Principles and Differences between GC-MS and LC-MS (2024)

Metabolomics requires the analysis of a complex and diverse array of metabolites in samples, which necessitates support from high-throughput, highly sensitive, and stable analytical technologies. Mass spectrometry, a powerful tool in metabolomics, enables the precise identification and quantification of metabolites. However, mass spectrometry typically produces reliable qualitative results for single components, and the diverse and chemically complex nature of metabolites can introduce significant interferences. Consequently, a single mass spectrometry instrument may not suffice to meet the analytical demands of metabolomics.

Chromatography-mass spectrometry (GC-MS or LC-MS) techniques address these challenges by leveraging the robust separation capabilities of chromatography to compensate for the limitations in mass spectrometric separation. Chromatographic separation, or chromatography, effectively separates various components within complex mixtures based on the principle of "like dissolves like." Components in a sample distribute differently between a stationary phase and a mobile phase, eluting at different times. The concept of chromatography was formally introduced in 1903 by Russian botanist M.S. Tswett, initiating the study of liquid chromatography. In the 1940s, A.J.P. Martin and R.L.M. Synge predicted the existence of gas chromatography, which came to fruition commercially in 1955. The first liquid chromatography instrument emerged in the 1960s.

From the 1950s to the 1970s, coupled techniques like GC-MS and LC-MS were developed. In addition, in 1967, Professor Csaba Horváth invented high-performance liquid chromatography (HPLC), significantly enhancing the separation capabilities of liquid chromatography. Around 2004, the introduction of <2µm particle size fillers in HPLC led to the emergence of ultra-high performance liquid chromatography (UHPLC). Currently, companies such as Shimadzu, Agilent, Thermo Fisher Scientific, and Waters dominate the chromatography market with their gas and liquid chromatography instruments.

Chromatographic separation techniques are primarily divided into two categories based on the mobile phase used: liquid chromatography (LC) and gas chromatography (GC). Since over 70% of metabolites typically lack volatility under normal conditions, liquid chromatography has become the predominant method for metabolomics analysis.

Domains of GC-MS and LC-MS techniques in the two dimensions of hydrophobicity and volatility(Bracket al., 2016).

Liquid Chromatography

Liquid chromatography (LC) utilizes a liquid as the mobile phase and typically a solid as the stationary phase. Depending on the type of stationary phase material used, it can be classified into paper chromatography, thin-layer chromatography, and column chromatography. In metabolomics analysis, high-performance liquid chromatography (HPLC) and ultra-high performance liquid chromatography (UHPLC) are frequently employed. HPLC is an advanced form of classical column chromatography that uses smaller particle sizes for the stationary phase, typically around 3.5-5µm, and operates under high pressures up to 5000 psi, significantly increasing the contact area between the solution and the stationary phase. This allows for the efficient separation of different components in a short amount of time. UHPLC builds upon HPLC by using even smaller particles, about 1.7-2µm, and higher pressures exceeding 25,000 psi to achieve separation in shorter chromatographic columns.

Based on different separation principles, HPLC/UPLC can be categorized into various types such as normal phase chromatography (NPLC), reverse phase chromatography (RPLC), ion-exchange chromatography (IEC), and size exclusion chromatography (SEC). Among these, RPLC is the most widely used in metabolomics, accounting for over 60% of chromatographic applications. It is particularly useful for analyzing lipid metabolomes and general metabolomes. In reverse phase chromatography, the stationary phase consists of non-polar materials such as C18, phenyl, C8, C3, etc. The mobile phase typically includes 1% acetonitrile for conventional metabolomes and 60% acetonitrile for lipid metabolomes, often mixed with isopropanol (in a 9:1 ratio with acetonitrile). During elution, more polar metabolites interact strongly with the mobile phase, while less polar components bind more to the hydrophobic stationary phase, resulting in the more polar substances eluting first.

In conventional metabolome analysis, the elution begins with a higher proportion of aqueous phase, gradually increasing the proportion of the organic phase as elution proceeds, allowing for the effective separation of more polar metabolites. In contrast, lipid metabolome analysis starts with a higher proportion of organic phase, with increasing amounts of aqueous phase over time, which aids in the elution of less polar substances such as lipids. Besides RPLC, hydrophilic interaction chromatography (HILIC) is also commonly used in metabolomics. HILIC primarily analyzes more polar substances, utilizing highly hydrophilic materials like silica for the stationary phase. The mobile phase usually consists of 95% acetonitrile. During elution, the hydrophilic stationary phase strongly adsorbs polar substances, and they interact less with the hydrophobic mobile phase, causing less polar metabolites to elute first, which is the reverse order compared to RPLC. Thus, HILIC is sometimes referred to as a reverse-reverse phase chromatography. In practice, while RPLC is the most widely used and capable of eluting most substances, HILIC often serves as a complementary method to RPLC, targeting the elution of more polar metabolites. Combining RPLC and HILIC techniques followed by mass spectrometric analysis can significantly enhance the identification of metabolites.

