Comprehensive Guide to the Application Methods of GC and GC-MS

I. Sample Preparation

(I) Collection and Sampling

1.Determination of Sample Sources

• It is determined according to the research purpose. For example, in environmental analysis, samples are collected from rivers, soils, the atmosphere, etc.; in food testing, various food samples are obtained. Gaseous samples can be collected using gas sampling bags, syringes or specialized gas sampling devices. Liquid samples can be directly collected with appropriate containers, and solid samples may require pretreatment such as grinding and crushing.

2.Consideration of Sampling Representativeness

• The internal components of solid samples may be unevenly distributed. For example, in ore analysis, useful minerals and impurities may be distributed differently in different parts of the ore. Multiple sampling points and mixing are required for analysis. Liquid and gaseous samples may have differences in composition at different depths, regions or spatial positions, so the sampling points should be set reasonably.

3.Determination of Sampling Purpose and Target Analytes

• Before sampling, it is necessary to clarify the purpose of sampling. For example, it may be to detect pollutants in the environment, evaluate product quality, or study metabolites in organisms. The target analytes are determined according to the sampling purpose, which will directly affect the subsequent sampling methods and tool selection. For example, if volatile organic compounds in the air are to be detected, the target analytes may include benzene, toluene, xylene, etc., and appropriate sampling equipment and methods need to be considered when sampling, taking into account the characteristics of these organic compounds.

4.Determination of Sampling Locations and Times

• Sampling Locations: For environmental samples, the selection of sampling locations is crucial. For example, when monitoring the water pollution of a river, factors such as the location of pollution sources and the direction of water flow need to be considered, and sampling points are set upstream, midstream and downstream of the pollution sources respectively to fully understand the degree and range of pollution. For industrial product quality testing, sampling locations may be different links on the production line, such as raw material warehouses, production workshops, and finished product warehouses.
• Sampling Times: The time factor is also very important. For samples with seasonal variations, such as pesticide residues in crops, sampling needs to be carried out in different growth seasons and pesticide application periods. For ambient air quality monitoring, different meteorological conditions (such as wind direction, temperature, humidity) and times (such as day and night) will affect the concentration and distribution of pollutants, so a reasonable sampling schedule needs to be formulated according to the actual situation.

5.Sampling Records

• Basic Information Recording: Record the source of the sample in detail, including the specific location of the sampling site (such as geographical coordinates, detailed address), sampling time, sample name and other basic information. This information is very important for subsequent analysis and result interpretation. For example, when analyzing the quality of a batch of imported food, accurate source information can help trace the production area and transportation process of the food, and timely measures can be taken when problems are found.
• Sample State Recording: Record the state of the sample at the time of sampling, such as the appearance (color, shape, texture) of solid samples, the transparency of liquid samples, whether there are precipitates or suspended substances, and the odor of gaseous samples. This state information can provide a reference for subsequent analysis and help judge whether the sample is contaminated or has changed during the sampling process. For example, when collecting water samples, if the color of the water sample is abnormal or there is an abnormal smell, it may indicate that there are pollutants in the water or chemical reactions have occurred.

6.Consideration of Sample Preservation Conditions

• Temperature Control: According to the nature of the sample, an appropriate preservation temperature is selected. For some samples that are volatile and easy to decompose, such as enzymes and volatile organic compounds in biological samples, low-temperature preservation is usually required. For example, when collecting blood samples for detecting hormone levels in the blood, the samples need to be stored in a low-temperature environment (such as 4 °C or -20 °C) to prevent hormone decomposition. For some samples that need to maintain their activity, such as microbial samples, specific temperature conditions are also required. Some microorganisms need to be stored at low temperatures, while others need to be stored at temperatures suitable for their growth.
• Light Protection: For samples that are sensitive to light, such as drugs containing photosensitive compounds and certain natural pigments, light-proof containers, such as brown glass bottles, need to be used for preservation. Light may trigger chemical reactions in the samples, resulting in changes in sample components. For example, when analyzing plant extracts containing chlorophyll, since chlorophyll is sensitive to light, light should be avoided during preservation to prevent chlorophyll degradation.
• Prevention of Oxidation or Reduction Reactions: For some samples that are prone to oxidation or reduction reactions, measures need to be taken to prevent these reactions from occurring. For example, when collecting water samples containing ferrous ions, in order to prevent ferrous ions from being oxidized, an appropriate amount of reducing agents such as ascorbic acid can be added to the water samples, and at the same time, well-sealed sample bottles are used to reduce the contact between the water samples and the air.

