Analytical techniques: Environmental samples. Lecture 2 Universidade do Algarve
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1 Analytical techniques: Environmental samples Lecture 2 Universidade do Algarve
2 Terms, definitions & applications Difference between technique and method: Analytical technique: Fundamental scientific application to acquire information from samples. Analytical method: Specific application of a technique to obtain specific information. Academic/research papers generally describe techniques, methods and procedures. Protocols are given in case of a new method to be established or submission of technical/analytical reports to law/policy making bodies. Difference between protocol and procedure: Analytical procedure: Written instructions for carrying out any analysesstandardized and general outline assuming that user has the basic knowledge of the analysis. Analytical protocol: Step-by-step description of any analytical procedure including all the necessary directions required to carry out the analysis. Usually, all scientific reports and documents submitted under any law/policy require analytical protocols.
3 NATURAL/INDUSTRIAL/UNKNOWN SAMPLES COLLECTION PRESERVATION ANALYSIS DATA INTERPRETATION PURPOSE IS TO OBTAIN INFORMATION WITH RESPECT TO 1. PHYSICAL CONDITION. 2. QUALIFICATION. 3. QUANTIFICATION. 4. DISTRIBUTION. 5. TURNOVER. THE INFORMATION MAY BE OF TECHNICAL, ACADEMIC OR POLICY/LAW INTEREST. THE DATA INFORMS US ABOUT 1. STATE OF THE ENVIRONMENT (PRESENT). 2. HEALTH OF THE ENVIRONMENT (OVER TIME & SPACE). 3. RICHNESS/DIVERSITY IN THE ENVIRONMENT (BIOLOGICAL). 4. PRODUCTIVITY OF THE ENVIRONMENT (BIOLOGICAL). 5. TURNOVER (EFFICIENCY OF THE ENVIRONMENT).
4 TYPES OF SAMPLES GAS LIQUID SOLID LITHOGENIC SEDIMENT/SOIL INORGANIC ORGANIC BIOLOGICAL TECHNIQUES ADOPTED FOR ANALYSIS OF SAMPLES DEPEND UPON: 1. STATE OF THE SAMPLE. 2. INFORMATION REQUIRED FROM THE SAMPLE. 3. AMBIENT CONCENTRATION/SIZE OF THE SAMPLES. 4. PRECISION LEVELS OF THE ANALYSIS. 5. NATURE OF THE SAMPLE (ORGANIC, LITHOGENIC, CHEMICAL). 6. NATURE OF INFORMATION REQUIRED.
5 Techniques widely used in research: Chemical samples: Spectroscopy- Spectrophotometry, Atomic spectrometry, IRS etc. Electrochemical: Voltammetry, Potentiometry, Coulometry etc. Chromatography: Gas, HPLC, Ion-exchange etc. Thermal analysis, Hyphenated techniques Biological samples: Microscopy. Culture techniques. Molecular biology. Enzyme based analysis. Radioactive tracers. Stable isotopes. Other methods: Remote-sensing. Data buoys. CTD. Electronic probes. Model-based approaches.
6 Chemical assays: Elemental analysis: Carbon, Nitrogen, Sulphur, oxygen analyzer Heavy metal/trace metal analysis: Pb, Hg, Cd, Fe, Mn, Co etc. Molecular Characterization: Sugars, amino acids, organopollutants etc. Biological assays: Tracers: Radioactive isotope and stable isotope based tracers. Fluorescent substrates: Enzymatic estimation. Biomarkers : Population diversity, evolution, dsitribution.
7 Elemental analyzer: Principle: Separation of elements like C, O, H, N & S as combustion gases on a gas chromatographic column and detected using a Thermal Conductivity Detector (TCD). Nature of sample: Solids (POM, sediments, soil etc.). Carrier gas & Combustion gas are used. O 2 as combustion and He as carrier gas for C/H/N/S analysis. For O-analysis, He is carrier gas, samples are pyrolyzed at 1080 C. O 2 measured as CO produced during pyrolysis C C 90 C 60 C Copper filings: To reduce NO x to N 2. Cobalt Oxide: Converts CO to CO 2. Ag-wool: To remove chlorides/bromides. Mg-perchlorate: Moisture trap. Tungsten oxide: Dehydrating agent. AgO: SO 2 trap.
