1、Angela Chang Mausami Desai Katie Sovik,ENVIRONMENTAL ANALYTICAL CHEMISTRY,Winter 1999,PRINCIPLES To experience and practice a variety of techniques useful in analyzing natural environmental processes. This includes complex biological, chemical, geological, and physical phenomena.This laboratory util
2、izes some of the state-of-the-art instrumentation currently available, noting the accuracy of results that can be obtained.The focus is split between a lesson on instrumentation and results analysis.,OBJECTIVEThis course specifically focuses on characterizing naturally occurring organic matter (NOM)
3、 because of its influence on the bioavailability and activity of pollution. The following analyses provide an introduction to important laboratory instrumentation while addressing a significant environmental material.,CONTENTS,Characterization of Total Organic CarbonCapillary ElectrophoresisPotentio
4、metric MethodsGlucosidic & Proteinaceous Fractions of DOMDOM Fingerprinting by PY-GC-MS,CHARACTERIZATION OF TOTAL ORGANIC CARBON,TOTAL ORGANIC CARBON,OBJECTIVE:Quantify overall organic carbon concentrations, and the dissolved and particulate fractions. This is a generalized starting point in analyzi
5、ng naturally occurring organic matter. Subsequent procedures determine more specific characterizations of the types of organic material or carbon.,TOTAL ORGANIC CARBON ANALYSIS,by Automated Carbon Analyzer (UV Persulfate Oxidation)by UV Spectroscopy,Automated Carbon Analyzer,2 STEP TOC ANALYSIS PROC
6、EDURE: Principals By UV persulfate oxidation the sodium persulfate and phosphoric acid reagents convert all organic matter CO2Measuring CO2 concentrations suggests organic carbon concentration The infrared absorbance detector measures and quantifies this CO2 as ppm total C,UV PERSULFATE OXIDATION,RE
7、ACTIONS:Excitation by UV light produces the primary oxidants (sulfate and hydroxide radicals) S2082- + v 2SO4- H20 + v H+ + OHUV light also breaks down the organic material into radical functional groups. R + v R,UV PERSULFATE OXIDATION,The combination of these 2 types of radicals oxidizes the organ
8、ic matter releasing CO2. R + SO4- + H20 nCO2 + . Ultimately a measure of the amount of CO2 produced quantifies the TOC,Dohrman DC-180 Carbon Analyzer Flow Diagram,See next page for system operations explanation,System Operations,A pump fills the pickup loop with sampleSpecific amounts of sample and
9、acid are injected into the spargerAcidification with H3PO4 in the sparger strips the inorganic (IC) and purgeable carbon (PuOC) from the sample. Separation of these fractions is aided by a bubbling flow of O2(g)The nonparticulate organic carbon (NPOC) remaining in the liquid sample is sent to the UV
10、 reactor by another injection loopUV radiation and the persulfate reagents oxidize all organics in the sample,System Operations (continued),The CO2(g) and OH-(g) are directed to the Gas/Liquid separator and bubbled with acidified water. A pH of 3 is maintained to aid the elimination of water from th
11、e CO2.The infrared absorbance of water significantly overlaps with our focus, CO2. The removal of water in an osmotic pressure dryer is thus important. In the Nondispersive Infrared Detector (NDIR) the absorbance of infrared radiation measures CO2.The computer calculates and displays this as ppm C.,
12、Interferences,There are 3 significant types of interferences related to the instrument procedure and components of the samples :The incomplete removal of inorganic and purgeable carbon in the spargerThe incomplete oxidation of the organic material in the UV reactorChloride present in the sample abso
13、rbing UV radiation,Calibration Curve counts = (15,500 +/- 102.4)conc - 540.6 +/- 1213,Calibration,5 standards of known C-concentration were made from KHP (K-acid phtalate) These concentrations ranged from 1-20 ppm2 blank samples were also analyzed and used to zero the calibrationThe error on the int
14、ercept is larger than the actual intercept estimate and insignificant with respect to the origin This intercept value can be disregarded Considering this was our first time doing error analysis, we included all error estimates in our calculations.,Organic Carbon Calculations,Calculations are based o
15、n average values of triplicate readings from the machine for each sample,Organic Carbon Calculations,Dissolved particles are defined as that smaller than 0.45 m by the filters used Suspended/colloidal materials ineffectively separated by filtration can thus be misrepresented as dissolvedThis is a po
16、ssible explanation for the large DOC values, misleadingly close to the TOCThe resultant small POC calculations suggest large amounts of colloidal materialThe error carried over from the total and dissolved carbon values is greatly amplified in the POC calculations making them essentially insignifica
17、nt,TOC - Sheboygan River,Corporate PCBs,Kohler Company,TOC - Lake Depue,Trends,Sheboygan River : The organic carbon levels are greatest upstream of the PCBs input The Kohler Co. does not seem to effect the carbon levels Overall there is about a 2 ppm downstream decrease in TOCLake Depue : No seasona
