Mastering Gas Chromatography in 7 Comprehensive Steps!

When dealing with an unknown sample, it is essential to first understand its origin, nature, and the analytical objectives. Based on this foundation, a preliminary assessment of the sample can be made. Combining this knowledge with information about known pure substances or relevant qualitative chromatographic reference data, one can employ specific methods for qualitative identification. This approach helps in navigating gas chromatography (GC) analysis of samples effectively. 

01 Sample Source and Preprocessing Methods

Samples that can be directly analyzed by GC are typically gases or liquids. Solid samples should be dissolved in an appropriate solvent before analysis, ensuring that the sample does not contain components that GC cannot analyze, such as inorganic salts, which may damage the chromatographic column. Therefore, when receiving an unknown sample, it is crucial to understand its source to estimate the potential components it may contain and the boiling point range of the sample. If the sample system is simple and sample components can vaporize, direct analysis is possible. However, if there are components in the sample that cannot be directly analyzed by GC or if the sample concentration is too low, necessary preprocessing steps must be taken. These may include adsorption, fractionation, extraction, concentration, dilution, purification, derivatization, and other methods to handle the sample appropriately.

02 Determining Instrument Configuration

Instrument configuration refers to the selection of the injection device, carrier gas, chromatographic column, and detector for analyzing the sample.

In general, it is advisable to first determine the type of detector. Hydrocarbons are often analyzed using Flame Ionization Detector (FID), substances with a higher content of electronegative groups (such as F, Cl, etc.) and lower carbon-hydrogen ratio are typically analyzed using Electron Capture Detector (ECD). Thermal Conductivity Detector (TCD) is a suitable choice when high detection sensitivity is not a requirement, or when non-hydrocarbon components are present. For samples containing sulfur or phosphorus, Flame Photometric Detector (FPD) can be selected.

For liquid samples, membrane pad injection can be chosen as the method, while for gas samples, a six-port valve or adsorption-thermal desorption injection method can be employed. Typically, gas chromatographs are equipped with membrane pad injection methods only, so for gas samples, analysis can be carried out using the adsorption-solvent desorption-membrane pad injection method.

Choose a chromatographic column based on the nature of the components to be tested, generally following the principles of similar compatibility. For separating non-polar substances, select a non-polar chromatographic column; for separating polar substances, choose a polar chromatographic column. Once the chromatographic column is selected, determine the working temperature based on the differences in the distribution coefficients of the components to be tested in the sample. For simple systems, use an isothermal method, while for complex systems with large differences in distribution coefficients, employ a temperature-programmed method for analysis.

Commonly used carrier gases include hydrogen, nitrogen, helium, and others. Hydrogen and helium, with their smaller molecular weights, are often used as carrier gases in packed column gas chromatography. Nitrogen, with a larger molecular weight, is commonly employed as a carrier gas in capillary gas chromatography. For gas chromatography-mass spectrometry (GC-MS), helium is typically used as the carrier gas.

03 Determining Initial Operating Conditions

Once the sample is prepared, and the instrument configuration is established, preliminary separation experiments can begin. It is essential to determine the initial separation conditions, mainly including the injection volume, injection port temperature, detector temperature, chromatographic column temperature, and carrier gas flow rate.

The injection volume should be determined based on the sample concentration, chromatographic column capacity, and detector sensitivity. For sample concentrations not exceeding 10 mg/mL, the injection volume for packed columns is typically 1 to 5 μL, while for capillary columns, if the split ratio is 50:1, the injection volume generally does not exceed 2 μL.
In principle, a slightly higher injection port temperature is advantageous. It should generally approach the boiling point of the highest boiling component in the sample but remain below its decomposition temperature.

04 Optimization of Separation Conditions

The purpose of optimizing separation conditions is to achieve satisfactory separation results in the shortest possible analysis time. When changing the column temperature and carrier gas flow rate fails to achieve baseline separation, it is advisable to consider replacing the chromatographic column with a longer one, or even switching to a column with a different stationary phase. In gas chromatography (GC), the chromatographic column is a critical factor in the success or failure of separation.

05 Qualitative Identification

Qualitative identification involves determining the attribution of chromatographic peaks. For simple samples, qualitative identification can be achieved through reference to standard substances. This involves injecting standard samples and actual samples separately under the same chromatographic conditions. The component to be analyzed is identified based on the retention time. It’s essential to note that different compounds may have the same retention time on the same chromatographic column. Therefore, relying on a single retention time for qualitative identification of unknown samples is not sufficient. Using retention indices on two or more columns is a more reliable method in GC because the likelihood of different compounds having the same retention time on different columns is much lower. If conditions permit, gas chromatography-mass spectrometry (GC-MS) can be employed for qualitative identification.

06 Quantitative Analysis

To determine the content of the target component, one must decide on a quantitative method. Common chromatographic quantitative methods include peak area (or peak height) percentage method, normalization method, internal standard method, external standard method, and standard addition method (also known as the overlay method). The peak area (or peak height) percentage method is the simplest but least accurate. It is only suitable when the sample is composed of homologous compounds, or when rough quantification is acceptable.

In comparison, the internal standard method offers the highest quantitative precision. This method quantifies using the response values relative to a standard substance (referred to as the internal standard), which is added separately to standard samples and unknown samples. This approach helps offset errors introduced by fluctuations in operational conditions, including injection volume.

As for the standard addition method, it involves quantitatively adding a standard substance of the analyte to the unknown sample. The quantification is then based on the increase in peak area (or peak height). While the sample preparation process is similar to the internal standard method, the calculation principle is derived entirely from the external standard method. The quantitative accuracy of the standard addition method should fall between that of the internal standard method and the external standard method.

07 Method Validation

Method validation aims to demonstrate the practicality and reliability of the developed method. Practicality generally refers to whether the instrument configuration is commercially available, whether the sample handling method is simple and easy to operate, whether the analysis time is reasonable, and whether the analysis cost is acceptable to peers, etc. Reliability includes the quantitative range, detection limit, method recovery, repeatability, reproducibility, and accuracy.