Sample extraction techniques Introduction Plants, animal tissues and food Aqueous samples
Introduction Different concerns with organic and inorganic analysis 1. Organic analysis use extraction and elemental analysis use digestion 2. Almost all trace organic analysis need clean-up and enrichment. Elemental analysis in many cases can do direct instrumental determination. Only metal, geological sample and sea water need enrichment/clean up. 3. A clean environment is very critical for ultra trace elemental analysis but has much less effect on organic trace analysis 4. In case of organic trace analysis, the result need qualitative confirmation using a different method. Sample extraction techniques Sample collection Plants, animal tissues and food Solvent extraction with a Warning Blender Extraction/ Digestion Aqueous samples Separatory funnel liquid-liquid extraction; solid-phase extraction (SPE); solid-phase microextraction (SPME); Purge & Trap (volatile analytes only). Cleanup/ Enrichment Solid samples (soil) Soxhlet extraction; supercritical fluid extraction (SFE), solvent extraction by tumbling or shaking, microwave assisted extraction. Instrument analysis Result Confirmation Dr. Wan Hai Bin CM4241- Trace Analysis 1 Dr. Wan Hai Bin Sample preparation for organics Blend with solvent Suitable for plants, animal tissues, and food. Sometime used for soil samples Normal procedure: 1) Blend 50-100 g sample with 100-200 mL of solvent for 1-3 min; 2) Suction-filter the mixture in a Buchner porcelain funnel ; 3) Blend the filter cake a second time with another 100-200 mL solvent; 4) Combine the two filtrates for further treatment. Extraction techniques Cleanup techniques Enrichment techniques Integrated techniques Requirements: understand the principles, advantages and disadvantages of important techniques. CM4241- Trace Analysis 3 Sample extraction techniques Dr. Haibin Wan email: [email protected] or [email protected] Tel: 65616602 (home) Dr. Wan Hai Bin CM4241- Trace Analysis 2 Dr. Wan Hai Bin CM4241- Trace Analysis 4 Sample extraction techniques Sample extraction techniques Extraction solvents Use solvents that are miscible with water (acetonitrile, acetone) which is easier to penetrate into the cell and get the analytes out. Solvents of low polarity (e.g. hexane, toluene) will give low extraction efficiency. In case of high fat sample, low polarity solvents can be used Soxhlet extraction For animal tissue, some Celite (a type of inorganic powder) or cellulose can be added to facilitate filtration of the sticky mixtures. Exhaustive extraction and gives highest extraction efficiency Advantages Often used as bench mark for evaluation of other extraction methods This technique is suitable for solid samples only. Suitable analytes are semi volatile organic compounds, pesticides, PCBs Advantages Well established technique Fast, simple, low cost, and widely used. Disadvantages Disadvantages Very time consuming (12-48 hours) In case of some stubborn analytes, this extraction may not be complete. Consumes more solvents than other extraction techniques. Dr. Wan Hai Bin CM4241- Trace Analysis Some thermal labile analytes may be decomposed due to the long extraction time and hot solvent 5 Sample extraction techniques Dr. Wan Hai Bin CM4241- Trace Analysis 7 Sample extraction techniques Soxhlet Extraction 1) Sample is placed inside a cellulose or ceramic thimble and placed in the extractor. Supercritical Fluid Extraction Above certain temperature and pressure, a substance will behave neither like a gas nor a liquid. This point on a phase diagram is called critical point for this substance. The temperature at the critical point is called critical temperature. The area above and to the right of the critical point is called supercritical region. Condenser 2) Solvent in the flask is boiled and the vapor rises to the condenser through the bypass arm. 3) Solvent vapor condense to become warm liquid and percolate through the sample in the thimble and returns to the flask. 