Labortty techinque

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Instrumentation and Laboratory Analysis Techniques
1. LABORATORY SAFETY
1. General Precautions
As with any place of work, safety is an important consideration in soil, plant and water analysis
laboratories, and one that is frequently overlooked. A safe working in a chemical laboratory needs
special care, both in terms of design and construction of the laboratory building, and handling and
use of chemicals.
For chemical operations, the release of gases and fumes in some specific analytical operation
are controlled through a fume hood or trapped in acidic/alkaline solutions and washed through
flowing water. Also, some chemical reactions during the process of analysis, if not handled
well, may cause an explosion.
Analytical processes normally carried out at room temperature can be affected by differences
in temperature so that an analysis performed in a “cold” room can give a different result to
one performed in a “hot” room. Many chemicals are affected by the temperature and humidity
conditions under which they are stored, particularly if these conditions fluctuate. The air
temperature of the laboratory and working rooms should ideally be maintained at a constant
level (25°C). Humidity should be kept at about 50 %.
All staff, irrespective of grade, technical skill or employment status, should be briefed on all
aspects of safety upon commencement of work. Periodic reminders of such regulations should
be given to encourage familiarity with respect to regulations. Ideally, posters relatively to
laboratory safely should be prominently displayed in the laboratory.
Personal and General laboratory safety
Never eat, drink, or smoke while working in the laboratory.
Read labels carefully.
Do not use any equipment unless you are trained and approved as a user by your supervisor.
Wear safety glasses or face shields when working with hazardous materials and/or equipment.
Wear gloves when using any hazardous or toxic agent.
Clothing: When handling dangerous substances, wear gloves, laboratory coats, and safety shield or glasses.
Shorts and sandals should not be worn in the lab at any time. Shoes are required when working in the
machine shops.
If you have long hair or loose clothes, make sure it is tied back or confined.
Keep the work area clear of all materials except those needed for your work. Coats should be hung in the
hall or placed in a locker. Extra books, purses, etc. should be kept away from equipment, that requires air
flow or ventilation to prevent overheating.
Disposal - Students are responsible for the proper disposal of used material if any in appropriate containers.
Equipment Failure - If a piece of equipment fails while being used, report it immediately to your lab
assistant or tutor. Never try to fix the problem yourself because you could harm yourself and others.
If leaving a lab unattended, turn off all ignition sources and lock the doors.
Never pipette anything by mouth.
Clean up your work area before leaving.
Wash hands before leaving the lab and before eating.

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2. General Attitude
1. Develop a positive attitude towards laboratory safety
2. Observe normal laboratory safety practices
3. Maintain a safe and clean work environment
4. Avoid working alone
Common Sense
Good common sense is needed for safety in a laboratory. It is expected that each student will work in a responsible
manner and exercise good judgement and common sense. If at any time you are not sure how to handle a particular
situation, ask your Teaching Assistant or Instructor for advice. DO NOT TOUCH ANYTHING WITH WHICH
YOU ARE NOT COMPLETELY FAMILIAR!!! It is always better to ask questions than to risk harm to yourself
or damage to the equipment.
2. Lab safety rules for students
 Report all accidents, injuries, and breakage of glass or equipment to instructor immediately.
 Keep pathways clear by placing extra items (books, bags, etc.) on the shelves or under the work tables. If under the
tables, make sure that these items can not be stepped on.
 Long hair (chin-length or longer) must be tied back to avoid catching fire.
 Wear sensible clothing including footwear. Loose clothing should be secured so they do not get caught in a flame or
chemicals.
 Work quietly — know what you are doing by reading the assigned experiment before you start to work. Pay close
attention to any cautions described in the laboratory exercises
 Do not taste or smell chemicals.
 Wear safety goggles to protect your eyes when heating substances, dissecting, etc.
 Do not attempt to change the position of glass tubing in a stopper.
 Never point a test tube being heated at another student or yourself. Never look into a test tube while you are heating
it.
 Unauthorized experiments or procedures must not be attempted.
 Keep solids out of the sink.
 Leave your work station clean and in good order before leaving the laboratory.
 Do not lean, hang over or sit on the laboratory tables.
 Do not leave your assigned laboratory station without permission of the teacher.
 Learn the location of the fire extinguisher, eye wash station, first aid kit and safety shower.

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 Fooling around or "horse play" in the laboratory is absolutely forbidden. Students found in violation of this safety rule
will be barred from particpating in future labs and could result in suspension.
 Anyone wearing acrylic nails will not be allowed to work with matches, lighted splints, bunsen burners, etc.
 Do not lift any solutions, glassware or other types of apparatus above eye level.
 Follow all instructions given by your teacher.
 Learn how to transport all materials and equipment safely.
 No eating or drinking in the lab at any time!
Emergency Response
 It is your responsibility to read safety and fire alarm posters and follow the instructions during an
emergency
 Know the location of the fire extinguisher, eye wash, and safety shower in your lab and know how to use
them.
 Notify your instructor immediately after any injury, fire or explosion, or spill.
 Know the building evacuation procedures.
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3. Instrument Operation
i. Follow the safety precautions provided by the manufacturer when operating instruments.
ii. Monitor instruments while they are operating.
iii. Atomic Absorption Spectrophotometer must be vented to the atmosphere. Ensure that the
drain trap is filled with water prior to igniting the burner.
iv. Never open a centrifuge cover until machine has completely stopped.
v. Use of balances:
 The warming-up time of the balances is 30 minutes
 Spilled chemicals should be removed immediately
 Never blow away the spilled product
 Brushes are supplied with the balances
4. Accidents
i. Learn what to do in case of emergencies (e.g., fire, chemical spill, etc.). Fire-fighting
equipment must be readily accessible in the event of fire. Periodic maintenance
inspections must be conducted.
ii. Learn emergency First Aid, such supplies are a necessity and laboratory staff should be
well trained in their use. Replacement of expended supplies must take place in a timely
fashion.
iii. Seek medical attention immediately if affected by chemicals, and use First Aid until
medical aid is available.

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iv. Access to eye-wash fountains and safety showers must not be locked. Fountains and
showers should be checked periodically for proper operation.
5. Handling of Chemicals
1. Use fume hoods when handling concentrated acids, bases or other hazardous chemicals.
2. Do not pipette by mouth; always use a suction bulb.
3. When diluting, always add acid to water, not water to acid.
4. Some metal salts are extremely toxic and may be fatal if swallowed. Wash hands thoroughly
after handling such salts or indeed any chemical regardless of toxicity.
5. Chemical spills should be cleaned promptly and all waste bins emptied regularly.
6. All reagent bottles should be clearly labeled and must include information on any particular
hazard. This applies particularly to poisonous, corrosive, and inflammable substances.
7. For the preparation of reagents, only distilled water (DI) is used.
8. Note that volatile acids, ammonia, nitrite, chlorine and carbon dioxide have to be removed by
means of a column containing resin (deionizer) which will exchange the charged ions, is
needed.
Chemical safety
9. Treat every chemical as if it were hazardous.
10. Make sure all chemicals are clearly and currently labeled with the substance name, concentration, date, and
name of the individual responsible.
11. Never return chemicals to reagent bottles. (Try for the correct amount and share any excess.)
12. Comply with fire regulations concerning storage quantities, types of approved containers and cabinets,
proper labeling, etc. If uncertain about regulations, contact the building coordinator.
13. Use volatile and flammable compounds only in a fume hood. Procedures that produce aerosols should be
performed in a hood to prevent inhalation of hazardous material.
14. Never allow a solvent to come in contact with your skin. Always use gloves.
15. Never "smell" a solvent!! Read the label on the solvent bottle to identify its contents.
16. Dispose of waste and broken glassware in proper containers.
17. Clean up spills immediately.
18. Do not store food in laboratories.
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6. Furnaces, Ovens, Hot Plates
Use forceps, tongs, or heat-resistant gloves to remove containers from hot plates, ovens or muffle
furnaces.
7. Handling Gas
Cylinders of compressed gases should be secured at all times. A central gas facility is preferred.
8. Protective Equipment
Body Protection - Use laboratory coat and chemical-resistant apron
Hand Protection - Use gloves, particularly when handling concentrated acids, bases & other
hazardous chemicals.

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Dust Mask - A mask is needed when grinding soil, plant samples, etc.
Eye Protection - Use safety glasses with side shields.
Full Face Shield - Wear face shields over safety glasses in experiments involving corrosive
chemicals.
Foot Protection - Proper footwear should be used; sandals should not be worn in the
laboratory.
9. Lab. Contamination
 Contamination is a most serious problem in any laboratory; therefore, its sources must be
identified and eliminated. Some common sources of contamination are:
 External dusts blown from the surrounding environment
 Internal dust resulting from cleaning operations
 Cross-contamination derived from while handling many samples at the same time (e.g.,
handling plant and soil samples together)
 Failure to store volatile reagents well away from the samples
 Washing materials, particularly soap powder
 Smoking in the laboratory
Additional Safety Guidelines
 Never do unauthorized experiments.
 Never work alone in laboratory.
 Keep your lab space clean and organized.
 Do not leave an on-going experiment unattended.
 Always inform your instructor if you break a thermometer. Do not clean mercury yourself!!
 Never taste anything. Never pipette by mouth; use a bulb.
 Never use open flames in laboratory unless instructed by TA.
 Check your glassware for cracks and chips each time you use it. Cracks could cause the glassware to fail
during use and cause serious injury to you or lab mates.
 Maintain unobstructed access to all exits, fire extinguishers, electrical panels, emergency showers, and eye
washes.
 Do not use corridors for storage or work areas.
 Do not store heavy items above table height. Any overhead storage of supplies on top of cabinets should be
limited to lightweight items only. Also, remember that a 36" diameter area around all fire sprinkler heads
must be kept clear at all times.
 Areas containing lasers, biohazards, radioisotopes, and carcinogens should be posted accordingly.
However, do not post areas unnecessarily and be sure that the labels are removed when the hazards are no
longer present.
 Be careful when lifting heavy objects. Only shop staff may operate forklifts or cranes.
 Clean your lab bench and equipment, and lock the door before you leave the laboratory.
Hazardous Property Explanation Example
Toxic Chemicals that at very low levels cause
damage to health.
 Acrylamide
 Cyanide
Respiratory Sensitiser  Animal allergen