(II) Sample Pretreatment

1.Extraction

• For complex samples, it is necessary to extract the target analytes from the sample matrix. For example, in the analysis of pesticide residues in plants, liquid-liquid extraction is often carried out using organic solvents such as acetonitrile and n-hexane. For solid samples, the accelerated solvent extraction (ASE) technique can also be used to make the solvent better penetrate into the samples under high temperature and high pressure, thereby improving the extraction efficiency.

2.Purification

• After extraction, the samples are purified using methods such as solid-phase extraction (SPE). For example, in the detection of drug components in biological samples, SPE columns are used to remove impurities such as proteins and fats, so that the target compounds can be enriched and purified.

3.Concentration

• If the concentration of the target analytes in the extract is low, concentration can be carried out. Usually, a rotary evaporator is used to make the solvent volatilize under reduced pressure, thereby increasing the concentration of the target analytes.

4.Derivatization

• Principle: Derivatization is to convert the target compounds in the sample into derivatives that are more suitable for analysis through chemical reactions. This is mainly because some target compounds may have properties that are not easy to detect, such as poor volatility, too strong or too weak polarity, and poor thermal stability. Through derivatization, these properties can be changed, enabling them to be better separated and detected in GC or GC-MS.

Application Scenarios and Examples:

• Improvement of Volatility: For some compounds with relatively strong polarity, such as organic acids and organic amines, their volatility is low, and it is difficult to directly conduct GC analysis. Taking carboxylic acids as an example, they can react with alcohols (such as methanol) to form esters. The volatility of ester compounds is usually better than that of carboxylic acids. For example, when analyzing organic acids in wine, the organic acids can be derivatized into corresponding methyl esters, which can make it easier to conduct gas chromatography analysis.
• Enhancement of Detection Sensitivity: Some compounds themselves have a low response on the detector. After derivatization, the signal of these compounds on the specific detector can be enhanced. For example, when using an electron capture detector (ECD) to analyze certain halogen-containing compounds, the target compounds can be derivatized into derivatives containing more halogen atoms, which can significantly improve their response signals on the ECD and thus improve the detection sensitivity.

1. Digestion

• Principle: Digestion is mainly used to process samples containing metal elements, especially some complex solid or liquid samples, such as soils, biological tissues, and foods. Its purpose is to destroy the organic substances in the samples so that the metal elements are released in the form of ions for subsequent analysis. The digestion process is usually carried out under high temperature and high pressure using strong oxidants or acids.

Application Scenarios and Examples:

• Soil Heavy Metal Analysis: When detecting the heavy metal content in soil, the metal elements in the soil are often combined with organic substances or wrapped in the crystal lattice of minerals. Through digestion, these metal elements can be released. For example, by using a mixed acid of nitric acid and perchloric acid to digest soil samples, heavy metal elements such as lead, cadmium, and copper in the soil can exist in the solution in the form of ions, and then can be detected by methods such as atomic absorption spectrometry (AAS) or inductively coupled plasma mass spectrometry (ICP-MS).
• Detection of Metal Elements in Biological Samples: When analyzing metal elements (such as iron, zinc, mercury) in biological tissues (such as liver, blood), digestion is also required. Because the metal elements in biological tissues are combined with organic components such as proteins, digestion can destroy these organic components and release the metal elements. For example, by using a mixed solution of hydrogen peroxide and nitric acid to digest liver tissues in a microwave digestion instrument, the metal elements in them can be conveniently quantified for analysis.

