FOR ANACHEM FINALS

LECTURE 1: INTRODUCTION TO ANALYTICAL CHEMISTRY

ANALYTICAL CHEMISTRY - a branch of chemistry that deals with the separation, identification & determination of analytes (components) in a sample

AREAS OF ANALYTICAL CHEMISTRY :

  • Industrial Quality Control - requirements of companies for product quality

  • Bioanalytical Chemistry and Analysis - detection or analysis of biological components (e.g., proteins, DNA, RNA, carbohydrates)

  • Environmental Analysis - monitoring of pollutants, soil and water analysis

  • Forensic Analysis - criminology, DNA finger printing, finger-print detection, blood analysis (crime detection and court testimonies)

  • Clinical Analysis - analysis of blood, urine, feces, cellular fluids (used in medical diagnosis)

  • Pharmaceutical Analysis - analysis of physical properties, toxicity, metabolites, quality control, etc.

TERMS

Validate - prove that the method works and define its limitations (e.g., sample type, detection limits, interferences, concentration, sensitivity, etc)

Verify - ensure that analysts are able to obtain correct results using the method

Sample - representative of the population or gross sample

Analyte - substance to be identified, detected, or separated in some manner

Matrix - all other constituents in a sample except for the analyte

Interference - a specific component identified to be causing an effect

CHEMICAL ANALYSIS

Qualitative Analysis - an analysis to identify the material(s) present in a sample

Quantitative Analysis - an analysis to determine the amount of a material is present in a sample

  • Complete Analysis - amount of each constituent of the sample is determined

  • Partial Analysis - amount of a certain selected constituent in a sample is determined

CLASSIFICATION OF CONSTITUENTS IN A SAMPLE

  • Major (more than 1%)

  • Minor (0.1 to 1%

  • Trace (less than 0.1%)

  • Ultra trace (a few ppm or less)

CLASSIFICATION OF ANALYTICAL METHODS

  1. Instrumental Methods

    • Spectroscopic Methods - measure the interaction between analyte and electromagnetic radiation or the production of radiation by an analyte

    • Separation/Chromatographic Methods - measure peak areas of the separated components of a sample

      • High Performance Liquid Chromatography

      • Gas Chromatography

    • Electroanalytical Methods - measure an electrical property which is chemically related to amount of analyte

  2. Classical Methods

    1. Gravimetric Methods - measure mass of the analyte or a compound chemically related to the analyte

    2. Volumetric Methods - measure volume of a solution containing sufficient reagent to react with the analyte

EXPRESSIONS OF CONCENTRATION

Concentration - the amount of solute in a known amount of solution (solute + solvent) with the formula: amount of solute / amount of solution

STEPS OF ANALYTICAL PROCESS
THE ANALYTICAL TRAIN:

  1. PROBLEM DEFINITION

    • identify problems and formulate questions

    • need to translate general questions into specific questions which will be answered by chemical measurement

  2. LITERATURE REVIEW / SEARCH

    • availability of methods or “standard methods” (e.g., journals, APHA, EPA, BS, ASTM Book of standards, methods of the AOAC, SIRIM, NIOSH Methods)

    • IN HOUSE METHOD - adopted from standard method with minor changes

    • COOKBOOK - analysis run by the instrument purchased

  3. METHOD SELECTION

    • sample type, sample size & preparation required

    • skill and training of analyst

    • tools/instruments, standard solutions available

    • selectivity, precision, sensitivity required

    • cost and speed

    • time required

    • need In -situ testing?

    • IN-SITU Testing - refers to testing methods that are carried out directly on the ground, rock, or soil at a particular site

  4. SAMPLING

    • the process to obtain a small representative and homogenous people

    • the MOST CRITICAL STEP because it can limit the accuracy of measurements

    • REPRESENTATIVE - content of analytical sample reflects content of bulk sample

    • HOMOGENOUS - content is the same throughout the whole sample

    • requires storage and preservation steps

    • sampling methods depends on the samples’ type, size and homogeneity, physical, and chemical states

      SAMPLING STEPS:

      1. Identify population

      2. Collect a gross representative

      3. Reduce gross sample to laboratory sample

      COMMON SAMPLING METHODS:

    • GRAB SAMPLE - A portion of sample removed from the population

    • COMPOSITE - several grab samples combined to form a single sample

    • HETEROGENEOUS PARENT SAMPLES - several samples have to be taken

      SAMPLING SOLIDS:

      PROBLEM: Solid materials are heterogenous making sampling difficult

      SOLUTION: Homogenization of Solid Samples

      HOW? : By crushing, pulverizing, grinding, or rendering the sample into a thoroughly mixed powder

      WHY? : The smaller the particle size of the sample, the lower the error in analysis. The more homogenous a sample is, the easier for digestion and extraction.

