# Defining Blood Plasma and Serum Metabolome by GC-MS
## 5 Workflow of GC-MS Analysis of Blood Metabolome
### Gas Chromatography
#### 5.2.1 Injection  
Several microliters of the sample are uploaded into the GC system through an injection port (Figure 5). The port is generally heated to a high temperature sufficient for instantaneous sample evaporation but not exceeding its decomposition temperature. Depending on the purpose of the experiment and the concentrations of the target components, injection of the sample can be effected in three modes: direct, split, and splitless. As expected, each mode has its area of applicability, advantages, and drawbacks. Thus, the direct mode is used for thermally labile compounds to avoid contacting the hot injection port and direct the sample directly to the column. In splitless mode, the entire sample is fed to the chromatographic column, but it is vaporized at the injection port. This approach allows the analysis of low-copy compounds. In split mode, only a part of the total sample volume after evaporation enters the column. Dilution of the highly concentrated analyte with gas is intended to make the peaks well resolved and prevent overloading of the column. As expected, at a high split rate (1:400, when 400 parts of the carrier gas dilute one part of the vaporized sample), a low amount of sample injected into the column leads to the low sensitivity of the method. On the other hand, when the split ratio is low (for example, 1:30), the introduction rate into the column increases as the sensitivity increases.  
The prepared sample enters the chromatographic system through the injection port, which is heated to a high temperature sufficient for instant vaporization of the sample. The majority of GC systems perform two types of sample introduction: split (when the sample is \"diluted\" by a highly pure carrier gas) and splitless (when most of the sample is uploaded into the column). Alternatively, injection directly into the chromatographic column can be applied to thermally unstable samples and trace analytes. The column itself is a long silica tube filled from the inside with various polymeric substances of different polarity (due to different functional groups in polysiloxanes) acting as a solid or liquid stationary phase. The column is placed in an oven, the temperature in which is maintained constant (isothermal mode) or changes during the wound (temperature-programmed mode). Two capillary columns with different characteristics can be connected in series through a modulator for advanced separation. After separation, eluents get into the detector.  
Highly pure H\({}_{2}\), N\({}_{2}\), and He are used as a mobile phase, carrying a gaseous sample through the column with a minimal effect on the process of chromatographic separation and subsequent mass spectrometric detection. The required level of chromatographic separation primarily determines the choice of carrier gas. Among the three popular gases, 99.9999% He has a priority for complex plasma and serum matrices; N\({}_{2}\) is also widely used due to its low operational cost. H\({}_{2}\) is less popular because it is flammable and not entirely inert: it can react with mixture components at high temperatures.