Food Analysis-5

Density and Sp Gravity

Density: The density, , is elementary physical property of matter.
 = m / V (the ratio of its mass m to its volume V), SI, Kg/m3
It is commonly used as
 a means of categorizing
 identifying different materials.
From qualitative point of view, Density defined as the measure of
the relative "heaviness" of objects with a constant volume.
It also refers how closely packed or crowded the materials.
Density increases with increasing temperature
Exception is water in temperature range between 0-40C
 Specific gravity
 Relative density: is the ratio of the density (mass of
a unit volume) of a substance to the density of a
given reference material
 Specific gravity usually means relative density
with respect to water.
 i.e., the ratio of the density of a substance to the density
of water.
Relative density = s / H2O
Relative density is preferred in scientific usage whereas Sp
gravity is common in industry.
Measurement of density/Sp gravity
 M/V if the geometry is high
 Archimedes wet/dry
 Sink float
 Pycnometry
 Density gradient
 Archimedes wet/dry
 An object of density, ρ, in a fluid has a buoyant force, B,
equal to the weight of the displaced fluid.
 Define:

Wa = Apparent weight in fluid of density ρo

W = Weight of object determined in air
Then Wa = W – ρoVg
and Wa = W – ρo* (W/ρg)*g
ρ = ρo* (W/W-Wa)
Density=density of water*wt of
object/apparent wt
 Sink Float
 Density is determined by floating the flakes in
calibrated miscible fluids
 Acetone;0.78 g/cc
 Diiodomethane; 3.32 g/cc

 Not suitable of samples having higher density

 Temperature is very crucial
 Small amount sample is enough
 Pycnometry
 There are two calibrated volumes:
the reference (Vr) and cell (Vc)
volumes.
 Sample volume is defined by Vs.
 Uses to determine both liquid and
solid.
 Temperature of 20-25° C is
crucial.
 Refractometer

Air Air
Light Sample Light

Angle to which light is reflected or bend

Typically used to determine angle of reflection

Angle of reflection Reflective index (RI), n

RI is unit less and varies between 1.3 to 1.7 for most compounds
Temperature

RI Na D line at 589nm
 Refractive Index (RI)
 RI is commonly determined as part of the
characterization of liquid samples
 It helps to identify or confirm the identity of a
sample
 It helps to assess purity of a sample
 It helps to determine the concentration of solutes in
a solution
 Principles of Refractometer

 A refractometer consists of a light source, filtered to a single wavelength,

which is directed towards the prism-sample interface by a converging lens.
This creates a range of incidence angles, some of which (those less than
the critical angle) will be completely reflected.
 A Charge-coupled Device (CCD) sensor precisely measures the intensity
of the reflected light and determines the exact angle α critical at which
light begins to be completely reflected. Because this angle is dependent on
the ratio of the refractive index of the prism to that of the sample, the
refractive index of the sample can be determined using the known
refractive index of the prism.
 Sample to be measured is in direct contact with the measuring prism. The
incoming light of angles less than the critical angle of total reflection is
partly refracted through the sample, while incoming light of angles greater
than the critical angle is totally reflected. A high-resolution sensor array
measures the amount of this reflected light.
 Principles of Refractometer

RI of unknown substance can be determined if

❖1. RI of prism is known
❖2. Angle of incidence and angle of reflection
are known

Nearly all refractometer utilize this principle, but may differ in their optical design
 Principles of Refractometer

Speed of light in vacuum is max

What will be in compressed gas?

Snell’s law:
Light refraction - incident (α) and
refracted (β) rays.

Light refraction - the shortest path

and the shortest travel path
Factors affecting RI measurement
 Temperature of the liquid
 Inverse relation
 For organic liquid, for every 10°C RI decreases 0.00045 &
0.0001 for water
 Wavelength of radiation source
 Inverse relation
 RI of transparent medium decreases with increase in
wavelength
 Shorter wavelength are reflected more than the longer

 Pressure
 Direct relation
 Types of refractometer

 Two types of instruments are used to determined RI

 1. Refractometer
◼ Abbe’s refractometer
◼ Pulfrich refractometer
◼ Immersion/dipping refractometer

 2. Interferometers (Differential refractometer)

◼ Deflection refractometer
◼ Reflection refractometer
 Abbe’s refractometer

1869 Ernst Abbe’s designed the first refractometer

1881 Carl Zeiss make it commercially available

1893 Temperature jacketed prism

Until Modification is still continuing but principles is the same.