      EXAMPLE: QUARTERING PROCESS

      SAMPLING LIQUIDS:

      Liquids are mostly homogenous and are easier to sample

      REMINDERS:

    • If necessary, add antibacterials (to prevent sample decomposition by bacteria), antioxidants (to prevent sample oxidation), and acidify (to prevent precipitation of metals from water samples.

    • If sample is non-homogenous and in small quantity, SHAKE AND SAMPLE IMMEDIATELY

    • If sample is from large stationary liquids such as lakes, sample at DIFFERENT DEPTHS using a thief sample

    • THIEF SAMPLER: a special device for obtaining aliquots at different level

    • If sample are biological fluids, the TIMING of sampling is very important (e.g., blood sample after fasting to analyze sugar)

      SAMPLING GASES:

      Gases tend to be homogenous and a large volume of a sample is required

      WHY LARGE VOLUMES?: because of low density

      EXAMPLE:

    • Air Analysis - use “Hi-Vol” sampler that contain filters to collect PM10

    • Activated carbon as adsorbent

    • Liquid displacement method: sample must be slightly soluble in the liquid and does not react with it

    • Breath sample: subject blows into an evacuated bag

    • USE TEDLAR BAGS to collect gas samples

    SAMPLE STORAGE AND PRESERVATION - gross sample must be transported from sampling site to lab without any physical changes (adsorption, diffusion, volatilisation) or chemical changes (oxidation and microbiological degradation)

    SAMPLE PRE-TREATMENT FOR SOLIDS: (1) Grind and sieve (2) Dry samples to remove moisture

  5. SAMPLE PREPARATION

    • different for inorganic analyte and organic analyte

    • please see lecture 7 for the analysis preparation of inorganic analytes (acid digestion, microwave digestion, dry and wet ashing) and lecture 13 for the analysis and preparation of organic analytes (LLE, SPE, Soxhlet)

    • basically the step where the sample is prepared before used in various instruments (AAS and ICP for inorganic and GC and HPLC for organic)

  6. ANALYSIS

    • External Standard Calibration Method

    • Standard Addition Method

    • Internal Standard

  7. CALCULATION AND REPORTING

    • determine the concentration of the analyte in the analytical sample solution

    • use results to calculate the amount of analyte in the original (bulk) sample

      • Evaluate the results thru the appropriate use of statistics (must be reasonable, reliable and related to the problem

    • report results with accuracy and precision (include SD and mean) as well as conclusions

    • verify reports

LECTURE 2: INTRODUCTION TO SPECTROSCOPY

Spectroscopy - the study of the properties of matter through its interaction with different frequency components of the electromagnetic radiation

By describing light as waves, the optical properties or characterization of light are explained. This corresponds to frequency and wavelength: v = c/lambda, where v is frequency, c is the velocity of light, and lambda is the distance of one cycle

By describing light as particles, or photons (have no mass yet carry a specific amount of energy), interactions between radiation and matter (absorption and emission) are described.

  • EMR consists of quantum of energy: E = hv = hc/lambda, where h is the Planck’s constant

  • The shorter the wavelength, the higher the E

INTERACTIONS OF MATTER WITH ELECTROMAGNETIC RADIATION

  • Absorption (to take up) - a transition (EXCITATION) from a lower energy state (level) to a higher state with transfer of energy from radiation field (flame) to an absorber (atoms of a metal)

  • Emission (to give off) - a transition (RELAXATION) from a higher energy state to a lower level with transfer of energy from the emitter to the radiation field.