18、l effects on TOC are noted There is evidence that the lake is highly colloidal,UV SPECTROSCOPY,Principle : Different compounds at certain wavelengths show unique and specific absorbances. The following methods attempt to quantify the fractions or concentrations of different types of organic matter f
19、rom absorbance spectra.,Correlation methods in particular, have been used as estimates in characterizing : Humidification % Aromaticity TOC The UV-254 correlation with TOC useful for specific water types has continued to be mentioned and documented because of the simplicity of the procedure and the
20、portability of spectroscopy equipment. Even though automated carbon analyzers are more widely accurate, this method has shown some advantages.,UV SPECTROSCOPY,UV SPECTROSCOPY,Transmittance is the fraction of incident light transmitted by a solution This cannot be measured directly in the lab due to
21、reflective interferences with any container used to hold the sample Beers Law (For use with dilute solutions only) Absorbance = - log T = bc = molar absorptivity L/mole*cm b = the path length through the solution c = concentration,Spectrophotometer,1 - D2 lamp 2 - Grating 1 3 - Entrance Slit 4 - Gra
22、ting 2 5 - Exit Slit 6 - Chopper 7 - Sample & Reference Positions 8 - Chopper 9 - Photo Tube,Spectrophotometry,Mirrors and gratings redirect and disperse the radiationThe slits limit the radiation range allowing successively isolated wavelengths to be selected The rotating chopper wheels alternately
23、 direct the light beam through the sample and reference A distilled water reference is required to zero the interference effects of the cuvetteOther Interferences include :chloride absorbanceparticulate scatteringnon-absorbing organic material,Absorption Ratios : Characterizations,Although negative
24、values are useless, the ratios developed have been used to characterize soil type and degree of humidification,E4/E6 & E2/E3 Ratios : Humic Substances,Constitute a large portion of the organic matter in soilsProduct of the degradation of plant and animal materials & microorganism activity Aromatic a
25、cidic Hydrophilic Flexible PolyelectrolytesLignin is the second most abundant polymer synthesized by plants and a structural unit for humics,Biochemistry & Significance,The aromatic building blocks of humic substances are connected by flexible low energy bonds Reactions and voids aggregate/trap othe
26、r materialsMetals ions and toxic organic pollutants are stabilized in complexes,Humidification Analysis E4/E6 & E2/E3 Ratios,Even though our results are inconclusive low E4/E6 ratios have been found to indicate a high degree of aromatic humic constituency High E4/E6 ratios indicate low aromaticity,
27、or a high degree of aliphatic structure,Humic Acids,Fulvic Acids,E4/E6,3.8 - 5.8,7.6 - 11.5,Humidification Analysis E4/E6 & E2/E3 Ratios,Less data has been compiled for E2/E3 ratios and thus they are less reliable although certain characterizations have been documented,E2/E3,Strongly humic and oligo
28、trophic lakes,Chlorolignin,Lignin,4.0,4.2,5.7,Absorption Ratios : Characterizations,Aromaticity,Aromaticity of organic matter is a specific structural factor significant to interactions with pollutants, and their stabilization The higher the aromatic fraction of DOM, the higher the xenobiotic bindin
29、g capacityA simple equation for % Aromaticity has been developed that is dependant on molar absorptivity = A/bc Aromaticity = 0.05 + 6.74Primary assumption : all organic matter absorbs the same at any wavelength and that also absorbs as the KHP standard, i.e. the of all organic matter is the same. T
30、his assumption in actuality is not valid, as varies for different types of organic matter.,TOC Surrogate,UV absorbance at 254 nm is documented as a widely used substitute for TOCWe analyzed the filtered samples in the spectrophotometer and thus ultimately compared DOC approximations from the 2 metho
31、ds,Non-Acidified Pseudo Calibration Curve abs =(0.01953 +/- 0.002089)(ppm C) - 0.01740 +/- 0.01393,Non-Acidified Pseudo Calibration Curve,Only 3 standards solutions ranging from 5 - 20 ppm C, and a blank were analyzedthe standards were diluted from a KHP stockthe samples were zeroed by the spectroph
32、otometerthe 10 ppm standard introduced error,TOC - Comparisons,The ppm C derived by the UV-254 correlation is doubly overcompensated. Greater error values must also be noted as a result of the limited calibration.,TOC Comparisons - Sheboygan River,TOC Comparisons - Lake Depue,Note,The effects of the
33、 colloidal particles noted in the POC calculations is greatly amplified in the UV-254 methodThe scattering action of the colloidal material is one explanation for high absorbance readings and the overcompensation for TOC It is common belief that UV persulfate oxidation and automated carbon analysis
34、is the more accurate method in determining TOCAlthough this exercise allowed a realization of the potential advantages and real limitations of experimental procedures,CAPILLARY ELECTROPHORESIS,Capillary Electrophoresis,OBJECTIVE:Determination of concentration of specified ions in sample waters,Intro
