Organic Geochemistry Methods I - Marine Organic Geochemistry

Marine Organic Geochemistry Practical considerations regarding sample storage, preparation, and safety (an organic geochemical perspective) Sample storage • Many organic compounds are biologically or chemically labile. • Susceptibility to: Enzymatic hydrolysis (e.g., DNA, Phospholipid Fatty Acids - PLFA) Photolysis (e.g., certain PAH - anthracene). Oxidation (e.g. polyunsaturated lipids) • Ideally, samples should be kept frozen, in the dark and under N2. < -80°C : minimal enzymatic activity - optimal. < -30°C: a good compromise. 4°C (refridgeration): OK for short periods of time room temp – ok once sample has been dried (for many compounds). *total lipid extracts are more stable than purified compounds/fractions Sample preparation • Drying of samples will deactivate enzymes, but can lead to oxidation of selected compounds. Options: • air-dry (40°C): OK for non-volatile, chemically inert compounds (may promote microbial activity) • freeze-dry under vacuum: good for semi-volatile compounds extract wet: good for thermally labile, and highly polar compounds • Practical considerations Potential Contamination Sources Materials compatible with organic geochemical analysis: • Glass • Teflon Aluminum foil (combusted, UV irradiated, or solvent-rinsed) • Stainless steel • Materials to avoid (contaminants): Parafilm (hydrocarbons) • Polystyrene (dissolves in many common solvents) • • Plasticizers (phthalates) (e.g., Tygon tubing) Silicone grease (silicones) • Finger grease (fatty acids) (you can detect lipids in 1/100th of a fingerprint by GC!) • Cleaning of reagents/labware • Combustion @ 450°C > 3hr (pyrex, quartz, aluminum) • Sulfuric acid based oxidizing solutions “Nochromix” (thermally “fragile” glassware or glassware for or volumetric measurement, teflon) • Certain Detergents • • Solvent rinsing (DCM, MeOH) Practical considerations Common solvents/reagents • Dichloromethane/methylene chloride (DCM) • Methanol (MeOH) • Hexane (Hex) Safety Issues • Many of solvent and reagents commonly used are toxic, carcinogenic, flammable, or exhibit the potential to form unstable products (e.g. peroxides) during storage (e.g. diethyl ether) that may lead to spontaneous combustion/explosions. • Always work with solvents/reagents in fume hood • Nitrile gloves offer good protection against most solvents • Safety glasses Building blocks of natural organic matter Lignin Protein Nucleic acids Cutin Polysaccharides Nucleic acids Tannin Polysaccharides Biopolymers Biopolymers Lignin Cutin Tannin Uncharacterized Protein Lipids Lipids Altered biopolymers “Geomacromolecules” Biochemicals Sedimentary organic matter Modes of Sample Analysis Bulk measurements • Optical assessement (identification of spores, pollen, algae, higher plant debris etc.) • TOC (%) • Elemental analysis (C,H,N,O,S) Isotopic composition (δ13C, δ15N, δD, δ34S, ∆14C) of bulk organic matter. • Advantages: • Potentially all inclusive Rapid, "easy" measurements, quick • No fractionation of the sample (except removal of inorganic species) • Disadvantages • Low information content Insensitive to subtle (yet often important) chemical variations • Modes of Sample Analysis Molecular-level measurements Advantages: • High biological information content based on molecular structure. Often can simultaneously isolate and quantify more than one molecular type. • • Ability to assign isotopic compositions to biomolecules from specific sources. Disadvantages: • Slow, and often procedurally difficult - multistage isolations. Intrinsically selective and biased. • • Very complex mixtures encountered - often can't identify all compounds isolated. Accurate quantification challenging. • Characterization of Solvent-Extractable Organic Matter Typical Procedure for Isolation and Characterization of Lipids Addition of recovery standards Sample Solvent Extraction Key steps • a. Extraction of Soluble Components with Solvents (total lipid extract, TLE). • b. Saponification (hydrolysis of ester-bound lipids, e.g. wax esters, phospholipids) • c. Separation