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 Latex
 Isocyanates
Carcinogenic Chemicals that may cause cancer or increase
its incidence.
 Methyl nitroso urea
 Formaldehyde
Mutagenic Chemicals that induce heritable genetic defects
or increase their incidence.
 Ethidium bromide
Substance Toxic to
Reproduction
Chemicals that produce or increase the
incidence of non-heritable effects in progeny
and/or an impairment in reproductive functions
or capacity.
 Halothane
 Ethylene oxide
Harmful Chemicals that may cause damage to health.  Bleach
Irritant Chemicals that may cause inflammation to the
skin or other mucous membranes.
 Ammonia
Corrosive Chemicals that may destroy living tissue on
contact.
 Strong acids and bases
Explosive Chemicals that explode.  Hexane
 Hydrogen
Oxidising Chemicals that react exothermically with other
chemicals.
 Hydrogen peroxide
Flammable Chemicals that have low flash points and may
catch fire on contact with air or ignition source.
 Di-ethyl ether
 Acetone
 Alcohols
Dangerous to Environment Chemicals that may present an immediate or
delayed danger to one or more components of
the environment.
 Mercury
3. SOIL ANALYSIS
Laboratory analyses are performed on small samples of soil taken from relatively large areas of
land. If the sample does not truly represent the soil you intend to treat, all the precision of the
analytical process is useless. Improperly collected samples not only make test results less
informative than they might be, but the results also may lead to erroneous recommendations that
reduce yields, waste money and resources, and pollute the environment.
Soil testing on regular bases is an important part of nutrient management. From the farmer’s point
of view, nutrient management is the process to maximize the proportion of applied nutrients that
is used by the crop; in other words, maximizing the “Nutrient Use Efficiency (NUE)”. Soil tests
are used to evaluate soil fertility, which ultimately measures the soil nutrient content. It focuses on
the measurement of available nutrients for the plants and excludes the total nutrient content. Total
nutrient content value of the soil is useless for the farmer, because only a small quantity of them
is available for the plant. Therefore it cannot provide information for fertilizer calculations. Soil
testing program is an analysis of the soil physical and chemical properties and an evaluation of the
soil nutrient-supplying capacity at the time of sampling. It contains four activities:
 Taking soil samples
 Analysis of soil samples
 Interpreting the results of the sample analysis
 Making recommendations for soil management and plant nutrition practices

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4. SOIL SAMPLING
1. How do I take a representative soil sample?
First, make a detailed map of your land. Divide your map into individual soil-test areas of a few
(1–5) acres each. Label each area clearly on the map by using a combination of letters and numbers
that make sense and thus are easy to remember. Each test area should consist of only one soil type
or variation. Areas with different slope, color, drainage, texture, or management history should be
sampled separately.
2. Sampling Procedure
The sampling process starts with the cleaning of the surface area then removing the top litter from
the surface to approximately 1 cm deep. The first rule in taking soil samples is to use clean and
proper tools. Soil auger is the most convenient, but a shovel can also be used. Clean auger or
shovels made of stainless steel are preferred. Remove a shovelful of soil to the depth you wish to
sample, then cut a one-inch section from the wall of the hole you have just dug. Place it in your
mixing bucket. Care should be taken that an equal amount of soil is taken at each of the sampling
sites, so that the resulting composite sample will represent all the sample sites equally. Mixing
buckets should be durable but light (preferably made of plastic) and clean. A small amount of lime
or fertilizer residue left in the bucket can severely distort your results. Clean and well-labelled
containers must be used to store the samples. If analysis for boron is desired, the soil samples
should not be stored in grocery (brown) paper bags, because such paper can release boron to the
sample.
3. Depth of Sampling
The depth of the sampling is important because the mobility of the nutrients varies with the
nutrient content in the different soil zones. The mobility of each nutrient in the soil is also
varying from each other. The recommended depth for sampling is the following:
 Samples should be three inch cross-sections of the soil (called cores) taken to a specified
depth, normally 0–15 cm for no-till fields or established pasture and turf and 0–30 cm for
conventionally tilled fields.
 For trees and fruit crops, two samples at different depths should be taken wherever possible:
a surface sample from 0–30 cm and a subsoil sample from 30–60 cm even 60-120 cm.
 Each sample to be tested should be a thorough mix of 10–15 cores taken randomly or in a
scientifically determined pattern. The depth of the sampling also varies according to the crop
in use.

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4. Soil Sampling Designs
The sampling area is no bigger than two hectares and is a field or part of it. The area should be
uniform to avoid a mix of different kinds of soil. The area should have the same kind of vegetation
or crop. The most common samplings collection designs are the following:
i). Grid sampling
A grid with suitable spacing is placed on the map and measured. The sampling will be taken at the
intersections of the grid or from inside of the grid cells. Grid sampling provides equally spaced
observations and it reveals any systematic variation across the tract under study.
ii). Random sampling
Sample locations are selected at random, with equal probabilities of selection and independently
from each other. The sample produced from one sampling area consists of 10-20 sub-samples
collected randomly throughout the sampling area using a zigzag pattern. The sub-samples should
only be collected from representative sites, avoiding areas like anthills, bunds, boundaries, etc.
Zigzag sampling pattern or a variant of it is often adequate for obtaining a reasonably
representative soil sample.

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iii). Random stratified sampling
The area is first divided into a number of subsections, called strata, and then random sampling
design is applied to each of the strata separately. The random sampling method is not a systematic
collection technique; meanwhile the stratified random sampling method provides a kind of mixture
of the systematic and non-systematic soil sampling collection methods.
iv). Transects
Soil samples are taken along straight lines across the targeted area. The spacing between sampling
points might be equal, nested, or random.
v). Target sampling
Based on specific attributes (e.g. slope, aspect, plan or profile curvature, color, etc.) the technician
identifies homogeneous and heterogeneous patterns of the targeted area, which will allow the

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fixation of representative sampling points where the sampling will be taken. This technique
minimizes the effort and cost and maximizes the information content.
5. Sampling Time
Soil nutrient content varies with the season. Therefore we should take samples as close as possible
to planting or the time when the crop needs the nutrient. It can be 2-4 weeks before planting or
applying fertilizer. In so doing, you will get your test results in plenty of time to plan your soil
amendment and fertilizer needs. Allow adequate time (at least two weeks) for the laboratory to
complete the analysis of your soils. Soils should be retested to confirm the effects of soil
amendments applied. Subsequent tests of actively managed soils should be done to warn of nutrient
buildup or depletion—perhaps once every two years or even more frequently, depending on the
cropping activity.
Avoid taking samples when the soil is very wet, dry or frozen. These conditions will not affect the
result of the analysis, but they will make difficult to take and handle the samples. We might test
the soil each year or at least once during each crop rotation cycle. The results of the analysis should
be maintained for each field separately to evaluate long-term trends in nutrient levels and other
properties.
5. PLANT SAMPLING
Different plant species may require different tissue parts for meaningful sampling and
interpretation. To ensure a representative sample, sample as many plants as practical. Generally,
the youngest fully matured leaves on main branches or stems are sampled. They should be taken
just prior to or at the onset of flowering. Do not collect tissue that is covered with soil or dust. Do
not collect from plants that are damaged by insects, mechanically injured, or diseased. Dead plants
or senescent tissues should not be sampled. Also, sampling is not recommended when plants are
under moisture or temperature stress. Samples must be protected from dirt and fertilizer materials
and should be placed in clean plastic bags.
i). How is plant-tissue samples tested?
Samples are cleaned, placed in a forced-draft oven at 65°C in hot air oven for at least 12-24 hours
and ground to pass a 2-mm sieve with a Wiley mill. A 0.50-g sample is dry-ashed in a porcelain
crucible for 4–6 hours at 500°C in a muffle furnace. (If the ashing is judged incomplete, then the
ash is cooled, dissolved in 1 M nitric acid, evaporated to dryness, then ashed again for 1 hour.)
The residue is dissolved in 25 ml of 1 M hydrochloric acid.
ii). Routine plant tissue analyses
Analyses for P, K, Ca, Mg, Fe, Cu, Zn, Mn, Mo, Al, and Na are done on the ash solution, using
an ICP spectrophotometer. Boron is measured by the azomethine-H colorimetric method.
6. COLLECTING AND PREPARING THE SAMPLE
If you suspect a nutrient deficiency:

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1. Sample when the symptom first appears (see table 2 for deficiency symptoms).
2. In the same field or area, collect similar samples of plant materials from plants that appear
abnormal.
3. Make sure that the symptoms are not due to a factor unrelated to plant nutrition.
The parts of plants to sample depend on the plant and its growth stage. Table 3 lists the best parts
to sample for common crops (see also fig. 1). More specific sampling strategies may be necessary
for cotton and peppers (chile). Also, many devices are available for a “quick test” of the plant
nitrogen status. Chlorophyll meters for certain crops can be used to predict the cost/benefit of
additional nitrogen fertilizer.
Instructions for petiole or leaf sampling may differ. Also, comparing samples from both a “good”
and a “bad” area often helps in determining corrective action. If specific sampling guidelines are
not given here, collect recently mature leaves just below the growing point from at least 10 plants.
Things to Avoid
Do not sample the following:
 Young, emerging leaves; old, mature leaves and seeds: These plant parts usually are not
suitable because they are not likely to reflect the nutrient status of the whole plant.
 Diseased or dead plants
 Plants that have insect or mechanical damage.
 A single plant showing visual deficiency symptoms, unless it is possible to sample normal
plants from an adjacent area in the field. Normal plants give a reference to help interpret
the chemical analysis of the deficient plant sample

