GC-MS Sample Preparation

Sample preparation methods for GC-MS

Gas chromatography mass spectrometry (GC-MS) sample preparation is performed on smaller and more volatile samples including environmental pollutants, industrial byproducts, food contaminants, pesticides, and metabolites of illicit and designer drugs. These molecules are more challenging to ionize and separate (i.e., resolve) using liquid chromatography mass spectrometry (LC-MS), and so are subjected to GC-MS instead.

GC-MS sample preparation can sometimes occur using LC-MS, as the following figure shows. Furthermore, advances in GC-MS technology, such as the introduction of the Orbitrap mass analyzer, have enabled analysis of larger and more complex compounds, propelling GC-MS into traditional LC-MS fields such as metabolomics.

GC-MS/LC-MS sample types overlap

Gas chromatography (GC) is first employed to volatize the sample, after which is it injected into the mobile phase, which is typically composed of a gas such as helium or argon. This mobile phase “carries” the sample until the stationary phase inside the GC column is reached. This stationary phase often consists of a chemical that is formulated to preferentially interact with select sample compounds.

Once at the stationary phase, the GC sample separates. This occurs because the GC column is ramped, or gradually heated, and compounds with lower boiling points are eluted first. Likewise, the pressure of the mobile phase can be varied to fine-tune separation. Finally, there are the chemical interactions between the sample compounds and the stationary phase to consider; weaker interactions will dissociate faster and elute earlier than stronger ones. A compound can be measured from the time of its injection until the time of its elution from the GC column; this is called the compound’s retention time.

Lower molecular weight compounds will elute from a GC column sooner than those with a higher molecular weight because of boiling point differences.

After this process of elution is complete, compounds undergo electron ionization (EI) or chemical ionization (CI) and become charged. They then undergo mass analysis within a mass spectrometer, and their unique mass (m) and charge (z) information is reported as numerical m/z ratios.

Once these values are reported, they are typically displayed as ion peaks. The values are also compared against previously compiled libraries of known mass spectra using analytical software programs. Matching spectra are identified and characterized. Mass spectrometry analysis enables the determination of compound molecular weight and formula, as well as functional groups.

GC-MS sample preparation protocols

GC-MS samples often contain dirty, labile, and volatile compounds that sometimes need further processing before they are introduced into the gas chromatograph. Different manual and automated sample extraction processes are often used prior to gas chromatography, and will differ based on the degree of selectivity required during sample preparation as well as the initial cleanliness of the samples.

Non selective -> Highly selective
  • Filtration/Dilute and shoot
  • Protein precipitation
  • Phospholipid depletion
  • Non-selective SPE (hydrophobic only)
  • Liquid-liquid extraction
  • C18 silica based SPE
  • C8 silica based SPE
  • C2 silica based SPE
  • Supported liquid extraction (SLE)
  • Mixed-mode SPE
  • Ion exchange SPE
  • Immunoaffinity, MIPs
Dirty Extracts -> Clean Extracts

Headspace sampling: In this technique, the liquid or solid sample is added to a glass vial until it establishes equilibrium. Some of the analytes vaporize from the liquid or solid and enter the headspace above the sample. If this gas phase is then directly injected into the gas chromatograph for separation, the method is referred to as static headspace sampling. If an inert gas is passed into the sample and the vaporized analytes accumulate on an absorbant surface or cryogenic trap, the method is termed dynamic headspace sampling or purge and trap. This method of analysis is often used with blood, cosmetics, plastics, solid, and materials with high water content.

Pyrolysis: This step is performed prior to GC-MS and involves heating samples to 120 C in either an inert environment or in air, resulting in their breakdown to smaller fragments. Samples that typically undergo pyrolysis include plastics, paints, dyes, resins, cellulose, wood, oils, and rubber, as well as larger samples that might be heavily crosslinked or insoluble. Because pyrolysis negates the need to use solvents, it is useful for identification of additives, solvents, and additives.

