MAP 2023- Sampling & LLE.pdf

Sample handling & liquid-liquid extraction Govert W. Somsen Sectie BioAnalytische Chemie Vrije Universiteit Amsterdam [email protected] MAP 17 November 2023 GC: wall-coated columns wall-coated open tubular (WCOT): standard format in GC stationary phase film fused-silica capillary thin film of s...

Sample handling & liquid-liquid extraction Govert W. Somsen Sectie BioAnalytische Chemie Vrije Universiteit Amsterdam [email protected] MAP 17 November 2023 GC: wall-coated columns wall-coated open tubular (WCOT): standard format in GC stationary phase film fused-silica capillary thin film of stationary phase (0.05 - 5 µm) on inner column wall gas flow plate height H for open tubular columns Van Deemter equation: no particles (no Eddy diffusion; no A term): H  A H  B  C  u0 u0 B  C  u0 u0 u0: velocity of mobile phase H = HB + HC HB  2 D m u0 HC Dm: diffusion coefficient of analyte in mobile phase d 2f d c2 u0  g( k ) u0  f ( k ) Dm Ds dc: Ds: df: f(k),g(k): column inner diameter diffusion coefficient of analyte in stationary phase film thickness stationary phase constants depending on analyte retention factor k Golay equation for open tubular GC columns d 2f 2 Dm d c2 H   f ( k ) u0  g( k ) u0 u0 Dm Ds u 0: linear velocity of mobile phase Dm: diffusion coefficient of analyte in mobile phase dc: column inner diameter Ds: diffusion coefficient of analyte in stationary phase df: film thickness stationary phase f(k): constant depending on analyte retention factor k g(k): constant depending on analyte retention factor k effect of column diameter dc on H d 2f 2Dm d c2 H   f ( k ) u0  g( k ) u0 u0 Dm Ds lowest H for small dc at higher flow rates: narrow peaks in short analysis times! effect of carrier gas on H diffusion constant Dm of analyte is carrier gas dependent d 2f 2Dm d c2 H   f ( k ) u0  g( k ) u0 u0 Dm Ds optimum H for He and H2 at higher flow rates: shorter analysis times! Dm,N2 < Dm,He < Dm,H2 He and H2 are preferred carrier gases column length L H depends on k 1  6 k  11 k 2 f(k ) 96 ( 1  k ) 2 g( k )  2k 3( 1  k ) 2 d 2f 2Dm d c2 H   f ( k ) u0  g( k ) u0 u0 Dm Ds H (cm) k = 5.0 k = 1.0 k = 0.5 u0 (cm/s) low retention factor k is favorable for low H (more narrow peaks) decrease k by using higher oven temperature: use temperature gradient ! column oven temperature programming final T T T-gradient cooling down start T time • separation of analytes with wide range of boiling points • sharp peaks (high N) for all analytes • high peak capacity (room for many peaks) temperature programming column: 15 m x 0.32 mm; 0.25 µm carrier gas: helium, 30 cm/s column oven temperature 100 °C 1. 2. 3. 4. 5. 6. 7. n-decane (Tb, 174 ˚C) n-undecane n-dodecane n-tridecane n-tetradecane n-pentadecane n-hexadecane (Tb, 287 ˚C) column oven temperature 50 °C for 1 min 50-120 °C at 20 °C/min sharp peaks for all compounds in much shorter analysis times! temperature programmed GC high resolution and fast GC long columns for high peak capacity column: 30 m x 250 µm T-gradient: 50-290 oC, 23 oC/min 93 peaks in 16 min short columns for fast GC column: 30 cm x 50 µm T: constant 9 peaks in 0.64 s flame ionization detector (FID) most used GC detector • carrier gas mixed with hydrogen • mixed with air and ignited: flame • combustion of organic compounds produces free electrons • signal proportional to flux of C-atoms • almost universal response (anything that contains C) • uniform sensitivity for all organics (98 – 102%) • quantification without calibration possible • sensitive: low LOD’s • wide linear range (> 105) GC vs. HPLC GC HPLC • for volatile analytes (or made volatile by derivatization) • non-volatile and thermo-labile analytes • very high peak capacity (capillary GC, T-programmed) • high detector sensitivity • main application areas: petrochemichal industry environmental analysis • better selectivity (more options) • larger sample volumes possible • main application areas: pharmaceutical industry biomedical and clinical analysis food analysis polymers chemical analysis analysis process of identifying and/or quantifying one or more compounds in a sample a chemical analysis object question analysis results sample sampling analysis information data processing interpretation answer analytes are part of samples analyte the compound to be determined (target compound) in sample analysis of target compound (analyte) often requires sample preparation (or sample pretreatment) time spent on a typical analysis sample pretreatment sample with analyte (is often a mixture) sample extract 1 determination of analyte requires highly selective method determination of analyte requires medium selective method interferents matrix sample extract 2 determination of analyte requires less selective method sample components that would cause incorrect results for the analyte the ’environment’ of the analytes; the bulk of the sample example: chocolate analysis question: does chocolate keep you awake? analytical question Chocolate: - cocoa beans - cocoa butter - sugar - milk powder how much caffeine en theobromine does a chocolate bar contain? Thiamine (B1) Riboflavin (B2) Niacin (B3) Vitamin B12 Vitamin E Calcium Iron Magnesium Manganese Phosphorus Potassium Selenium Zinc theobromine caffeine selected analytical method determine cafeïne en theobromine content by HPLC • Caffeine Theobromine HPLC can separate theobromine • and caffeine scheiden • HPLC can quantitatively detect theobromine and cafeine fat in chocolate interferes with the HPLC analysis • need for clear