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7. SAMPLING AND SAMPLE HANDLING
Representative sample: A representative sample is one that truly reflects the composition of the
material to be analyzed within the context of a defined analytical problem.
The importance of obtaining a representative sample for analysis cannot be overemphasized.
Without it, results may be meaningless or even grossly misleading. Sampling is particularly crucial
where a heterogeneous material is to be analyzed. It is vital that the aims of the analysis are
understood and an appropriate sampling procedure adopted. In some situations, a sampling plan
or strategy may need to be devised so as to optimize the value of the analytical information
collected. This is necessary particularly where environmental samples of soil, water or the
atmosphere are to be collected or a complex industrial process is to be monitored. Legal
requirements may also determine a sampling strategy, particularly in the food and drug industries.
A small sample taken for analysis is described as a laboratory sample. Where duplicate analyses
or several different analyses are required, the laboratory sample will be divided into sub-samples
which should have identical compositions.
Homogeneous materials (e.g., single or mixed solvents or solutions and most gases) generally
present no particular sampling problem as the composition of any small laboratory sample taken
from a larger volume will be representative of the bulk solution. Heterogeneous materials have to
be homogenized prior to obtaining a laboratory sample if an average or bulk composition is
required.
Conversely, where analyte levels in different parts of the material are to be measured, they may
need to be physically separated before laboratory samples are taken. This is known as selective
sampling. Typical examples of heterogeneous materials where selective sampling may be
necessary include:
 Surface waters such as streams, rivers, reservoirs and seawater, where the concentrations
of trace metals or organic compounds in solution and in sediments or suspended particulate
matter may each be of importance;
 Materials stored in bulk, such as grain, edible oils, or industrial organic chemicals, where
physical segregation (stratification) or other effects may lead to variations in chemical
composition throughout the bulk;
 Ores, minerals and alloys, where information about the distribution of a particular metal or
compound is sought;
 Laboratory, industrial or urban atmospheres where the concentrations of toxic vapors and
fumes may be localized or vary with time.
Repeated sampling over a period of time is a common requirement. Studies of seasonal variations
in the levels of pesticide, herbicide and fertilizer residues in soils and surface waters, or the
continuous monitoring of drinking water supplies are two examples.
Having obtained a representative sample, it must be labeled and stored under appropriate
conditions. Sample identification through proper labeling, increasingly done by using bar codes
and optical readers under computer control, is an essential feature of sample handling.

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8. SAMPLE STORAGE:
Due to varying periods of time that may elapse between sample collection and analysis, storage
conditions must be such as to avoid undesirable losses, contamination or other changes that
could affect the results of the analysis.
Samples often have to be collected from places remote from the analytical laboratory and several
days or weeks may elapse before they are received by the laboratory and analyzed. Furthermore,
the workload of many laboratories is such that incoming samples are stored for a period of time
prior to analysis. In both instances, sample containers and storage conditions (e.g., temperature,
humidity, light levels and exposure to the atmosphere) must be controlled such that no significant
changes occur that could affect the validity of the analytical data. The following effects during
storage should be considered:
 Increases in temperature leading to the loss of volatile analytes, thermal or biological
degradation, or increased chemical reactivity;
 Decreases in temperature that leads to the formation of deposits or the precipitation of
analytes with low solubilities.
 Changes in humidity that affect the moisture content of hygroscopic solids and liquids or
induce hydrolysis reactions;
 UV radiation, particularly from direct sunlight, that induces photochemical reactions,
photodecomposition or polymerization;
 Air-induced oxidation;
 Physical separation of the sample into layers of different density or changes in
crystallinity.
A particular problem associated with samples having very low (trace and ultra-trace) levels of
analytes in solution is the possibility of losses by adsorption onto the walls of the container or
contamination by substances being leached from the container by the sample solvent. Trace metals
may be depleted by adsorption or ion-exchange processes if stored in glass containers, whilst
sodium, potassium, boron and silicates can be leached from the glass into the sample solution.
Plastic containers should always be used for such samples. Conversely, sample solutions
containing organic solvents and other organic liquids should be stored in glass containers because
the base plastic or additives such as plasticizers and antioxidants may be leached from the walls of
plastic containers.

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9. SAMPLE PRE-TREATMENT
Preliminary treatment of a sample is sometimes necessary before it is in a suitable form for analysis
by the chosen technique and method. This may involve a separation or concentration of the
analytes or the removal of matrix components that would otherwise interfere with the analysis.
Samples generally need to be brought into a form suitable for measurements to be made under
controlled conditions. This may involve dissolution, grinding, fabricating into a specific size and
shape, pelletizing or mounting in a sample holder.
Samples arriving in an analytical laboratory come in a very wide assortment of sizes, conditions
and physical forms and can contain analytes from major constituents down to ultra-trace levels.
They can have variable moisture content and the matrix components of samples submitted for
determinations of the same analyte(s) may also vary widely. A preliminary, or pre-treatment, is
often used to condition them in readiness for the application of a specific method of analysis or to
pre-concentrate (enrich) analytes present at very low levels. Examples of pretreatments are:
 Drying at 100°C to 120°C to eliminate the effect of a variable moisture content;
 Weighing before and after drying enables the water content to be calculated or it can be
established by thermo-gravimetric analysis
 Separating the analytes into groups with common characteristics by distillation, filtration,
centrifugation etc.
 Removing or reducing the level of matrix components that are known to cause interference
with measurements of the analytes;
 Concentrating the analytes if they are below the concentration range of the analytical
method to be used by evaporation, distillation, co-precipitation, ion exchange, solvent or
solid phase extraction or electrolysis.
Sample clean-up in relation to matrix interference and to protect specialized analytical equipment
such as chromatographic columns and detection systems from high levels of matrix components
is widely practiced using solid phase extraction (SPE) cartridges. Substances such as lipids, fats,
proteins, pigments, polymeric and tarry substances are particularly detrimental.

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10. CHEMICAL STANDARDS/STANDARDS SOLUTIONS
A chemical standard is a material or substance of very high purity and/or known composition that
is used to standardize a reagent or calibrate an instrument.
Materials or substances suitable for use as chemical standards are generally single compounds or
elements. They must be of known composition, and high purity and stability. Many are available
commercially under the name AnalaR. Primary standards, which are used principally in titrimetry
to standardize a reagent (titrant) (i.e. to establish its exact concentration) must be internationally
recognized and should fulfil the following requirements:
1. Be easy to obtain and preserve in a high state of purity and of known chemical composition;
2. Be non-hygroscopic and stable in air allowing accurate weighing;
3. Have impurities not normally exceeding 0.02% by weight;
4. Be readily soluble in water or another suitable solvent;
5. React rapidly with an analyte in solution;
6. Other than pure elements, to have a high relative molar mass to minimize weighing errors.
Primary standards are used directly in titrimetric methods or to standardize solutions of secondary
or working standards (i.e. materials or substances that do not fulfill all of the above criteria that
are to be used subsequently as the titrant in a particular method). Chemical standards are also used
as reagents to effect reactions with analytes before completing the analysis by techniques other
than titrimetry.
A reference material: A reference material is a material or substance, one or more properties of
which are sufficiently homogeneous and well established for it to be used for the calibration of
apparatus, the assessment of a measurement method or for assigning values to materials.
Reference materials are used to demonstrate the accuracy, reliability and comparability of
analytical results. A certified or standard reference material (CRM or SRM) is a reference material,
the values of one or more properties of which have been certified by a technically valid procedure
and accompanied by a traceable certificate or other documentation issued by a certifying body
such as the Bureau of Analytical Standards. CRMs or SRMs are produced in various forms and
for different purposes. Mostly they are pure substances or solutions for calibration or
identification;
They have a number of principal uses, including
 Validation of new methods of analysis;
 Standardization/calibration of other reference materials;
 Confirmation of the validity of standardized methods;
 Support of quality control and quality assurance schemes

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 Preparation of standard solutions/molar and normal solutions will be demonstrated and
practical examples will be given for practice of students.
11. INSTRUMENT CALIBRATION AND LINER REGRESSION
i). Calibration:
Calibration or standardization is the process of establishing the response of a detection or
measurement system to known amounts or concentrations of an analyte under specified conditions,
or the comparison of a measured quantity with a reference value.
With the exception of absolute methods of analysis that involve chemical reactions of known
stoichiometry (e.g., gravimetric and titrimetric determinations), a calibration or standardization
procedure is required to establish the relation between a measured physico-chemical response to
an analyte and the amount or concentration of the analyte producing the response. Techniques and
methods where calibration is necessary are frequently instrumental, and the detector response is in
the form of an electrical signal. An important consideration is the effect of matrix components on
the analyte detector signal, which may be supressed or enhanced, this being known as the matrix
effect. When this is known to occur, matrix matching of the calibration standards to simulate the
gross composition expected in the samples is essential (i.e. matrix components are added to all the
analyte standards in the same amounts as are expected in the samples). There are several methods
of calibration, the choice of the most suitable depending on the characteristics of the analytical
technique to be employed, the nature of the sample and the level of analyte(s) expected. The most
common method of calibration for instruments is called external standardization.
ii). External standardization:
A series of at least four calibration standards containing known amounts or concentrations of the
analyte and matrix components, if required, is either prepared from laboratory chemicals of
guaranteed purity (AnalaR or an equivalent grade) or purchased as a concentrated standard ready
to use. The response of the detection system is recorded for each standard under specified and
stable conditions and additionally for a blank, sometimes called a reagent blank (a standard
prepared in an identical fashion to the other standards but omitting the analyte). The data is either
plotted as a calibration graph or used to calculate a factor to convert detector responses measured
for the analyte in samples into corresponding masses or concentrations.
Instruments and apparatus used for analytical work must be correctly maintained and calibrated
against reference values to ensure that measurements are accurate and reliable. Performance should
be checked regularly and records kept so that any deterioration can be quickly detected and
remedied. Microcomputer and microprocessor controlled instrumentation often has built-in
performance checks that are automatically initiated each time an instrument is turned on.
Some examples of instrument or apparatus calibration are
 Manual calibration of an electronic balance with certified weights;