Solid phase extraction (SPE)

Solid phase extraction (SPE): This manual extraction technique typically involves the use of a solid packing material, often contained within a cartridge, to separate sample components, remove interferences, or fractionate the sample matrix. The sample itself will typically (although not always) be in liquid form. Samples frequently extracted via SPE include biological samples such as urine, saliva, and plasma, environmental samples such as water, and food products such as beverages.

Solvent extraction: Many analytes, such as pesticides, are highly polar and therefore amenable to extraction with solvents such as acetonitrile. Upon addition of salts such as magnesium sulfate, phase separation of acetonitrile from water occurs. This is the guiding principle behind the QuEChERS technology.

Solid phase micro extraction (SPME): Used before both gas chromatography or HPLC, SPME is a sample extraction technique that does not use solvent. Instead, a fused silica fiber, which is coated with a stationary (polymer) phase, is added to the sample. This fiber can be exposed to the vapor phase (headspace) above the liquid or solid or immersed into the liquid sample directly. The analyte(s) of interest diffuse onto the stationary phase; afterwards, the fiber is placed into the injection port of the GC-MS system and the analytes are transferred to the analytical column after thermal desorption. SPME is a fast and simple process that has obvious advantages for field application work. It is frequently used on high background samples such as food.

Stir bar sorptive extraction (SBSE): This method consists of exposing a magnetic stirring rod that is coated with a sorptive layer to the sample. Following exposure, the bar is removed, rinsed with deionized water, and dried. It is then placed inside the desorber tube and undergoes thermal desorption. During this process, the analytes are transferred onto the chromatographic system. SBSE has the advantage of higher sensitivity due to the larger amount of sorptive phase used; the trade-off is a longer extraction time and mostly manual procedure.

The following sample preparation protocols enable faster throughput and lowered costs as a result of automation:

Automated SPE: Automated extraction systems can process many SPE samples in just a few hours, enabling quick analysis of compounds of interest including pesticides, flame retardants, semivolatiles, nitrosamines, and steroids.

Accelerated solvent extraction (ASE): Automated batch sample processing of solids and semi-solid samples including pesticides, oils, nutritional supplements, and biofuels is frequently accomplished using automated extraction systems. In this way, compounds are quickly extracted from the sample using a minimum amount of time, solvent, and overall cost.

The following SPE columns offer a range of phases for the retention of polar, non-polar, ionic, and non-ionic compounds.

Compound of interest

organic soluble
water soluble
Non-polarModerately polarPolarAnionicCationic
Reversed phaseNormal phase & Reversed phaseNormal phaseAnion exchangeCation exchange

Polymeric (mixed mode) phase chemistries

Polymeric (mixed mode) phase chemistries

Polymeric or mixed mode columns contain spherical particles composed of porous graphitic carbon (PGC). This material is highly suited to polar compounds and performs better than silican or resin. Retention is determined by the strength of interaction between analytes and the Hypercarb surface.

HyperSep Retain PEP: This column contains polystyrene divinylbenzene material that is surface-modified with urea groups and is ideal with drugs and metabolites in biological matrices, environmental samples, and desalting of peptides in serum, plasma, or biological fluids.

HyperSep Retain-CX: With a versatile polymeric material that retains basic compounds, this column is typically used for the retention of drugs of abuse from biological matrices.

HyperSep Retain-AX: This column contains a versatile polymeric material that retains acidic compounds and is typically used in the analysis of acidic drugs of abuse (e.g., THC) from biological matrices.

HyperSep HypercarbSPE: This column contains a unique material that retains highly polar compounds. Typical applications of this column include the retention and separation of highly polar and challenging species.

Reversed phase silica phase chemistries

Reversed phase silica columns contain an alkyl-bonded phase for nonpolar to moderately polar compounds. The hydrophobic reversed phase material retains many nonpolar compounds and most organic analytes from aqueous matrices.

HyperSep C18: This column contains a highly retentive alkyl-bonded silica phase for non-polar to moderately polar compounds. Column applications include drugs and their metabolites in biological matrices, trace organics in environmental water samples, and toxins in food samples.

