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ENVIRONMENTAL ANALYTICAL CHEMISTRY Winter 1999 Angela Chang Mausami Desai Katie Sovik ENVIRONMENTAL ANALYTICAL CHEMISTRY Winter 1999 Angela Chang Mausami Desai Katie Sovik

PRINCIPLES To experience and practice a variety of techniques useful in analyzing natural environmental PRINCIPLES To experience and practice a variety of techniques useful in analyzing natural environmental processes. This includes complex biological, chemical, geological, and physical phenomena. F This laboratory utilizes some of the state-of-the -art instrumentation currently available, noting the accuracy of results that can be obtained. F The focus is split between a lesson on instrumentation and results analysis.

OBJECTIVE This course specifically focuses on characterizing naturally occurring organic matter (NOM) because of OBJECTIVE This course specifically focuses on characterizing naturally occurring organic matter (NOM) 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 n Characterization of Total Organic Carbon n Capillary Electrophoresis n Potentiometric Methods n CONTENTS n Characterization of Total Organic Carbon n Capillary Electrophoresis n Potentiometric Methods n Glucosidic & Proteinaceous Fractions of DOM n DOM Fingerprinting by PY-GC-MS

CHARACTERIZATION OF TOTAL ORGANIC CARBON CHARACTERIZATION OF TOTAL ORGANIC CARBON

TOTAL ORGANIC CARBON OBJECTIVE: Quantify overall organic carbon concentrations, and the dissolved and particulate TOTAL ORGANIC CARBON OBJECTIVE: Quantify overall organic carbon concentrations, and the dissolved and particulate fractions. n This is a generalized starting point in analyzing naturally occurring organic matter. Subsequent procedures determine more specific characterizations of the types of organic material or carbon.

TOTAL ORGANIC CARBON ANALYSIS n by Automated Carbon Analyzer (UV Persulfate Oxidation) n by TOTAL ORGANIC CARBON ANALYSIS n by Automated Carbon Analyzer (UV Persulfate Oxidation) n by UV Spectroscopy

Automated Carbon Analyzer 2 STEP TOC ANALYSIS PROCEDURE: Principals n By UV persulfate oxidation Automated Carbon Analyzer 2 STEP TOC ANALYSIS PROCEDURE: Principals n By UV persulfate oxidation the sodium persulfate and phosphoric acid reagents convert all organic matter CO 2 n Measuring CO 2 concentrations suggests organic carbon concentration F The infrared absorbance detector measures and quantifies this CO 2 as ppm total C

UV PERSULFATE OXIDATION REACTIONS: n n Excitation by UV light produces the primary oxidants UV PERSULFATE OXIDATION REACTIONS: n n Excitation by UV light produces the primary oxidants (sulfate and hydroxide radicals) S 2082 - + v 2 SO 4 - H 20 + v H+ + OH UV 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 organic UV PERSULFATE OXIDATION The combination of these 2 types of radicals oxidizes the organic matter releasing CO 2. R + SO 4 - + H 20 n. CO 2 +. . . Ultimately a measure of the amount of CO 2 produced quantifies the TOC

Dohrman DC-180 Carbon Analyzer Flow Diagram See next page for system operations explanation Dohrman DC-180 Carbon Analyzer Flow Diagram See next page for system operations explanation

System Operations n A pump fills the pickup loop with sample n Specific amounts System Operations n A pump fills the pickup loop with sample n Specific amounts of sample and acid are injected into the sparger n n n Acidification with H 3 PO 4 in the sparger strips the inorganic (IC) and purgeable carbon (Pu. OC) from the sample. Separation of these fractions is aided by a bubbling flow of O 2(g) The nonparticulate organic carbon (NPOC) remaining in the liquid sample is sent to the UV reactor by another injection loop UV radiation and the persulfate reagents oxidize all organics in the sample

System Operations (continued) n n The CO 2(g) and OH- (g) are directed to System Operations (continued) n n The CO 2(g) and OH- (g) are directed to the Gas/Liquid separator and bubbled with acidified water. A p. H of 3 is maintained to aid the elimination of water from the CO 2. The infrared absorbance of water significantly overlaps with our focus, CO 2. The removal of water in an osmotic pressure dryer is thus important. In the Nondispersive Infrared Detector (NDIR) the absorbance of infrared radiation measures CO 2. The computer calculates and displays this as ppm C.