4) The solvent re-boils, and the cycle is repeated until the sample is completely extracted, and the extract is in the lower flask. Dr. Wan Hai Bin Supercritical region A liquid in this region is called supercritical fluid. CM4241- Trace Analysis 6 Dr. Wan Hai Bin CM4241- Trace Analysis 8 Sample extraction techniques Sample extraction techniques Properties of supercritical fluid Application Solvent strength that can dissolve many substances For solid matrix and analytes of low polarity. Still not widely used. Reason: There is no urgent need as other familiar techniques can meet most of the needs. The instruments are still not robust enough for routine analysis. High diffusivity to allow fast mixing Low viscosity to allow fast penetration of solid matrix Application to sample extraction Many substance can become supercritical fluids at their supercritical points (ethylene, 9.3 degrees/50 bar; water 374 degrees/220 bar). Advantages of SFE No waste disposal problem and environment friendly Fast extraction (<30 minutes) The extraction power can be adjusted by changing the pressure or adding modifiers (such as methanol). CO2 becomes the best choice due to its low cost, safety, and convenience (31 degrees and 73 atm is easy to achieve). Disadvantages The instruments available are still not robust enough Extraction is mainly for analytes of low polarity Dr. Wan Hai Bin CM4241- Trace Analysis 9 Dr. Wan Hai Bin 11 CM4241- Trace Analysis Sample extraction techniques Sample extraction techniques Solid-phase extraction (liquid-solid extraction) A technique for extracting organics from water Normal Steps 1. Adjust pH of the sample if necessary 2. Conditioning with a solvent compatible with the sorbent (e.g. methanol) 3. Add water sample to the cartridge 4. After all the sample has gone through the cartridge, blow away extra water 5. Rinse with a solvent to bring the analytes down 6. Concentrate the eluate if necessary Supercritical Fluid Extraction Instrumentation Dr. Wan Hai Bin CM4241- Trace Analysis 10 Dr. Wan Hai Bin Sorbent Vacuum CM4241- Trace Analysis 12 Solid phase extraction Sample extraction techniques Why adjust pH? Most common sorbent is silica bonded with C18. This sorbent has stronger adsorption to nonpolar compounds (it is like a reverse phase C18 column) Some analytes have different retention at different pH. The pH can affect stability of some analytes The extraction material (normally bonded with C18 or other groups) may break down if the pH is not appropriate. Graphtized carbon black is used for trapping polar compounds Why conditioning? The extraction material is normally highly hydrophobic (does not like water). You need a solvent to bring the two together (better reaction). It also help to remove contamination from the system. There are also some cartridges packed with silica gel, florisil, or alumina. They are normally used for cleanup purpose Dr. Wan Hai Bin CM4241- Trace Analysis 13 Dr. Wan Hai Bin Sample extraction techniques CM4241- Trace Analysis 15 Sample extraction techniques Solid phase extraction using disk extraction media Advantages of solid phase extraction 1. Assemble the apparatus. Add filter aid (small fibrous particle) to prevent clogging Consume very little organic solvent 2. Adjust pH of sample if necessary There is no emulsification problem (often occurred in liquid/liquid extraction) 3. Condition the extraction disk with an organic solvent (methylene chloride followed by a water miscible solvent such as methanol and acetonitrile) Easy to be automated 4. Add sample continuously. The resulting sample is of very small volume, so concentration is fast or unnecessary 5. After all the sample has passed the disk, blow dry the disk Disadvantages 6.Rinse the analytes down with organic solvents. Use a water miscible solvent first then a less polar solvent. In case of dirty water, the sorbent can be easily saturated or blocked. Some components in natural water may reduce the recovery 7. Concentrate the eluate if necessary Dr. Wan Hai Bin CM4241- Trace Analysis 14 Dr. Wan Hai Bin CM4241- Trace Analysis 16 Sample extraction techniques Sample extraction techniques Microwave assisted extraction Microwave assisted extraction normal steps Microwave is a radiation with wavelength between infrared and radio wave. Dry the sample by air dry or mixing with anhydrous sodium sulphate It can heat up materials by acting on the dipoles of molecules and movement of ions Allow the sample to cool down Extraction for ca 10 min at preset temperature and pressure Advantages Materials of high polarity is most effective absorber of microwave energy (e.g. water, acetone, methanol) Fast extraction, recovery comparable to Soxhlet extraction, much less solvent consumption (10% of amount used in Soxhlet or shaking extraction). In microwave assisted extraction, the extraction is accelerated by fast and homogenous heating. Disadvantages Pressurized container allows higher extraction temperature. Sample drying process can be long (several hours). Cooling