Water Pollution W10

Techniques for Separation and Analysis

qualitative analysis

to identify or detect the presence of a species

quantitative analysis

to measure amount of substance present

structural analysis

detailed determination of molecular

problems for analytical chemists

• range of analyses very wide (simple inorganic species to complex bio-molecules)

• sample matrices are often complex and unknown (solid, liquid, or gaseous)

• environmental programs often generate large numbers of samples - think about costs, space, automation

• some analysis can only be made at or close to sampling sites (need portable / robust equipment)

analytical method

• based on chemical principles e.g., precipitation, distillation, titration

• to get reliable information from an env investigation, mastery of techniques is required

• needs unified overview of whole process

Unified overview of techniques

• Definition of aims

• Selection/development of an appropriate method

• Samplingplan;Sample:collection,handling,pre-treatment

• Separations

• Finalmeasurements

• Methodvalidation

• Assessmentandinterpretationofresults

• Health & safety

gravimetric

Weight of pure analyte or of stoichiometric compound of it

Volumetric

Volume of a solution containing a known amount of reagent reacting with the analyte

spectrometric

Wavelengths and intensity of EM radiation emitted or absorbed by the analyte

Mass spectrometric

Abundance of atomic ions or molecular fragments derived from the analyte

Electrochemical

Electrical properties of analyte solutions

Chromatographic

Physico-chemical properties of analytes following separation

Nuclear or radiochemical

Energy and intensity of nuclear radiations emitted by the analyte

thermal

Physico-chemical properties of the analyte as heat is applied to it

range of factors to select appropriate method

• What is the analyte?

• Detectionlimits

• Precision

• Accuracy

• Turn-aroundtime

• Are onsite analyses required?

• Number of samples and any consequent automation

• Costs

• Health & safety

• Are methods specified by regulatory or funding bodies?

Precision

closeness of an agreement between replicate determinations

(if replicates have almost identical values then there is high precision)

Doesn't always mean high accuracy though, may have been systematic error.

standard deviation

accuracy

Refers to closeness of agreement between experimental results and “true result” – the ultimately correct value

scientists strive for best estimate of true result

can use number of different analytical methods for same determination and by checking analytical methodologies

errors

any measurement is subject to error which may be reduced, but not completely eliminated

Result will only be of limited value until magnitude of error can be quantified using appropriate statistical techniques

analytical errors

All analytical determinations are subject to error

E = R - t

E = error

R = experimental value

t = true value

relative error

Er = (R - t)/t

% of relative error

Ep = 100(R - t)/t

determinate errors

have a definite value + can be measured

indeterminate errors

no definite values + fluctuate in a random manner

determinate errors include:

• personal error

• instrumental error

• method error

indeterminate errors include:

uncertainties in measurement

• Unknown
• Cannot be controlled
• Slight variations in successive measurements

• Assumed to lie on a normal distribution

how to minimise errors

• Thorough preparation; piloting

• Use of calibration and application of corrections

• Use of blanks

• Use of control samples (placebos)

• Use of certified and standard reference materials (CRMs and SRMs)

• Running of parallel determinations (ie duplicates and triplicates, “repeat” questions, “blind” analyses, etc)

• Use of different, independent methods

• Proper maintenance of equipment

• Use of skilled staff, staff training and development

detection limit

lowest mass or concentration of analyte that can meaningfully be detected (ie discriminated from zero or the blank)

look up equation

Quality assurance + control aims

• Accurate, precise and credible measurements

• Data representative of ambient conditions
• Comparable and traceable results
• Measurements that are consistent over time
• High data capture, evenly distributed
• Optimal use of resources

QA covers pre-measurement phase

• Defining monitoring objectives

• Network design

• Management and training systems

• Site selection and establishment

• Equipment evaluation and selection

QC covers outputs

• Routine site operations

• Calibration procedures

• System maintenance and support

• Data review and management

• Periodic system review and development

QC/QA paperwork

must be:

