Sample preparation techniques
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1 Sample preparation techniques Dragana Mutavdžić Pavlović, PhD, associated professor, Department of Analytical Chemistry, Faculty of Chemical Engineering and Technology University of Zagreb
2 Sample preparation Analytical procedure: Sampling Sample preparation Separation Detection Data analysis 2
3 Sample preparation Sampling GOAL: Obtain representative sample sample which contains all the ingredients in the same proportions as unit from the sample was taken for analysis Unit (1 big pool of wastewater or 1 t of sludge) 3
4 Sample preparation Sample preparation WHY is so IMPORTANT??? Problem with complex samples 4
5 5 Sample preparation
6 The basic concept of sample-preparation methods is to convert a real matrix into a sample suitable for analysis. Goals of sample preparation procedure: increasing the initial concentration of target analyte interference removal if necessary, to convert an analyte into a more suitable form; and, to provide a robust, reproducible method that is independent of variations in the sample matrix 6
7 Sample preparation Good sample preparation provides the ability to Simplify the chromatographic separation Removing matrix and co-eluting species enables better, more consistent quantitation Reduce analytical variability Higher, more consistent recovery Minimize matrix effects Less rework Increase column lifetime Fewer columns need to be replaced Reduce system downtime Less time spent with wrenches or waiting for service 7 Taken from Waters Corporation 2016
8 Sample preparation Concentrate target analytes More consistent quantification with higher signal to noise Quantitative low levels of analyte accommodating different detection methods and instruments (UV, DAD, FLD, MS) Increase signal to noise ratio Minimize matrix effects Elimination of ion suppression/enhancement Elimination of interference peaks Reduction of background noise SAMPLE PREPARATION is a prerequisite for most analytical procedures. Analysts have responded to this challenge. 8
9 Sample preparation Example: Baseline interferences 9
10 Sample preparation Example: Matrix causes ion suppresion in case of MS detection 10
11 Sample preparation How can turn these? In to this? 11
12 Sample preparation Sample preparation techniques: Which one? Sample preparation can be achieved by employing a wide range of techniques, but all methods have the same goal Although many traditional sample-preparation methods are still in use, there have been trends in recent years towards: 1. use of smaller initial sample sizes, small volumes or no organic solvents; 2. greater specificity or greater selectivity in extraction; and, 3. increased potential for automation. SAMPLE PREPARATION PROCEDURE depends about physical state of sample (liquid, solid and gas sample) 12
13 Extraction of water samples (river, drinking and waste water) Dragana Mutavdžić Pavlović, PhD, associated professor, Department of Analytical Chemistry, Faculty of Chemical Engineering and Technology University of Zagreb
14 Extraction of water samples Extraction techniques (water samples): Application rates to environmental analysis (1994 -) 11% 25% 64% SPE: the most widely applied extraction technique SPME: few applications to the analysis of EDCs (but increasing) 14 LLE: few applications but still prescribed in some official methods
15 Extraction of water samples Solid-phase extraction a big advantage over LLE extraction of analytes is achieved its distribution between the solid and liquid phases affinity to the solid phase is much higher than for the matrix sample sorbent is the most important parameter sorbent = the solid phase in which target analyte goes 15
16 Extraction of water samples 16 Pictures were taken from Gilson
17 Extraction of water samples SPE strategies 1. Pass through mode SPE = sample extract is passed through sorbent without retention of analytes; matrix interferences are retained 2. Retention- Cleanup- Elution mode = sample extract is loaded on SPE sorbent; analytes are retained on sorbent; matrix interferences are washed 17 Pictures were taken from Waters Corporation 2016
18 Extraction of water samples Comparison of the SPE strategies PASS-THROUGH SPE RETENTION-CLEANUP- ELUTION Analyte Non-retained Initially retained and then eluted Matrix Mostly retained Non-retained or removed by washing Sorbent Selection Minimize: analyte retention Maximize: matrix retention Enrichment Limited by solvent evaporation Yes Minimize: matrix retention Maximize: analyte retention Analysis Multiresidue analysis Compounds with similar structures and/or properties Cleanup More effective than DSPE in general Most effective and very selective 18