17. 17
 Calibration of volumetric glassware by weighing volumes of pure water;
 Calibration of the wavelength and absorbance scales of spectrophotometers with certified
emission or absorption characteristics;
iii). Internal Standardization:
This is a calibration procedure where the ratio of the instrument response for an analyte to that of
an added standard is measured for a series of analyte standards and samples.
iv). Calibration of an Instrument
Many quantitative analytical procedures rely on instrumental measurements where a property of
the analyte(s) is monitored by a suitable detection system. The detector generates an electrical
signal, the magnitude of which is determined by the mass or concentration of the analyte. Before
using a particular analytical procedure to analyze samples, it is first necessary to establish the
detector responses to known amounts of the analyte (calibration standards) over a selected mass
or concentration range, a process known as calibration. The relation between the two variables is
often linear (directly proportional), but there is generally an upper limit to the range of values
beyond which a curved or curvilinear relation is observed. In some instances, there may be no
direct linear relation at all, or a logarithmic or more complex mathematical correlation may be
found.
Calibration data are generally used to construct a calibration graph, where detector response is
plotted on the ordinate axis (y-values) and mass or concentration of the analyte on the abscissa
axis (x-values) as shown in Figure 1.
The graphs are often linear, being defined by the equation
y = bx + a (1)
Where, ‘b’ is the slope and ‘a’ the intercept on the y-axis. In some cases, it is preferable to plot a
logarithmic function of the detector response or analyte concentration to obtain a linear calibration
curve.

18. 18
Unknown levels of the analyte are determined from the graph by interpolation. Where a linear
relation has been established, a calibration factor can be used to convert detector response to mass
or concentration of analyte when analyzing samples.
Theoretically, the graph should pass through the origin, but frequently in practice there is a small
positive intercept due to traces of analyte in the reagent blank or contributions to the detector signal
by other components in the standards. Calibration points also show a degree of scatter due to the
effects of experimental errors in preparing the standards, or noise in the measuring circuitry. A
line of best fit through the points, known as a regression line, is therefore drawn or computed.
Calibration graphs may show curvature, particularly at higher mass or concentration levels, but
this does not invalidate their use if the data are reproducible (See Figure-2). However, it is
advisable to prepare additional standards to define the curve more closely, and the use of a factor
to compute analyte levels in samples is precluded.
v). Correlation Coefficient
The correlation coefficient (r), indicates the degree of linearity between x and y and the range of
possible values for ‘r’ is -1 < r <+1. A value of unity indicates a perfect linear correlation between
x and y, all the points lying exactly on a straight line, whilst a value of zero indicates no linear
correlation. Values may be positive or negative depending on the slope of the calibration graph
(Figure 2 a-c). Most calibration graphs have a positive slope, and correlation coefficients
frequently exceed 0.99. They are normally quoted to four decimal places. (Note that graphs with
a slight curvature may still have correlation coefficients exceeding about 0.98 (Fig. 2(d)), hence
great care must be taken before concluding that the data shows a linear relation. Visual inspection
of the plotted points is the only way of avoiding mistakes.)

19. 19
vi). Linear Regression
When inspection of the calibration data and the value of the correlation coefficient show that there
is a linear relation between the detector response and the mass or concentration of the analyte, it
is necessary to draw a line of best fit through the plotted points before they can be used as a working
curve. Although this can be done by eye, a more accurate method is to employ linear regression.
It is invariably the case that, due to the effects of indeterminate errors on the data, most of the
points do not lie exactly on the line, as shown in Figure 1. Linear regression enables a line of best
fit through the points to be defined by calculating values for the slope and y-axis intercept ‘b’ and
‘a’ respectively in equation (1).
Example
A calibration graph was prepared as part of a validation procedure for a new method to determine
an active constituent of a sun cream by UV spectrophotometry. The following data were obtained:
Analyte conc. (ppm) 0 20 40 60 80 100 120
UV absorbance at 325 nm 0.095 0.227 0.409 0.573 0.786 0.995 1.123
The data is first checked for linearity by calculation of the correlation coefficient, ‘r’ and visual
inspection of a plotted curve. Some calculators and computer software can perform the
computation from the raw data.
r = 0.9989
Figure-3 and the correlation coefficient of 0.9989 shows, that there is a good linear relation
between the measured UV absorbance and the analyte concentration. The slope and y-axis
intercept of the regression line are b = 0.00878 and a = 0.0686. The y-axis intercept, slope and
analyte masses or concentrations calculated by interpolation from the regression line are all
affected by errors.
vi). Detection Limits
For any analytical procedure, it is important to establish the smallest amount of an analyte that can
be detected and/or measured quantitatively. In statistical terms, and for instrumental data, this is
defined as the smallest amount of an analyte giving a detector response significantly different from
a blank or background response (i.e. the response from standards containing the same reagents and
having the same overall composition (matrix) as the samples, where this is known, but containing
no analyte). Detection limits are usually based on estimates of the standard deviation of replicate
measurements of prepared blanks.
A detection limit of two or three times the estimated standard deviation of the blanks above their
mean, x-b, is often quoted, where as many blanks as possible (at least 5 to 10) have been prepared
and measured. This is somewhat arbitrary, and it is perfectly acceptable to define alternatives
provided that the basis is clear and comparisons are made at the same probability level.

20. 20
12. ANLALYTIC TECHNIQUES
Analytical techniques consist of a range of techniques and methodologies to obtain and assess
qualitative, quantitative and structural information on the nature of matter.
1. Qualitative analysis is the identification of elements, species and/or compounds present in a
sample.
2. Quantitative analysis is the determination of the absolute or relative amounts of elements,
species or compounds present in a sample.
3. Structural analysis is the determination of the spatial arrangement of atoms in an element
or molecule or the identification of characteristic groups of atoms (functional groups).
 An element, species or compound that is the subject of analysis is known as an analyte.
 The remainder of the material or sample of which the analyte(s) form(s) a part is known as
the matrix.
SCOPE AND APPLICATIONS
Analytical data are required in a wide range of disciplines and situations that include not just
chemistry and most other sciences, from biology to zoology, but the arts, such as painting and
sculpture, and archaeology. Space exploration and clinical diagnosis are two quite disparate areas
in which analytical data is vital. Important areas of application include the following.
1. Quality Control (QC). In many manufacturing industries, the chemical composition of raw
materials, intermediates and finished products needs to be monitored to ensure satisfactory
quality and consistency. Virtually all consumer products from automobiles to clothing,
pharmaceuticals and foodstuffs, electrical goods, sports equipment and horticultural products
rely, in part, on chemical analysis. The food, pharmaceutical and water industries in particular
have stringent requirements backed by legislation for major components and permitted levels of
impurities or contaminants. The electronics industry needs analyses at ultra-trace levels (parts
per billion) in relation to the manufacture of semi-conductor materials.
2. Monitoring and control of pollutants. The presence of toxic heavy metals (e.g., lead, cadmium
and mercury), organic chemicals (e.g., polychlorinated biphenyls and detergents) and vehicle
exhaust gases (oxides of carbon, nitrogen and sulfur, and hydrocarbons) in the environment are
health hazards that need to be monitored by sensitive and accurate methods of analysis, and
remedial action taken.
3. Clinical and biological studies. The levels of important nutrients, including trace metals (e.g.,
sodium, potassium, calcium and zinc), naturally produced chemicals, such as cholesterol, sugars
and urea, and administered drugs in the body fluids of patients undergoing hospital treatment
require monitoring. Speed of analysis is often a crucial factor and automated procedures have
been designed for such analyses.
4. Geological assays. The commercial value of ores and minerals is determined by the levels of
particular metals, which must be accurately established. Highly accurate and reliable analytical
procedures must be used for this purpose, and referee laboratories are sometimes employed where
disputes arise.
5. Fundamental and applied research. The chemical composition and structure of materials used
in or developed during research programs in numerous disciplines can be of significance. Where
new drugs or materials with potential commercial value are synthesized, a complete chemical
characterization may be required involving considerable analytical work.

21. 21
13. LAB ANALYTICAL TECHNIQUES AND METHODS
There are numerous chemical or physico-chemical processes that can be used to provide
analytical information. The processes are related to a wide range of atomic and molecular
properties and phenomena that enable elements and compounds to be detected and/or
quantitatively measured under controlled conditions. The underlying processes define the various
analytical techniques. Most common and widespread techniques use in labs are;
1. Atomic and Molecular Spectrometry Techniques may involve either the emission or
absorption of electromagnetic radiation over a very wide range of energies, and can provide
qualitative, quantitative and structural information for analytes from major components of a
sample down to ultra-trace levels. The most important atomic and molecular spectrometric
techniques and their principal applications are listed in Table 2.
2. Chromatographic Techniques provide the means of separating the components of
mixtures and simultaneous qualitative and quantitative analysis, as required.
3. Electrophoresis is another separation technique with similarities to chromatography that is
particularly useful for the separation of charged species.
4. The linking of chromatographic and spectrometric techniques, called HYPHENATION,
provides a powerful means of separating and identifying unknown compounds.
 Selection of the most appropriate analytical method should take into account the following
factors:
1. The purpose of the analysis, the required time scale and any cost constraints
2. The level of analyte(s) expected and the detection limit required;
3. The accuracy required for a quantitative analysis;
4. The availability of reference materials, standards, chemicals and solvents,
instrumentation and any special facilities;
5. Quality control and safety factors