of neutral compounds (alcohols and hydrocarbons) from acids • d. Acidification of carboxylic acid salts (convert to protonated forms) • e. Isolation of protonated acids using apolar solvent • f. Purification (column or thin-layer chromatography) • g. Derivatization (acids and alcohols) • h. Chromatographic separation of individual compounds. • i. Identification and quantification of compounds. Extract Residue + H or _ OH Other (estces) "Free" lipids "boond" lipids Liquid chromatogrophy ( S1=A1 ) etc Addition of Quantification Standards etc F1 Least Polar F2 F3 F4 Derivatization Gas chromatogrophy or High pressure liquid chromatogrophy Mass Spectrometry F5 More Polar Lipid Extraction Methods Soxhlet extraction • Recycling distillation of solvent(s) followed by condensation, percolation through sample and return to distillation flask Advantages: • Mechanically gentle process yet quite efficient extraction • Can perform elemental sulfur removal simultaneously (activated Cu) Cold water Thimble Disadvantages: • Lengthy process (>24 hrs) • Require azeotropic mixtures* to ensure efficient extraction. *Definition of an azeotrope – A liquid mixture of two or more substances that retains the same composition in the vapor state as in the liquid state when distilled or partially evaporated. sample Heat Heat Lipid Extraction Methods Ultrasonic extraction • Vibrational disruption of samples Advantages: • Cold extraction - good for thermally labile compounds Quick (a few hours) • • A sequence of solvents may be used Disadvantages: • Physically violent process - may cause breakdown of macromolecules and clays. • Labor intensive. Lipid Extraction Methods Supercritical Fluid Extraction (SFE) • Flow through of supercritical CO2 through sample. Advantages: • Ideal (in theory) - Supercritical fluid has the solvating power of a liquid (will readily dissolve compounds) and viscosity of a gas (will fully permeate the sample). • Quick (<1 hr). • Does not use large amounts of solvent to extract samples (environmentally sound). • Good for extraction of non-volatile components. Disadvantages: • Requires expensive equipment. • In practice it is difficult to extract a wide range of polarities. • Use of "modifiers" (e.g. Methanol) with CO2 partially but not completely overcomes this problem. Lipid Extraction Methods Accelerated Solvent Extraction (ASE) • Extraction of samples are elevated temperature and pressure • (e.g., 100°C, 1000 psi). Advantages: • Quick (ca. 15 min. / sample). • Low solvent consumption. • Solvent systems similar to Soxhlet extraction, so easy to optimize. • Automated (>20 samples can be processed as a batch). Disadvantages: • May not be appropriate for thermally labile compounds. • Expensive. Figure courtesy of Dionex Corporation. Used with permission. solvent sand GFF sample GFF sand extract Chromatographic Separation, Purification and Analysis Definition of Chromatography : "The resolution of material on two phases using chromatographic apparatus" Nomenclature: Technique • GC: Gas Chromatography* CC: Column (gravity chromatography)# • • TLC: Thin Layer Chromatography# • HPLC: High Performance/Pressure Liquid Chromatography* *Typically (but not exclusively) used as analytical tools (molecular level separation) #Typically used as screening or semi-preparative tools (compound class separation) Chromatographic Separation, Purification and Analysis Mobile phase: • developer (TLC) eluent (CC) • • carrier gas (GC) Stationary phase: • adsorption chromatography: • partition chromatography: surface active solid material fixed liquid (e.g, film coated on solid support) Solute: • A mixture of substances to be separated Chromatograph: • Instrument used for chromatographic separation Chromatogram • Trace showing separation achieved in a chromatographic analysis Principles of chromatography Chromatography uses two phases one of which is stationary and the other mobile. Chromatography is based upon use of different physical properties to induce separation of compounds or compound classes: • solubility • adsorption • volatility • size Polarity • Polarity is a relative term applied to solvents, solutes and adsorbents • • Compound(s) water • alcohols, esters, ketones • aromatic hydrocarbons • aliphatic hydrocarbons Properties strong dipole strong H-bonding weaker H-bonding Polarity very polar no dipole, but polarisable electron cloud less polar intermediate polarity least polar/apolar Principles of chromatography • • • • Adsorbent Alumina (Al2O3) Silica (SiO2) Charcoal Solute type basic/neutral acid/neutral Polarity polar polar non-polar/apolar In general: • (1) The polarity of organic compounds increases with an increase in the # of functional groups (e.g. sugars - polar; n-alkanes - apolar). • (2) The polarity decreases with increasing molecular weight • (3) Partition and adsorption chromatography both primarily depend on polarity differences between solutes. Partition depends on solubility in two liquids, but also on molecular size. Therefore members of a homologous series (e.g. n-alkanes) are best separated by partition chromatography. • (4) Partition chromatography has more resolving power than adsorption chromatography Chromatography Types • • • • • • • Mobile phase Gas Gas Gas Liquid Liquid Solid Stationary phase Gas Liquid Solid Liquid (film on a solid support) Solid Solid Chromatography type Not important Partition (GLC, abbrev. GC) Adsorption (packed column GC) Partition (RP-HPLC) Adsorption (TLC, CC, NP-HPLC) Not important Column (Gravity) Chromatography (CC) SOLVENT (ELUENT) Advantages: • Can perform large scale separations (several hundreds of milligrams of solute) Disadvantages: • Lower resolution than TLC or HPLC (see below) • Separation is performed "blind" Methodology: • The stationary phase is gravity packed into a glass column plugged with glass wool. • The sample is applied to the top of the column followed by the eluent(s) • The separated compound(s) are collected after they have passed through the column (eluant). SAMPLE ADSORVENT or Al2O3) (SiO2 SAND GLASS HOOZ ELUENT Column (Gravity) Chromatography (CC) Elution techniques: 1. Frontal analysis • Continual addition of two components (A+B) to column. Only is A is retrieved (B is adsorbed). • • Limited by capacity of the adsorbent to retain B 2. Displacement analysis/development A+B introduced onto the column followed by a more polar eluent. • Induces sequential elution of A , A+B and B • (*1&2 are used in Solid Phase Extraction (SPE) methods) 3. Elution development (most popular) Sequential addition of solvents increasing in polarity • • Weak eleunt (apolar)-> weak + strong -> strong eluent (polar) This is done by addition of strong to weak eluent over time. • • Gradient elution is a modification of above used in HPLC Factors limiting column efficiency Column Dimensions • Size of column determined by the difficulty of separation and amount of solute • For adsorption chromatography usually adsorbent : solute = 30:1 to 100:1 • For partition chromatography usually adsorbent : solute = 50:1 to 500:1 • Column efficiency usually increases with length/width ratio Particle Size • Smaller the particle size (i.e., > surface area) = better separation. CC mesh size typically 60-100 µm • • TLC mesh size 5 or 10 µm • HPLC mesh size 3 µm Pressure • The smaller the particle mesh size the more pressure is required in solvent delivery • Small particle sizes used in HPLC require pressures up to 5000 p.s.i. Time • The length of time required to afford separation Flow rate • Typically 0.3 - 3 column volumes per hour is optimal Thin-Layer Chromatography (TLC) Advantages: • Higher resolution than CC due to smaller particle size and larger surface area. • More reproducible (although sensitive to ambient temperature/humidity) • Can assess separation at end of development by spraying with fluorescent dye and visualizing under UV irradiation. Can run standards in separate "lanes" to check separation. Disadvantages: • Volatility losses due to exposure of sample on surface • Difficult to perform quantitative recovery of sample (requires scraping off of bands) • Limited