  • Scattering - a redirection of light due to its interaction with matter

SPECTROSCOPY AND SPECTROMETRY METHODS

  1. MOLECULAR

    • study of the light interactions with molecules

    • molecules can absorb (absorbed by bonding electrons), emit and scatter light

    • all these interactions allow us to know the identity and structure of substance (e.g., IR, NMR)

    • EXAMPLE: UV-Vis Spectroscopy

  2. ATOMIC

    • measurement of the wavelength or intensity of light that is emitted or absorbed by free atoms (absorbed by valence electrons)

    • to do this type of measurement, a sample must be converted into atoms

    • EXAMPLES: AAS, AES, ICP

CHARACTERISTICS OF WAVELENGTHS ABSORBED AND EMITTED

  • depends on the identity of the compound

  • depends on the amount of analyte present in the light path

  • remains the same regardless of the quantity of the analyte present

INTERFERENCES

  • Spectral - absorption or emission of other components of the matrix that occur at the same wavelength of use for analysis

  • Chemical - materials determined are not in the same chemical or suitable form

  • Instrumental - excess illumination due to imperfections in the instrument


REMEMBER:

  • The color transmitted or the color we see is the complement of the absorbed light in the color wheel

  • Only transmissions within the visible region (350 - 750 nanometer) is visually seen with colors

  • The absorbed radiation is electronically measured

BEER’S LAW

FORMULAS:

Transmittance (T) = P (transmitted radiation, power out) divided by P0 (incident radiation, power in)

Absorbance = - log of T = log of [P0/P]

A = ebc

  • where e is molar absorptivity with unit L/mol-cm

  • A is the absorbance (unitless)

  • b is the path length (cm)

  • c is the concentration (mol/L)

As light passes through an analyte in solution, the intensity of the light is reduced. The lesser the transmittance, the higher the absorbance.

Absorbance is proportional to pathlength and concentration. The longer the pathlength and the higher concentration, the higher the absorbance.

DEVIATIONS FROM BEER LAMBERT LAW

Deviations occur when plotting Abs vs Conc does not produce a straight line

SPECTRAL DEVIATION

  1. Polychromatic Radiation - Beer’s only apply when measurements are made with monochromatic source radiation.

  • If analyte absorbs at 635 nm with 10 nm bandwidth, it will absorb between 630-640 nm.

  • If slit width is large, allowing wavelength between 610-660 nm, matrix will absorb at wavelength outside the bandwidth.

  • This will result in an unintended higher absorption since it would also absorb matrices at the additional wavelength

  • Higher absorbance than normal will result to a positive deviation from the curve

  1. Stray Radiation - results from scattering, and reflection off the surface of gratings, lenses, etc. The radiation may not have passed through the sample but detected by detector. This will result in an unintended increase in P and a decrease in absorbance. Such behavior results in a negative deviation.

CHEMICAL DEVIATION

  • concentration changes

  • arise when an analyte dissociates, associates, or reacts

LECTURE 3: PRINCIPLES OF UV-VIS

  • Vacuum UV has a wavelength of less than 200 nm

  • UV has a wavelength of between 200 to 380 nm

  • Visible region has a wavelength of between 380 to 780 nm

SUBSTANCES can detect and quantify UV because the chromogen has chromophore.

CHROMOPHORE - substituent (atom or group of atoms) in a molecule that absorb light (radiation is not absorbed by all atoms in the molecule)

  1. Chromophores in which the groups have pi electrons undergo pi - pi star transitions. (ex: ethylenes, acetylenes)

  2. Chromophores having both pi electrons and n (non-bonding electrons undergo two types of transitions: pi to pi star and n to pi star. (carbonyls, nitriles, azo compounds and nitro compounds)

FACTS TO REMEMBER:

  • Color arises when a molecule absorbs certain wavelengths of visible light and transmits or reflects others

  • UV and visible light that hits the chromophore can thus be absorbed by exciting an electron from its ground state into an excited state

  • CHROMOGEN - molecule containing chromophore

  • Molar absorptivity (e) may be very large for strongly absorbing chromophores (>10 000) and very small if absorption is weak (10-100). In other words, e increases as absorption of chromophores increases.

  • Conjugation increases molar absorptivity. One of the most important factors affecting the wavelength of absorption by a molecule is the extent of conjugation. The increase in size of the conjugated system gradually shifts the absorption maxima (λmax) to longer wavelength

  • CONJUGATION - having alternating double and single bonds

  • ELECTRONS are responsible for most UV-Vis electronic transitions

UV-VIS ELECTRONIC TRANSITIONS:

  1. n to pi star transitions

    • an electron of unshared electron pair is excited to pi star antibonding orbital

    • this transition involves the least amount of energy than all the transitions

    • therefore, this transition gives rise to an absorption band at longer wavelengths

  2. pi to pi star transitions

    • this transition is available in compounds with unsaturated centres (e.g., simple alkenes, aromatics, carbonyl compounds)

