Luminescence transitions Fluorescence spectroscopy Advantages: High sensitivity (single molecule detection!) Measuring increment in signal against a dark (zero) background Emission is proportional to excitation intensity, therefore can use high energy sources (lasers) to enhance sensitivity High specificity Emission spectrum is compound specific (analogous to absorption spectra being compound specific Disadvantages: Limited number of natively fluorescent chemicals Quenching of fluorescence by matrix components
X-Ray Fluorescence Process Bohr model of atom X-ray energy spectrum for lead
Potential interferences in XRF XRF spectroscopy Advantages Good sensitivity(low microgram) (Relatively) non-destructive Applicable to solid samples Obtain measurements from multiple elements simultaneously Disadvantages Provides no information on the chemical form of the element Primarily of use for metals and inorganic compounds, not applicable to organics Uses ionizing radiation (radioactive source or high E electrical discharge)!safety issues
XRF spectroscopy Applications Field screening for metals in solid samples (e.g. Niton XRF) Sensitive detection of elements in air particulate (collected on filters) Multivariate data collected in this way is used to deduce sources of particulate air pollution Pie chart shows the major sources of PM in Seattle. The source strengths were deduced in large part from XRF measurements of multiple elements on filter samples Chromatography ENVH 431 Reading: Chapters 23-25 in Harris, Quantitative Chemical Analysis
Learning objectives: What is chromatography, what are the underlying principles? Why (and when) is chromatography performed? What are the common modes of chromatography? How do you interpret chromatographic data? Chromatography The separation of analytes for individual detection in a complex matrix Chromatography was invented Russian botanist Mikhail Tswett (~1900) for separation of plant pigments Passed solutions of chlorophylls and xanthophylls through glass columns packed with calcium carbonate Separated species appeared as colored bands
Liquid-Liquid Extraction of Analytes like dissolves like Partition Coefficient (K) K = C S / C M Partitioning of the analyte (chloroform) between S: Organic Solvent (MtBE) M: Water Principles of Chromatography Stationary phase is fixed in place in a column or on a planar surface Mobile phase (or Eluent) is moving over or through the stationary phase, carrying the analyte along with it Chromatography: components of a mixture are separated based on rates at which they are carried through the stationary phase by a gaseous or liquid mobile phase Elution: process of an analyte being washed through a stationary phase by the movement of a mobile phase Chromatogram: the plot of analyte signal (function of concentration) versus elution time
Column Chromatography Detector Signal B A Time Liquid Chromatography (HPLC) uses liquid mobile phase
Gas Chromatography (GC) Desirable features of a chromatographic separation Adequate retention Capacity factor: Sharp, symmetrical peaks Narrow peak width No tailing Adequate separation between peaks Resolution:
Signal Time (minutes) Peak Resolution Change Column phase or dimensions (e.g., Length) Change Flow Rate Mobile phase (gas or liquid) Change Gradient Temperature or pressure Mobile Phase Composition Chromatographic Parameters Stationary Phase (solid or solid-liquid) Adsorption Partition Molecular exclusion Ion-Exchange Affinity (protein lock to antibody key) Mobile Phase (gas or liquid) Flow rate Composition Column Dimensions Length, width (ID), particle size or film thickness GC HPLC
Quantifying chromatographic peaks Amount is proportional to the integrated signal across the peak ( area under the curve ) Proportionality constant comes from calibration using standard solutions Peak areas can be approximated: Peak height* Triangle area* *Requires constant peak shape Electronic signal integration is now standard with instrument software packages Interpreting Chromatograms Analysis of Trihalomethanes in Drinking Water
Disinfection Byproducts (DBPs) in Drinking Water + Cl 2! Trihalomethanes + Br - (THMs) Natural Organic Matter found in water and soil (structure proposed by Kleinhempel, D. Albrecht Thaer Arch., 1970, 14:3) and other DBPs Potential Carcinogens: Regulated DBPs in Drinking Water Trihalomethanes (sum of 4) CHCl 3 + CHBrCl 2 + CHBr 2 Cl + CHBr 3 US EPA! 80 µg/l WHO! 100 µg/l of each Haloacetic Acids (sum of 5) US EPA! 60 µg/l Bromate US EPA and WHO! 10 µg/l