Interferences There are 3 significant types of interferences related to the instrument procedure and Interferences There are 3 significant types of interferences related to the instrument procedure and components of the samples : F The incomplete removal of inorganic and purgeable carbon in the sparger F The incomplete oxidation of the organic material in the UV reactor F Chloride radiation present in the sample absorbing UV

Calibration Curve counts = (15, 500 +/- 102. 4)conc - 540. 6 +/ - Calibration Curve counts = (15, 500 +/- 102. 4)conc - 540. 6 +/ - 1213

Calibration n 5 standards of known C-concentration were made from KHP (K-acid phtalate) F Calibration n 5 standards of known C-concentration were made from KHP (K-acid phtalate) F n n These concentrations ranged from 1 -20 ppm 2 blank samples were also analyzed and used to zero the calibration The error on the intercept is larger than the actual intercept estimate and insignificant with respect to the origin This intercept value can be disregarded F Considering this was our first time doing error analysis, we included all error estimates in our calculations. F

Organic Carbon Calculations are based on average values of triplicate readings from the machine Organic Carbon Calculations are based on average values of triplicate readings from the machine for each sample

Organic Carbon Calculations n Dissolved particles are defined as that smaller than 0. 45 Organic Carbon Calculations n Dissolved particles are defined as that smaller than 0. 45 m by the filters used F F n Suspended/colloidal materials ineffectively separated by filtration can thus be misrepresented as dissolved This is a possible explanation for the large DOC values, misleadingly close to the TOC The resultant small POC calculations suggest large amounts of colloidal material F The error carried over from the total and dissolved carbon values is greatly amplified in the POC calculations making them essentially insignificant

TOC - Sheboygan River Corporate PCB’s Kohler Company TOC - Sheboygan River Corporate PCB’s Kohler Company

TOC - Lake Depue TOC - Lake Depue

Trends Sheboygan River : n n n The organic carbon levels are greatest upstream Trends Sheboygan River : n n n The organic carbon levels are greatest upstream of the PCB’s input The Kohler Co. does not seem to effect the carbon levels Overall there is about a 2 ppm downstream decrease in TOC Lake Depue : n n No seasonal 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. 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 from absorbance spectra.

UV SPECTROSCOPY n Correlation methods in particular, have been used as estimates in characterizing UV SPECTROSCOPY n Correlation methods in particular, have been used as estimates in characterizing : F Humidification F% Aromaticity F TOC n 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 portability of spectroscopy equipment. Even though automated carbon analyzers are more widely accurate, this method has shown some advantages.

UV SPECTROSCOPY n Transmittance is the fraction of incident light transmitted by a solution UV SPECTROSCOPY n Transmittance is the fraction of incident light transmitted by a solution F This cannot be measured directly in the lab due to reflective interferences with any container used to hold the sample n Beer’s Law (For use with dilute solutions only) Absorbance = - log T = bc F = molar absorptivity [L/mole*cm] F b = the path length through the solution F c = concentration

Spectrophotometer 1 - D 2 lamp 2 - Grating 1 3 - Entrance Slit Spectrophotometer 1 - D 2 lamp 2 - Grating 1 3 - Entrance Slit 4 - Grating 2 5 - Exit Slit 6 - Chopper 7 - Sample & Reference Positions 8 - Chopper 9 - Photo Tube

Spectrophotometry n n Mirrors and gratings redirect and disperse the radiation The slits limit Spectrophotometry n n Mirrors and gratings redirect and disperse the radiation The slits limit the radiation range allowing successively isolated wavelengths to be selected The rotating chopper wheels alternately direct the light beam through the sample and reference A distilled water reference is required to zero the interference effects of the cuvette Other Interferences include : chloride absorbance particulate scattering non-absorbing organic material