    • this transition requires lesser energy than transition in a simple alkene


CONJUGATION

  • Bathochromic (red shift) - to longer wavelength

  • Hypsochromic (blue shift) - to shorter wavelength

  • Hyperchromic - to greater absorbance

  • Hypochromic - to lower absorbance


ABSORPTION OF UV by INORGANIC COMPOUNDS

By inorganic species - ions and complexes of elements in first two transition series

  • Absorb broad bands of visible radiation in at least one of their oxidation states, and are colored,

  • Absorption is between filled and empty d-orbitals

Charge Transfer Absorption - by charge transfer complexes (CTC)

  • Absorption has large molar absorptivity (>10000)

  • CTC consists of an electron-donor group bonded to an electron transfer.

  • Electron pair from donor is transferred to acceptor orbital

    • e.g., Iron III complex with 1,10 phenanthroline, Iron III with thiocyanate

  • In metal ion complexes, the metal ion serves as the electron acceptor

AUXOCHROMES - substituent groups which are not themselves optically active in the UV-Vis energy range but interacts with other chromophores to increase intensity and shift to higher wavelength. (e.g., hydroxyl, amines, halogens)

They provide unshared electrons that interact with pi electrons in the chromophore (n to pi conjugation)


INSTRUMENTATION

  1. LIGHT SOURCES

    • Deuterium and hydrogen lamp - covers UV: 200 - 330 nm

    • Tungsten lamp - covers UV-Vis: 330 - 700 nm

    • Xenon arc lamp - covers UV-Vis: 330 - 700 nm

  2. MONOCHROMATOR / WAVELENGTH SELECTOR

    • Photometer

      • uses filters to select wavelength

      • only detect a single wavelength at a time

      • has two types: Interference and Absorption

      • INTERFERENCE: remove selected wavelength by internal destructive interference and reflection

      • ABSORPTION: absorb unwanted wavelength

      • provide low resolution wavelength selection

      • suitable for quantitative work

      • wavelength cannot vary continuously

    • Spectrophotometer

      • uses monochromator to scan various wavelengths

      • input may be a continuum source or a line source

      • has two types: Grating and Prism

      • GRATING: uses diffraction, often producing a sharper line spectrum

      • PRISM: uses refraction, creating a continuous spectrum

      • produce high resolution

      • qualitative and quantitative work

      • wavelength can vary continuously

  3. SAMPLE CELL

    • Cells (cuvettes) are selected appropriately based on wavelength requirements

      • Quartz (fused silica) - UV/Visible (>210 nm)

      • Silicate glass - Visible (>350 nm)

      • Plastic - Visible (>300 nm)

    • PRECAUTIONS ON USING THE CUVETTE:

      • Keep the cuvette clean

      • Don’t clean with paper products - use optical paper

      • Store dry. Store carefully and gently

      • Don’t get fingerprints on them.

      • Use the same sample cell during running the blank, standard and sample.

    • QUESTION: How does the following affect the sensitivity of the analysis?

      • Different pathlength size and shape - A longer path length allows more opportunities for the light to interact with the sample, resulting in higher absorbance values

      • Different material - Quartz has the capability of more light transmission and is more transparent. So, it is used for sensitive experiments. Besides, quartz is more temperature resistant. At the same time, glass and plastic cuvettes are not ideal for concentration and purity measurements.

      • Old or new - cuvettes used should be consistent especially when running the reference and sample whether it is new or old

    • THE SAMPLE SOLVENT - How does solvent choice impact UV-Vis spectroscopy readings?

      • SOLUBILITY - undissolved solutes can form aggregates or precipitates that can distort the absorption spectrum. Solute should be highly soluble in chosen solvent.

      • HYDROGEN BONDING - Hydrogen bonding or polar solvents interact more strongly with unshared electron pairs of the ground state molecule. This results to a greater stabilization of ground state molecules than excited ones. Thus, needing more energy required for the n to transition to pi star. MORE POLARITY, MORE ENERGY NEEDED. This also results in a blue shift.

      • SOLVENT ABSORPTION - Solvent must not absorb light in the same region as the solute. Solvent must be transparent in this region so that it will not interfere with the measurement and will result to a more accurate result.

      • IONIC STRENGTH - increasing ionic strength (increasing the concentration of solvent) shifts the maximum absorption peak towards blue while decreasing molar absorptivity, which is why the concentration of solvent is typically less than 50 mM.

