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Analytical Chemistry
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JoVE Science Education Analytical Chemistry
Gas Chromatography (GC) with Flame-Ionization Detection
  • 00:00Overview
  • 01:06Principles of Gas Chromatography
  • 03:54Instrument Initialization
  • 05:37Running the GC
  • 06:32Representative Results: Quantification of Caffeine and Palmitic Acid in Coffee
  • 07:28Applications
  • 08:59Summary

气相色谱 (GC) 与火焰电离检测

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Overview

资料来源: 实验室的博士 B.吉尔 Venton-弗吉尼亚大学

气相色谱 (GC) 用来分离和检测小分子化合物,在气相中。该示例是一种气体或液体蒸发在进样口。通常情况下,分析了化合物都是小于 1,000 Da,因为它很难蒸发较大的化合物。GC 是受欢迎的环境监测和工业应用的因为它是非常可靠,可以几乎连续运行。GC 通常用于在应用程序中地方小,易挥发的分子检测,并与非水溶液。高效液相色谱法测量水样中更受欢迎,可以用来研究更大的分子,因为分子不需要蒸发。而 LC 是更常见的分离极性物的 GC 青睐为非极性分子。

气相色谱流动相载气通常氦已,因为是其低分子量和化学惰性。施加压力和流动相移动通过列分析物。分离是使用涂层与固定相柱来完成的。开管毛细管柱是最受欢迎的专栏,有涂层的毛细管墙上固定相。固定相往往衍生物的聚二甲基硅氧烷,5 — — 10%的官能化来优化分离的组。典型的功能组是苯基、 氰基丙基或 trifluoropropyl。毛细管柱通常是 5-50 米长。窄列有更高的分辨率,但需要较高的压力。也可以在固定相涂在珠包装在列中用填料的塔。填料的塔是短,1-5 m.开放管状毛细血管具有一般首选因为他们允许更高的效率,更快的分析,并且有更高的能力。

火焰离子化检测 (FID) 是碳的检测样品中含量的气相色谱中有机物的良好一般探测器。后列,样品被烧热,氢空气火焰中。碳离子是由燃烧产生的。而过程的整体效率较低 (仅有 1 中 105碳离子生产中火焰离子) 的离子总量是碳的样品中含量成正比。电极用于测量从离子电流。FID 是破坏性的探测器,热解的完整示例。FID 是受不可燃气体和水。

Principles

Procedure

1.初始化的气相色谱法 打开的氦载气和空气,调整仪器上的压力表。 打开列烤箱到很高的温度 (通常 250 ° C 或以上) 烤列中。不超过列中的最高温度。这将删除任何污染物。让它在运行示例前烘烤至少 30 分钟。 2.制作方法文件 在软件中控制仪,输入方法文件所需的值。首先,设置自动进样器设置。设置的预运行的冲洗,冲洗,运行后,冲洗与?…

Applications and Summary

GC is used for a variety of industrial applications. For example, it is used to test the purity of a synthesized chemical product. GC is also popular in environmental applications. GC is used to detect pesticides, polyaromatic hydrocarbons, and phthalates. Most air quality applications use GC-FID to monitor environmental pollutants. GC is also used for headspace analysis, where the volatiles that are evaporated from a liquid are collected and measured. This is useful for the cosmetic and food and beverage industries. GC is used for forensic applications as well, such as detecting drugs of abuse or explosives. In addition, GC is useful in the petroleum industry for measuring hydrocarbons. The extensive applications makes GC a billion dollar per year worldwide market.

Figure 3 shows an example of how GC could be used in the food industry. Figure 3 shows a chromatograph of artificial vanilla (black) and real vanilla (red). GC can be used to identify the real sample, which contains a large peak for vanillin but does not contain a second peak for ethylvanillin.

Figure 3

Figure 3. GC-FID chromatogram of vanilla samples. Both imitation and real vanilla show large peaks at 4.7 min due to vanillin, the principle component of vanilla. However, imitation vanilla also has a large peak at 5.3 min, which is due to ethylvanillin, a compound not present in large quantities in real vanilla.

Transcript

Gas Chromatography, or GC, is a technique that is used to separate, detect, and quantify small volatile compounds in the gas phase.

In GC, liquid samples are vaporized, then carried by an inert gas through a long, thin column. Analytes are separated based on their chemical affinity with a coating on the inside of the column.

Because GC requires that analytes are vaporized to the gas phase, the instrument is ideally suited for volatile, nonpolar chemicals less than 1,000 daltons in mass. For larger, aqueous, or polar molecules that are difficult to vaporize, liquid chromatography is a useful alternative. This video will introduce the basics of gas chromatography, and illustrate the steps required to analyze the chemical species in a non-aqueous mixture sample using a gas chromatograph.

