Instrumental Analysis Text: Principles of Instrumental Analysis, 5th Ed., Skoog, Holler, Nieman, Harcourt Brace, 1998

Classification of Analytical Methods Classical Also called wet-chemical methods Separation of component of interest (analyte) from the sample by precipitation, extraction, or distillation Followed by gravimetric or titrimetric measurement for quantitative analysis Instrumental Use of new methods for quantitative analysis

Involve interactions of analyte with EMR Instrumental Methods Involve interactions of analyte with EMR Radiant energy is either produced by the analyte (eg., Auger) or changes in EMR are brought about by its interaction with the sample (eg., NMR) Other methods include measurement of electrical properties Potentiometry, voltammetry, amperometri

Instruments Converts information stored in the physical or chemical characteristics of the analyte into useful information Require a source of energy to stimulate measurable response from analyte Data domains Methods of encoding information electrically Nonelectrical domains Electrical domains (Analog, Time, Digital)

Detector Device that indicates a change in one variable in its environment (eg., pressure, temp, particles) Can be mechanical, electrical, or chemical Sensor Analytical device capable of monitoring specific chemical species continuously and reversibly Transducer Devices that convert information in nonelectrical domains to electrical domains and the converse

Selecting an Analytical Method What accuracy is required How much sample is available What is the concentration range of the analyte What components of the sample will cause interference What are the physical and chemical properties of the sample matrix How many samples are to be analyzed

Accuracy vs. Precision Accuracy Describes the correctness of an experimental result Absolute error Relative error Precision Describes the reproducibility of results Standard deviation Variance CV

Figures of Merit Precision Degree of mutual agreement among data that have been obtained in the same way A measure of the random, or indeterminate error of an analysis FOM Absolute standard deviation Relative standard deviation Coefficient of variation Variance

Bias A measure of the systematic, or determinate, error of an analytical method Bias = µ - xt In developing an analytical method, sources of bias should be identified and eliminated or corrected for with use of blanks or instrument calibration

Standard Reference Materials (SRM) Provided by National Institute of Standards and Technology (NIST) Specifically prepared for validation of analytical methods Concentration of constituents has been determined by A previously validated reference method 2 or more independent, reliable measurement methods Analyses from a network of cooperating labs

Sensitivity Of an instrument or method is its ability to discriminate between small differences in analyte concentration 2 factors limit sensitivity Slope of calibration curve Precision of measuring device

Detection Limit The minimum concentration or mass of analyte that can be detected at a known confidence level Sm = Sbl + ksbl

Dynamic Range Extends from the lowest concentration at which quantitative measurements can be made (LOQ), to the concentration at which the calibration curve departs from linearity (LOL) An analytical method should have a dynamic range of at least 2 orders of magnitude

Selectivity Of an analytical method refers to the degree to which the method is free from interference by other species contained in the sample matrix No method is totally free from interference from other species

Calibration of Instrumental Methods Analytical methods require calibration Process that relates the measured analytical signal to the concentration of analyte 3 common methods Calibration curve Standard addition method Internal standard method

Calibration Curve Standards containing known concentrations of the analyte are introduced into the instrument Response is recorded Response is corrected for instrument output obtained with a blank Blank contains all of the components of the original sample except for the analyte Resulting data are then plotted to give a graph of corrected instrument response vs. analyte concentration An equation is developed for the calibration curve by a least-squares technique so that sample concentrations can be computed directly

Standard Addition Method Usually involves adding one or more increments of a standard solution to sample aliquots of the same size (spiking)

Lab 1: Spectrophotometric Analysis of a Mixture of Absorbing Substances Purpose is to determine the individual concentrations of a mixture of absorbing substances Gain experience working with a UV-Vis Spectrophotometer Practice several analytical techniques Understand absorbance and application of the Beer-Lambert Law

Background: Absorption of Radiation Absorption – A process in which electromagnetic energy is transferred to the atoms, ions, or molecules composing a sample Promotes particles from their normal room temperature state (ground state) to one or more higher-energy states. Atoms, molecules or ions have a limited number of discrete energy levels For absorption to occur, the energy of the exciting photon must exactly match the energy difference between the ground state and an excited state of the absorbing species

Absorbing solution of concentration, c Absorption Methods Absorbance A of a medium is defined A = -log10T = log10P0/P Beer-Lambert Law is defined A = Єbc P0 P b Absorbing solution of concentration, c