Gas Chromatography

Gas chromatography (GC) employs a gas as the mobile phase and a liquid or solid as the stationary phase. Unlike LC, the mobile phase in GC is typically an inert gas such as helium. GC primarily utilizes different liquid or solid stationary phases in the chromatographic column to adsorb metabolites. When a sample is introduced into the chromatographic column by the inert gas, the stationary phase exhibits varying affinities for different substances, causing different gas components to move at different speeds through the column. Substances with weaker adsorption are separated first, while those with stronger adsorption elute later. Gas chromatographic columns can be classified into non-polar, intermediate-polarity, and polar columns depending on their applications.

Common non-polar gas chromatographic columns include DB(HP)-1, DB(HP)-5, AC1, AC5, SPB-1, SPB-5, DM-1, DM-5, SP-2100, HP-101, etc. These columns primarily use stationary phases such as 100% polydimethylsiloxane, 5% diphenyl-95% polydimethylsiloxane, and 5% diphenyl-1% vinyl-94% polydimethylsiloxane, with increasing polarity corresponding to increasing proportions of diphenyl and vinyl groups. Non-polar columns are commonly used for separating hydrocarbons, amino acids, nitro compounds, phenols, esters, alcohols, etc. Intermediate-polarity gas chromatographic columns include DB(HP)-1701, AC10, SPB-1701, RT-1701, CP-Sil19CB, etc. These columns use stationary phases such as 50% diphenyl-50% polydimethylsiloxane, 14% cyanopropylphenyl-86% polydimethylsiloxane, and 50% cyanopropylphenyl-50% polydimethylsiloxane, with increasing polarity corresponding to increasing proportions of diphenyl, cyanopropyl, and phenyl groups. Intermediate-polarity columns are commonly employed for separating herbicides, insecticides, drugs, and amines, with widespread applications in pesticide residue analysis. Polar gas chromatographic columns, such as AC20, FFAP, PEG-20M, C-2000, etc., utilize stationary phases like polyethylene glycol, HP-INNOWAX (where FFAP is its reaction product with nitroterephthalic acid), carbon molecules, etc., and are often used for separating free fatty acids, phenols, amines, pesticides, etc.

Difference between GC-MS and LC-MS

In 1952, J.C. Holmes and F.A. Morrell developed the first gas chromatography-mass spectrometry (GC-MS) instrument. However, analyzing liquid substances with mass spectrometry required vaporization and ionization, presenting limitations to liquid chromatography-mass spectrometry (LC-MS) until the advancement of atmospheric pressure ionization (API) technology. It wasn't until 1972 that the first LC-MS instrument emerged, enabling the vaporization and ionization of liquid substances into the mass spectrometer. Subsequent developments in ionization techniques like electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI), along with enhancements in chromatographic separation technologies such as ultra-high performance liquid chromatography (UPLC) and high-performance liquid chromatography (HPLC), propelled chromatography-mass spectrometry coupling towards higher throughput and greater versatility.

When chromatography and mass spectrometry are integrated, the chromatography system segregates metabolites into a gradient series of substances, subsequently analyzed and identified by the mass spectrometer. The chromatographic elution time correlates with the mass spectrometry's operational duration. Presently, chromatography-mass spectrometry coupling encompasses gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS), each complementing the other with distinct focal points in practical applications.

In terms of sample applications, LC-MS is typically favored for analyzing biological samples such as animal blood plasma, tissues, cells, various plant parts, and microbial fermentations, where metabolites are predominantly non-volatile and readily ionizable. Conversely, GC-MS is preferred for volatile and thermally stable compounds, serving as a supplementary tool to liquid chromatography-mass spectrometry, particularly for volatile fatty acids, aromatic hydrocarbons, and flavor compounds. GC-MS necessitates derivatization of less volatile metabolites to lower their boiling points, while LC-MS sample preparation is comparatively simpler.

Regarding instrumental configuration, LC-MS commonly employs reverse-phase chromatographic columns (RPLC), hydrophilic interaction chromatographic columns (HILIC), and ionization methods like electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). LC-MS utilizes both positive and negative ion modes during ionization for comprehensive mass spectrometric analysis. Conversely, GC-MS utilizes weakly polar, moderately polar, or strongly polar chromatographic columns, along with ionization methods like electron ionization (EI), chemical ionization (CI), and negative chemical ionization (NCI), typically employing only one ionization mode and providing data in either negative or positive ion mode. While GC-MS offers superior separation efficiency due to faster gas molecule diffusion, LC-MS exhibits higher sensitivity, reaching levels as low as femtograms (10^-15), and has a broader dynamic range for detecting trace substances.

Concerning database applications, LC-MS primarily utilizes databases such as METLIN, MassBank, mzCloud, Lipidmaps, MS-DIAL, and company-built spectral and MS/MS databases. However, due to the complexity of metabolites detected, LC-MS databases may lack comprehensive information, resulting in incomplete annotations for detected metabolite ions. In contrast, GC-MS commonly employs databases like NIST, Fiehn, GMD, which provide more comprehensive metabolite information due to the relatively fewer types of volatile substances. Additionally, the availability of standard substances facilitates accurate quantitative analysis by referencing their spectral information. In practical applications, combining LC-MS and GC-MS enables comprehensive metabolome profiling, providing information on all detectable metabolites in samples.

Reference

1. Brack, Werner, et al. "Effect-directed analysis supporting monitoring of aquatic environments—an in-depth overview." Science of the Total Environment 544 (2016): 1073-1118.

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