22. 22

23. 23
14. DRYING AND HEATING
Drying can be done in single or many stages.
 To remove water, the filtered solid in its container is placed in a desiccator and left for a few
hours.
 A vacuum desiccator is even more efficient for removing solvents at low temperature.
 Heating in ovens, furnaces or directly with burners will raise the temperature to remove
materials or to decompose the precipitate to a more stable form.
Example: ‘basic aluminum succinate’ is a good precipitate for aluminum, but must be ignited to
constant weight at about 1200 C to convert to aluminum oxide. Similarly soil moisture content
are accurately determined by weighing and drying the pre weighed soil sample to constant
temperature and then weighing again for calculation of moisture loss or its contents in soil.
15. GRAVIMETRY
Gravimetry is the analytical technique of obtaining a stable solid compound, of known
stoichiometric composition so that the amount of an analyte in the sample may be found by
weighing. Gravimetry is one of the ‘classical’ techniques of analysis, and although less frequently
used now, it is of value when an accurate reference method is required for comparison with an
instrumental technique. If an element is present in a mixture, for example, silver in a sample of
nickel, one way of separating it is to dissolve the metal completely in a suitable solvent. In this
example, the metal mixture could be dissolved in concentrated nitric acid and a reagent added that
would react with the silver to produce a precipitate, which for silver might be a sodium chloride
solution:
Ag(s) +Ni(s) +HNO3(sol) = AgNO3(sol) +Ni(NO3)2(sol)
AgNO3(sol) +NaCl(sol) = AgCl(s) +NaNO3(sol)
The silver chloride is precipitated completely, and may be filtered off since both nickel nitrate and
sodium nitrate are very soluble in water. The precipitate will be wet and may contain traces of
nickel in solution, so must be thoroughly washed and dried.

24. 24
Since weighing may be carried out readily and accurately in almost all laboratories, gravimetry is
often used as a reference method. Analysis of major components of metal samples such as steel,
and of minerals and soils may be carried out by gravimetric methods, but they often involve
lengthy separations and are time consuming. Newer instrumental methods may determine several
components simultaneously, rapidly and are generally applicable down to trace levels.
16. TITRIMETRY
Titrimetry is an analytical technique for the determination of the stoichiometry of a reaction by
the addition of controlled amounts of a standard reagent. Titrations usually involve the addition of
controlled volumes of a standard solution, whose concentration is known accurately, to a solution
of reactant of unknown concentration. In order to obtain accurate quantitative data for a reaction
in solution, it is necessary that the reaction be fast, complete and occur in fixed, reproducible
amounts. The requirement for fast reaction is achieved readily when ionic species are involved,
although in some other cases, it is necessary to warm the solutions or add a catalyst. The reaction
will be complete provided the equilibrium constant is large.
The technique of volumetric analysis is the simplest type of titrimetry, and involves the addition
of controlled volumes of a reagent solution (titrant) to a known volume of another solution (the
titrand) in a volumetric titration. This procedure may be automated, and the changes detected
instrumentally. In some cases, excess of a reagent is added and the excess measured by back
titration. The volumes and concentrations can be measured with high accuracy.
Application:
There are many applications for acid-base titrations, several of which are routinely used in
analytical methods
 The determination of the concentration of acid in foods and pharmaceuticals.
 The measurement of acid number (or base number) during the course of a reaction. For
example, in the production of polyester resins by the reaction of a glycol with maleic and
phthalic acids, the total acid remaining is determined by titration of a weighed sample with
potassium hydroxide using phenolphthalein as indicator.
 The Kjeldahl method for nitrogen determination is a good example of a back titration. The
sample (plant material) is digested in concentrated acid and then excess sodium hydroxide
solution is added and the ammonia released is carefully distilled off and captured into a
known volume of standard acid, such as 0.1 M boric acid. The excess acid is then titrated
with standard alkali to know the nitrogen contents.
Automated titrations are important in producing rapid, reproducible results in commercial and
research laboratories. Samples may be prepared and added using mechanical pipets or direct
weighing methods. Titrant is added to the sample titrand solution using peristaltic pumps or burets
driven by pressure or piston systems. The addition is very reproducible after accurate calibration.

25. 25
17. ATOMIC AND MOLECULAR SPECTROMETRY
The intensity of the spectral emission or the reduction of intensity by absorption is related to the
concentration of the species producing the spectrum.
i). Principles of Flame Photometry (FFM)
When the atoms of samples are excited to higher electronic energy levels in flames they emit
radiation in the visible and UV regions of the electromagnetic spectrum. Emission intensities
may be measured to analyze for metals, especially alkali and alkaline earth elements.
The processes that occur to transfer the sample to the flame may be summarized as follows:
i. Production of an aerosol from solution (nebulization)
ii. Removal of solvent MA(aq) →MA (solid)
iii. Vaporization of sample MA(solid) →MA(vapour)
iv. Atomization MA → A• +A•
v. Excitation M• →M*
vi. Emission M* →M•
The wavelength of the light emitted from the flame is characteristic of the particular element. The
intensity of this light is, in most cases, proportional to the absolute quantity of the species present

26. 26
in the flame at any moment, i.e. the number of atoms returning to the ground state is proportional
to the number of atoms excited, i.e. the concentration of the sample. The emitted radiation is
isolated by an optical filter and then converted to an electrical signal by the photo detector.
Figure: Basic components of a simple flame photometre.
ii). Components of FES
A simple flame photometer consists of the following basic components:
1. A flame that can be maintained in a constant form and at a constant temperature:-
“The Burner”
2. A means of transporting an homogeneous solution into the flame at a steady rate:-
“Nebuliser and mixing chamber”
3. A means of isolating light of the wavelength to be measured from that of extraneous
emissions:- “Simple colour filters” (interference type)
4. A means of measuring the intensity of radiation emitted by the flame:- “Photo Detector”

27. 27
iii). Instrumentation:
Flame atomic emission spectrometers have similar optical systems to those of UV-visible
spectrometers, but the source of radiation is provided by the sample itself. A flame photometeris a
simpler instrument employing narrow bandpass optical filters in place of a monochromator (Fig.
1). The sample is prepared as a solution, which is drawn into a nebulizer by the effect of the flowing
oxidant and fuel gases. The fine droplets produced pass into the flame where sample atoms are
progressively excited. The emitted radiation passes through the monochromator or filter and is
detected by a photocell or photomultiplier tube.
iv). Interferences:
 Interferences may affect the linearity of the calibration due to the emission lines produced
by other species close to those of the analyte. They may be minimized by selecting a
different spectral line for the analysis, or by altering the spectral resolution or filter.
 The presence of anions that form very stable compounds with the metal ions, such as sulfate
and phosphate may interfere with some determinations.
 At high analyte concentrations, the concentration of atoms in the flame may be high enough
to cause self-absorption. That is, the emission is reabsorbed by the ground state atoms in
the cooler outer layers of the flame. This sometimes causes a loss in sensitivity at higher
concentrations.
v). Applications:
Flame atomic emission spectrometry (FAES) and flame photometry are used widely for the
determination of alkali and alkaline earth metals. The rapid determination of Na, K and Ca in
biological and clinical samples is one of the most important applications; for example, calcium in
beer, milk or biological fluids. The usual solvent is water, but organic solvents may be used to
enhance the intensity, since they produce smaller droplets, and have a smaller cooling effect on
the flame.
The advantages of FAES and flame photometry are that the instrumentation is relatively simple
and measurements can be made quickly. A disadvantage is the sensitivity of the emission
intensities to changes in flame temperature due to variations in gas flow, or cooling by the
solvent

28. 28
18. ATOMIC ABSORPTION AND ATOMIC FLUORESCENCE
SPECTROMETRY
A. Principle of Absorption Spectrometry: (Beer–Lambert absorption law)
When metals are exposed to heat, they absorb light. Each metal absorbs light at a characteristic
frequency. For example:
Metal Zn Fe Cu Ca Na
λ (nm) 214 248 325 423 589
The metal vapor absorbs energy from an external light source, and electrons jump from the ground
to the excited states. The ratio of the transmitted to incident light energy is directly proportional to
the concentration of metal atoms present. Then, a calibration curve is constructed [Concentration
(ppm) vs. Absorbance to get final reading in ppm (conc.). Only a very small number of the atoms
in the flame are actually present in an excited state at any given instant. Thus there is a large
percentage of atoms that are in the ground state and available to be excited by some other means,
such as a beam of light from a light source. AA takes advantage of this fact and uses a light beam
to excite these ground state atoms in the flame. Thus AA is very much like molecular absorption
spectrophotometry in that light absorption (by these ground state atoms) is measured and related
to concentration. The major differences lie in instrument design, especially with respect to the
light source, the "sample container," and the placement of the monochromator.
B. Instrumentation
The light source, called a hollow cathode tube, is a lamp that emits exactly the wavelength required
for the analysis (without the use of a monochromator). The light is directed at the flame containing
the sample, which is aspirated by the same method as in FP. The flame is typically wide (4-6
inches), giving a reasonably long path length for detecting small concentrations of atoms in the
flame. The light beam then enters the monochromator, which is tuned to a wavelength that is
absorbed by the sample. The detector measures the light intensity, which after adjusting for the
blank, is output to the readout, much like in a single beam molecular instrument. Also as with the
molecular case, the absorption behavior follows Beer's Law and concentrations of unknowns are
determined in the same way. All atomic species have an absorptivity, a, and the width of the flame
is the path length, b. Thus, absorbance (A) of standards and samples are measured and
concentrations determined as with previously presented procedures, with the use of Beer's Law (A
= ἐbc).