down also need half an hour or longer Some thermal labile analytes can be decomposed Only suitable for dry solid samples Dr. Wan Hai Bin CM4241- Trace Analysis 17 Dr. Wan Hai Bin Sample extraction techniques CM4241- Trace Analysis 19 Sample cleanup techniques Purpose of cleanup Microwave assisted extraction Instrumentation requirement To remove substances that will adversely affect analysis (it is not for getting pure analytes). Can control temperature and pressure of the extraction These substance can either affect performance of the instruments Stir during the extraction or interfere with the separation/detection of analytes Safety feature like solvent leak sensor and over pressure vent Considerations in selection of cleanup techniques Properties of sample matrix (type of interference) properties of analytes Concentration level to be analyzed final instrumental analysis Dr. Wan Hai Bin CM4241- Trace Analysis 18 Dr. Wan Hai Bin CM4241- Trace Analysis 20 Sample cleanup techniques Sample cleanup techniques Selection of the two phases Available cleanup techniques The extracts are normally polar, water miscible but can also dissolve in non polar solvents liquid/liquid partition Column chromatography using various adsorbents The other solvent for partitioning has to be less polar and not miscible with water Gel permeation chromatography Co-distillation To allow phase separation, the extracts need to be diluted with water. NaCl may be added to salt out the analytes and facilitate the phase separation. Ideal situation Weak solvent Strong solvent The extraction strength need to be considered also. Dr. Wan Hai Bin CM4241- Trace Analysis 21 Sample cleanup techniques Dr. Wan Hai Bin CM4241- Trace Analysis 23 Sample cleanup techniques The extraction strength of solvents Liquid/liquid partition ethyl acetate>CHCl3>CH2Cl2>toluene>hexane>petroleum ether Separation is based on the difference in solubility between the analytes and the interference. Commonly used solvents for partitioning Advantages CH2Cl2 easily concentrated due to low boiling point (+). Easy to remove as it stays at the bottom of the funnel (+), appropriate strength for most analytes (+). Much higher density than water make the phase separation fast and clear (+). It may affect instrument analysis as some GC detectors does not like it (-) . Simple technique, low cost, widely used. Disadvantages Cannot remove interfering substances of similar solubility as analytes. Hexane easily concentrated due to low boiling points (+), appropriate strength for non polar analytes (+), low toxicity (+), not good for extraction of polar analytes. May become difficult to separate due to emulsification The shaking duration and extent affect the recovery. Petroleum ether (saturated hydrocarbon with boiling points 30-60 degrees or 60-90 degrees) Low cost (+), mainly for non polar analytes, extraction rate not as good as hexane. Dr. Wan Hai Bin CM4241- Trace Analysis 22 Dr. Wan Hai Bin CM4241- Trace Analysis 24 Sample cleanup techniques Sample cleanup techniques Commonly used adsorbents Cleanup by gel permeation chromatography Alumina (Basic) Good for basic and neutral analytes(+), can cause polymerization and hydrolysis to some compounds, cannot use acetone and ethyl acetate as eluate. The separation is based on size of molecules. Large molecules come out first. Alumina (neutral) Good for neutral and reactive compounds (aldehydes, ketones, esters; less active than basic and acidic Alumina. A very effective method for removing lipids, fats, proteins, polymers, resins and other large molecules Alumina (acidic) Good for acidic analytes (they may be strongly adsorbed by neutral or basic alumina) These material can block HPLC or GC columns and contaminate the GC inlet system. Florisil A magnesium silicate type adsorbent with acidic properties (Florisil is the trade name from Floridin Co.); widely used for pesticides, chlorinated hydrocarbons, phthalate esters, nitrosamines, notroaromatics. (start with Florisil if not sure what adsorbent to use). Can be used with almost any type of organic analytes (no matter polar or non polar) The sample after this step is normally ready for instrumental analysis Silica gel A slightly acidic adsorbent, good for phenolic compounds and PCBs. Dr. Wan Hai Bin CM4241- Trace Analysis flow 26 Dr. Wan Hai Bin 28 CM4241- Trace Analysis Sample cleanup techniques Sample cleanup techniques Packings for the GPC column Alumina and Florisil: heat at 450-500 degrees overnight and then