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

2. 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

3. 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

4. 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)

5. 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

6. 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

7. 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

8. 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

9. 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

10. 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

11. 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

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

13. 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

14. 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

15. 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

16. 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

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

18. 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

19. 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

20. 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

21. Lab Report Write-up
Introduction to spectroscopy, instrument basics, absorption principles and Beer-Lambert Law
Experimental section
Specific instrumention (
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

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

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

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

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

28. 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

29. 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

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

31. 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)

32. 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)

33. 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

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

35. 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

36. 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

37. 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

38. 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

39. 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

40. 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

41. 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)

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

43. 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

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

45. 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

46. 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

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

48. 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

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

50. 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

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

52. 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

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

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

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

56. 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

57. 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

58. 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

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

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

61. Infrared Sources Inert solid electrically heated to 1500-2200K
Nernst Glower
Rare earth oxides formed into a cylinder
Formed into a resistive heating element, K
Globar Source
Silicon carbide rod, also electrically heated, K
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

62. Chromatographic Separations

63. 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

64. 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

65. Migration Rates of Solutes
Distribution constant
Amobile ↔ Astationary
Retention Factor

66. 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

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

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