Absorption Ratios : Characterizations Although negative values are useless, the ratios developed have been Absorption Ratios : Characterizations Although negative values are useless, the ratios developed have been used to characterize soil type and degree of humidification

E 4/E 6 & E 2/E 3 Ratios : Humic Substances n n Constitute E 4/E 6 & E 2/E 3 Ratios : Humic Substances n n Constitute a large portion of the organic matter in soils Product of the degradation of plant and animal materials & microorganism activity F F n Aromatic acidic Hydrophilic Flexible Polyelectrolytes Lignin is the second most abundant polymer synthesized by plants and a structural unit for humics

Biochemistry & Significance n The aromatic building blocks of humic substances are connected by Biochemistry & Significance n The aromatic building blocks of humic substances are connected by flexible low energy bonds F Reactions and voids aggregate/trap other materials èMetals ions and toxic organic pollutants are stabilized in complexes

Humidification Analysis E 4/E 6 & E 2/E 3 Ratios n Even though our Humidification Analysis E 4/E 6 & E 2/E 3 Ratios n Even though our results are inconclusive F low E 4/E 6 ratios have been found to indicate a high degree of aromatic humic constituency F High E 4/E 6 ratios indicate low aromaticity, or a high degree of aliphatic structure E 4/E 6 Humic Acids 3. 8 - 5. 8 Fulvic Acids 7. 6 - 11. 5

Humidification Analysis E 4/E 6 & E 2/E 3 Ratios n Less data has Humidification Analysis E 4/E 6 & E 2/E 3 Ratios n Less data has been compiled for E 2/E 3 ratios and thus they are less reliable although certain characterizations have been documented E 2/E 3 Strongly humic and oligotrophic lakes 4. 0 Chlorolignin 4. 2 Lignin 5. 7

Absorption Ratios : Characterizations Absorption Ratios : Characterizations

Aromaticity n Aromaticity of organic matter is a specific structural factor significant to interactions Aromaticity n Aromaticity of organic matter is a specific structural factor significant to interactions with pollutants, and their stabilization F n The higher the aromatic fraction of DOM, the higher the xenobiotic binding capacity A simple equation for % Aromaticity has been developed that is dependant on molar absorptivity = A/bc Aromaticity = 0. 05 + 6. 74 Primary 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. This assumption in actuality is not valid, as varies for different types of organic matter.

TOC Surrogate n UV absorbance at 254 nm is documented as a widely used TOC Surrogate n UV absorbance at 254 nm is documented as a widely used substitute for TOC F We analyzed the filtered samples in the spectrophotometer and thus ultimately compared DOC approximations from the 2 methods

Non-Acidified Pseudo Calibration Curve abs =(0. 01953 +/- 0. 002089)(ppm C) - 0. 01740 Non-Acidified Pseudo Calibration Curve abs =(0. 01953 +/- 0. 002089)(ppm C) - 0. 01740 +/ - 0. 01393

Non-Acidified Pseudo Calibration Curve n Only 3 standards solutions ranging from 5 - 20 Non-Acidified Pseudo Calibration Curve n Only 3 standards solutions ranging from 5 - 20 ppm C, and a blank were analyzed F the standards were diluted from a KHP stock F the samples were zeroed by the spectrophotometer F the 10 ppm standard introduced error

TOC - Comparisons The ppm C derived by the UV-254 correlation is doubly ercompensated. TOC - Comparisons The ppm C derived by the UV-254 correlation is doubly ercompensated. Greater error values must also be noted as result of the limited calibration.