8 Carbon/Nitrogen analyzer for water samples: Total Organic Carbon (TOC) analyzer measures both total carbon and inorganic carbon. Difference between the two gives TOC. Principle: Samples are acidified, then Photochemically/Thermochemically/Catalytically/electrolytic ally oxidized and then detected using Conductivity/Non- Dispersive Infra-red (NDIR) detector. Sample types: Water, marine, sewage, effluents etc. Oxidation techniques: HTCO: 680 C, Pt catalyst. Combustion: 1350 C. UV: Photolytic method. UV-persulfate: OM is excited by UV. Persulfate reacts with excited OM.
9 Carbon/Nitrogen analyzer for liquids. Methods of detection- Conductivity cell: Membrane conductivity & Direct conductivity. Limitations- Low accuracy, interferences from other gases, changes in ph can alter sensitivity and analytical time. NDIR: Non-invasive, based on absorptive properties of CO 2 in the IR range, very specific, results are integrative in nature, highly sensitive. Nitrogen in the sample is estimated using Chemiluminescence detection after oxidation. Chemiluminescence involves reaction of NO with ozone resulting in fluorescence that gets amplified in a photomultiplier tube.
10 Chromatography: Principle: Miscible solutes (mobile phase) are separated on a charged surface (stationary phase), the solutes separate out along the stationary phase after undergoing repeated interactions (partitioning) and are eluted out with increasing partitioning. Partitioning requires differences in physical/chemical properties of the solutes. Chromatography Liquid Chromatography (LC) Liquid mobile phase LC (LLC) Solid stationary phase LC (SLC) Ion-Exchange (IEC) Gas Chromatography (GC) Gas Liquid (GLC) Gas Solid (GLC) Exclusion Chromatography (EC)
11 Factors affecting Chromatography Retention Behaviour: Reflects distribution of solutes between mobile & stationary phases. V r = t r F c ; where Vr is retention volume, &t r & F c are retention time and Flow rate of the mobile phase. Partition Ratio: Also called capacity ratio, it is the measure of time a solute spends in stationary compared to mobile phase. k = (t r t m )/t m = (V r V m )/V m. Partition coefficient: Average velocity of solute zone within the mobile phase. V r V m = KV s Column efficiency & Resolution: Under linear partitioning between mobile and stationary phase, K and k are independent of solute concentrations. It means peaks are sharp, clearly defined. Otherwise, peaks are broad, height is reduced and affects resolution of closely eluted solutes.
12 Some examples of chromatograms: Case 1: Good selectivity, resolution, efficiency, poor baseline. Case 2: Good efficiency, resolution, poor selectivity/low k. Case 3. Poor resolution, Poor separation Detectors: For GCs Thermal conductivity detector (TCD): Amount of heat lost from heated filament by gas stream. Most compounds can be detected. Sensitivity. Flame Ionization Detectors (FID): Heated ionizable gas (C-H bonds) conducts current across two electrodes. Eg., CH 4. Insensitive to most compounds. Thermionic Emission Detectors (TED): Filament based, low temperature flame, works like FID but only for N 2 & P-compounds. Electron Capture Detectors (ECD): One radioactive electrode emits β-particles, bombards N-gas, generates a plasma of positive ions & electrons, changes in current is detected. Detects halogens, organometals, nitriles etc. Flame Photometric detectors (FPD): Works like flame emission photometer, used to detect volatile S & P.
13 Continued.. Photoionization detectors (PID): Uses UV-lamp to ionize solute molecules, the current generated is then measured. Non-destructive, used to detect hazardous chemicals, air quality etc. Electrolytic Conductivity Detectors (ELCD): Solutes are burned in a miniature furnace producing readily ionizable molecules that contribute to conductivity of deionized water. Halogens, S, N etc. TED ELCD PID ECD TCD FPD FID ATOMIC ABSORPTION SPECTROMETRY Most widely used method to analyze environmental samples. It has two components- production of free atoms from a sample and absorption of specific wavelengths from an external light source ng Ionization takes place at high temperature ( C). Complete ionization depends on fuel/oxidant ratio and varies for different elements. Atomization is done using 1. Graphite/Carbon/Tungsten furnace. 2. Chemical vaporization: Involves hydride formation of volatile elements using discharge lamps.
14 Flame Emission Spectrometry Sample is nebulized and introduced into the flame to rapidly desolvated, vaporized and atomized. Excited atoms emit characteristic radiation specific for different elements. The emitted energy is then detected photodetector that measures the power and wavelength. Both these techniques are widely used to quantify heavy metals and trace metals. Schematic diagram of AES
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