EPA Method 551.1 for Trihalomethanes (THMs) Liquid-liquid extraction of water sample with Methyl tert-butyl Ether (MtBE) Addition of an internal standard IS (1,2-Dibromopropane) to the extraction solvent Analysis of extracts by gas chromatography with electron capture detection (GC-ECD) Quantification by plotting relative area (THM/IS) vs concentration and drawing a best-fit line through a range of calibration points. Identify Peaks in Chromatograms Internal Standard (IS) 1,2-Dibromopropane Four Trihalomethanes Chloroform (CHCl 3 ) Bromodichloromethane (CHBrCl 2 ) Dibromochloromethane (CHBr 2 Cl) Bromoform (CHBr 3 ) Compare chromatograms to Method 551.1
Trihalomethanes by GC-ECD EPA Method 551.1 Detector Response Internal Standard (IS) Retention Time (minutes) MtBE
Extraction Solvent: IS/MtBE THM standards in IS/MtBE
Trihalomethanes by GC-ECD EPA 551.1 Detector Response Internal Standard (IS) Retention Time (minutes) THMs detected in an extract of chlorinated water
GC vs HPLC GC Highly efficient separations Analytes must be (semi) volatile Analytes must be thermally stable Non-polar, small molecules Limited control over mobile and stationary phases HPLC (Slightly) less efficient separations Small and large molecules, polar and non-polar Useful for thermally unstable analytes Many choices available for mobile and stationary phases Gas Chromatograph (GC) Chromatogram
Manual GC injection port GC Operating Variables Mobile Phase (gas) N 2, He, H 2 Stationary Phase in GC Column Packed Columns Silica (short & wide) Open Tubular Columns Liquid-bonded siloxanes Long & narrow
GC Operating Variables Temperature gradient Increase the temperature at a set rate ( C/min) in order to sharpen peaks and decrease retention time on column. Pressure gradient Increase the inlet pressure to increase carrier gas flow rate and decrease retention time of late-eluting peaks. Signal Peak Resolution! Change Temperature Gradient 1 Time (minutes) Temperature Program 2 Temperature (C) 1 2 Time (minutes)
Quantitative GC Detectors Thermal Conductivity Detector (TCD) Flame Ionization (FID) Electron Capture (ECD) Flame Photometric (FPD) Qualitative and Quantitative Infrared Spectrometer (FTIR) Mass Spectrometer (MS or MS/MS) GC vs HPLC GC Highly efficient separations Analytes must be (semi) volatile Analytes must be thermally stable Non-polar, small molecules Limited control over mobile and stationary phases HPLC (Slightly) less efficient separations Small and large molecules, polar and non-polar Useful for thermally unstable analytes Many choices available for mobile and stationary phases
High Performance Liquid Chromatograph (HPLC) High Performance Liquid Chromatograph (HPLC) Fluorescence detector Diode array (UV) detector Column heater + micro switching valve Autosampler Solvents Solvent degasser Sampler cooling unit High pressure pump unit
HPLC Mobile Phase Choices Composition of solvent water, phosphate buffer, methanol, acetonitrile Isocratic elution single solvent or constant solvent mixture Solvent gradient Increase eluent strength to elute the strongly retained analytes Reversed phase column: start with 100% aqueous phase (A) and gradually increase the % of organic solvent phase (B) running through the column as mobile phase Signal Peak Resolution! Change Mobile Phase Composition 1 Time (minutes) 2 Percent Organic Mobile Phase Gradient Program 1 2 Time (minutes)
Choice of Stationary Phase Principles of Reversed Phase Partitioning HPLC Analyte partitions between stationary and mobile phase Changes in mobile phase composition (e.g. gradient elution) alter the equilibrium between mobile and stationary phases All compounds spend the same amount of time in the mobile phase (void volume). Retention is therefore due to differing amounts of time spent in/on the stationary phase
HPLC Detectors Quantitative Ultraviolet (UV) Refractive Index Conductivity Light-Scattering Fluorescence Post-column Electrochemical Derivatization Qualitative and Quantitative Photodiode array (DAD): UV spectrum of each peak Mass spectrometer Selectivity differences in HPLC detectors