19 Extraction of water samples Solid phase = sorbent Could be: - disks mg - cartridges - SPEC microcolumns 5-56 mg Disk- advantage: the consumption of organic solvents according to LLE <90% according to cartridges <20% = depends on the sorbent mass 19 Pictures were taken from Waters Cataloque
20 Extraction of water samples The lack of discs use over the cartridges is a smaller breakthrough volume, mainly for polar components. SPEC microcolumns advantage: sorbent in the form of a disc avoiding the formation of voids which are present in the cartridges reduced loss of analyte increased efficacy 20 Pictures were taken from Waters Cataloque
21 Extraction of water samples Sorbent important proper selection (selectivity, affinity and capacity) depends on the desired analyte and possible interactions of sorbent with functional groups of the analyte, depends about matrix of the sample and on its interactions with the sorbent and analyte the physico-chemical properties of an analyte (e.g., log K ow, pk a, solubility) log K ow is an indicator of the lipophilicity of the compound (a high log K ow is typical for hydrophobic compounds, whereas a low log K ow signifies a compound soluble in water) 21 Analyte polarity: apolar: log K ow 3 moderately polar: log K ow 1 3; adequate hydrophobicity (log K ow 1.5 4) polar: log K ow 1
22 Extraction of water samples Factors that influence on SPE performance Interaction between Solvent, Analyte, Sorbent and Matrix determine the SPE method performance Interactions of all factors need to be considered together when developing and controlling an SPE method Optimized SPE method maximize selective interactions while minimizing unwanted interactions 22 Taken from Waters Corporation 2016
23 Extraction of water samples Analyte and Matrix Considerations Key consideration of the sample matrix Key considerations of the sample matrix 23 Taken from Waters Corporation 2016
24 Extraction of water samples Analyte and Matrix Considerations Key functions of the solvent Key consideration for SPE Sorbents 24 Taken from Waters Corporation 2016
25 Extraction of water samples Key consideration for SPE sorbents selection 25 Taken from Waters Corporation 2016
26 Extraction of water samples SPE sorbents: Normal-Phase sorbents (NP sorbents) 26 Silica, Alumina, Florisil, Aminopropyl silica (NH 2 ), Diol silica (2OH), cyanopropyl (CN) Reversed-Phase sorbents (RP sorbents) C2, C8, C18 (alkyl bonded silica), black graphitized carbon (GCB), Ion exchange SCX, SAX Oasis HLB, Strata-X, Lichrolut EN, SDB Mixed Mode (ion-exchange/reversed-phase) MAX, WAX (strong and weak anion-exchange) MCX WCX (strong and weak cation-exchange)
27 Extraction of water samples 27 Oasis HLB is the backbone of all Oasis sorbents Stable across ph 1-14 Polar retention No silanol interactions High recoveries for acids, bases and neutrals Water-wettability allows the elimination of conditions and equilibration steps Taken from Waters Corporation 2016
28 Extraction of water samples 28 Picture was taken from Waters Cataloque
29 Extraction of water samples Examples: offline SPE Sample: Surface water, Effluent wastewater Compounds: pesticides and their degradation products, pharmaceuticals and their metabolites, perfluorinated compounds, drugs of abuse and life-style products as caffeine and nicotine 200 ml of water samples prefiltering the collected water (ph 7) to eliminate the suspended matter solid-phase extraction ( Oasis HLB 200 mg/3 ml cartridges) (Waters, Milford, MA, USA ) conditioning the cartridges with 4 ml of methanol + 8 ml Milli-Q water sample loading procedures air-dried disc eluting the cartridges with methanol (2x 4 ml) evaporating the extract under gentle nitrogen stream (Turbo Vap LV, Zymark, Hopkinton) with water bath temperature of 37 C dissolving the residues in 1 ml of MeOH/water (20:80) LC-TOFMS analysis 29
30 Extraction of water samples SPE: advanced techniques Immuno-affinity sorbent Molecular imprinted polymers (MIPs) Restricted access materials (RAM) Extraction of water Selectivity sensitivity On-line coupling Cartridge reuse Clean-up of liquid/solid samples extracts 30
31 Extraction of water samples Immunosorbents Immunosorbents = sorbent with immune attractiveness covalent binding of the antibody to an appropriate solid support selective extraction long-term preparation (weeks to months) Examples in literature PAHs in sediments and sewage sludge (S Pérez et al., Analyst (2000) 125, ) PAHs en rain water (T Schrnweber et al., Field Anal. Chem. Technol. (2000) 4, nonylphenol in water (N Masqué et al., Anal. Chem. (2000) 72, ) Atrazine in water (DH Thomas et al., Anal. Chem. (1994) 66, Carbendazim in water (DH Thomas et al., J. Chromatogr. A (1996) ) 31