sample capacity (<20 mg) Thin-Layer Chromatography (TLC) Methodology: • The adsorbent (e.g. silica gel ) is deposited as a thin layer (0.1 - 2mm) on a flat supporting surface (usually glass). The adsorbent is held with a binding agent (e.g. CaSO4.xH2O) • Silica plates are "activated" by removal of water from active sites (e.g. 120°C, 1hr) • The sample mixture (in a suitable solvent) is applied to the plate in a series of spots or a line and the plate is "developed" in a solvent of choice to afford a chromatographic separation. • By using mixtures of solvents can modify separation to optimize for particular compounds of interest. • "Iatroscan" - rapid version of TLC with Flame Ionization Detection Retention data • Rf = distance of compound from origin/distance of solvent from origin RS RC2 Time RO Rf = ( RC2 RO ) ( RS ) RO 2-Dimensional TLC Solvent D Solvent C Solvent B System Components: • Solvent Reservoir(s) • Pump • Injection loop • Column(s) • Detector(s) Solvent A High Performance Liquid Chromatography (HPLC) injector pump column Post-column pump collector Modes of operation • Isocratic operation: • Gradient elution: detector single solvent system binary, ternary or quaternary gradient system Chromatography Type • Normal phase: mobile phase less polar than stationary phase • Reverse phase: mobile phase more polar than stationary phase High Performance Liquid Chromatography (HPLC) Detectors • UV/Vis Photodiode array • • Fluorescence Refractive Index • • Chemiluminescence Evaporative Light Scattering (ELSD) • • Mass spectrometer (MS) HPLC separation of pigments Chlorophyll alteration products (RP-HPLC, MeOH/acetone) NH Carotenoids (RP-HPLC) N NH HN Pyropheophorbide Cholesteryl ester O C O 0 10 20 30 40 50 B-carotene IS Diatoxanthin Diadinoxanthin Fucoxanthin Chl -c Diatoms 19 Bu Fxn HPLC of PCEs in Black Sea sediments. After King and Repeta (1991). Ch1-c3 Absorbance 1+2 Ch -a 19 Hx Fxn O Prymnesiophytes Minutes (NB these compounds are too non-volatile for analysis by GC) NP-HPLC-ELSD of marine particulate lipid classes Polar Non-polar AB C D E H F I G 0 10 20 30 Time (min) Separation of lipid classes extracted from seawater spring-bloom particulate material (0.7-100 µm). A = nonpolar lipids like hydrocarbons, sterol esters and wax ester; B = triacylglycerols; C = sterols; D = chlorophyll a; E = monogalactosyldiacylglycerols; F = digalactosyldiacylglycerols; G = sulphoquinovosyldiacylglycerols; H = phosphatidyglycerols; I = phosphatidylcholine Gel Permeation Chromatography (GPC) or Size Exclusion Chromatography (SEC) Principle • Separates solutes according to molecular size • Use columns packed with gels of different nominal pore sizes • Molecules larger than this pore size are excluded from pores and therefore move with solvent and elute first . Smaller molecules follow a more tortuous route in and out of the pores and elute later. Methodology: • Similar instrumental set-up to HPLC • Use only isocratic solvent elution Gas Chromatography (GC) System Components • Gas supplies carrier gas detector gases • Injector • Column • Oven • Detector injector detector det. gases Carrier gas Data station column GC Oven Basic theory of Gas Chromatography A gas chromatogram usually represents a plot of detector response versus time (or volume of carrier gas) Intensity • Inj. Air W½ t’R tR Peak ht. Baseline Peak width Time Definitions • Baseline: The response of the detector to zero solute Peak width: obtained by drawing tangents to the peak and measuring at the baseline • Peak height: from baseline to top of peak • • Width at half height - self explanatory Retention time (uncorrected, tR): Time from injection to peak max. • • Retention time (corrected, t'R): Time from the air peak to peak max. Retention volume, VR = tR x flow rate • Relative retention time (α) = retention time of peak relative to another peak (usually a standard) • Chromatographic resolution Relative retention time (α) = retention time of peak relative to another peak (usually a standard) If α = 1 then A and B are inseparable If α >> 1 then still might