deactivate to required activity by adding certain amount of water (2-6%) Porous particles. Sephadex LH-20 Silica gel: Heat at 130-140 degrees overnight followed by deactivation with water (up to 10%). SX-3 Biobeads (a cross-linked divinylbenzens-styrene copolymer) They are all polar material. The separation of the analytes from the interfering substances is based on the difference in polarity. The columns can be used many times (allowing automation) The eluant must be organic solvent and dissolve the sample extracts well. As they are all very polar and have strong adsorption to polar compounds, the recovery of polar analytes could be low. Procedures Swell the packing and then pack the column (dimension 0.6m x 25mm id); calibrate the column; add sample extract (c.a. 1 mL); elute the column; collect the fraction containing the analytes; concentrate if necessary. Limits GPC cleanup cannot remove interfering substances of similar molecular size as the analytes. The interference may be removed by using suitable column or selective detectors. Dr. Wan Hai Bin CM4241- Trace Analysis 27 Dr. Wan Hai Bin 29 CM4241- Trace Analysis Integrated techniques Sample enrichment techniques Application of SPME Aqueous samples or headspace samples. The extracted analytes can be introduced into a GC or a HPLC system. Concentrate using Rotary evaporator Fast (+), Low temperature (+), easy to dry out (-), may lose sample if too fast (-) Advantages Snyder column Almost solvent free, easy to be automated, easy operation, normally no further cleanup is needed, low cost. Concentrate using Kuderna-Danish concentrator High recovery (+), can go down to very small volume (0.5 mL), slower than rotary evaporator (-) Concentration flask Concentration by blowing nitrogen can be used to reduce volume further (0.2 mL). Tedious (-) Receiver Water bath SPE also includes enrichment Dr. Wan Hai Bin Headspace Disadvantages CM4241- Trace Analysis The sorbent only absorb a fraction of the analytes from the sample. The quantitation is based on the distribution equilibrium of analytes between the aqueous phase and the sorbent. Not very good for quantitation (more suitable for pre screening), the results can be affected by the sample matrix, carryover from previous run if not treated properly. 30 Dr. Wan Hai Bin Integrated techniques SPME Procedures: SPME using a stirring bar 1. Immerse the sorbent segment in the water sample and stir the sample for 20-40 min. 2. Pull back the sorbent segment into the needle and insert the needle into a hot GC inlet. The stir bar is coated with adsorbent. During fast stirring process, the analytes are quickly caught by the sorbent. The extraction is near exhaustive as the surface of the sorbent is much larger than conventional SPME 3. Analytes desorbed from the sorbent and are focused at the head of the column. Advantages Fast, more quantitative than conventional SPME, better sensitivity. 4. Remove the needle from GC inlet and start GC run. Disadvantages Need to use thermal desorption unit, thermal labile analytes may decompose 5. Heat or rinse the sorbent to remove any organics left from previous run 31 CM4241- Trace Analysis 32 CM4241- Trace Analysis Integrated techniques Dr. Wan Hai Bin Liquid sample Sorbent Polymer Magnet bar Thermal desorption unit Carrier gas GC Dr. Wan Hai Bin 33 CM4241- Trace Analysis Integrated techniques Integrated techniques Headspace sampling Headspace sampling --- How to increase the detection limit? The technique is based on the following equation: Add salt to aqueous sample -- Example: P i = Ki Xi ; P i = k Ai ; Xi=(k/K)Ai septum Xi concentration of compound i in liquid phase. headspace Ai Peak area given by the headspace sample. Pi Vapor pressure of compound i in headspace. Liquid sample K Henry constant To establish the equilibrium between the liquid and the gas phase, a closed container must be used. Add water to organic solution to reduce solubility of analytes Add water to DMF (N,N-dimethyl formamide) containing 120 ppm styrene can increase the response of styrene: 0% water, 4; 10% water, 9; 50%, 144; 90%, 504. Increase the temperature A compound with higher volatility and lower solubility in the liquid phase will have higher concentration in the headspace. The response may double if temperature is increased by 10 degrees. If the temperature is too high, decomposition may occur. Maximum is 150-180 o C Increase the temperature also increase the amount of analytes in the headspace Dr. Wan Hai Bin Addition of NaCl to Aqueous sample containing 2% ethanol increases the response by 3 times. Replace the NaCl with K2CO3, the increase become 5 times. This approach is not very effective to non polar analytes or analytes of low polarity (e.g. toluene, hexane). 