1. Filtration and Centrifugation

Principle:

• Filtration: Filtration separates the solid particles in the sample from the liquid part through devices such as filter paper, filter membranes or filters. It is based on the fact that the size of the solid particles is larger than the pore size of the filtering medium, so they are retained on the filtering medium, while the liquid part passes through.
• Centrifugation: Centrifugation uses the centrifugal force generated by the centrifuge to separate different components in the sample according to the difference in density. Components with higher density will gather at the bottom of the centrifuge tube, while components with lower density will be close to the tube mouth.

Application Scenarios and Examples:

• Filtration: When analyzing dissolved organic matter in water samples, the water samples may contain solid impurities such as silt. By filtering through filter paper, these impurities can be removed to obtain clear water samples containing dissolved organic matter for subsequent operations such as organic matter extraction. In the analysis of pharmaceutical preparations, for drug solutions containing insoluble excipients, the excipients can also be removed by filtration to analyze the drug components.
• Centrifugation: In biochemical experiments, the separation of cells from cell culture medium is a common application of centrifugation. For example, in the process of microbial fermentation, microbial cells can be separated from the fermentation broth by centrifugation, and then the metabolites or cell components in the cells can be analyzed. In blood analysis, centrifugation can separate blood cells from plasma, facilitating the detection of various components in the plasma (such as proteins, drug components).

II. Selection of Appropriate Chromatographic Columns

1.Selection of Stationary Phase According to Analyte Properties

• For non-polar analytes, such as alkane compounds, non-polar or weakly polar stationary phases, such as polydimethylsiloxane (PDMS), are selected. This stationary phase can separate analytes according to their boiling point differences. For polar analytes, such as alcohol and aldehyde compounds, polar stationary phases, such as polyethylene glycol (PEG), need to be selected, which can achieve separation through the polar interaction between analytes and the stationary phase. Consider the chemical structure and functional groups of analytes. For example, compounds containing aromatic rings may have better separation effects on stationary phases containing phenyl groups because of the π-π interaction between them.

2.Determination of Chromatographic Column Specifications

• The length of the chromatographic column is generally between 15 and 60 meters. Longer chromatographic columns can provide higher separation degrees, but the analysis time will also be correspondingly extended. For example, in the analysis of complex multi-component mixtures, chromatographic columns with a length of 30 - 60 meters may be required to ensure good separation of each component. The inner diameter is usually between 0.25 and 0.53 millimeters. Chromatographic columns with smaller inner diameters require less sample volume and have higher requirements for instrument sensitivity; chromatographic columns with larger inner diameters can handle larger volumes of samples, but the separation efficiency may be slightly lower. For information on how to select chromatographic columns according to the properties of analytes and the specifications of chromatographic columns, please refer to this article.

III. Optimization of Instrument Parameters

1.Carrier Gas Selection and Flow Rate Setting

• Commonly used carrier gases include helium, nitrogen, and hydrogen. Helium has high versatility and safety and is one of the commonly used carrier gases. Nitrogen has a good separation effect in some cases and has a relatively low cost. Although hydrogen has a high separation efficiency, it has certain risks. The carrier gas flow rate is generally between 1 and 10 mL/min. If the flow rate is too high, the separation degree may decrease, and if the flow rate is too low, the analysis time will be prolonged. The optimal flow rate can be determined through experiments. For example, standard mixtures can be analyzed at different flow rates, and changes in separation degree and analysis time can be observed.

2.Optimization of Injection Modes and Injection Volumes

• Injection Modes: The main injection modes include split injection and splitless injection. Split injection is suitable for high-concentration samples and can prevent column overload. Splitless injection is suitable for low-concentration samples and can introduce all samples into the chromatographic column to improve the detection sensitivity.
• Injection Volumes: The injection volume is usually between 0.1 and 10 μL, and the specific injection volume needs to be determined according to the sample concentration, chromatographic column capacity and detection sensitivity requirements. For example, for the analysis of high-concentration pure substances, the injection volume can be smaller, such as 0.1 - 0.5 μL; for complex trace analysis, a larger injection volume may be required, such as 5 - 10 μL.