not get good separation if peaks are v. broad • • RESOLUTION is the critical factor Resolution = 2xAB/w+y Intensity • • • Inj. AB Air x • • • y Time Resolution can be improved by: (a) increasing the efficiency of the liquid phase (solvent) (b) improving the efficiency of the column (increases # of theoretical plates) Improving Gas Chromatographic resolution SE-30 140°C 80°C 3 Intensity Intensity 3 2 1 2 1 Retention time Retention time 3 Carbowax 2 1 Retention time 80°C Intensity 140°C Intensity (a) Increase solvent efficiency • Increased temperature reduces efficiency by reducing time spent by solute in liquid phase • Use lower temps - more liquid phase interaction - more separation - longer time • Change liquid phase • e.g. SE-30 vs carbowax 3 2 1 Retention time The increased efficiency in the first solute is due to boiling point differences The peak order in the second solute is changed by differing solubilities Improving GC resolution (b) Column efficiency Key parameter: • HETP = Height Equivalent to the Theoretical Plates tR Intensity t’R Inj. Air w Time tn+1 Intensity Definition of plate • Plate = length of the column required for the solute to come into equilibrium in the distribution between gas and liquid phase. • Number of theoretical plates of a column, n = 16 x tR2/w2 • Number of effective theoretical plates, neff = 16 x t'R2/w2 • HETP = l/n (l = length of column (cm) • Require column height (length) to be small relative to the # plates • Separation number (Trennzahl value) Tz = the number of completely separated peaks that can be obtained between 2 homologous n-alkanes • Tz = [(tn+1 - tn)/(w½)n + (w½)n+1]-1 • Good columns give Tz values up to 40 tn (w½)n (w½)n+1 Time Improving GC resolution Column Efficiency (cont'd) • 3 main factors infuence column efficiency (band broadening) • These are known as the A,B and C terms in the Van Deemter equation: HETP = A+B/υ+C υ where υ = linear gas velocity Therefore υ works in 2 opposing ways, both working to make HETP large (detrimental) and small (beneficial). Optimal Separation HETP • Max. # of plates, Minimum HETP C term B term A term υ (increasing flow rate) Improving GC resolution A Term - Eddy diffusion (multiple path term) • Caused by different paths that solute takes along column. • Not affected by υ (flow rate) • N.B. This only applies for packed columns. • Minimize by using: (i) small particle size (ii) regular packing B Term - Molecular (longitudinal) diffusion • Due to longitudinal diffusion of the solute in the column • i.e. get peak spreading at low flow rates • Minimize by using: (i) increased υ (flow rate) (ii) increase molecular weight of gas Improving GC resolution C Term - resistance to mass transfer • Describes the difficulty of transferring a solute into and out of the liquid phase. This term should be minimized to induce rapid transfer between phases. • A function of: thickness of stationary phase (film thickness) film viscosity amount of stationary phase in the column • Minimize by using: (i) Thin film columns (ii) low viscosity phases (iii) lower flow rate to allow equilibrium Gas Chromatography using multicapillary columns Van Deemter Curve Comparison 1.0 0.9 HETP (mm) 0.8 SE-54 Multicapiltary Column 0.7 0.6 0.5 Conventional 5% diphenyl 95% dimethylpolysiloxane 0.4 0.3 0.2 0.1 0.0 0 30 50 90 120 150 Linear Velocity (cm/sec) 180 210 Relationship between log10 tR vs Carbon # Isothermal run • • Kovat's Retention Index (Kovat's Indices, I). Based on linear relationship between log10 tR vs Carbon #. I of any alkane on any liquid phase = 100 x Carbon # of alkane. This index system can be used for compound identification. Ix = 100z + 100[(log10t'Rx-log10t'Rz)/(log10t'Rz+1 log10t'Rz). For fatty acids, a different expression is often used, the Equivalent Chain Length (ECL). All the above is for ISOTHERMAL chromatography. However most analyses are done on a LINEAR TEMPERATURE PROGRAM. Ix(program) = 100z + 100 m'/y”. • • • • • • • Log10 tR Carbon # (homologous series) I = 1000 Intensity • • 300°C I = 1100 Inj. Cz x Cz+1 Time y‘ Intensity Retention