Currently digital refractometer is available

Works on the principle of critical angle

 Abbe’s refractometer

Works based on the principles of critical angles

• Sample is sandwich betn IP & RP
•Light is projected through IP
•Bottom surface of IP is ground

RP is made of glass with the highest

RI (1.75) and sample should be lower .
Anatomy of a refractometer
Hand Refractometer

• HR is most popular, simple,

instant, less tedious and low
cost
• IP is replaced with
illuminating flap
•Light through sample enter
measuring prism & falls on
scale
• Its scale graduated as
Brix or %
Digital Refractometer
 Concentration of solution

In most cases the refractive index is

linearly (or nearly linearly) related to
the percentage of dissolved solids in a
solution (Figure 2). By comparing the
value of the refractive index of a
solution to that of a standard curve the
concentration of solute can be
determined with good accuracy. Many
refractometers contain a "Brix" scale
that is calibrated to give the
percentage (w/w) of sucrose dissolved
Brix scale: Brix scale is defined as the
in water.
number of grams of pure cane sugar
dissolved in 100grams of pure water grams
sugar/100g water
 Brix
°Brix is the ratio of Total Soluble Solids (TSS) to water in solution
25° Bx = 25% TSS by weight (e.g. 25 grams of solids to 75 grams of water)

 Higher Brix – higher nutrient density (assumption)

 Higher Brix – better taste (widely acknowledged)
 Higher Brix – resistance to rotting
 Higher Brix – resistance to disease
 Higher Brix – resistance to frost
 Higher Brix – Higher Quality!
Brix chart
Brix chart
Brix chart
 Application
 Refractometer for sugar milling,  Flavor, Fragrance and Cosmetic
refining and processing  Perfumes
 Cane sugar milling and refining  Skin cleaners
 Beet sugar milling and refining  Lotions
 Invert sugar  Lemon, limes, orange

