phase as they reach the detector.
Consider Detector Stability
Too low = condensation → noisy baseline and low response
Too high = risk of thermal degradation or reactive losses
Note: Refer to the Instrument and column manufacturer specifications to avoid any failure
Case Study: Example of detector temperature selection
Final oven temperature: 260°C
FID Temp: 300°C
TCD Temp: 280°C
ECD Temp: 280°C
MS Transfer Line: 280–290°C
12.0 Develop Injection Parameters
Injection Mode: Choose between split, splitless, or on-column injection. Split injections are suitable for concentrated samples, while splitless is ideal for trace analysis.
Injection Volume: Select an appropriate injection volume (typically between 0.1 µL to 2 µL) to avoid overloading the column and affecting resolution.
13.0 GC parameters optimisation
Stationary phase polarity: Select a nonpolar column for nonpolar compounds, an intermediate polar column for for sample mixture containing different polarities and a polar column for polar compounds
The film thickness of the stationary phase: As film thickness increases, peak sharpness decreases and vice versa
Internal diameter: As the column internal diameter decreases, peak sharpness increases and vice versa
Column length: As the column length increases, a separation between peaks increases and vice versa
14.0 SST (system suitability testing) selection in GC method development
In GC, SST is decided by using two or more of the following chromatographic parameters:
Theoretical plate or column efficiency or N
Tailing factor or USP tailing or T
Resolution or R
HETP or Height equivalent to theoretical plate
Source:pharmaknowledgeforum.com
15.0 Develop a Calculation Method
Based on the method requirement, select either of the following methods for calculation:
Area normalisation method or area percentage method: Use for purity test and ipurity test at intemediate stages
External standard method: Use for assay, content test and related substances test
Internal standard method: Use for content test
16.0 Verification/validation of the method
Perform method validation or mini-validation so that the method can be validated when needed and any surprises can be avoided. The verification parameters can be selected based on the sensitivity of the method, which may include:
Specificity: Ability to separate analytes from matrix components.
Linearity: The method’s ability to produce a straight-line response over the concentration range.
Accuracy: The closeness of measured values to the true value.
Precision: Repeatability and reproducibility of results.
Detection Limit (DL) and Quantification Limit (QL).
Robustness: The method’s reliability under small variations in conditions (e.g., slight changes in temperature or mobile phase composition).
17.0 Documentation and STP (Standard Test Procedure) Preparation
Once the method is developed, then report the experimental result and prepare the STP (Standard Test Procedure)
Case Study: GC Method Development to separate methanol, ethanol, Isopropyl alcohol and toluene in a sample mixture
Choice: Dimethyl sulfoxide (DMSO) — good solvent for polar and non-polar analytes, high boiling point (will elute after all solvents)
3.0 Column Selection
Column: DB-624 (30 m × 0.32 mm × 1.8 µm)
Reason: Designed for volatile and semi-volatile organics (specially residual solvents), good for polar/non-polar separation
4.0 Injection Parameters
Injection Mode: Split mode (1:10 split ratio)
Injection Volume: 1 µL
Liner: Deactivated liner with glass wool (to aid in vaporisation and protect the column)
Injector Temperature: 200°C (to vaporise solvents quickly but not degrade API)
5.0 Oven Temperature Program
Initial Temp: 40°C, hold 5 min
Ramp: 10°C/min to 200°C
Final hold: 5 min
Total Run Time: ~20 min
6. Carrier Gas & Flow Conditions
Carrier Gas: Helium
Flow Rate: Constant flow at 1.5 mL/min
Rationale: Helium is inert and compatible with FID; good balance of resolution and speed
7. Detector Selection and Settings
Detector: Flame Ionisation Detector (FID)
Detector Temp: 250°C
Hydrogen/Air Flows: Set per instrument manufacturer specs
Rationale: FID is sensitive to organic solvents, provides a linear response in the ppm range
8.0 System Suitability and Validation
System Suitability Test (SST):
Resolution between methanol and dichloromethane: > 2
RSD for replicate injections: < 2%
6.0 Elution pattern
Methanol elutes early (~3.4 minutes), Ethanol (~4.5 minutes), Isopropyl alcohol (~6.5 minutes), and Toluene at the end (~10.2 minutes)
This program avoids co-elution and provides sharp, well-resolved peaks
Elution pattern of Methanol, Ethanol, Isopropyl alcohol, and Toluene
Conclusion
Developing a GC method involves choosing the right column, detector, carrier gas, and temperature program, followed by careful sample preparation and calibration. The key to success lies in continual optimisation, validation, and troubleshooting to refine your method for accuracy, precision, and reproducibility.
GC Method Development is the systematic process of selecting and optimising the appropriate chromatographic conditions, including the choice of column, detector, carrier gas, diluent, temperature program, and calculation procedure, to create a robust, reproducible, and validatable analytical method for the analysis of a volatile pharmaceutical