TOC Comparisons - Sheboygan River TOC Comparisons - Sheboygan River

TOC Comparisons - Lake Depue TOC Comparisons - Lake Depue

Note n The effects of the colloidal particles noted in the POC calculations is Note n The effects of the colloidal particles noted in the POC calculations is greatly amplified in the UV-254 method F n The 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 is the more accurate method in determining TOC F Although this exercise allowed a realization of the potential advantages and real limitations of experimental procedures

CAPILLARY ELECTROPHORE SIS CAPILLARY ELECTROPHORE SIS

Capillary Electrophoresis OBJECTIVE: Determination of concentration of specified ions in sample waters Capillary Electrophoresis OBJECTIVE: Determination of concentration of specified ions in sample waters

Introduction n Electrophoresis is the migration of ions in solution under influence of electric Introduction n 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 k. V to separate the components inside a fused silica capillary tube. Since different solutes have different mobilities, they will migrate through the capillary at different speeds This gives the extraordinary resolution and separation of many ionic species.

Electrophoresis n n When an ion with charge q is placed in an electric Electrophoresis n n When an ion with charge q is placed in an electric field E, the force on the ion is: F = q*E In solution, the other major force on the ion is the retarding frictional force f*vep, where vep is the electrophoretic velocity and f is the coefficient of friction: vep= q*E/f = µep. E The constant of proportionality between speed of ion and the applied electric field is: µep is proportional to the charge on the ion and inversely proportional to the friction coefficient.

Electroosmosis n n The inside surface of the silica capillary is covered with silanol Electroosmosis n n The inside surface of the silica capillary is covered with silanol (Si-OH) groups which carry a negative charge above p. H=2 These negative charges on surface induce cations to neutralize some of the surface charge The constant of proportionality between electroosmotic velocity (veo) and applied field is the electroosmotic mobility: µeo A relationship for the electrophoretic effect is: veo=µeo*E

Diagram: Hydrodynamic Velocity Profile n n (a) Positive charges move toward cathode, absorbed on Diagram: Hydrodynamic Velocity Profile n n (a) Positive charges move toward cathode, absorbed on surface of glass (b) More dispersion created by velocity profile because pushed from middle

Apparent Mobility n n The apparent (or observed) mobility ( app) of an ion Apparent Mobility n n 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+ eo For a cation moving in the same direction as the electroosmotic flow, the mobilities have the same sign and then app is greater than ep

Diagram: Solute Mobilities n n n (a) Optimize electrolyte conditions to make separation larger Diagram: Solute Mobilities n n n (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 n n n n Both ends of capillary placed into electrolyte Sample Diagram: Apparatus n n n n Both ends of capillary placed into electrolyte Sample injected by siphon effect Insert capillary into vial and elevate After injection, vial returned to normal height Apply voltage of 15 k. V Ions migrate through electrolyte Indirect detection

Br- SO 42 - Cl- NO 3 - Br- SO 42 - Cl- NO 3 -

Sample Data Sample Data

Cl- SO 42 - NO 3 - Cl- SO 42 - NO 3 -

1. DASS 2. WDNR 3. RP 4. KCL 5. EP 1. DASS 2. WDNR 3. RP 4. KCL 5. EP

Analysis n n n [NO 3] drops significantly from May to November [Cl-] and Analysis n n n [NO 3] drops significantly from May to November [Cl-] and [SO 4] increased overall in Lake Depue [Cl-] a bit higher along entire Sheboygan River compared to other ions

POTENTIOMETRI C METHODS POTENTIOMETRI C METHODS

Potentiometric Methods OBJECTIVE: Determination of acid/base properties of samples. Potentiometric Methods OBJECTIVE: Determination of acid/base properties of samples.

Alkalinity & CT n Representation of the buffer capacity of a water sample or Alkalinity & CT n Representation of the buffer capacity of a water sample or the ability of the water to neutralize strong acid. F Alk n = 2[CO 32 -] + [HCO 3 -] + [OH-] - [H+] Measure of alkalinity due to carbonate system F CT = [H 2 CO 3] + [HCO 3 -] + [CO 32 -]

Computer Automated Titration System ME-10 Analyzer Components n Automatic burette n Potentiometer Ô Glass Computer Automated Titration System ME-10 Analyzer Components n Automatic burette n Potentiometer Ô Glass n n 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 Glass Electrode Titrant (w/ reference electrode and ion selective membrane) Computer Titration System Setup Glass Electrode Titrant (w/ reference electrode and ion selective membrane) Computer Sample & mixer Burette