Data 200°C m‘ Inj. Cz x Cz+1 Time GC Column Characteristics Column Types Packed columns • Typical dimensions: 5'-20' long x 1/16-1/4" diameter • Total effective plates: ca. 5,000 • Not used so much today, except for gas-solid chromatography (GSC) • Good for resolving gases and light hydrocarbons (e.g. methane, ethene, ethane etc.) Adsorbents in GSC: molecular sieve, silica gel, charcoal, alumina • Capillary columns • Typical dimensions: 10 m (30') – 100 m (300') long x 0.1, 0.25, 0.32 or 0.53 mm i.d. • Total effective plates: up to 90,000 • Support coated open tubular (SCOT) columns (walls of column coated with silica with liquid phase) • Wall coated open tubular (WCOT) *most commonly used today • • • Capillary columns Initially made of stainless steel Made of glass or, more recently, polyimide-externally coated fused silica Polyimide provides additional strength and flexibility. GC Column Characteristics • • • • • • • • • • Liquid phase Requirements: Non-volatile over chromatographic temperature range Thermally stable Must not chemically react with solute Should exhibit appropriate solubility characteristics for solutes Liquid phases are designated as “polar” or “non-polar” Non-polar phases ("boiling point" phases) Silicones CH3 CH3 CH3 e.g. methylsilicone CH3 Si O Si CH3 • • • where n=40,000-55,000 (mol wt. 3-4 × 106) Commercial names: OV-1, SE-30, HP-1, DB-1, BP-1 This is the most popular apolar phase O Si CH3 CH3 CH3 n GC Column Characteristics Polar phases A. Silicones 5% phenyl (SE-52, DB-5, HP-5) • • 5% phenyl, 1% vinyl (SE-54) • 50% phenyl (OV-17) • 75% phenyl (OV-25) B. Carbowax • 2 common polar liquid phases • Carbowax 6000 (Mw = 6000) • Carbowax 20M (Mw = 20,000) CH3 CH3 CH3 Si CH3 O Si O Si CH3 CH3 CH3 n H O CH2 CH2 OH n Polyethylene glycol Example gas chromatogram of waxes (alkane fraction) from Tobacco leaves FID INTENSITY C 31 C 33 C 29 C 27 53 55 57 59 61 63 65 RETENTION TIME (min.) This figure shows a typical gas chromatography trace of a hydrocarbon (alkane) fraction extracted and purified from a higher plant leaf sample. Note the predominance of long-chain (>C24) odd-carbon­ numbered n-alkanes (marked with circles) that is highly characteristic of higher plant leaf waxes. The chain-length distribution of these compounds is indicative of growth temperature. Gas Chromatography conditions • • • • • • • • • • • • • • • • • Carrier Gas Must be inert hydrogen, helium, nitrogen, argon Hydrogen is generally favored for most applications Helium is the usually the best alternative (esp. for GC/MS) Constant pressure vs constant flow Injectors Four main types: Split Split/splitless PTV On-column Temperature Control Two modes of operation isothermal (analogous to isocratic HPLC) temperature program Typical temperature program 50°C - 320°C @ 4°C/min. (≥ C40) Maximum temperature limited by stability of stationary phase. New columns can attain temperatures of 450°C (≥ C90) GC Detectors Thermal conductivity detector (TCD) • advantages: • simple • universal sensitivity (H2, CO2) disadvantages • • low sensitivity (10-10g/s) narrow linear range (104) • Flame Ionization detector (FID) • Advantages: • detects anything that burns high sensitivity (10-12g/s) • • broad linear range (107) Disadvantages • will not detect some materials (e.g. CO2) • GC Detectors Electron Capture Detector (ECD) Responds to halides, NO2 • Advantages: • Very high sensitivity for some compound types (10-14g/s) Very selective • • Disadvantages: Very selective (highly variable response factors) • • Poor linearity Flame Photometric Detector (FPD) Element selective detection of Sulfur and Phosphorus • Advantages: • High selectivity for S and P High sensitivity (10-11 to 10-12 g/s for S and P, respectively) • Disadvantages • • Non-linear (quadratic) detector response Quenching at high concentrations • GC Detectors Atomic Emission Detector (AED) • "GC-ICP" • Advantages: • Will detect emission lines from a wide variety of elements (presently up to 4 in any run) • High selectivity Good linear response • • Sensitivity generally as good as