      • PI STACKING - increases the absorption wavelength (red) since the intermolecular overlap of pi-orbitals causes excitation delocalization.

      • SOLVENT must also be inert or unreactive with the analyte

  4. DETECTORS

    • PHOTOMULTIPLIER TUBE (PMT)

      • detects one wavelength at a time

      • Photon of radiation enters tube and strikes the cathode.

      • Cathode strike causes the emission of several electrons.

      • Electrons are accelerated towards first dynode (90 V more positive than the cathode)

      • Electrons strike the 1st dynode and emits several electrons for each incident electron

      • This electrons are accelerated towards second dynode to produce more electrons which will be accelerated towards third dynode, and so on.

      • The electrons will eventually be collected at the anode. By this time, each original photon has produced 106-107 electrons.

      • The resulting current is amplified and measured

    • There are detectors that can do full spectra detection such as CCD and diode arrays:

      • SOURCE - SAMPLE - MONOCHROMATOR - AREA DETECTOR

    • There are also detectors that do single wavelength detection such as the PMT

      • SOURCE - MONOCHROMATOR - SAMPLE - POINT DETECTOR

  5. RECORDER / READOUT

    • UV - VISIBLE SPECTRUM - a plot of absorbance versus wavelength of a sample at different concentrations

    • SINGLE BEAM

      • employs a single source to supply radiation to the sample and then the background in turn

      • ADVANTAGE - single set of components required (complex devices may be incorporated)

      • DISADVANTAGE - separate correction must be done for background spectrum due to solvent or matrix interferences (longer time)

    • DOUBLE BEAM

      • a single source sup[plies equal intensity beams through the sample and reference, which are then dispersed and detected alternately.

REMINDERS IN GENERAL PROCEDURE OF UV-VIS SPECTROSCOPY

  1. Run a blank. This is done because the matrix, solvent, buffer might have their own absorbance.

  2. Run standards for calibration plot for quantification analysis

  3. Run quality control to check for accuracy

  4. Run samples

  5. In DUAL BEAM: simultaneous measurement of reference cell eliminates absorbance of background

  6. IN SINGLE BEAM: it is required to measure the reference spectrum and subtract it from sample spectrum

  7. Use the same cuvette because cuvettes have different sizes and lengths. Consistency is very crucial when it comes to reading for result accuracy. Using the same cuvette will ensure the same optical conditions.


HOW TO DO BACKGROUND CORRECTION:

LECTURE 4: PRINCIPLES OF AAS AND AES

Atomic Spectrometry - techniques for determining the elemental composition of an analyte by its electomagnetic or mass spectrum

ATOMIC SPECTROMETRY METHODS

  1. Optical Spectrometry

  • AAS

    • F-AAS

    • GF-AAS

    • HG-AAS

  • AES

    • F-AES

    • ICP-OES

  1. Mass Spectrometry

    • ICP-MS


INSTRUMENTATION OF ATOMIC SPECTROMETERS

  1. EXTERNAL LIGHT SOURCES

    • Hollow Cathode Lamp (HCL)

      • the source of light is made from tungsten anode and cathode is composed of the element being measured

      • each analyzed element requires a different lamp

      • can be a single lamp or multielemental

    • Electrodeless Discharge Lamp (EDL)

      • made of metal/salt of interest sealed in a quartz tube filled with Ne or Ar at low pressure

      • energized by a field of radio frequency or microwave radiation for ionization of Ar to give a high frequency accelerated component

      • EDL has two advantages: (1) able to use light-generating substances that would react with metal electrodes in normal lamps, (2) have extended bulb life

      • EDL is much more intense and sensitive than HCL. Hence, it has better precision and lower detection limit for an intensity limited analysis

  2. LIGHT CHOPPER

    • placed between the HCL and the flame

    • light is “chopped” with a rotating half-mirror beam-splitter device

    • this helps differentiate between the light absorbed by the sample and the emission from the flame, which can interfere with measurements

    • this also modulates the light beam improving its sensitivity and selectivity

  3. SAMPLE INTRODUCTION

    • samples are dissolved in water

    • a flame is created, using ethyne and oxygen fuel (aka oxidant fuel)

    • aspirator sucks the liquid into the small tube from the sample container

    • fine aerosol is created and is mixed with fuel and oxidant for introduction into flame

    • the liquid is transferred to the flame where the sample undergoes atomization

    • the metals then absorb light from the source

  1. MONOCHROMATOR

  2. DETECTOR