34 Dr. Wan Hai Bin CM4241- Trace Analysis 36 CM4241- Trace Analysis Integrated techniques Integrated techniques Headspace sampling Headspace sampling --- How to calibrate? Application : volatile analytes in liquid or solid samples. (volatile organic compounds in water, residue solvent in pharmaceutical products, alcohol in blood, flavor components in beverages, trace organic material in solid material). External standard calibration: 1) Make a set of solution of similar composition as the sample matrix; 2) Add different amount of analytes to these solutions; 3) Carry out analysis under the same condition as those for sample analysis; 4) draw calibration curve using the response and the concentration data. Manual injection: low cost (+) but the temperature of the syringe must be maintained during the injection. To use this calibration method, you must know the composition of your sample matrix (e.g. QC samples, drinking water). Automatic headspace sampler: more reproducible, can transfer the headspace sample directly to a GC inlet Method of standard addition 1) Put sample in 4-5 sampling vials; 2) add different amount of analyte to these vials (volume added should be the same); 3) carry out Headspace-GC analysis; draw calibration curve based on the response and the added concentration. Injection Fill loop Dr. Wan Hai Bin 35 CM4241- Trace Analysis Dr. Wan Hai Bin 37 CM4241- Trace Analysis Integrated techniques Integrated techniques Headspace sampling --- How to calibrate? Instrumentation for purge and trap Method of standard addition This approach is suitable to samples whose composition is unknown. (waste water, wine sample given by a customer). Must remember the equation Xi=(k/K)Ai is correct only when the concentration of analytes is low. If the concentration is too high, you may not get a linear relationship. In such case, you can dilute the sample and repeat the analysis. Peak area 25 20 15 10 5 60 40 20 0 20 40 60 80 100 Concentration (ug/mL) 38 Dr. Wan Hai Bin CM4241- Trace Analysis Integrated techniques Integrated techniques Purge and Trap sampling Construction of the trapping tube Applicable to analytes of boiling points below 200 degrees and insoluble or slightly soluble in water. For compounds of high solubility in water, the recovery may be low. Sample matrix can be aqueous or soil (dispersed with water). This is a very sensitive method ( can go done to 1 ppb level). To prevent cross contamination, sample should be pre- screened using HeadspaceGC approach. Activated charcol: for Freon or compounds of similar volatility Trapping tube High purity He or N2 40 CM4241- Trace Analysis Glass wool Silica gel: for compounds with boiling points below 35 degrees and higher than Freon Tenax (diphenylene oxide polymer): for compounds of boiling points above 35 degrees. OV-1 (methyl silicone) coated on inert powder: for heavy hydrocarbons. It can extend the lifetime of the trap. Purging tube The trap can be reused after conditioning at 180 degrees for ca. 10 min. Activated charcol Silica gel Desorb direction Adsorption activity Dr. Wan Hai Bin Glass wool Tenax Tenax 3% OV-1 3% OV-1 Analyte inlet Dr. Wan Hai Bin 39 CM4241- Trace Analysis Dr. Wan Hai Bin 41 CM4241- Trace Analysis Sample cleanup techniques Cleanup by column chromatography Integrated techniques Comparison of Headspace sampling and Purge and Trap technique Headspace Design of the column: Purge&Trap must be easy to clean Reservoir ---------------------------------------------------------------------------------------Sample Matrix aqueous/solid aqueous/solid Analytes volatile volatile Normal procedures: Na2SO4 Change the solvent to a non polar type Principle equilibrium in G and L total extraction Detection limit above 1 ppm above 1 ppb Cost less more Add the concentrated extract to the column Sample handling easier more complicated Continue the elution with the eluting solvent Analysis time shorter longer Collect the fraction that contain the analytes Requirement to GC simpler more Concentrate the eluate if necessary Dr. Wan Hai Bin Sample Concentrate the extract from the extraction Slide 42 Pre-elute the column with the eluting solvent adsorbent Cotton wool 42 CM4241- Trace Analysis CM4241- Trace Analysis
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