32 Extraction of water samples Molecularly Imprinted Polymers (MIPs) substitute for immunosorbent (simple and fast preparation) artificial antibodies functional monomer template for print (analyte) very promising type of sorbent 32
33 Extraction of water samples Example: target compound (template molecule): sulfaguanidine; polar compounds Previously publish literatures shown that sulfaguanidine makes a problem during extraction procedure from water: L.J. Zhou et al., J. Chromatogr. A 1244 (2012) ; M.J. García-Galán et al., Talanta 81 (2010) ; W.L. Shelver et al., J. Chromatogr. A 1217 (2010)
34 Extraction of water samples Restricted access materials (RAMs) The principle - preventing access of macromolecules (like a proteins) to sorbent in which a retention of analytes This is achieved by: preventing the physical diffusion in the pores of the internal surface with reverse phase sorbent or chemical diffusion preventing by network polymer particles on the outside surface of semipermeable material Macromolecular matrix components Analyte Successful application in analysis of biological fluids (plasma, blood, urine, saliva, supernatants of cell cultures and tissue) Seldom applied in environmental analysis 34
35 Extraction of water samples Solid-phase microextraction (SPME) a mechanism similar to the SPE the difference is in the amount of sorbent - fiber for the gaseous, liquid and solid samples Advantages: a small amount of solvent required, simplicity, speed, ability to connect directly with GC, HPLC and CE Disadvantages: lack of robustness the needle can be easily bent fiber has limited time of usage extraction is incomplete (due to the small volume of fibers bonded phase) 35
36 Extraction of water samples Stir-bar sorptive extraction (SBSE) improve SPME sorbent = phase which is coated of magnetic core material (stir bar); same material as well as in SPME (PDMS is the most popular) advantage: times increased sorption surface (typical SPME phase: <0.5 L stirs and extracts in one step disadvantage: difficulties in removing the stir-bar from the samples and typical Twister SBSE phase: L) 36 its rinsing (extraction phase is greater; slower desorption method- desorption combined with cold trapping and reconcentration is required)
37 Extraction of water samples Comparison SBSE with SMPE Greater SBSE capacity gives higher recovery of analytes relative to SPME as polarity increases Lower detection limits due to higher analyte recoveries 37
38 Extraction of solid samples (soil, sediment, sludge) Dragana Mutavdžić Pavlović, PhD, associated professor, Department of Analytical Chemistry, Faculty of Chemical Engineering and Technology University of Zagreb
39 Extraction of solid samples Solid samples = complex samples completely different from the homogeneous water samples 39 Schematic representation of the process during the extraction of analytes from solid samples: 1. entrance solvents in solid matrix 2. desorption of analytes from the active sites of solid particle (matrix) 3. diffusion through the organic matter 4. solvation in a solvent on the surface of the solventsolid matrix 5. diffusion through immobile solvent in the pores of the solid matrix 6. diffusion through the stationary layer of solvent out of the particles 7. transfer through interparticle spaces by solvent
40 Extraction of solid samples Solid samples: extraction Pressurized liquid extraction (PLE) Pressurized fluid extraction (PFE), Enhanced solvent extraction (ESE), Assisted solvent extraction (ASE) Microwave-assisted extraction (MAE) Supercritical fluid extraction (SFE) Ultrasonic solvent extraction (USE) Soxhlet Matrix-solid phase dispersion (MSPD) 40
41 Volume (ml) Time (min) Extraction of solid samples Comparison of extraction techniques in terms of solvent consumption and duration of extraction Soxhlet (SOX) Automated Soxhlet (ASOX) = Soxtec Ultrasonic solvent extraction (USE) Microwave-assisted extraction (MAE) Supercritical Fluid Extraction (SFE) Matrix solid-phase dispersion (MSPD) Pressurized Liquid Extraction (PLE) Solvent consumption Analysis time Personnel qualification Automatization Energy consumption Solvent Consumption Time per sample SCFE MAE PLE ASOX SOC SOX Technique PLE MAE SOC SCFE ASOX SOX Technique
42 42 Extraction of solid samples
43 Extraction of solid samples Sample pretreatment is needed to assure good contact between the solvent and the matrix air-dried, heating or lyophilization Freeze Drying (lyophilization) High vacuum ( 200 mbar) and low temperature (-40 ºC) Main Advantages 43 Avoid denaturation caused by heating by maintaining the sample frozen throughout drying Speed and completeness of rehydration Main drawbacks High capital cost (~3 folds more expensive) High energy cost (2-3 folds higher) Long process time (> 24 h cycle)