29. 29
The Sketch diagram for AAS instrument
C. Main Component
1. The light Source
2. Monochromator
3. Atomizer
4. Detector
5. Amplifier/Readout System
I. Light Source: (Hollow Cathode Lamb):
II. An HCL usually consists of a glass tube containing a cathode, an anode, and a
buffer gas (usually a noble gas). A largevoltage across the anode and cathode will cause
the buffer gas to ionize, creating a plasma. The buffer gas ions will then be accelerated into
the cathode, sputtering off atoms from the cathode. Both the buffer gas and the sputtered
cathode atoms will in turn be excited by collisions with other atoms/particles in the plasma.
As these excited atoms decay to lower states, they will emit photons, which can then be
detected and a spectrum can be determined. Either the spectrum from the buffer gas or the
sputtered cathode material itself, or both, may be of interest. [1]
The source is often a hollow cathode lamp (HCL) as shown in Figure. This has a glass envelope
with a quartz window and a cathode and an anode. It contains a gas such as argon, which is excited
by an electric discharge. The excited argon atoms bombard the cathode, which is made of the
element to be determined and the atoms of that element are then excited in the discharge too. The
excited atoms decay back to their ground state, emitting the characteristic radiation. A turret with
several lamps allows multi-element determinations. The following steps occurs when AAS is
powered “ON’
1. A large voltage across the anode and cathode will cause the inert gas to ionize.

30. 30
2. The inert gas ions will then be accelerated
into the cathode, sputtering off atoms from
the cathode.
3. Both the inert gas and the sputtered cathode atoms will in turn be excited by collisions
with each other.
4. Both the inert gas and the sputtered cathode atoms will in turn be excited by collisions
with each other.
5. When these excited atoms decay to lower energy levels they emit a few spectral lines
characteristic of the element of interest.
6. The light is emitted directionally through the lamp's window, a window made of a glass
transparent in the UV and visible wavelengths.
7. The light can then be detected and a spectrum can be determined.
Fuel Oxidant Temperature
(K)
Acetylene air 2400 – 2700
Acetylene oxygen 3300 – 3400
Acetylene nitrous oxide 2900 – 3100

31. 31
III. Sample Compartment/Nebulizer:
For flame vaporization, the sample is usually prepared as a solution that is sprayed into the burner.
A flow spoiler removes large droplets, and the sample undergoes a similar sequence of events,
converting it into gaseous atoms. The signal reaches a constant value proportional to the
concentration of the analyte element in the sample.
Nebulizer do the following functions in AAS;
1. Sucks up the liquid sample (= aspiration)
2. Creates a fine aerosol (fine spray) for introduction into flame
3. Mixes aerosol, fuel and oxidant thoroughly, creates a heterogenous mixture
4. The smaller the size of the droplets produced, the higher the element sensitivity
Hydrogen air 2300 – 2400
most commonly used: air / acetylene

32. 32
The flame is generally produced using one of the gas mixtures given in Table below. Air-acetylene
gives a flame temperature of about 2400 K, while air-propane is cooler (~1900 K), and nitrous
oxide-acetylene hotter (~ 2900 K). The structure and temperature of the flame is most important,
as is the alignment of the optical path with the region of the flame in which an optimum
concentration of the atoms of analyte is present. Variations in flame temperature, including those
caused by cooling due to the sample, will affect the sensitivity of the technique. Flame sources
have advantages for analysis where large volumes of analyte are available.
IV. The Monochromator
The monochromator and detector are similar to those used in other forms of spectrometry.
Monochromator do the isolation of the absorption line from background light and from molecular
emissions originating in the flame, i.e. tuned to a specific wavelength. AAS is essentially a single-
beam technique. Since the flame and matrix may produce background radiation in the region of
interest, correction for this is important.
V. Detector
A photomultiplier measures the intensity of the incident light and generates an electrical signal
proportional to the intensity.
VI. Readout Components
As with molecular spectrophotometry, the readout of the absorbance and transmittance data can
consist of either a meter, a recorder or digital readout. The meter can be calibrated in either %
transmittance (or % absorption 100 %T) or absorbance, or possibly both. If %T or % absorption
are displayed, these of course must first be converted to absorbance (-log T) before plotting.

33. 33
Method of Standard Additions
Due to the effects of other constituents in a sample, it is always desirable to match the blank and
standards to the sample as much as possible. With AA, the sample preparation is frequently so
simple that samples to be tested are aspirated directly into the flame and measured.
The solution to this problem is to use the method of standard additions. In this method, small
amounts of a standard solution of the element being determined are added to the sample and the
absorbance measured after each addition. In this way, the sample matrix is always present, and
interfering sample components affect the observance equally with each measurement. Therefore
there is no effect on the outcome and the total sample composition need not be known.
The plotting procedure and the use of the graph for obtaining the sample concentration is altered
somewhat, however, The Beer's Law plot is a graph of A vs. concentration added. Since the
element being determined is present in the sample from the start, a bonafide absorbance reading is
measured for the sample to which nothing has been added.
As more and more analyte is added, the absorbance reading simply increases (linearly) so that a
graph, which does not intersect zero (at zero added concentration) is plotted. (See Figure below)
Extrapolation of the graph to zero absorbance, as shown, results in a length of x-axis, on the
negative side of zero added, which represents the concentration in the unknown.
[Pb+2] (ppm) Absorbance Calculated Pb (II) concentraions (ppm) Absorbance
0.000 0.000 0.357 0.340
0.100 0.116
0.200 0.216
0.300 0.310
0.400 0.425
0.500 0.520
y = 1.0505x
R² = 0.9988
0.000
0.100
0.200
0.300
0.400
0.500
0.600
0.000 0.100 0.200 0.300 0.400 0.500 0.600
Absorbance
[Pb+2] (ppm)
Lead (II) Calibration Curve
Disadvantages
 Only solutions can be analysed
 Only 5 – 15 % of the nebulized sample reaches the flame
 Relatively large sample quantities required (1 – 2 mL)
 Less sensitivity (compared to graphite furnace)

34. 34
 Problems with refractory elements
 Samples which are viscous require dilution with a solvent
Advantages
 Inexpensive (equipment, day-to-day running)
 High sample throughput
 Easy to use
 High precision
Interferences
Interferences may be troublesome in AAS. Background absorption by smoke particles or solvent
droplets may be removed as detailed above. Matrix interference, such as any reaction that prevents
the sample getting into the flame, may reduce the sensitivity. It is always preferable to run the
standards in the same matrix, or to use standard addition procedures.
1. Chemical Interference
Elements that form stable compounds that are not completely atomized at the temperature of the
flame or graphite furnace. Chemical interference due to the production of thermally stable
compounds, such as in-volatile phosphates of calcium, may sometimes be dealt with by adding a
releasing agent such as EDTA, or by using a hotter flame or a reagent that preferentially forms
stable, volatile compounds.
Example: calcium in the presence of phosphate forms stable calcium phosphate
3Ca2+
+ 2PO4
3
→Ca3 (PO4)2
 Higher flame temperature (nitrous oxide / acetylene instead of air / acetylene)
 Addition of release agents
 Addition of chelating agent
Addition of a chelating agent for the analysis of calcium:
Ca3 (PO4)2
+ 3EDTA →3Ca (EDTA) + 2PO4
3-
Addition of a release agent for the determination of calcium:
for example: addition of 1000 ppm LaCl3
Ca3 (PO4)2
+ 2LaCl3→ 3CaCl2+ 2LaPO4
2. Ionisation Interference
Ionization interference due to the production of ions is most troublesome with alkali metals
because of their low ionization potentials. Occasionally, ionization suppressors such as lithium or
lanthanum salts, which are easily ionized, are added.
M(g) → M+(g) + e-
Problem in the analysis of alkali metal ions: alkali metals have lowest ionisation energies and are
therefore most easily ionised in flames.
Example: 2450 K, p = 0.1 Pa Na 5 % ionised
K 33 % ionised

35. 35
Ionisation leads to reduced signal intensity, as energy levels of ions are different from those of
the parent ions. Ionisation of the analyt element can be suppressed by adding an element that is
more easily ionised. Ionisation of the added element results in a high concentration of electrons
in the flame.
Example: Addition of 1000 ppm CsCl when analysing for Na or K
4. Spectral interference
i. Spectral Overlap
Spectral interference is rare because of the sharpness of the atomic elemental lines, but is difficult
to overcome. For example, the zinc line at 213.856 nm is too close to the iron line at 213.859 nm,
but the iron line at 271.903 nm could be used to determine iron instead. Similarly, avoid the
interference by observing the aluminum line at 309.27 nm.
Cu = 324.754 nm Eu = 324.753 nm
Al = 308.215 nm V = 308.211 nm Al = 309.27 nm
Zn = 213.856 nm Fe = 213.859 nm Fe = 271.903 nm
ii. Light scatter
Metal oxide particles with diameters greater than the wavelength of light. When sample contains
organic species or when organic solvents are used to dissolve the sample, incomplete combustion
of the organic matrix leaves carbonaceous particles that are capable of scattering light.
The interference can be avoided by variation in analytical variables, such as flame temperature and
fuel-to –oxidant ratio
4. Interferences by Physical properties of the solution
The amount of sample that reaches the flame depends on
 Viscosity
 Surface tension
 Density
 Solvent or vapour pressure of the solution.
Physical properties of sample and standard solutions for calibration curve should match as
closely as possible.
Application of Atomic Absorption Spectrophotometry:
The techniques are used for many industrial and research purposes, especially:
 Agricultural samples, particularly the analysis of soils -metal pollutants in soil and water
samples are often determined by AAS;

36. 36
 Clinical and biochemical determinations, e.g., the measurement of sodium, potassium,
lithium and calcium in plasma and serum, and of iron and lead in whole blood (Ca, Mg,
Li, Na, K, Fe).
 Metallurgical samples may be assayed to measure impurities;
 Oils and petrochemical sample scan be analyzed for metals in feed-stocks and to detect
metals in used oils due to corrosion and wear. Analysis of additives in lubricating oils and
greases (Ba, Ca, Na, Li, Zn, Mg) also done.
 Water samples are extremely important (e.g. Ca, Mg, Fe, Si, Al, Ba content), since
pollution may be a health hazard. Nickel, zinc, mercury and lead are among the metals
determined.
Graphite Furnace AAS
An alternative vaporization method is to use a graphite furnace, which is an open-ended cylinder
of graphite placed in an electrically heated enclosure containing argon to prevent oxidation.
Temperatures in the region of 2500 K are achieved, and the heating program is designed to heat
the sample, deposited on a smaller tube or L’ Vov platform, by radiation. The graphite furnace
produces a peak signal whose area is proportional to the total amount of vaporized element in the
sample. Samples are placed directly in the graphite furnace which is then electrically heated. Beam
of light passes through the tube.
Three stages of sample processing:
1. Drying of sample
2. Ashing of organic matter
3. Vaporization of analyte atoms to burn off organic species that would interfere with the
elemental analysis. Molecules have broad absorption bands!
Stages in Graphite Furnace
Typical conditions for Fe:
1. Drying stage: 125 C for 20 sec
2. Ashing stage: 1200 C for 60 sec
3. Vaporization: 2700 C for 10 sec
Advantages over flame atomic absorption spectroscopy:
1. Solutions, slurries and solid samples can be analysed.
2. Greater sensitivity- It gives increased sensitivity because of the longer residence time of
the sample in the beam from the source. The sensitivity is further increased because a
higher proportion of atoms are produced.
3. Smaller quantities of sample (typically 5–50 L) - It has the ability to handle small
volumes of samples, down to 0.5-10 ml, such as clinical specimens.