Glass Electrode Measures p. H n n The indicator electrode measures potential difference across Glass Electrode Measures p. H n n 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 èreference electrode èion selective glass membrane

Reference Electrode Within a tube in the indicator glass electrode: n n The reference Reference Electrode Within a tube in the indicator glass electrode: n n The reference electrode contains a small volume of dilute HCl and Ag. Cl. 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 strength 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 Ion Selective Membrane The ion selective glass membrane is sealed into one end of the glass tube. n When hydrated, it allows for the interaction between singly charged cations (electric conductivity) in the glass and protons from the solution. F n 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. F H+Gl- H+ + Gl. F (between glass and sample solution) (between internal solution and glass)

Typical Electrode System for Measuring p. H Typical Electrode System for Measuring p. H

Measurement through Electrode n n The equilibrium position of these 2 reactions are determined Measurement through Electrode n n 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. èA boundary potential Eb develops across the membrane which is sensed by the analyzer and recorded by the computer. n The potential change is recorded in m. Volts along with the corresponding volume of acid added

Measurement through Electrode n n Since constant temperature and ionic strength are maintained, the Measurement through Electrode n n Since constant temperature and ionic strength are maintained, the system obeys the Nernst equation. Eb is Emv where Emv= EG + k. T ln [H+] EG = Potential normal of the glass electrode for [H+]=1 M Includes reference potential and corrects for departure from ideal behavior k = R/F (R = Gas constant, F = Faraday’s constant) T = Room temperature in Kelvin

Calibration of Electrode System n n Titrated solution of 5 m. L 0. 1 Calibration of Electrode System n n Titrated solution of 5 m. L 0. 1 M KCl (to maintain ionic strength) and distilled water with 0. 1 M HCl at T = 22 o C or 295 K Verifies Nernst Equation by obtaining linear relationship by plotting p. H vs. . change in potential where p. H is ln [H+] = ln [(Vad * t. HCl)/(Vo + Vad)] Note: t. HCl = [HCL]=0. 1 M n Theoretically under these conditions should be k. T = 58. 54, the experimentally obtained value was k. T = 57. 51 a variation of less than 2%

Error Sources in Potentiometric Method n n Variation in temperature of solution possibly due Error Sources in Potentiometric Method n n Variation in temperature of solution possibly due to constant stirring Variation in ionic strength The Eppendorf was not sealed properly and additions of KCL to samples may have varied n Junction Potential: Potential develops from the difference in composition between sample and titrant. This potential arises from the unequal distribution of cations and anions and the different rates at which the species migrate. As long as ionic strength is maintained this potential is reduced.

Gran Method Priniciple: Graphical procedure based on knowledge that added increments of strong acid Gran Method Priniciple: Graphical procedure based on knowledge that added increments of strong acid linearly increase [H+] or decrease [OH-], likewise added increments of strong base decrease [H+] or increase [OH-]. n A titration curve is obtained by plotting volume added vs. potential E in m. V. In this lab strong acid is added to determine the alkalinity of our samples so we know that the lower part of the curve is composed of base, while the upper part of the curve is composed of acid.

Potentiometric Titration-DASS sample acidic V 2 = 2. 503+/- 0. 0159 m. V 200 Potentiometric Titration-DASS sample acidic V 2 = 2. 503+/- 0. 0159 m. V 200 180 160 140 120 100 80 60 40 20 0 -40 -60 -80 -100 -120 -140 -160 0. 2 0. 4 0. 6 0. 8 1 1. 2 1. 4 1. 6 1. 8 2 2. 4 2. 6 2. 8 basic V 1= 0. 1204+/-0. 0074 Volume Added 3 3. 2 3. 4 3. 6 3. 8 4