44 Extraction of solid samples Homogenization Mortar grinding Ultrasonic system Sieving 44 Manual Automated
45 Extraction of solid samples Soxhlet extraction Standard and reliable method for decades Multiple extraction by distilling and condensing vapors of solvent Reuses solvent for multiple extractions Solvent near boiling point during extraction Requirees hours of extraction Typical solvents used: Hexane 45 Hexane/acetone Methylene chloride Toluene Benzene Ethanol
46 Extraction of solid samples Sonication (or ultrasonic assisted extraction) ultrasound creates microscopic bubbles that expand and contract creates localized turbulence and increases mass transfer can breakup a solid surface and microscopic cracks relatively quick method (10-30 min) but the extraction efficiency is not so high as efficiencies reached with other methods small sample size (0.1-2 g), small solvent volume (5-25 ml) 46
47 Microwave Assisted Extraction 47 Extraction of solid samples Microwave energy is a nonionizating radiation that causes molecular motion by migration of ions and rotation of dipoles The couple forces of electric and magnetic components change direction rapidly. Polar molecules try to orient in the changing field direction and hence get heated Microwave radion interacts with dipole of polar and polarizable materials Only solvents which adsorb microwaves (polar solvents) can be used as well as solvent mixture with and without dipoles provides of potential solvent In non-polar solvents without polarizable groups, the heating is poor (dielectric absorption only because of atomic and electronic polarizations); certain solvents like methylene chloride may not be compatible High pressures and temperatures to enhance extraction Fast extraction Metal pieces in the solid can be a problem as well as content of organic matter in one MAE vessel (not higher than 0.5 g)
48 Extraction of solid samples Example: comparison of Soxhlet, UAE and MAE 48
49 Pressure Extraction of solid samples Supercritical Fluid Extraction Above the critical point, density of a gas and liquid is the same solid liquid Critical point Supercritical fluid gas Triple point Temperature GAS SUPERCRITICAL FLUID LIQUID Density (g/cm 3 ) (0.6-2) x Diffusion coefficient (cm 2 /s) (1-4) x (0.2-2) x 10-5 Viscosity (g/cms) (1-3) x 10-4 (1-3) x 10-4 (0.2-3) x 10-2 Solvent effectiveness of fluid can be controlled by small changes in pressure 49 and by temperature
50 Extraction of solid samples Advantages of supercritical fluid extraction Fast extraction (typically minutes) Variable solvent strength Easy recovery of the extracted analytes SFE extract does not require additional filtration Safe Disadvantage of supercritical fluid extraction Requires specially dedicated equipment capable of providing the conditions needed high pressure, high temperature 50
51 Choices of Extraction Medium Supercritical fluid CO 2 is a nonpolar solvent, that is nonflammable and safe Other supercritical fluid (Table) 51 Extraction of solid samples Supercritical fluid CO 2 an excellent solvent for non-polar species, such as alkanes and terpenes a moderately good extraction medium for moderately polar species, such as polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), organochloro pesticides and fats Supercritical fluid CO 2 with methanol improve properties of CO 2 addition of small quantities (1-10%) strongly polar modifiers (such as low MW alcohols, dichoromethane, propylene carbonate, 2-methoxyethanol) is essential for extraction of highly polar compounds T c ( C) P c (atm) CO N 2 O SF NH H 2 O n-butane n-pentane Xe CCl 2 F CHF modifiers reduce analyte-matrix interactions; improving extraction efficiency
52 Extraction of solid samples Pressurized liquid extraction PLE has become a well-established method and has proven its advantages in the determination of different compounds in solid samples due to high extraction efficiency within a short period, low solvent consumption, and the possibility of automation. conventional solvents temperature ( C) pressure ( psi) which are below the critical point of the solvent sample amounts typically range g samples are often mixed with an inert material as sand, aluminum oxide, diatomaceous earth, or Hydromatrix as commercially available material to increase the exposure surface area 52