Lab Report Write-up Introduction to spectroscopy, instrument basics, absorption principles and Beer-Lambert Law Experimental section Specific instrumention (www.oceanoptics.com) Experimental procedures Results Abs vs. wavelength spectra Plots of concentration vs. absorbance, including equations of lines and R2 Red at λ1 and at λ2 Yellow at λ1 and at λ2 Tables Dilutions Red absorbance by concentration Yellow absorbance by concentration Є values Equations and unknown concentrations Conclusions References

Signals and Noise Analytical measurements consist of 2 components Signal to noise ratio S/N = x/s = mean / standard deviation

Sources of Noise in Instrumental Analysis Chemical Noise Instrumental Noise Thermal noise Shot noise Flicker noise Environmental noise

Signal to Noise Enhancement Hardware Software Ensemble Averaging Boxcar Averaging Digital Averaging Fourier transformation

An Introduction to Spectrometric Methods Spectroscopy Interactions of various types of radiation with matter Electromagnetic radiation (light, X-Rays) Ions and electrons

Properties of EMR Described by means of sine wave Wavelength, frequency, velocity, amplitude Particle model of radiation is necessary Represented as electric and magnetic fields that undergo sinusoidal oscillations at right angles to each other and the direction of propogation

vi = n li Frequency is determined by source and remains invariant Velocity depends on medium Velocity (air or vacuum) = c = 3.00 x 108 m/s = l n

Transmission of Radiation Refractive index A measure of the interaction of radiation with the medium it travels through hi = c/vi

Scattering of Radiation Small fraction of radiation is scattered as it passes through a medium Rayleigh Scattering (elastic) Scattering by molecules with wavelengths smaller than wavelength of radiation Its intensity is proportional to 1/l4 Raman Scattering (inelastic)

Diffraction of Radiation All types of EMR exhibit diffraction Is a consequence of interference A parallel beam of radiation is bent as it passes a barrier or slit nl = BC sin q (Bragg Equation)

The Photoelectric Effect Experiments revealed that a spark jumped more readily between 2 charged spheres when their surfaces were illuminated with light EMR is a form of energy that releases electrons from metallic surfaces Below a certain frequency, no additional sparks (electrons) are observed

E = hn (Einstein) eV0 = hn - w E = hn = eV0 + w

Emission of Radiation EMR is produced when excited particles (atoms, ions, or molecules) relax to lower energy levels by giving up their excess energy as photons Excitation can be brought about by Bombardment with electrons Irradiation with a beam of EMR

Radiation from an excited source is characterized by an emission spectrum Plot of relative power of emitted radiation vs wavelength or frequency Types of spectra Line Band Continuum

Absorption of Radiation In absorption, EM energy is transferred to atoms, molecules comprising the sample Absorption promotes these particles from RT state to a higher-energy excited state For absorption to occur, the energy of the exciting photon must exactly match the energy difference between the ground state and one of the excited states of the absorbing species

Atomic Absorption Passage of radiation through a medium that consists of monoatomic particles results in absorption of a few frequencies Simplicity is due to small number of possible energy states for the absorbing particles

Molecular Absorption More complex because the number of energy states is large compared to isolated atoms The energy, E, associated with the molecular bands: E = Eelectronic + Evibrational + Erotational

Components of Optical Instruments Stable source of radiant energy Transparent sample container Device that isolates a restricted region of the spectrum Radiation detector Signal processor and readout

Sources of Radiation Source must generate a beam of radiation with sufficient power Output must be stable for reasonable periods Radiant power of a source varies exponentially with the voltage of its power supply Continuum (tungsten) Line (lasers)

Wavelength Selectors Narrow bandwidth is required Filters Monochromators, consisting of Entrance slit Collimating lens (or mirror) Grating (or prism, historical) Focusing element Exit slit

Radiation Transducers Convert radiant energy into an electrical signal Photon transducers Photomultiplier tube (PMT) Contain a photoemissive surface Emit a cascade of electrons when struck by electrons Useful for measurement of low radiant power

Component Configuration for Optical Absorption Spectroscopy Source Lamp Sample Holder Wavelength Selector Photoelectric Transducer Signal Processor and Readout

Atomic Absorption Spectrometry Most widely used method for determination of single elements in analytical chemistry Quantification of energy absorbed from an incident radiation source from the promotion of elemental electrons from the ground state Technique relies on a source of free elemental atoms electronically excited by monochromatic light

