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

Instrumental Analysis

Classification of Analytical Methods

•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

Classical

•Use of new methods for quantitative analysis

Instrumental

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

• Methods of encoding information electrically• Nonelectrical domains• Electrical domains (Analog, Time, Digital)

Require a source of energy to stimulate measurable response from analyte

Data domains

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

•Describes the correctness of an experimental result•Absolute error•Relative error

Accuracy

•Describes the reproducibility of results•Standard deviation•Variance•CV

Precision

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)

• A previously validated reference method• 2 or more independent, reliable measurement methods• Analyses from a network of cooperating labs

Specifically prepared for validation of analytical methods

Concentration of constituents has been determined by

Sensitivity

Of an instrument or method is its ability to discriminate between small differences in analyte concentration

• Slope of calibration curve• Precision of measuring device

2 factors limit sensitivity

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

• Calibration curve• Standard addition method• Internal standard method

Process that relates the measured analytical signal to the concentration of analyte

3 common methods

Calibration Curve

Standards containing known concentrations of the analyte are introduced into the instrument

• Blank contains all of the components of the original sample except for the analyte

Response is recorded

Response is corrected for instrument output obtained with a blank

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

Absorption Methods

• Absorbance A of a medium is definedA = -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

Spectrophotometric Analysis of a Mixture of Absorbing Substances

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

300 500 700 900 1100

Wavelength, nm

Ab

so

rba

nc

e

Red 6 ppm

Badger Red

0

0.1

0.2

0.3

0.4

0.5

0.6

0 2 4 6 8 10 12

Conc, ppm

Ab

s abs at l1

abs at l2

Signals and Noise

• Analytical measurements consist of 2 components– Signal– Noise

• 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 = h n (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 HolderWavelength

Selector

PhotoelectricTransducer

Signal Processorand 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 16March 10, 2005

• Theory of Infrared Spectroscopy• Components• Read Sections 16A, 16B• Homework: 16-2

IR Spectral Regions, Table 16-1

RegionWavelength 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

21

21

mm

mm

21

21 )(

2

1

2

1

mm

mmkkvm

m

kvm 2

1 (16-7)

(16-9)

(16-8)

k

xk

cv 12103.5

2

1 (16-14)

Vibrational Modes

• Linear molecules3N-5 (number of possible molecular vibrations)

• Polyatomic molecules3N-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 constantAmobile ↔ Astationary

• Retention Factor

m

s

c

cK

M

MRA t

ttk

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

2

2

16 Rt

LWH

Column Resolution

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

BA

ARBRs WW

ttR

])()[(2