The document describes several different analytic techniques used in chemistry and biochemistry, including amino acid analysis, spectrophotometry, atomic absorption spectrometry, titrimetry, gravity separation, polarimetry, and fluorometry. Amino acid analysis uses ion exchange liquid chromatography to separate and quantify amino acids. Spectrophotometry measures light absorption to determine chemical concentrations. Atomic absorption spectrometry analyzes metals using flame or furnace atomic absorption. Titrimetry determines concentrations via acid-base or redox reactions with a standard solution. Gravity separation separates components by specific weight. Polarimetry measures sample rotation of polarized light. Fluorometry has greater sensitivity than spectrophotometry in detecting fluorescent compounds.

1. Different Analytic Techniques
Dr. Gitanjali
Assistant professor cum Junior Scientist

3. Amino acid Analyser
• Its technique is based on ion exchange liquid chromatography, used
for qualitative and quantitative compositional analysis of protein.
• The sample containing a mixture of amino acids is loaded onto a
column of cation-exchange resin and buffers of varying pH and
ionic strength are then pumped through the column to separate
the various amino acids.
• The column temperature is accurately controlled and can be varied,
as necessary, to produce the required separation.
• The column eluent is mixed with the ninhydrin reagent, this
mixture being passed through the high temperature reaction coil.
In the reaction coil ninhydrin reacts with the amino acids present in
the eluate to form coloured compounds. The amount of coloured
compound produced is directly proportional to the quantity of
amino acid present in the eluate.

4. Cont:
• The eluate/ninhydrin mixture is fed to the photometer unit where
the amount of each coloured compound is determined by
measuring the amount of light absorbed. The light absorption is
measured at two wavelengths, 570nm and 440nm, because
amino acids produce coloured compounds which absorb light
with a wavelength of 440nm, whereas other amino acid coloured
compounds absorb light at 570nm.
• Amino acid analyser is used for the determination of the amino
acid composition or content of proteins, peptides and other
pharmaceutical or biological preparations or samples containing
compounds that contain primary or secondary amino groups
within their molecular structure.
• Amino acid analysis allows for amino acid quantification or amino
acid identification of free amino acids, as well as amino acids
released from macromolecules such as peptides, proteins or
glycoproteins.

35. Spectrophotometry
• Spectrophotometry is a method to measure how much
a chemical substance absorbs light by measuring the
intensity of light as a beam of light passes through
sample solution.
• The basic principle is that each compound absorbs or
transmits light over a certain range of wavelength.
• It can also be used to measure the amount of a known
chemical substance.
• Spectrophotometry is one of the most useful methods
of quantitative analysis in various fields such as
chemistry, physics, biochemistry, material and
chemical engineering and clinical applications.

36. Cont:
• A spectrophotometer is an instrument that measures
the amount of photons (the intensity of light) absorbed
after it passes through sample solution. With the
spectrophotometer, the amount of a known chemical
substance (concentrations) can also be determined by
measuring the intensity of light detected. Depending
on the range of wavelength of light source, it can be
classified into two different types:
• UV-visible spectrophotometer: uses light over the
ultraviolet range (185 - 400 nm) and visible range (400 -
700 nm) of electromagnetic radiation spectrum.
• IR spectrophotometer: uses light over the infrared
range (700 - 15000 nm) of electromagnetic radiation
spectrum.

37. Atomic Absorption Spectrometer AAS
• The AAS is used to analyze metals / minerals at very low
concentrations, typically in ppm or ppb ranges.
• A liquid sample containing dissolved material whose
concentration is to be measured is aspirated into a thin, wide
AA flame, or is introduced into a small carbon furnace which is
heated to a high temperature.
• Basic Principle:
• AAS is the measurement of absorption of radiation by free
atoms. The total amount of absorption depends on the number
of free atoms present and the degree to which the free atoms
absorb the radiation.
• At the high temperature of the AA flame, which may be either
oxy-acetylene or nitrous oxide/acetylene.
• The sample is broken down into atoms and it is the
concentration of these atoms that is measured .