• appropriate

• practical

• sustainable

general lab rules

• Always wear eye protection

• Always wear a laboratory coat

• Wash your hands regularly

• Keep your work area clear and tidy

• Never eat or drink in the laboratory

• Carry out a risk assessment before starting work

• Clean up all spillages immediately

• Keep long hair tied back

• Wear appropriate clothing that does not leave skin exposed (e.g. shorts, skirts,

  sandals)

lab safety equipment available

• A shower, for use if clothing catches fire or in the event of a large spill of caustic chemicals on an individual

• Facilities for flushing chemicals out of an individual’s eye with cold water or saline solution

• Fire buckets, blankets and extinguishers

• Spill kits, to prevent the spread of spilled chemicals that may be hazardous

• Health & safety noticeboard that displays information such as chemical data

  sheets, location of fire exits, first aid procedures and contact points for staff

  qualified in first-aid

• Safety spectacles or goggles, full face-shields, disposable gloves and heat-

  resistant gauntlets, for use as appropriate

• Dustpan, brush and mop, for clearing broken glassware and water spills

  respectively

• Telephone, so emergency services can be called if necessary

GLP (Good Laboratory practice)

• formulated by OECD in 1981

• analytical data collected in one OECD should be accepted in another OECD country

• GLPMA is part of MHRA

• MHRA administer UK GLP Compliance

  Monitoring Programme

• principles are not codes of conduct, they regulatory requirements

notebook purposes

• traceable record

• personal reference book

• can repeat or revise experiment exactly

• enables tutor to see if any mistakes were made or calculations wrong

classical analysis

depends on chemical properties of sample

• Reagentreactscompletelywithanalyte

• Relationship between measured signal and analyte concentration is determined by chemical stoichioimetry

useful for precise measurements

instrumental analysis

physical property of sample measured

• Potential difference between 2 electrodes in a solution of sample
• Ability of the sample to absorb light

can detect individual atoms or molecules in sample

advantages of instrumental techniques

1. Ability to perform trace analysis

2. Large numbers of samples may be analyzed very quickly

3. Many instrumental methods can be automated

4. Most instrumental methods are multi-channel techniques

5. Less skill and training usually required to perform instrumental analysis than classical analysis

single-channel techniques

generate a single number for each analysis of a sample

e.g., gravimetry

multi-channel techniques

generate a series of numbers for a single analysis

• ability to obtain measurements while changing independently controllable parameter

• e.g, absorption spectrum where absorbance of sample is monitored as function of wavelength of light transmitted through sample

absolute analytical techniques

analyte concentration calculated directly from measurement of a sample

No other measurements required (other than measurement of sample mass or volume)

relative analytical techniques

measurement of sample compared to measurements of additional samples that are prepared with the use of analyte standards (e.g. solutions of known analyte concentration)

resolution in microscopy

• ability to distinguish between 2 closely spaced objects in a specimen – is best estimate of microscope’s utility

types of light microscopes

• Bright field microscope - area observed appears brightly lighted and microorganisms appear dark

• Dark field microscope - dark background against which objects are brightly illuminated

• Phase contrast microscope - light passing through one material and into another of different refractive index

  and/or thickness will undergo in change in phase

• Fluorescence microscope - the specimen acts as light source. Specimens used either fluorescent materials or stained with fluorescent dye

electron microscopy

• uses electromagnetic lenses to focus a beam of electrons

• used when higher resolution necessary

• Source of light in EM is electron gun or electron beam

• magnify by about 200,000 x

Transmission Electron Microscope (TEM)

electrons that pass through the specimen are imaged

Scanning Electron Microscope (SEM)

electrons reflected from the specimen (secondary electrons) are collected, and the surfaces of specimens are imaged

Steps in Gravimetry

• Sample dried and triplicate portions weighed

• Preparation of the solution
• Precipitation
• Digestion

• Filtration
• Washing
• Drying or igniting • Weighing
• Calculation

What is chromatography used for in forensics

• Analyse blood and urine samples for drugs

• Analyse products such as paints, cosmetics and oils

• Analyse environmental samples

• Test for presence of explosives

Chromatography phases

• stationary phase (fixed in place)