dedicated detectors • Disadvantages: Very expensive (ca. $100,000) • • Costly to operate and maintain Infrared Detector (IR) Mass Spectrometer (MS) • More to follow…… Pyrolysis-GC-AED of Miocene sediment Graph removed due to copyright restrictions. Gas chromatography Quantitation Methods • Usually better to measure peak area rather than peak height Detector responses vary depending on compound type • • Determination of response factors Internal standards • External standards • Other chromatographic methods • Preparative Capillary Gas Chromatography (PCGC) • Comprehensive 2-dimensional GC (GCxGC) (Capillary) Electrophoresis (CE) • Ion Exchange Chromatography (IEC) • • Supercritical Fluid Chromatography (SFC) Other Methods of Sample Sub-fractionation • Molecular sieves Urea/thiourea adducts • Derivatization • • Ion exchange chromatography Derivatization • Organic compounds with readily exchangeable hydrogens (acids, alcohols) generally exhibit poor chromatographic behavior on apolar GC stationary phases. Derivatization serves to “protect” polar group. Advantages Provides improved chromatography (better sensitivity and quantification). Prevents GC column degradation. Disadvantages • Derivatives often unstable • Cleavage of derivative group during MS analysis can dominate mass spectrum (good & bad) • Additional carbon introduces error in isotopic measurements from GC-irMS. Common derivatization methods: Fatty Acids • Methylation (BF3/MeOH or MeOH/HCl) to yield Fatty Acid Methyl Esters (FAMES) • Silylation (BSTFA) to yield trimethylsilyl (TMS) esters Alcohols (n-alkanols, sterols) • Acylation (pyridine, acetic anhydride) to yield acetates. • Silylation (BSTFA) to yield trimethylsilyl (TMS) ethers Recipe for Molecular Analysis of Lipids in Marine Particles & Sediments Sediment (dry) ASE extraction (e.g., DCM:Hex, 4:1, 100°C, 1000 psi) Residue Total Lipid Extract NH2 SPE F2 (Hex:DCM, 3:1) F1 (Hex) Hydrocarbons Ag+ SiO2 TLC Alkanes Urea adduction n-Alkanes GC-FID/GC-MS F3 (Hex:DCM, 9:1) Ketones/esters Saponification Alcohols Silylation (BSTFA) F4 (2% Formic acid in DCM) Fatty Acids Methylation (BF3/MeOH) Ketones TMS ethers FAMES GC-FID/GC-MS GC-FID/GC-MS GC-FID/GC-MS Recipe for Analysis of Bacterial Signature Lipids (Phospholipid-derived Fatty Acids, PLFA) Sediment (wet) Ultrasonic extraction MeOH:DCM, 2:1, 0°C Residue Total Lipid Extract SiO2 CC Hydrocarbons Diglycerides Phospholipids Mild methanolysis (NaOCH3) PLFA FAMES GC-FID/GC-MS Analysis of Pigments (chlorophylls, carotenoids) Sediment (wet) Stored frozen under N2 Ultrasonic extraction (Isoprop:CHCl3:MeOH, 0°C) Residue Total Lipid Extract GPC Chlorophylls RP-HPLC/DAD Carotenoids RP-HPLC/DAD Analysis of Polychlorinated Biphenyls (PCBs) in Sediments Dry sediments (~1g) is spiked with PCB-103 and PCB-198 and then extracted by pressurized fluid extraction (Hexane/Acetone; 1/1) (to extract the PCBs from the sediment) Reduce solvent volume to ~1 ml of hexane and react with ~1 ml of concentrated sulfuric acid (to remove most other organic compounds) Remove hexane layer from the acid and mix with activated copper (~1g) (to remove elemental sulfur) Charge hexane extract to a fully activated silica gel column (15 cm long x 6 mm width) and elute with 12 ml of hexane (to isolate a PCBs fraction) Reduce volume to ~100 ul, spike with PCB-110, and analyze on either a GC-ECD or GC-MS (to determine the concentration of each PCB congener) Key points: 1) There are 209 possible PCB congeners and for simplicity, each congener is assigned numbers 1 to 209. However, there are only ~150 congeners present in the commercial mixtures (Aroclors). This has been beneficial as those remaining compounds are used as recovery and quantification standards. In this flowchart, PCB-103 (pentachlorobiphenyl), -198 (octachlorobiphenyl), and -110 (pentachlorobiphenyl) are used as standards. 2) The sulfuric acid treatment is particularly helpful in removing color and interfering compounds as PCBs do not react with sulfuric acid. 3) This method could also be used for analyzing tissue samples as well.