37. 37
4. Provides a reducing environment for easily oxidized elements
5. It avoids interactions between the sample components and the flame since atomization
takes place in an inert gas stream.
6. The results are more reproducible than flame AAS.
Disadvantages
1. Expensive
2. Low precision
3. Low sample throughput
4. Requires high level of operator skill

38. 38
19. ULTRAVIOLET AND VISIBLE MOLECULAR SPECTROMETRY:
Absorption Spectrometery: Absorption in the ultraviolet and visible regions of the
electromagnetic spectrum corresponds to transitions between electronic energy levels and provides
useful analytical information for both inorganic and organic samples.
Instrumentation: The components of ultraviolet and visible spectrometers include a source of
radiation, a means of dispersion and a detector specific to this spectral region.
Applications: The structure of a molecule determines the nature of its UV or visible spectrum and
facilitates qualitative analysis of a sample. Measurement of the relation between concentration and
absorbance allows quantitative analysis using the Beer-Lambert Law.
Description of Technique:
The ultraviolet (UV) and visible region of the electromagnetic spectrum covers the wavelength
range from about 100 nm to about 800 nm. The vacuum ultraviolet region, which has the shortest
wavelengths and highest energies (100–200 nm), is difficult to make measurements in and is little
used in analytical procedures. Most analytical measurements in the UV region are made between
200 and 400 nm. The visible region occurs between 400 and 800 nm.
The energy levels involved in transitions in the UV-visible region are the electronic levels of atoms
and molecules. For example, although light atoms have widely spaced energy levels, some heavy
atoms have their outer orbitals close enough together to gives transitions in the visible region. This
accounts for the colors of iodides. Transition metals, having partly occupied d or f orbitals, often
show absorption bands in the visible region and these are affected by the bonding of ligands. For
example, iron (III) reacts with the thiocyanate ion to produce an intense red color due to the iron
(III) thiocyanate complex, which may be used to determine iron (III) in the presence of iron (II).
Principle
Many molecules absorb ultraviolet (UV), visible (Vis) or near infrared (NIR) radiation. In terms
of the electromagnetic spectrum, UV radiation covers the region from 190–350 nm, visible
radiation covers the region 350–800 nm and NIR radiation covers the region 800–2500 nm (and
maybe a little higher). Absorption of UV and/or Vis radiation corresponds to the excitation of outer
electrons in the molecule. Typically, radiation with a specific intensity is passed through a liquid
sample, often in a quartz cuvette. When the radiation emerges on the other side of the cuvette, it is
reduced in intensity owing to losses from a) reflection off the cuvette windows, b) scattering and
c) absorption by the sample itself. Often, a reference solution which has no analyte is also analysed
to account for the losses due to reflection and scattering; thereby the intensity attenuation due to
absorption alone can be worked out by simple subtraction. In organic molecules, this absorption
is restricted to certain functional groups (chromophores) that contain electrons of low excitation
energy.

39. 39
Aromatic molecules, for example, mostly absorb UV in the 200–300 nm region. An absorption
spectrum is usually a plot of absorbance versus wavelength and is normally continuous and broad
with little fine structure (Figure 2.2). The broad spectrum is due to the fact that the higher energy
radiation involved means that vibrational and rotational transitions co-occur as well as electronic
transitions; all of these are superimposed on each other resulting in broad bands rather than sharp
peaks. In UV–Vis absorption spectrometry, concentration of the species is related to absorbance
by the Beer–Lambert Law (see Equation-1).
A = ɛλcl
Where; Aλ = absorbance at a particular wavelength (λ), ελ= extinction coefficient at a particular
wavelength (λ), c = concentration and l = path length. During most experiments, ε and l remain
constant, so absorbance is proportional to concentration, a relationship that is exploited for
quantitative analysis.
Absorption of NIR radiation corresponds to certain vibrations of the molecule and is due to
overtones and combinations of parent absorption bands in the mid-infrared (IR) region. Generally,
these absorptions are weaker than the parent absorptions in the IR but the decrease in intensities is
not the same for all molecules. It can be seen as a complementary technique to conventional IR,
exploiting a different region of the electromagnetic spectrum. For example, water has less
absorption in the NIR compared with mid IR, so NIR spectra of aqueous samples are often sharper.
A NIR absorption spectrum is usually a plot of absorbance versus wavelength and has more fine
structure than a UV or UV–Vis spectrum. NIR absorbance also follows the Beer–Lambert Law,
so can be used as a quantitative technique.
For absorption spectrometry the intensity of the incident (exciting) radiation is reduced when it
interacts with the atoms or molecules, raising them to higher energy levels. In order to interact, the
radiation must come into contact with the species. The extent to which it does this will depend on
the concentration of the active species and on the path length through the sample, as shown in
Figure.
As the radiation of a particular wavelength passes through the sample, the intensity decreases
exponentially, and Lambert showed that this depended on the path length, (l), while Beer showed
that it depended on the concentration (c). The two dependencies are combined to give the Beer–
Lambert absorption law:
It =Io exp (-k’cl)
where Io and It are the incident and transmitted intensities, respectively.
Converting to the base 10 logarithmic equation:
log ( Io/ It) = A = ɛ c L

40. 40
where A = the absorbance and ɛ = the molar absorptivity.
The value of ɛ (sometimes incorrectly referred to as the ‘extinction coefficient’), is most usually
quoted for a concentration of 1 M and a path length (l) of 1 cm. However, if the concentration is
expressed in mol m-3 (=1000 × M) and l is expressed in meters (=cm/100) then the units of ɛ are
m2
mol-1
, suggesting that the absorptivity depends on the effective capture area of the species. This
indicates that ɛ combines the transition probability and the nature of the absorbing species. (Note
that ɛ in m2
mol-1
= ɛ in (cmM)-1
/10.
Very high values of e, for example over 10 000 for the UV, π– π*
absorption of conjugated
polyenes, indicate a favored transition. Lower values, for example, ɛ is less than 100 for the π– π*
absorption of ketones, show that the transition is less favored or ‘forbidden’
ii). Types of UV-Vis Spectrophotometer
Single-Beam spectrometers
The simplest type of spectrometer employs a single source to supply radiation to the sample and
then to the background in turn; for example, in some UV visible absorbance measurements or in
plasma emission spectrometry.
The advantages of this system are that only a single set of components is required and that complex
sampling devices may be incorporated. The main disadvantage is that correcting for the
background spectrum, due to the solvent, matrix or interferences must be done separately, adding
to the analysis time.
Double-Beam spectrometers
In order to make rapid, accurate comparisons of a sample and a reference, double-beam
instruments are frequently used. Since it is essential that the two beams are as similar as possible,
a single source is used and the optics arranged to pass equal intensities of the beam through the
sample area and through the reference area, and then to disperse and detect them alternately. This
is shown schematically in Figure 1for an infrared spectrometer.

41. 41
The source is reflected equally onto mirrors so that beams pass through the sample and reference
areas. These beams are then selected alternately by a rotating mirror and each beam follows a
common path to the diffraction grating, which disperses the radiation and directs it onto the
detector. The width of the beams is controlled by slits, which determine the resolution. In a UV
spectrometer the beam is dispersed before passing through the sample to avoid irradiating the
sample with high energy UV radiation which could cause decomposition.
Figure: block diagram of dispersive double-beam spectrometer
Figure: block diagram of dispersive single beam spectrometer
Instrumentation
Source:
1. The source of visible light (400-800 nm) is generally a tungsten filament lamp or a tungsten
halogen bulb.
2. For the UV (200-400 nm) region, the source most often used is a deuterium lamp and
arrangements are made to switch between these sources at an appropriate wavelength, often
around 380 nm. Xenon arc lamps may also be used.