Gran Method cont. n n n At the midpoint the [base A-] = [ Gran Method cont. n n n At the midpoint the [base A-] = [ acid HA]. All base is titrated at the endpoint or equivalence point of the titration and is represented by an inflection point. Thus, using this method, it is possible to determine the carbonate system equivalence points, assuming that the alkalinity of our samples is due to the carbonate system. Ü Determine equilibrium constant for carbonate system

Gran Plots n F 1 For volumes > than the second equivalence point, volume Gran Plots n F 1 For volumes > than the second equivalence point, volume v 2: [H+] >> [HCO 3 -], [CO 32 -], and [OH-] together n So the following relationship is true F 1 = (vo+ vad)* [H+] = (vad - v 2)*t. HCl Note: t. HCl = [HCL]=0. 1 M n When plotted against vad, the function is linear beyond v 2 so F 1 = 0 for vad = v 2

Gran Plots F 2 n Between the first and second equivalence points volumes, v Gran Plots F 2 n Between the first and second equivalence points volumes, v 1 and v 2: [H 2 CO 3] >> [H+] - [CO 32 -] - [OH-] [HCO 3 -] >> [CO 32 -] + [OH-] - [H+] n n Similarly the following relationship holds F 2 = (v 2 - vad)*[H+] = (vad - v 1)*K 1 F 2 is linear for volumes less than v 2 when plotted against vad and F 2 = 0 for vad = v 1, with the slope of F 2 = K 1 the equilibrium constant for H 2 CO 3 HCO 3 - + H+

K= 6. 2512+/-2. 65 E-3 K= 6. 2512+/-2. 65 E-3

Alkalinity & CT Results n Determined by the following relationships [Alk] = (v 2* Alkalinity & CT Results n Determined by the following relationships [Alk] = (v 2* t. HCl)/ v 0 CT = ((v 2 - v 1)* t. HCl)/ v 0 DASS WDNR RP KCL EP MAY NOV Alkalinity 5. 0007 E-03 5. 0060 E-03 5. 0271 E-03 5. 0620 E-03 5. 0870 E-03 2. 7100 E-03 4. 4810 E-03 CT 4. 7601 E-03 4. 8310 E-03 4. 7971 E-03 4. 8210 E-03 4. 8160 E-03 2. 5910 E-03 4. 2230 E-03 both are molar values M

Analysis n Sheboygan River: Alkalinity increases moving downstream along the, while CT remains essentially Analysis n Sheboygan River: Alkalinity increases moving downstream along the, while CT remains essentially constant varying around 4. 8 E-3 M n Lake Depue: Alkalinity and CT increase in the winter nearly doubling, most likely due to the course of productivity throughout the year

GLUCOSIDIC & PROTEINACEOUS FRACTIONS OF DOM GLUCOSIDIC & PROTEINACEOUS FRACTIONS OF DOM

GLUCOSIDIC & PROTEINACEOUS FRACTIONS OF DOM OBJECTIVE: Qualitative analysis of simple sugars and amino GLUCOSIDIC & PROTEINACEOUS FRACTIONS OF DOM OBJECTIVE: Qualitative analysis of simple sugars and amino acid fractions of DOM through colorimetric and fluorescence methods. These residuals of biological activity and decay are significant in determining COD.

GLUCOSIDIC FRACTION COLORIMETRY/SPECTROSCOPY: n Reaction of phenol and H 2 SO 4 (Dubois reagents) GLUCOSIDIC FRACTION COLORIMETRY/SPECTROSCOPY: n Reaction of phenol and H 2 SO 4 (Dubois reagents) with the samples breaks down complex sugars. n n The attachment of phenol to the reduced sugar monomers produces compounds of a stable yellowish color. Spectrometric measurement of the intensity of color quantifies the specific fraction of sugars.