35、duction,Electrophoresis is the migration of ions in solution under influence of electric field. In a typical capillary electrophoresis (CE) application, use an electric field of 15-30 kV to separate the components inside a fused silica capillary tube. Since different solutes have different mobilitie
36、s, they will migrate through the capillary at different speedsThis gives the extraordinary resolution and separation of many ionic species.,Electrophoresis,When an ion with charge q is placed in an electric field E, the force on the ion is:F = q*EIn solution, the other major force on the ion is the
37、retarding frictional force f*vep, where vep is the electrophoretic velocity and f is the coefficient of friction:vep= q*E/f = epEThe constant of proportionality between speed of ion and the applied electric field is: epep is proportional to the charge on the ion and inversely proportional to the fri
38、ction coefficient.,Electroosmosis,The inside surface of the silica capillary is covered with silanol (Si-OH) groups which carry a negative charge above pH=2These negative charges on surface induce cations to neutralize some of the surface chargeThe constant of proportionality between electroosmotic
39、velocity (veo) and applied field is the electroosmotic mobility: eoA relationship for the electrophoretic effect is: veo=eo*E,Diagram: Hydrodynamic Velocity Profile,(a) Positive charges move toward cathode, absorbed on surface of glass (b) More dispersion created by velocity profile because pushed f
40、rom middle,Apparent Mobility,The apparent (or observed) mobility (app) of an ion is the sum of the electrophoretic mobility of the ion and the electroosmotic mobility of the solution:app= ep+ eoFor a cation moving in the same direction as the electroosmotic flow, the mobilities have the same sign an
41、d then app is greater than ep,Diagram: Solute Mobilities,(a) Optimize electrolyte conditions to make separation larger and force ions out of system faster (b) Use TTAB as reversal compound to separate anions out first (c) Sum of all ions out of sides of capillary,Diagram: Apparatus,Both ends of capi
42、llary placed into electrolyte Sample injected by siphon effect Insert capillary into vial and elevate After injection, vial returned to normal height Apply voltage of 15kV Ions migrate through electrolyte Indirect detection,Br-,Cl-,SO42-,NO3-,Sample Data,Cl-,SO42-,NO3-,1. DASS 2. WDNR 3. RP 4. KCL 5
43、. EP,Analysis,NO3 drops significantly from May to NovemberCl- and SO4 increased overall in Lake DepueCl- a bit higher along entire Sheboygan River compared to other ions,POTENTIOMETRIC METHODS,OBJECTIVE:Determination ofacid/base properties ofsamples.,Potentiometric Methods,Alkalinity & CT,Representa
44、tion of the buffer capacity of a water sample or the ability of the water to neutralize strong acid. Alk = 2CO32- + HCO3- + OH- - H+Measure of alkalinity due to carbonate system CT = H2CO3 + HCO3- + CO32-,Computer Automated Titration System ME-10 Analyzer,Components Automatic burette Potentiometer G
45、lass Electrode Windows Interface program controls ME-10 Analyzer unit during titration and records data (volume additions and potential) Data analysis to determine the equivalent volumes and equilibrium constants,Titration System Setup,Computer,Burette,Sample & mixer,Glass Electrode (w/ reference el
46、ectrode and ion selective membrane),Titrant,Glass Electrode,Measures pH The indicator electrode measures potential difference across a glass membrane between 0.1 M HCl and the sample solution.The glass electrode has two key components reference electrode ion selective glass membrane,Reference Electr
47、ode,Within a tube in the indicator glass electrode:The reference electrode contains a small volume of dilute HCl and AgCl. The Ag wire forms a reference electrode creating a link to the potential measuring device. This electrode should obey Nernst equation when constant temperature and ionic strengt
48、h are maintained. The reference electrode provides a base potential from which changes in potential can be measured.,Ion Selective Membrane,The ion selective glass membrane is sealed into one end of the glass tube. When hydrated, it allows for the interaction between singly charged cations (electric
49、 conductivity) in the glass and protons from the solution. H+ + Na+Gl- Na+ + H+Gl-More specifically when Na+ is low, conduction within the hydrated layer involves the movement of hydrogen ions by the following reactions H+ + Gl- H+Gl- (between glass and sample solution) H+Gl- H+ + Gl- (between inter
50、nal solution and glass),Typical Electrode System for Measuring pH,Measurement through Electrode,The equilibrium position of these 2 reactions are determined by H+ in the solutions on the two sides of the membrane.The surface where greater dissociation occurs becomes negative w/respect to other surface with less dissociation. A boundary potential Eb develops across the membrane which is sensed by the analyzer and recorded by the computer.The potential change is recorded in mVolts along with the corresponding volume of acid added,
