Gas chromatography-mass spectrometry. Agilent 7820a Applications • Foods: GC:MS is extensively used to analyse free fatty acids, sterols, esters, aromatic hydrocarbons and aldehydes. • Perfumes and Essential oils: GC:MS can separate, closely related molecules and distinguish between volatile alcohols and terpenes. • Hydrocarbons: GC:MS separates isomers with the same molecular weight, distinguishing between structural isomers and even cis/trans isomers. • Environmental: GC:MS is routinely used in identifying pesticide, organic pollutants and plastic contamination. • Biofuels: GC:MS can analysis and quantify the components of biofuels to establish whether they are a good substitute for the petroleum industry. Food Industry. Analysis of Plant Oils. • • • • • • • • • Free Fatty Acid (FFA) and Sterol Analysis by GC:MS. Fatty acids and long chain sterols must be derivatized before injecting into the GC:MS. This procedure involves a TMS agent and heating. The TMS agent is chosen according to the availability of the oxygen atom to be derivatized. Hindered alcohol groups require a specialised agent and longer heating times. We use TMS reagents purchased from Regis Technologies. http://www.registech.com/Markets/Chromatography/GCDerivatizationReagents/Silylation.html We can advise on pre-analysis clean up of samples to achieve the best results. Quantification can be achieved by internal and external standards. Kovats index can be calculated for chromatographic peaks. Mass Spectra synchronised with reliable databases and our own database. Typical analysis of FFA and Sterols, on oils, nuts, seeds and other plant material. TMS Trimethyl silanes Cholesterol Essential Oils and Perfumes • • • • • • Analysis of essential oil components. By mass spectral fingerprint, Kovats Index and comparisons with reliable databases. These oils contain dozens if not hundreds of distinct compounds, some of which are incredibly similar. These molecules have the same molecular weight MW: 204.35 g/mol, but are different compounds. GC:MS will separate them. Calculation of the Kovaks Index and the individual mass spectra aids in their identification. Zingiberene Alpha cedrene Beta caryophyllene Characterization of Essential Oils. Chromatography peaks tell you when and how much compound you have. The Mass Spectrum shows you the compounds fragmentation pattern, which acts as a molecular finger print. The Kovats Index, provides a dimensionless unit for comparison with other databases. Table Showing GC:MS Analysis Of 2 Oils Dill Geranium Citronella Oil GC: Retention time 6.509 7.427 8.037 8.098 8.177 8.981 9.291 9.495 10.382 10.474 10.705 10.876 11.755 Lemon Oil GC: Retention Compound Name time Limonene 5.099 Linalool 5.197 neoiso-Isopulegol 5.411 Citronellal 5.769 Isopulegol 5.816 Citronellol 5.994 Geraniol 6.470 Geranial 6.517 Citronellyl Acetate 6.929 Eugenol 7.304 Geranyl Acetate 7.430 beta Elemene 7.489 Napthalene 7.882 11.83 beta Cubebene 7.937 11.989 12.215 12.466 12.749 13.274 alpha Muurolene delta Cadinene Hedycaryol endo-1-bourbonanol Guaiol 8.027 8.101 8.433 8.587 9.163 Compound Name alpha Thujene alpha Pinene Camphene Sabinene beta Pinene Myrcene o-Cymene Limonene gamma Terpinene alpha Terpinolene Linalool Nonanal cis-Limonene oxide trans-Limonene oxide Epoxyterpinolene Citronellal Terpinen-4-ol alpha Terpineol Citral Lemon Grass Kovats Index (KI). • • • A dimensionless quantity that characterizes each GC:MS eluted compound, according to a linear retention index and eradicates instrument variations e.g. oven temperature, gas flow etc…….but not column differences. The retention times of a known C7-C40 alkane standard is used to calibrate KI. We have a software program that then generates the KI from the retention times of the standard alkane above and below the analyte of interest . Retention Times of Alkane Standard Tea Tree Reference Retention Times Name Octane Nonane Decane Undecane Dodecane Triddecane Tetradecane Pentadecane Hexadecane Heptadecane Octadecane Nonadecane Icosane Henicosane Docosane Tricosane Number of Carbons Retention Time 8 3.267 9 4.688 10 6.097 11 7.417 12 8.646 13 9.795 14 10.872 15 11.887 16 12.848 17 13.759 18 14.625 19 15.451 20 16.239 21 16.993 22 17.715 23 18.406 Lavender KI Database Comparisons • • • • We have our own database. GC:MS, Wiley database. Pherobase. http://www.pherobase.com/ Nist Chemistry Webbook. http://webbook.nist.gov/chemistry/ • http://lipidlibrary.aocs.org • All excellent databases for comparison of mass spectrums and KI index's. Hydrocarbons • • • • We have various GC:MS methods for analysing hydrocarbons of different volatilities. Equivalent Carbon Length (ECL) can be generated, to establish the number of carbons in each chromatographic peak. Mass Spectra and databases can then be used to identify each chemical species. APPLICATIONS: Phthalates Biofuels, insect pheromones, pesticides and herbicides. Equivalent Carbon Lengths ECLx = n + RTx-RTn/RTN-RTn EQUIVALENT CARBON LENGTH. A dimensionless quantity that characterizes each GC:MS eluted compound, according to a linear retention index and eradicates instrument variations e.g. oven temperature, gas flow etc…….but not column differences. The retention times of a known C7-C40 alkane standard is used to calibrate ECL. We have a software program that then generates the ECL numbers from the retention times of the standard alkane above and below the chromatographic peak of interest. Reference Retention Times Name Octane Nonane Decane Undecane Dodecane Triddecane Tetradecane Pentadecane Hexadecane Heptadecane Octadecane Nonadecane Icosane Henicosane Docosane Tricosane Number of Carbons Retention Time 8 3.267 9 4.688 10 6.097 11 7.417 12 8.646 13 9.795 14 10.872 15 11.887 16 12.848 17 13.759 18 14.625 19 15.451 20 16.239 21 16.993 22 17.715 23 18.406 ECL = Equivalent Carbon Length RTn= lower molecular weight alkane retention time. RTN= higher molecular weight alkane retention time. RTx = retention time of species you are interested in finding ECL for. Retention Times of Alkane Standard Similar to the KI, but often used for hydrocarbons and sometimes fatty acids. GC:MS Detection Limit • Generally, the HP GC:MS can detect chemical species down to the 10ng per µL, quantities. • If your analyte is below this concentration we can get access to an, Agilent 5975 series MSD • Agilent 7820A GC System. • With a detection limit of the order, 1ng per µL, depending on the chemical species. Special Projects. Centrifuge • Garbage in Garbage OUT! • We can advise on the best pre GC:MS clean up procedures. • We have a number of solid phase extraction procedures (SPE) we use to chemically separate sample components. As well as liquid extractions. • Physical extraction can also be achieved by centrifuge techniques. • For larger projects we can train and assist you, to use the GC:MS instrument and achieve the best results. Vacuum manifold, SPE Procedures