42. 42
Discriminator
A monochromator is usually used as a wavelength selector. Monochromators are composed of a
dispersing medium to ‘separate’ the wavelengths of the polychromatic radiation from the source,
slits to select the narrow band of wavelengths of interest and lenses or mirrors to focus the chosen
radiation. The dispersing medium can be a diffraction grating, a prism or an optical filter. Those
based on a grating are most effective at producing spectra with reduced stray light. The more finely
separated the ruled lines on the grating are, the higher the resolution. However, especially for NIR,
interferometers are becoming more common in Fourier Transform (FT) instruments. FT is more
effective at longer wavelengths such as in IR and NIR but can be used for UV–Vis also.
Sample
The sample holder must be transparent in the wavelength region being measured. The sample
should also not be too concentrated as the Beer–Lambert Law starts to deviate at high absorbance
levels. The sample solution is contained in Quartz cuvettes, normally used for UV–Vis and NIR
measurements. For UV–Vis absorbance, cuvettes are usually 1 cm in path length in laboratory
based instruments, though shorter path lengths can be employed. A matched reference cell contains
the solvent alone or a blank of solvent and reagents is also used in some tests. If measurements are
restricted to the visible region, ordinary glass or plastic cells may be used, but these should not be
used for UV work.
All cuvettes and cells should be handled carefully to avoid leaving fingerprints. The sample
compartment must be able to prevent stray light and dust from entering because this will adversely
affect the absorbance readings. For practical purposes, it is important that the sample solution is
dilute and that it contains no particulate matter. More concentrated samples may reabsorb the
emitted radiation, either due to the sample itself or due to the presence of some other quenching
agent. Particles will cause the radiation to be scattered.
Detector
The detector is typically a photomultiplier tube (PMT), a photodiode array (PDA) or a charge-
coupled device (CCD). For detection in the UV-visible region, photomultipliers or other
photoelectric devices are used. Some instruments may use a multi-channel diode array detector.
An array of typically 300 silicon photodiodes detects all the wavelengths simultaneously with a
resolution of about ±1 nm. This provides a great saving in time and an improved signal/noise ratio
and throughput advantage, which allows the use of a single deuterium source for the whole UV–
Vis range from 200–780 nm. The recorded spectrum is generally displayed by plotting absorbance
against wavelength. This allows direct quantitative comparisons of samples to be made.
The greatest recent improvement in spectrophotometers has been in the detector. PMTs are
monochannel detectors and are still very popular. They consist of a photosensitive surface and a
series of electrodes (dynodes), each at an increased potential compared to the one before. When a

43. 43
photon strikes the photosensitive surface, a primary electron is emitted and accelerates towards the
first dynode. This electron impacts the dynode and causes the release of a number of secondary
electrons, which hit the next electrode and so on, until the signal is amplified many times over.
Even extremely small signals can now be detected. However, multichannel detectors are increasing
in popularity. These consist of arrays of diodes such as are found in PDAs, CCDs and charge-
injection devices (CIDs). They have the advantage over PMTs of being able to measure many
wavelengths simultaneously. Hence, these instruments have a different configuration with no
monochromator before the sample and, instead, a polychromator placed after the sample and
before the detector (see figure).
These multichannel detectors work by having hundreds of silicon photodiodes positioned side by
side on a single collision crystal or chip. Each photodiode has an associated storage capacitor that
collects and integrates the photocurrent generated when the photons strike the photodiode.
Periodically, they are discharged and the current read. A spectrum can be recorded if radiation
dispersed into its different wavelengths falls on the surface of the diode array.
A CCD is also based on semi-conductor technology. It is a two-dimensional array which stores
photo-generated charge. The electrons in each element are transferred out for reading until the
array has been fully read. For NIR, the detectors described above can cover the shorter wavelength
NIR spectrum but for the longer wavelength NIR spectrum lead sulfi de or indium/gallium/arsenic
(InGaAs) detectors are used. The InGaAs detector is about 100 times more sensitive than the mid
IR region detectors and therefore, with NIR measurements, there are very low noise levels. It can
also be array-based.
Output
The PC collects the data, converts it from transmission to absorbance and displays the spectrum.
The PC can often carry out baseline subtraction and smoothing and filtering tasks as well as
qualitative and quantitative analysis. It may have other capabilities, such as the ability to compare
a spectrum to those in a spectral library and to carry out peak purity checks.
Applications
There are a huge number of applications for UV, Vis and NIR instruments. UV–Vis is routinely
used for the determination of solutions of transition metals, which are often coloured, and highly
conjugated organic compounds. For example, determination of iron by forming a coloured
complex with 1,10-phenanthroline can be detected by visible spectrophotometry. The analysis of
nitrate nitrogen in water, phosphate in water and soil and lead on the surfaces of leaves can also
be determined colorimetrically. Many pharmaceuticals, dyes and other organic compounds can be
detected easily by UV due to their strong chromophores. Moisture, fat, sugars, fibre, protein and
oil can be determined in foodstuffs such as soy bean, corn, rice, milk, meat and cheese by NIR.

44. 44
ATOMIC EMISSION SPECTROSCOPY
Atomic emission spectroscopy (AES) is a method of chemical analysis that uses the intensity of
light emitted from a flame, plasma, arc, or spark at a particular wavelength to determine the
quantity of an element in a sample. The wavelength of the atomic spectral line gives the identity
of the element while the intensity of the emitted light is proportional to the number of atoms of the
element.
A sample of a material (analyte) is brought into the flame as either a gas, sprayed solution, or
directly inserted into the flame by use of a small loop of wire, usually platinum. The heat from the
flame evaporates the solvent and breaks chemical bonds to create free atoms. The thermal energy
also excites the atoms into excited electronic states that subsequently emit light when they return
to the ground electronic state. Each element emits light at a characteristic wavelength, which is
dispersed by a grating or prism and detected in the spectrometer. A frequent application of the
emission measurement with the flame is the regulation of alkali metals for pharmaceutical
analytics
Inductively coupled plasma atomic emission spectroscopy (ICP)
Inductively coupled plasma atomic emission spectroscopy (ICP-AES) uses an inductively coupled
plasma to produce excited atoms and ions that emit electromagnetic radiation at wavelengths
characteristic of a particular element.
Advantages of ICP-AES are excellent limit of detection and linear dynamic range, multi-element
capability, low chemical interference and a stable and reproducible signal. Disadvantages are
spectral interferences (many emission lines), cost and operating expense and the fact that samples
typically must be in solution.
Spark and arc atomic emission spectroscopy
Spark or arc atomic emission spectroscopy is used for the analysis of metallic elements in solid
samples. For non-conductive materials, the sample is ground with graphite powder to make it
conductive. In traditional arc spectroscopy methods, a sample of the solid was commonly ground
up and destroyed during analysis. An electric arc or spark is passed through the sample, heating it
to a high temperature to excite the atoms within it. The excited analyte atoms emit light at
characteristic wavelengths that can be dispersed with a monochromator and detected. in the past,
the spark or arc conditions were typically not well controlled, the analysis for the elements in the
sample were qualitative. However, modern spark sources with controlled discharges can be
considered quantitative. Both qualitative and quantitative spark analysis are widely used for
production quality control in foundries and steel mills.

45. 45
CHROMATOGRAPHY
Chromatography is the process of separating the components of mixtures (solutes) that are
distributed between a stationary phase and a flowing mobile phase according to the rate at which
they are transported through the stationary phase.
Chromatography was originally developed by the Russian botanist Michael Tswett in 1903 which
is now, in general, the most widely used separation technique in analytical chemistry having
developed into a number of related but quite different forms that enable the components of
complex mixtures of organic or inorganic components to be separated and quantified. A
chromatographic separation involves the placing of a sample onto a liquid or solid stationary phase
and passing a liquid or gaseous mobile phase through or over it, a process known as elution.
Sample components, or solutes, whose distribution ratios (vide infra) between the two phases differ
will migrate (be eluted) at different rates, and this differential rate of migration will lead to their
separation over a period of time and distance Chromatographic techniques can be classified
according to whether the separation takes place on a planar surface or in a column. They can be
further subdivided into gas and liquid chromatography, and by the physical form, solid or liquid,
of the stationary phase and the nature of the interactions of solutes with it, known as sorption
mechanisms (vide infra). Table-1 lists the most important forms of chromatography, each based
on different combinations of stationary and mobile phases and instrumental or other requirements.
Thin-Layer Chromatography
Thin-layer chromatography is a technique where the components of mixtures separate by
differential migration through a planar bed of a stationary phase, the mobile phase flowing by
virtue of capillary forces. The solutes are detected in situ on the surface of the thin-layer plate by
visualizing reagents after the chromatography has been completed.
Thin-layer chromatography is used primarily as a qualitative analytical technique for the
identification of organic and inorganic solutes by comparisons of samples with standards
chromatographed simultaneously. Quantitative analysis is possible but precision is relatively poor.
Paper chromatography (PC) is simple and cheap but lacks the separating power and versatility
of thin-layer chromatography (TLC) which has largely replaced it. Both require only inexpensive
equipment and reagents, and, unlike the various forms of column chromatography, comparisons
can be made between a number of samples and standards chromatographed simultaneously.
Gas Chromatography
Gas chromatography is a technique for the separation of volatile components of mixtures by
differential migration through a column containing a liquid or solid stationary phase. Solutes are
transported through the column by a gaseous mobile phase and are detected as they are eluted.
High-Performance Liquid Chromatography
High-performance liquid chromatography (HPLC) is a technique for the separation of components
of mixtures by differential migration through a column containing a microparticulate solid
stationary phase. Solutes are transported through the column by a pressurized flow of liquid mobile
phase, and are detected as they are eluted.

46. 46
Gas (GC) and high performance liquid chromatography (HPLC) are complementary techniques
best suited to the separation of volatile and nonvolatile mixtures, respectively. Both these
techniques are instrumentally-based and computer-controlled, with sophisticated software
packages and the ability to separate very complex mixtures of up to 100 or more components.
HPLC is particularly versatile, having several alternative modes suited to different types of solute.
Size-exclusion (SEC) and chiral chromatography (CC) are two additional modes used for
separating mixtures of high relative molecular mass solutes and enantiomers, respectively. Earlier
forms of liquid chromatography, used for relatively large scale separations and known as classical
column chromatography, are based on large glass columns through which the mobile phase flows
by gravity compared to the pressurized systems used in HPLC.
ELECTROPHORESIS AND ELECTROCHROMATOGRAPHY
Electrophoresis, in its classical form, is used to separate mixtures of charged solute species by
differential migration through a buffered electrolyte solution supported by a thin slab or short
column of a polymeric gel, such as polyacrylamide or agarose, under the influence of an applied
electric field that creates a potential gradient. Electrophoresis is a technique for the separation of
components of mixtures by differential migration through a buffered medium across which an
electric field is applied.
Electrochromatography is a hybrid of electrophoresis and HPLC