HyperSep C8: This column contains a less retentive alternative to C18 for non-polar to moderately polar compounds. It has the same applications as the HyperSep C18 column.

HyperSep Phenyl: This column offers an alternative selectivity for the retention of basic compounds. Applications include benzodiazepines in biological matrices and extraction of aromatic compounds.

Normal phase silica phase chemistries

These medium polarity sorbent columns are used in the isolation of polar analytes from nonpolar matrices such as hexane and oils. They are also used in reversed phase extractions of moderately polar compounds.

HyperSep Silica: Contains a polar sorbent used to retain analytes from non-polar matrices. Common applications include extraction of aldehydes, pesticides, carotenoids, aflatoxins, phospholipids, amines, herbicides, fat-soluble vitamins and fatty acids.

HyperSep Florisil: This column is ideal for the isolation of polar compounds from non-polar matrices. Its applications include the extraction of pesticides using AOAC and EPA methods, as well as PCBs in transformer oil.

HyperSep Cyano: Intended for the retention of polar compounds from non-polar matrices, this column is used for the retention of polar compounds from hexane and oil.

HyperSep Aminopropyl: This column contains a polar sorbent for both polar and anion exchange interactions. It is intended for petroleum fractionation, saccharide, and drug/drug metabolite applications.

HyperSep Diol: Intended for extraction of polar compounds, this column is used in normal phase extraction and purification of polar compounds applications.

Ion exchange phase chemistries

Ion exchange columns contain a strong ion exchange sorbent for the isolation of charged acidic and basic compounds including proteins, food pigments, phenols, antibiotics, pesticides, drugs, and nucleic acids.

HyperSep Strong Anion Exchanger (SAX): This column is used in the extraction of weak acids, with application areas including extraction of nucleic acids and surfactants, and the removal of acidic food pigments, and phenolic compounds.

HyperSep Strong Cation Exchanger (SCX): This column is used for the extraction of charged basic compounds, including extraction of antibiotics, organic bases, catecholamines, drugs, amino acids, and herbicides.

HyperSep Verify-CX: This non-polar and anionic phase column enables the analysis of drugs of abuse from biological matrices.

HyperSep Verify-AX: This non-polar and cationic phase column enables the analysis of acidic drugs of abuse from biological matrices (e.g., THC).

GC-MS column types

Gas chromatography is used to separate sample components and remove background contamination prior to mass spectrometry. Columns are typically made of metal or fused silica and coated with a variety of liquid films; the liquid film is called the stationary phase. Packed columns, which are composed of metal or glass, can also be used in GC.

The type of column and stationary phase used will differ depending on sample polarity and charge. GC-MS samples can be polar, nonpolar, or polarizable. They can also be non-ionic or ionic.  Going with the idea that “like isolates like,” columns that closely match the characteristics of the analyte should be chosen.

Polar GC Columns are designed for the separation of polar samples including alcohols, fatty acids, solvents, flavor compounds, aromatic isomers, herbicides and essential oils.

Mid-Polar GC Columns are intended for the separation of chlorinated and nitroaromatic compounds, hydrocarbons, polychlorophenyls (PCBs), drugs of abuse, pesticides, and essential oils.

Low-Polar GC Columns are composed of stationary phases such as 5% diphenyl/95% dimethyl polysiloxane and are commonly used to separate alcohols, phenols, aromatics, solvents, pesticides, amines, and free fatty acids.

Non-Polar GC Columns can be used in the separation of hydrocarbons, pesticides, essential oils, gasoline range organics (GROs), drugs of abuse, and refinery gases.

Ultrafast GC Columns are used in the separation of petrochemicals, flavorings, fragrances, ketones, alcohols, volatiles.

PLOT Columns are used to separate alkanes, hydrocarbon isomers, natural and refinery gases, and oxygenated compounds and solvents.

Application-specific Columns are used in the retention and analysis of specific compounds and metabolites including pollutants, dioxins, pesticides, herbicides, fatty acid methyl esters (FAMEs), amphetamines, codeine, morphine, and THC.

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