Sample Introduction in AAS Flame Method of supplying atom source Utilizes a nebulizer in conjunction with air/acetylene flame Solvent evaporates Metal salt vaporizes and is reduced to complete the atomization process Radiation source is a hollow cathode lamp

Graphite Furnace AAS Samples are atomized by electrothermal atomization Provide an increase in sensitivity and improved safety compared to Flame AAS instruments Applications

Mass Spectrometry Relies on separating gaseous charged ions according to their mass-to-charge ratio (m/z) Widely used in conjunction with other analytical techniques

Operating Principles Sample inlet Sample ionization Ion acceleration by an electric field Ion dispersion according to m/z Identification of ion mass

Mass to Charge Ratio Obtained by dividing the atomic or molecular mass of an ion, m, by the number of charges, z, of the ion Most ions are singly charged

Molecular Absorption Measurement of Transmission and Absorption Limitations to Beer-Lambert Law Concentration Chemical deviations Polychromatic Radiation

Fluorescence and Phosphorescence Following absorption Nonradiative relaxation Loss of energy in a series of small steps Energy of molecule is conserved Fluorescence Emission Excited State analyte molecule returns to the GS producing radiative emission (a photon is emitted) ~10-5 s Phosphorescence Emission Similar to fluorescence but process is > 10-5 s Due to relaxation from an excited triplet state

Units Wavenumbers (cm-1) are convention Easy to convert between wavelength and frequency

Infrared Spectroscopy, Chapter 16 March 10, 2005 Theory of Infrared Spectroscopy Components Read Sections 16A, 16B Homework: 16-2

IR Spectral Regions, Table 16-1 Wavelength Range, mm Wavenumber Range, cm-1 Frequency Range, Hz Near 0.78-2.5 12,800-4,000 3.8E14 – 1.2E14 Middle 2.5-50 4,000-200 1.2E14-6.0E12 Far 50-1,000 200-10 6.0E12-3.0E11 Most used 2.5-15 4,000-670 1.2E14-2.0E13

Dipole Changes During Molecular Vibrations IR radiation is not energetic enough to cause electronic transitions To absorb IR radiation, a molecule must undergo a net change in dipole moment due to its vibrational (or rotational) motion If n of EMR matches a vibrational frequency of the molecule, a net transfer of energy occurs Results in change in amplitude of vibration Absorption of radiation occurs

Types of Molecular Vibrations Stretching Continuous change in interatomic distance along axis of atomic bond Bending Characterized by a change in angle between 2 bonds Scissoring Rocking Wagging Twisting

Simple Harmonic Oscillator Model which approximates atomic stretching vibrations Vibration of a single mass attached to a spring hung from an immovable object (Figure 16-3a) : F = -ky

Vibrational Frequency (16-7) (16-8) (16-9) (16-14)

Vibrational Modes Linear molecules Polyatomic molecules 3N-5 (number of possible molecular vibrations) Polyatomic molecules 3N-6 (number of possible molecular vibrations)

Infrared Sources Inert solid electrically heated to 1500-2200K Nernst Glower Rare earth oxides formed into a cylinder Formed into a resistive heating element, 1200-2200K Globar Source Silicon carbide rod, also electrically heated, 1300-1500K Greater output than Nernst Glower below 5 mm Tungsten Filament Lamp Used in near-IR region of 4,000-12,800 cm-1 Infrared lasers

Chromatographic Separations

General Description In all chromatographic separations, the sample is transported in a mobile phase Gas, liquid, or supercritical fluid fundamental classification Mobile phase is forced through an immiscible stationary phase Column or solid surface As a consequence of differences in mobility, sample separates into bands or zones

Chromatograms Plot of analyte concentration vs. time Positions of peaks on time axis identify components of sample Areas under peaks provide quantitative measure of amount of each component Figure 26-4

Migration Rates of Solutes Distribution constant Amobile ↔ Astationary Retention Factor

Chromatographic Peak Shape Similar to normal error or Gaussian curve Attributed to additive combination of random motions of solute molecules in chromatographic zone Peak represents behavior of average molecule Breadth of band is directly related to residence time in column and inversely related to mobile phase velocity

Column Efficiency Plate height Plate count, N = L/H Maximum efficiency occurs at minimum H

Column Resolution Resolution, Rs, provides a quantitative measure of its ability to separate analytes