How must a compound be measured by gas chromatography (GC)?

Gas Chromatography Compound Measurement Requirements

For accurate compound measurement by gas chromatography, the compound must be volatile or rendered volatile through chemical derivatization, thermally stable during hot injection, and the entire analytical system—from injection needle to column—must be meticulously clean to prevent degradation and cross-contamination. 1

Essential Requirements for GC Measurement

Volatility and Derivatization

• Compounds must evaporate during the injection procedure to be measured by GC 1
• Non-volatile compounds require upfront chemical derivatization to increase stability and volatility, typically achieved through trimethylsilylation on completely dried samples 1
• The trimethylsilylation reaction works at room temperature with pyridine as catalyst and can be performed manually or automatically 1
• Derivatization success depends critically on solvent cleanliness, sample cleanliness, and complete dryness—not reaction time or temperature 1

Thermal Stability Considerations

• Hot injections preclude accurate quantification of thermolabile compounds such as di- and triphosphates (ATP, NADPH) 1
• Even after derivatization, thermal degradation can occur during injection, accelerated by degradation products from unclean injectors or the sample itself 1
• O-trimethylsilyl groups are stable, making GC-MS robust for sugars, phosphates, and hydroxy acids 1
• N-trimethylsilyl groups are less stable and may be lost from amines and amino acids, showing variable ratios of fully versus partially derivatized compounds across samples 1

Critical System Cleanliness Requirements

Injection System Maintenance

• Cleanliness of both sample and injector system is critical to GC-MS performance, from injection needle to involatile depositions in the liner and column beginning 1
• Liners must be kept meticulously clean and changed regularly; automatic liner exchangers help maintain analysis quality 1
• Empty guard columns should be employed to protect the analytical column, as involatile matrix components deposit on the guard column's start site 1
• The guard column can be cut during quality maintenance without compromising separations 1

Sample Preparation

• The sample is introduced into the inlet liner, volatilized, and the resulting vapor mixed with carrier gas 1
• Nonvolatile compounds like phosphatidylcholines can degrade in the injector, causing cross-contamination and catalytic degradation of target analytes 1
• Staff requires detailed training in maintenance protocols to ensure analysis quality 1

Optimal Compound Classes for GC

Best Suited Compounds

• Low-molecular-weight and volatile analytes including small species that don't retain well on LC and uncharged species that ionize poorly by electrospray 1
• Very-short-chain fatty acids and alcohols, hydroxy acids, sugars and monophosphorylated sugars, and sterols 1
• Amino acids, fatty acids, polyamines, aromatics, and catecholamines 1
• Essential oils and volatiles (GC is the only universally applicable method for these) 1

Alternative Derivatization Strategies

• Using multiple derivatization reactions enhances precision and accuracy when N-trimethylsilyl instability is problematic 1
• Trimethylsilylation for general metabolome profiling (sugars, hydroxy acids) 1
• Tertiary-butyldimethylsilylation specifically for amines and amino acids 1

Detection and Quantification

Mass Spectrometry Coupling

• GC typically uses electron ionization (hard ionization) yielding reproducible fragments for known compound identification 1
• Chemical ionization is softer and more commonly yields molecular ion peaks, observable even for high molecular-weight trimethylsilylated metabolites 1
• Single quadrupole detection is cost-effective and robust; TOF mass spectrometers yield better coverage and sensitivity 1

Quantification Robustness

• GC-MS does not suffer from ion suppression or differential adduct formation unlike LC-MS electrospray ionization 1
• Quantitative precision ranges from 2% RSD for sugars and acids to >20% RSD for low-abundance compounds affected by matrix composition 1

Common Pitfalls to Avoid

• Thermal decomposition during injection creates artifacts that may be misidentified as naturally occurring metabolites 1
• Matrix loads in samples affect N-trimethylsilyl linkage stability, causing variable derivatization ratios 1
• Inadequate system cleanliness accelerates analyte degradation 1
• Attempting to measure thermolabile compounds without appropriate method modifications leads to inaccurate quantification 1