53 Extraction of solid samples Example: extraction of sulfonamides by PLE 2 g of sludge sample digested under anaerobic conditions and dewatered by centrifugation; freeze-dried and kept at -30 C until analysis grinding and homogenized; samples were mixed in the extraction cells with hydromatrix dispersing agent PLE (ASE 300 accelerated solvent extractor (Dionex, Sunnyvale, CA)) ACN-water (25:75, v/v), 5 min of preheating period; 3 static cycles of 5 min each; total flush volume of 100 %, 60 s of nitrogen purge, presure of 1500 psi, extraction temperature was 50 C Recovery values at two spike levels and MLODs and MLOQs Oasis HLB: elution by 5 ml of MeOH in 50mM HCOOH and 5 ml of acetone in 50 mm HCOOH Extracted were purified by SPE the extract were dried under a gentle N 2 stream dissolving the residues in 1 ml of HPLC grade water-acn (75:25, v/v) 53 LC-MS/MS analysis
54 Extraction of solid samples Matrix solid-phase dispersion (MSPD) method which allows simultaneous extraction and the cleanup of analytes from solid samples advantages over other extraction methods (PLE, Soxhlet, etc.): simple usage, low cost, reduced extraction time, more selectively and more quickly extraction with similar or better recoveries room temperature, atmospheric pressure samples are blended with a suitable sorbent (silica-based material (C18), sand, Florisil, etc.) in ratio 1:2 or 1:4 (or other) to form a homogeneous packing material = looks like SPE column sample/sorbent column is eluted using similar procedure as SPE the most important parameters: the type of sorbent and the solvent polarity chosen sorbent is used not only as an adsorption separation material but also as a 54 blending solid support to disrupt and disperse the sample
55 Extraction of solid samples Example spiking with standard solution slurry was drying in dark 2 days, 1 month before analysis freeze drying sludge ready for analysis 0.2 g sludge were soaked with 100 L of aq KOH (60%, w/v) + 1 g anhydrous Na 2 SO 4 1 g Florisil mixing, blending iand disperdes n mortar 3 min introducing the dispersed sample into syringe body with 3 g silica as clean-up sorbent 15 ml of hexane/acetone = 1:2, v/v 15 ml of sludge extract concentrating by evaporation under a stream of N 2 reconstitution the residues with 500 L of MeOH and 500 L of ultrapure water extracts were filtered through 0,2 m GHP Acrodisc 13 mm syringe filters LC-QTOF-MS 55
56 Extraction of solid samples Sample cleanup natural organic matters, such as humic and fulvic acids, present in environmental samples are coextracted with the analytes and often complicate analytic detection most of the mentioned extraction methods used for the preconcentration of analyte from solid samples are not selective, which is why cleanup procedures are a necessary step in analytic methodology the most commonly used cleanup methods in environmental analysis are: solid-phase extraction (SPE) dispersive solid-phase extraction (DSPE) gel permeation chromatography (GPC) 56
57 On-line systems Dragana Mutavdžić Pavlović, PhD, associated professor, Department of Analytical Chemistry, Faculty of Chemical Engineering and Technology University of Zagreb
58 On-line systems Traditional SPE methods (off-line): time consuming, generate large amounts of hazardous chemical waste as well as expose laboratory personnel to dangerous chemicals and fumes. Advantages of on-line SPE a set of samples can be analyzed is a completely automated minimal amount of solvent required for extraction and small sample volumes the simplicity of the technique not requires highly qualified or experienced staff results are more reliable, in terms of reproducibility and accuracy, because there is minimal or no sample manipulation. sensitivity is improved because the whole sample transferred to the chromatographic system Disadvantages of on-line SPE unavailability of a final extract for parallel determinations, of flexibility as compared to off-line (for example: elution solvents must be compatible with the LC column), possible matrix interferences from loading the entire extracted sample 58
59 On-line systems Example: online SPE 59 Samples were automatically preconcentrated and separated on an UltiMate TM 3000 system (Dionex, Usa) coupled online SPE with HPLC The left pump control the first three steps (sampling, cleanup and SPE column regeneration) The right pump control elution and HPLC separation After the first part of procedure, the valve was switch to position 2; meanwhile, the SPE column have to regenerate for the next analysis
60 On-line systems On-line systems - On-line SPE (Prospekt, Symbiosis) - Dual column LC using RAM - Dual column LC EQuan technology and TurboFlow chromatography On-line Solid Phase Extraction Low sample volume requirements 0.5 ml easy sample storage (at 25 ºC) Minimum sample pre-treatment 15 min centrifugation and addition of the IS High throughput: simultaneous SPE (n+1) and LC-MS-MS (n) 60
61 61
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