39. Titrimetry
• Titration or Titrimetry is a common laboratory
method of quantitative chemical analysis to
determine the concentration of an
identified analyte (a substance to be analyzed).
• A reagent, termed the titrant or titrator, is
prepared as a standard solution of known
concentration and volume.
• The titrant reacts with a solution of analyte to
determine the analyte's concentration.
• The volume of titrant that reacted with the
analyte is termed the titration volume.

40. Cont:
• A typical titration begins with a beaker or Erlenmeyer
flask containing a very precise amount of the analyte and a small
amount of indicator (phenolphthalein) placed underneath a
calibrated burette containing the titrant.
• Small volumes of the titrant are then added to the analyte and
indicator until the indicator changes color in reaction to the
titrant saturation threshold, representing arrival at
the endpoint of the titration, meaning the amount of titrant
balances the amount of analyte present.
• The concentration of the analyte solution can thus be calculated
from its volume and the volume and concentration of the titrant
at the endpoint.

41. Gravity separation
• It is an industrial method of separating two components, either a
suspension, or dry granular mixture where separating the components
have different specific weight.
• The gravity separator lifts the material by vacuum over an inclined
vibrating screen covered deck. This results in the material being
suspended in air while the heavier impurities are left behind on the
screen and are discharged from the stone outlet.
• The most notable advantages of the gravitational methods are their
cost effectiveness and in some cases excellent reduction.
• Gravity separation is an attractive unit operation as it generally has low
capital and operating costs, uses few if any chemicals that might cause
environmental concerns and the recent development of new
equipment enhances the range of separations possible.

42. Cont:
• In agriculture gravity separation are used for the removal
of impurities, admixture, insect damage and immature
kernels from the wheat, barley, oilseed rape, peas, beans,
cocoa beans, linseed.
• It can be used to separate and standardize coffee beans,
cocoa beans, peanuts, corn, peas, rice, wheat, sesame
and other food grains.
• Can also be used to remove viable or valuable
components from the recycling mixture i.e. metal from
plastic, rubber from plastic, different grades of plastic and
these valuable materials are further recycled and
reutilized.

43. Polarimeter
• Polarimeter is a scientific instrument used to
measure the angle of rotation caused by
passing polarized light through an optically
active substance.
• Some chemical substances are optically active, and
polarized (uni-directional) light will rotate either to
the left (counter-clockwise) or right (clockwise)
when passed through these substances. The
amount by which the light is rotated is known as
the angle of rotation. The angle of rotation is
basically known as observed angle.

44. Cont:
• The angle of rotation of an optically active
substance can be affected by:
• Concentration of the sample
• Wavelength of light passing through the sample
(angle of rotation and wavelength tend to be
inversely proportional)
• Temperature of the sample
• Length of the sample cell)
• Filling conditions (bubbles, temperature and
concentration gradients)

45. Cont:
• Food, beverage and pharmaceutical industries
Concentration and purity measurements are especially important
to determine product or ingredient quality in the food &
beverage and pharmaceutical industries. Samples that display
specific rotations that can be calculated for purity with a
polarimeter include: Steroids, Diuretics, Antibiotics, Narcotics,
Vitamins, Analgesics, Amino acids, Essential oils, Polymers,
Starches , Sugars etc.
• Polarimeters are used in the sugar industry for determining
quality of both juice from sugar cane and the refined sucrose.

47. Fluorometry
• Fluorometry is superior to spectrophotometry in terms of
sensitivity and specificity.
• The sensitivity of fluorescence is 10–1000-fold higher in
comparison to absorbance measurements.
• Fluorometry was introduced in immunological assays to
improve immunoassay sensitivity. The potential
sensitivity of fluorometry is that the search for single-
molecule detection has been based almost exclusively on
the use of fluorescent compounds.

48. Cont:
• Fluorometric determination could combine several
parameters simultaneously, such as excitation
and emission wavelength and polarization. All
parameters are affected by changes in
the microenvironment of the
fluorescent compound.
• Fluorometric determination frequently allows the
direct study of molecular processes, such
as antigen-antibody interaction.
• This forms the basis for the development of
homogeneous fluorescence immunoassays.