The GC instrument has five essential components. First, an injection port is used to introduce the sample into the instrument. Next, a heating chamber vaporizes the sample and mixes it with an inert gas. The inert gas, such as helium or nitrogen, carries the vaporized sample through the system. Combined, the carrier gas and sample make up the mobile phase. Next, the mobile phase enters the heated column, separating the analytes as they flow through. Lastly, a detector records the gases as they exit the column, or elute, and sends data to a computer for analysis. The most critical component of the instrument is the column. The column is a capillary with a stationary phase matrix coating the inner walls. Alternatively, columns can be packed with matrix-coated beads. The stationary phase is usually modified polydimethylsiloxane, which is ideal for resolving nonpolar molecules. Its separation properties are refined by adding 5–10% phenyl, cyanopropyl, or trifluoropropyl groups.

Analytes with low chemical affinity for the stationary phase move quickly through the column, while molecules with high affinity are slowed as they adsorb to the column walls.The length of time a compound spends inside the column is called its retention time, or Rt, and allows compounds to be identified. The detector sits at the end of the column and records gases as they elute. Flame-ionization detection, or FID, is widely used because it senses carbon ions, allowing it to detect virtually any organic compound. In FID, analytes combust in a hydrogen-air flame as they exit the column, producing carbon ions that induce a current in nearby electrodes. The current is directly proportional to the carbon mass, thus, the concentration of the compound can be determined. The final result is a chromatogram, which is a plot of FID signal vs time, showing each eluted component as they exit the column. Ideally, each peak will have a symmetrical, Gaussian shape. Asymmetrical features, such as peak tailing and peak fronting, can be due to overloading, injection problems, or the presence of functional groups that stick to the column, such as carboxylic acids.

Now that the principles of gas chromatography have been discussed, let’s take a look at how to carry out and analyze a gas chromatography analysis in the laboratory.

Before running an experiment, turn on the helium gas tank. Open the software on the computer, then bake out the column to remove any potential contaminants. Set the oven to a high temperature, typically 250 °C or above, and bake the column for at least 30 min.

Next, adjust the autosampler settings. Set the number of pre- and post-run rinses to clean the column between samples.

Use a sample volume of 1 μL and set the split ratio setting to program the instrument to accept only a fraction of the input. Adjust the flow rate of the carrier gas, and use established settings or trial and error to find the ideal pressure.

Now enter the temperature settings for the experiment. For an isothermal run, enter the temperature and the time for the separation. Alternatively, for a temperature gradient, enter the starting temperature and hold time, the ending temperature and hold time, and the ramp speed in °C per min.

Set the time for the column to cool between runs for either a gradient or isothermal run.

Finally, set the sampling rate and the detector temperature. The detector must always be hotter than the column to prevent condensation. After all the settings are programmed, save the methods file.

Activate the detector by opening the hydrogen tank valve and ignite the flame of the FID. The instrument is now ready for sample analysis.

To run the sample on the GC, first fill a vial with a wash solvent, such as acetonitrile or methanol. Prepare the sample, being certain to use glass syringes and glass vials as plastic residues can contaminate the GC.

Now add the prepared sample to a vial with a pipette. Fill at least half way, so that the autosampler syringe will be fully submerged. Then, load the wash and sample vials into the autosampler rack. Before running the sample, zero the baseline of the chromatogram on the computer software. Data can be collected either as a single run or using a batch table for multiple runs. Press “start” to run the sample.

In this example, caffeine and palmitic acid levels in coffee were analyzed using GC with FID. Caffeine is smaller and less polar, so it is less attracted to the column, and elutes first. Palmitic acid, which has a long alkane chain tail, elutes later due to a higher affinity with the stationary phase.

Because peak dimensions are proportional to carbon mass, the concentration of each component can be determined from its respective peak area on the chromatograph and compared to standards of known concentration.

The effect of column temperature was also explored. At 200 °C, samples moved through the column twice as fast as the sample run at 180 °C. Note that while peak heights change, the area under the curve remains constant.

GC is an important technique for chemical analysis, and is widely used in scientific, commercial, and industrial applications.

Due to the simplicity of GC, chemists routinely use it to monitor chemical reactions and product purity. Reactions can be sampled over time to show product formation and reactant depletion. The chromatograph reveals product concentrations and also the presence of unintended or side products.

GC is commonly used in tandem with mass spectrometry, called GS-MS, to unambiguously identify chemicals in samples or air. Mass spectrometry, or MS, separates molecules based on their mass to charge ratio, and enables the determination of compound identities. GC-MS is a powerful tool, as GC first separates complex mixtures into individual components, and MS gives precise mass information and chemical identity.

GC is routinely used in air monitoring to detect volatile organic compounds, or VOCs, which may arise from environmental pollution, pesticides and explosives. GC can be used to track and identify VOCs both indoors for headspace analysis and outdoors, for health, safety, and security.

You’ve just watched JoVE’s introduction to gas chromatography with FID. You should now understand the basic principles of gas chromatography and FID detection.

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JoVE Science Education Database. JoVE Science Education. Gas Chromatography (GC) with Flame-Ionization Detection. JoVE, Cambridge, MA, (2023).