Spectrometer 1 - D 2 lamp 2 - Grating 1 3 - Entrance Slit Spectrometer 1 - D 2 lamp 2 - Grating 1 3 - Entrance Slit 4 - Grating 2 5 - Exit Slit 6 - Chopper 7 - Sample & Reference Positions 8 - Chopper 9 - Photo Tube

Spectrophotometry n n Mirrors and gratings redirect and disperse the radiation The slits limit Spectrophotometry n n Mirrors and gratings redirect and disperse the radiation The slits limit the radiation range allowing successively isolated wavelengths to be selected The rotating chopper wheels alternately direct the light beam through the sample and reference A reference sample is required to zero the interference effects of the cuvette. Other Interferences include : particulates scattering non-absorbing organic material

GLUCOSIDIC CALIBRATION @ 480 nm No distinction can be made between the 0 and GLUCOSIDIC CALIBRATION @ 480 nm No distinction can be made between the 0 and 10 u. M concentrations. Most of our data points fall within this range. The calibration from this method therefore does not lead to conclusive results.

GLUCOSIDIC CALIBRATION @ 490 nm GLUCOSIDIC CALIBRATION @ 490 nm

GLUCOSIDIC RESULTS 480 nm As noted, all of our data points refer to concentrations GLUCOSIDIC RESULTS 480 nm As noted, all of our data points refer to concentrations less than 10 u. M with the exception of samples EP and May. In general, this did not provide us with useful data for analysis.

GLUCOSIDIC RESULTS 490 nm GLUCOSIDIC RESULTS 490 nm

ANALYSIS n The glucosidic fraction of TOC increases downstream in the Sheboygan River. F ANALYSIS n The glucosidic fraction of TOC increases downstream in the Sheboygan River. F The TOC values previously determined show a slight decreasing trend downstream. n n The Lake Depue fraction is higher in May than November. Using glucose as a representation of all other sugars is not a good quantitative method.

Total Hydrolyzable Free Amino Acids (Proteinaceous Fraction) n n Samples reacted with OPAMERC solution Total Hydrolyzable Free Amino Acids (Proteinaceous Fraction) n n Samples reacted with OPAMERC solution to bind with aromatic compounds, such as proteins Fluorometric measurement quantifies proteinaceous fraction

FLUORESCENCE n n The fluorometer energy source excites electrons of organic compounds bound to FLUORESCENCE n n The fluorometer energy source excites electrons of organic compounds bound to OPA-MERC High energy state is unstable As electrons return to a more stable ground state, visible light is emitted From the intensity of emitted light, proteinaceous fractions can be determined

Fluorometer Fluorometer

Procedure n Simple, low-cost, and easy to use n Mercury lamp for fluorescence excitation Procedure n Simple, low-cost, and easy to use n Mercury lamp for fluorescence excitation n Source beam split near source into a reference beam and a sample beam Both beams pass through primary filter Sample beam causes emission of fluorescent radiation

Proteinaceous Calibration Like the glucosidic calibration the amount of scattering within the data shows Proteinaceous Calibration Like the glucosidic calibration the amount of scattering within the data shows that this method is unreliable at these concentrations.

Proteinaceous Results No apparent trends are noted. Proteinaceous Results No apparent trends are noted.

DOM FINGERPRINTING BY PY-GC-MS DOM FINGERPRINTING BY PY-GC-MS

DOM FINGERPRINTING OBJECTIVE: To characterize the constituents of dissolved organic matter, accomplished by breaking DOM FINGERPRINTING OBJECTIVE: To characterize the constituents of dissolved organic matter, accomplished by breaking down the DOM through a 3 -step process. The data obtained in this procedure is based on a wetland sample taken on August 6, 1998

PY-GC-MS METHOD STEP 1: PYROLYSIS n In an inert environment and at a controlled PY-GC-MS METHOD STEP 1: PYROLYSIS n In an inert environment and at a controlled temperature, the organic matter from the concentrated water samples is thermally degraded F Bonds within the OM are broken and rearranged F Predictable and reproducible fragments form

PY-GC-MS METHOD STEP 2: GAS CHROMATOGRAPHY n n The pyrolyzed fragments are drawn into PY-GC-MS METHOD STEP 2: GAS CHROMATOGRAPHY n n The pyrolyzed fragments are drawn into the GC column The fragments migrate through the column by action of a He mobile phase flushing the system F affinity to a stationary phase (the silica column) results in varied migrations rates for the different types of fragments