• mobile phase (gas or liquid which moves past stationary phase)

types of chromatography

• paper

• thin layer

• column (modern method)

• gas

• HLPC

chromatogram

A plot of the detector signal versus time

desirable features of chromatographic separation

• All analytes separated

• Well separated peaks

• Symmetrical, sharp peaks

• Short run times

Atomic Absorption Spectroscopy (AAS)

• measures concentrations of metallic elements in different materials

• Matter absorbs energy, which create a change in its state

• Atomic part refers to atoms in a material, which will absorb

  radiated energy from a light source

• Atoms each have own characteristics re: to absorbing energy

  - each element has unique electronic structure

spectroscopy

study of how energy and materials interact

spectrometry

refers to how you apply this as a measuring technique

benefits of AAS

• results normally fall between 0.5-5% accuracy

• highly sensitive method of analysis

• helped revolutionise practices e.g., trace toxins

• able to detect elements we were previously unaware existed in certain material

• comparatively inexpensive

Uses of AAS

• env testing (rivers, sea, drinking water)

• food and drink industry (contamination)

• pharmaceutical companies

• industry (raw materials)

• mining (before excavation)

• agriculture (plants + soil mineral content)

EM radiation

– Visible light that comes from a lamp – Radio waves from a radio station
– Microwaves
– Infraredlight
– Ultraviolet light
– X-rays
– Gamma-rays

What creates measurable signals?

• during spectroscopy absorbing energy moves to electrons with more energetic level

• atoms absorb light in excited state

• atomic absorption measures amount of light at a specific wavelength, which passes through cloud of atoms + is absorbed by them

• once excited atoms relax they release energy as photons

AAS measures

• Amount of a specified element present in a material is determined by measuring the amount of light absorbed and the energy emitted during the spectroscopy process

Why is AAS best for metals?

• Atoms in metal elements are more easily readable

• For AAS to be effective, atoms in material must be in isolation and free of contaminating lines from molecules

• Metals generally have narrow, single emission and absorption lines, which form brightly and clearly

• Allows for selective detection that AAS requires

How AAS destructs the sample

• Sample must be first turned into an atomic gas

• For liquid samples, this involves evaporation

• Solid samples are vapourised

• Then any compounds must be broken down into free atoms,

  and this process is atomisation

components of AAS instrumentation

• Atomization

• Hollow cathode lamp

• Monochromator

• Detector

• Recorder

two steps of AAS

1. atomisation

either flame or furnace used to convert metallic elements to atomic disassociated vapour

temperature must be controlled to avoid ionisation

2. absorption

of radiation from light source by free atoms

Hollow Cathode Lamp

• used in AAS

• gaseous ions bombard cathode + eject metal ions

• cathode concentrates most of these emitted ions into a beam that passes through a quartz window

• metal in cathode is same as metal being analysed

• lamp filled with noble gas at low pressure

monochromator

An optical device that transmits a narrow band of wavelengths of light or other radiation from a wider range of wavelengths

desired bands of line can be isolated with a monochromator

detector

convert light coming from a monochromator to a simplified electrical signal

recorder

can receive electrical signals from detector to convert them into a readable response

calibration curves

• known wavelength selected

• detector will only measure energy emitted at that wavelength

• As concentration of target atom in sample increases, absorption will increase proportionally

benefits of AAS

• Accurate, typically producing results in a 0.5-5% range

• Incredibly sensitive, measuring at ppm

• Can analyse specific elements because of unique light- absorbent qualities of their atoms

• Can determine concentrations of >65 elements

• Relatively simple process, with well-documented protocols

• Allows for high throughput of samples

• Inexpensive in comparison to other analytical techniques

• Supports broad range of industries and sectors

job of monochromator

• Isolate analytical lines' photons passing through the flame

• Remove scattered light of other wavelengths from the flame

• Hence only a narrow spectral line impinges on (enters) the PMT

job of photomultiplier tube (PMT)

• Acts as the detector

• Determines the intensity of photons of the analytical line exiting monochromator