solution (no solid particles) for HPLC analysis chocolate sample handling sample preparation (step 1)   petroleumether: mixture of light alkanes (contains no ethers) grind piece of accurately weighed chocolate dissolve chocolate fat in petroleumether, centrifuge and remove supernatant chocolate sample handling sample preparation (step 2)   transfer chocolate residue to flask dissolve theobromine en caffeine from residue in hot water   centrifuge solution and filter supernatant result: clear solution suitable for HPLC analysis sample preparation objective reproducibly provide a representative solution from the sample that: - contains all the analyte(s) - is clear and homogeneous - is suitable for analysis by the selected method reasons for sample pretreatment  remove particulate matter  remove interfering sample constituents  prevent damage to the analytical instrument  provide compatibility with intended analysis e.g. solvent switch, salt removal, or analyte derivatization  achieve pre-concentration (analyte enrichment) sample preparation aspects ideal sample preparation procedure  provides quantitative recovery of analytes (>99%)  has minimum number of steps  hardly affects the performance of the analytical method  is easily automated sample pretreatment in practice  often is time-consuming (>60% of total analysis time)  often requires manual handling  may significantly affect accuracy and/or precision of analytical result due to errors sample handling typical sample-preparation methods method dilution description sample is diluted with a miscible solvent that is compatible with subsequent analytical method evaporation lyophilization sample liquid is removed using gentle heating and flow of inert gas aqueous sample is frozen, and water is removed by sublimation (freeze drying) under vacuum centrifugation liquid sample is placed in tapered tube and spun at high speed, and supernatant is decanted (ultra)filtration liquid is passed through a paper or membrane filter to remove and/or concentrate suspended particulates or large molecules deproteinization acid, salt or organic solvent is added to the sample to precipitate proteins followed by centrifugation liquid-liquid extraction sample constituents are partitioned between two immiscible phases, and analytes are recovered from one of the two phases sample types sample nature  organic (biological)  inorganic sample state  solids  semi-solids (e.g. creams, gels, suspensions, tissues)  liquids  gases biological samples example: blood - most analysed human sample for clinical diagnostics and pharmacology - routine determination of hundreds of different constituents as biomarkers - therapeutic drug montoring (pharmacokinetics) blood has a complex composition serum and plasma removal of blood cells serum plasma - blood liquid that remains after blood - liquid, cell-free part of the blood clotting and precipitation of clots obtained after centrifugation - clotting takes 10-15 min at room - blood has been treated with anti- temperature coagulant to prevent clotting anticoagulant - heparine - citrate - EDTA centrifugation deproteinization of plasma determination of metabolites in plasma addition of agent causes denaturing, aggregation and precipitation of proteins centrifuge filtration removing solid particles from solution removing or concentrating small particles and large molecules(>10,000 Da) ultrafiltration through membrane paper - pore size > 1 µm - by gravity membrane - pore size 0.1 - 1 µm - requires light pressure membrane - pore size 1 - 100 nm - requires mechanical pressure extraction extraction: transfer of compound(s) from one phase to another phase e.g. from solid phase (coffee, tea) to liquid phase (water) extraction is a basic separation technique liquid-liquid extraction LLE (1) transfer of compound(s) from one liquid phase to another immiscible liquid phase by bringing the phases in close contact most often extraction of an aqueous solution with an immiscible organic solvent LLE in practice LLE: single droplet microextraction LLE: industrial macroextraction phase partitioning Partitioning is resultant of molecular interactions between A and the org A organic and water phase: - hydrophobic interactions (“disaffinity” with water) - dipole-dipole interactions aq A - H-bonds - vanderwaals forces P depends on: - properties compound (analyte) - properties organic phase - temperature P is partition coefficient logP for octanol/water extraction solvent immiscible with water density < 1 kg/l org toluene ethyl ether ethyl acetate aq butyl alcohol aq org density > 1 kg/l chloroform dichloromethane fraction extracted what amount of analyte A is extracted from the water phase to the organic phase? org aq ntot  norg  naq A A norg = mol A in organic phase ntot = total mol A present Vorg = volume of organic phase naq = mol A in water phase Vaq = volume of water phase fraction extracted norg ntot  P P  (Vaq / Vorg ) % E  100 norg ntot percentage extracted (extraction yield or extraction efficiency) repeated extraction fraction extracted after n extractions = 1‒  (Vaq / Vorg )     P  (V / V )  aq org   n separation of compounds X and Y by LLE assuming Vorg = Vaq requirements for at least 99% separation: org aq X Y org OR aq Y X separation of multiple-compound mixture by LLE in individual components is difficult: use chromatography! next Today Exercises Sampling & LLE Friday 24 November at 10:00 Short Quiz 6 (Canvas) Friday 24 November at 13:30 Lecture Measurement Statistics Exercises Significance Testing