Gas Chromatograph Gas Chromatograph

PY-GC-MS METHOD STEP 3: MASS SPECTROMETRY n n n A detector senses when the PY-GC-MS METHOD STEP 3: MASS SPECTROMETRY n n n A detector senses when the organic matter fragments reach the end of the column This signal is plotted versus time producing a chromatogram Specific fragments can be identified by their characteristic retention times

Chromatogram 1 1. Unknown aliphatic 2. Acetic Acid 3. Propanoic Acid 4. Dimethyl-Propanedioic Acid Chromatogram 1 1. Unknown aliphatic 2. Acetic Acid 3. Propanoic Acid 4. Dimethyl-Propanedioic Acid 5. Butanoic Acid 6. Hexanoic Acid 7. Butenoic Acid 8. Phenol 2 8 3 4 56 7

PY-GC-MS METHOD STEP 3 CONTINUED: MASS SPECTROMETRY n n n In the mass spectrometer PY-GC-MS METHOD STEP 3 CONTINUED: MASS SPECTROMETRY n n n In the mass spectrometer the fragments are ionized An alternating current through the 4 poles of the mass spec separates the ionized fragments by their mass/charge ratios The mass spec plots the spectrum of the ionized fragments; mass/charge ratio versus % abundance

Quadrupole Mass Spectrometer the fragments of the specific mass/charge ratio wanted at any one Quadrupole Mass Spectrometer the fragments of the specific mass/charge ratio wanted at any one time pass between the rods without being neutralized F the other fragments are neutralized by contact with the rod walls F

PY-GC-MS METHOD STEP 3 CONTINUED: MASS SPECTROMETRY n The software program used in conjunction PY-GC-MS METHOD STEP 3 CONTINUED: MASS SPECTROMETRY n The software program used in conjunction with this method performs an online library (NIST) search to match the mass spectra of a fragment to known compounds. n The compounds detected in our sample : unknown aliphatic acetic acid propanoic acid dimethyl-propanoic acid butanoic acid hexanoic acid butenoic acid phenol

Note: n n If we had samples leftover to analyze, the PY-GCMS method would Note: n n If we had samples leftover to analyze, the PY-GCMS method would have been beneficial in providing structural feature fingerprints serving as chemical markers within our samples. Trends may be depicted. Analysis methods can allow results comparisons with other methods. Such as with determination of the glucosidic fraction. PY-GC-MS analysis is enhanced when used in conjunction with data from other methods.

Evaluation n n We have improved our laboratory skills Through this course we have Evaluation n n We have improved our laboratory skills Through this course we have gained an understanding of analytical methods currently being used in the environmental field We not only have a more comprehensive understanding of scientific terminology, we are capable of analyzing data in a more applicable way In previous laboratory courses, error analysis was not required. We have gained an understanding of how results are obtained and how error can limit their relevance

References n C 45 Winter 1999 Lab Manual n Samuel Webb, Jill Kostel, Tanita References n C 45 Winter 1999 Lab Manual n Samuel Webb, Jill Kostel, Tanita Sirivedhin - technical advice n Leary, Skoog. Principles of Instrumental Analysis n Chen, Senesi, Schnitzer. “Information Provided on Humic Substances by E 4/E 6 Ratios, ” Soil Science Society of America. n n n Aiken, Chin, O’Loughlin. “Molecular Weight, Polydisperity and Spectroscopic Properties of Aquatic Humic Substances, ” Environmental Science & Technology. Dean, Dobbs, Wise. “The Use of Ultra-Violet Absorbance fo r. MOnitoring the Total Organic Carbon Content of Water and Wastewater, ” Water Resources. Kukkonen. “Effects of Lignin and Chlorolignin in Pulp Mill Effluents on the Binding and Bioavailability of Hydrophobic Organic Pollutants, ” Water Resources.