3.Temperature Program Setting

• The initial temperature is generally set according to the lowest boiling point component in the sample to ensure that this component can elute under appropriate conditions. For example, for a sample containing low-boiling organic solvents and high-boiling organic compounds, the initial temperature can be set to 40 - 50 °C. The heating rate is usually between 3 and 10 °C/min and is adjusted according to the complexity of the sample and the separation requirements. The final temperature should be higher than the boiling point of the highest boiling point component in the sample to ensure that all components can elute from the chromatographic column. For more information on how to optimize instrument parameters, please refer to this article.

If you want to know more about how to optimize instrument parameters, please read this article. How to Set Chromatography Instrument Parameters?

IV. Determination of Detection Methods (for GC-MS)

1.Ion Source Selection

• The electron impact ion source (EI) is one of the most commonly used ion sources. It can generate abundant fragment ions and provide structural information of compounds, and is suitable for the analysis of most organic compounds. The chemical ionization source (CI) can obtain a mass spectrum with a stronger molecular ion peak, which is useful for analyzing compounds that are prone to fragmentation or for determining the molecular weight of compounds.

2.Mass Analyzer Setting

• The quadrupole mass analyzer is widely used. It has the characteristics of fast scanning speed and moderate resolution and is suitable for the rapid analysis of multi-component samples. The time-of-flight mass analyzer (TOF) has high resolution and a wide mass range and can provide accurate molecular weight information. It has advantages in the analysis of complex mixtures and the identification of unknown substances. The parameters of the mass analyzer, such as scanning range and scanning speed, are set according to the analysis purpose and sample type.

3.Data Acquisition and Processing Parameters

• Determine the type of data to be collected. For example, the full scan mode can obtain a complete mass spectrum for compound identification; the selected ion monitoring (SIM) mode can improve the detection sensitivity and is used for the quantitative analysis of target compounds. Set the data acquisition frequency, time window, and data processing algorithms, such as peak identification, integration, and calibration.

V. Problems and Countermeasures in GC and GC-MS Sample Collection and Sampling

(I) Sample Representativeness Problems

1.Deviation Caused by Non-uniformity

• Solid Samples: If they are solid samples, such as ores and soils, their internal components may be unevenly distributed. For example, in ore analysis, useful minerals and impurities may be distributed differently in different parts of the ore. If samples are only collected from a certain part of the ore, the analysis results may not represent the composition of the entire ore.
• Liquid and Gaseous Samples: For liquid samples, such as industrial wastewater, the composition of wastewater at different depths or different regions may be different. The distribution of gaseous samples in space may also be uneven. For example, the concentration of pollutants in the atmosphere may be different at different heights and in different wind directions. If the sampling points are not set reasonably, the collected samples cannot accurately reflect the overall situation.

2.Insufficient or Excessive Sampling Amounts

• Insufficient Sampling Amount: If the collected sample amount is too small, the target analytes may not be detected. For example, in trace analysis, if the sample amount is too small, the content of the target substances may be lower than the detection limit of the instrument, and accurate analysis results cannot be obtained. When analyzing trace organic pollutants in water, too little water sample may result in too low a pollutant concentration to be detected.
• Excessive Sampling Amount: Excessive sampling amount may lead to difficulties in subsequent processing. For some samples that require complex pretreatment, excessive sample amounts will increase the workload and cost. For example, when extracting and analyzing a large amount of biological tissues, excessive tissues will require more organic solvents for extraction, increasing the cost and may also affect the analysis results due to incomplete extraction.

(II) Sample Contamination Problems

1.Contamination of Sampling Containers

• Residual Impurities: The sampling containers themselves may contain impurities, which will contaminate the samples. For example, plastic sampling bottles may release some organic compounds, such as plasticizers. If such bottles are used to collect water samples for organic matter analysis, the plasticizers may interfere with the detection of target organic matter.
• Incomplete Cleaning: If the sampling containers are not cleaned thoroughly, the substances remaining previously will be mixed into the newly collected samples. For example, if a container used for collecting food additives was previously used for other samples containing similar components and was not thoroughly cleaned, cross-contamination will occur.

2.Contamination Caused by Environmental Factors

• Airborne Pollutants: When collecting samples, especially for samples that are sensitive to airborne pollutants, such as high-purity chemical reagents or biological

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