 Liquid sugar  Palm

 Creams
 Confectionery sugar
 Waxes
 Molasses
 Natural oil
 Brown sugar
 Sandalwood
 Petroleum  Chemical Industry
 Waxes  Resins
 Fuels  Polymers
 Oils  Coolants
 Lubricants  Gels
 POLARIMETRY
Principle:
 Polarimetry measures the rotation of polarized light as it passes through an
optically active fluid. The measured rotation can be used to calculate the
value of solution concentrations; especially substances such as sugars,
peptides and volatile oils.
 POLARIMETRY
Technique:
 Polarimeters measure this by passing monochromatic light through
the first of two polarizing plates, creating a polarized beam.
 This first plate is known as the polarizer. This beam is then rotated
as it passes through the sample.
 After passing through the sample, a second polarizer, known as the
analyzer, rotates either via manual rotation or automatic detection of
the angle.
 When the analyzer is rotated such that all the light or no light can
pass through, then one can find the angle of rotation which is equal
to the angle by which the analyzer was rotated(θ) in the former case
or (90-θ) in the latter case.
 POLARIMETRY
Application:
 Research applications for polarimetry are found in industry, research institutes and
universities as a means of:
➢ Isolating and identifying unknowns crystallized from various solvents or separated by
high performance liquid chromatography (HPLC).
➢ Evaluating and characterizing optically active compounds by measuring their specific
rotation and comparing this value with the theoretical values found in literature.
➢ Investigating kinetic reactions by measuring optical rotation as a function of time.
➢ Monitoring changes in concentration of an optically active component in a reaction
mixture, as in enzymatic cleavage.
➢ Analyzing molecular structure by plotting optical rotatory dispersion curves over a
wide range of wavelengths.
➢ Distinguishing between optical isomers.
➢ In each of these applications, the AUTOPOL Polarimeter offers up to six discrete
wavelength selections to observe the effect of wavelength upon an optically active
substance.
 POLARIMETRY
 Using Polarimetry in Quality and Process Control Applications:
➢ Quality and process control applications, both in the laboratory or on-
line in the factory, are found throughout the pharmaceutical, essential
oil, flavor, food and chemical industries. A few examples are listed
below.
❖ Pharmaceutical Industry
✓ Polarimetery determines product purity by measuring specific rotation
and optical rotation of: Amino Acids,Antibiotics, Dextrose, Steroids,
Amino Sugars, Cocaine, Diuretics, Tranquilizers, Analgesics, Codeine,
Serums, Vitamins
❖ Polarimetry for Flavor, Fragrance, and Essential Oil Industry
✓ Utilizes polarimetry for incoming raw materials inspection of:
Camphors, Gums, Orange oil, Citric acid, Lavender oil, Spearmint oil,
Glygeric acid, Lemon oil
 POLARIMETRY
❖ Polarimetery for Food Industry Applications
✓ Polarimetery – Ensures product quality by measuring the concentration
and purity of the following compounds in sugar based foods, cereals and
syrups: Carbohydrates, Lactose, Raffinose, Various starches, Fructose,
Levulose, Sucrose, Natural monosaccharides, Glucose, Maltrose, Xylose
❖ Using Polarimetery in the Chemical Industry
✓ Polarimetery – Analyzes optical rotation as a means of identifying and
characterizing: Biopolymers, Natural polymer, Synthetic polymers
❖ Polarimetry of sugar solutions
✓ Polarimetry is frequently used for determining the quality of sugar
products. Measurements are made by polarimeters or saccharimeters with
the scale in angle degrees (0) and sugar degrees (0Z). Angle of rotation
depends linearly on concentration of sugar in the solution other
parameters (temperature, light source, length of the tube) being the same.
 DENSITOMETRY
 Densitometry is the quantitative measurement of optical density in
light-sensitive materials, such as photographic paper or photographic
film, due to exposure to light.
Principle:
 Optical density is a result of the darkness of a developed picture and
can be expressed absolutely as the number of dark spots (i.e., silver
grains in developed films) in a given area, but usually it is a relative
value, expressed in a scale.
 DENSITOMETRY
Types of densitometry:
 Single-line densitometry
 Multiple energy densitometry
 Absorption-edge densitometry
Applications:
 spot densitometry: the value of light absorption is measured at a single spot
 line densitometry: the values of successive spots along a dimension are
expressed as a graph
 bi-dimensional densitometry: the values of light absorption are expressed as
a 2D synthetic image, usually using false-color shading. Dual-energy X-ray
absorptiometry is used in medicine to evaluate calcium bone density, which is
altered in several diseases such as osteopenia and osteoporosis. Special
devices have been developed and are in current use for clinical diagnosis,
called bone densitometers.
 SPECTROPHOTOMETER
 A device that measures the absorption of light by a sample.
 It includes:
➢ Light
➢ Source
➢ Wavelength selector
➢ Electrical means of detecting light.
Types:
1. UV spectrophotometer
2. Visible spectrophotometer
3. IR spectrophotometer
4. Atomic absorption spectrophotometer
 SPECTROPHOTOMETER
Absorption process:
 Each different atomic and hence molecular structure of a
chemical substance has unique resonant frequencies.
 When the frequency of the incident radiation on a substance
coincides with this resonant frequency or energy level, its
radiant energy is imparted to the substance.
 This phenomenon of energy transfer is referred to as
absorption.
 The amount of radiant energy absorbed by a sample at a
certain wavelength depends upon the concentration of the
sample.
 SPECTROPHOTOMETER
 There are two major classes of spectrophotometers
1. Single beam and
2. Double beam
Single beam:
 A single beam spectrophotometer measures the relative light intensity of the
beam before and after a test sample is inserted.
 Although comparison measurements from double beam instruments are
easier and more stable, single beam instruments can have a larger dynamic
range.
 Optically simpler and more compact.
Double beam:
 A double beam spectrophotometer compares e light intensity between two
light paths reference sample and test sample
 ATOMIC ABSORPTION
SPECTROPHOTOMETER
Principle:
 In atomic absorption (AA) spectrometry, light of a specific
wavelength is passed through the atomic vapor of an element of
interest, and measurement is made of the attenuation of the intensity
of the light as a result of absorption.
 ATOMIC ABSORPTION
SPECTROPHOTOMETER
APPLICATIONS:
 Atomic absorption spectrometry has many uses in different areas of chemistry.
1. Clinical analysis: Analysing metals in biological fluids such as blood and urine.
2. Environmental analysis: Monitoring our environment – eg finding out the levels
of various elements in rivers, seawater, drinking water, air, petrol and drinks such
as wine, beer and fruit drinks.
3. Pharmaceuticals: In some pharmaceutical manufacturing processes, minute
quantities of a catalyst used in the process (usually a metal) are sometimes present
in the final product. By using AAS the amount of catalyst present can be
determined.
4. Industry: Many raw materials are examined and AAS is widely used to check that
the major elements are present and that toxic impurities are lower than specified –
eg in concrete, where calcium is a major constituent, the lead level should be low
because it is toxic.
5. Mining: By using AAS the amount of metals such as gold in rocks can be
determined to see whether it is worth mining the rocks to extract the gold.
 ATOMIC ABSORPTION
SPECTROPHOTOMETER
 ADVANTAGES  DISADVANTAGES
➢ The atomic absorption ➢ AA spectroscopy is highly
technique is specific because specific. Each element has to
the atoms of a particular be tested separately.
element can only absorb ➢ The samples and standards
radiation of their own have to be in solution, or at
characteristic wavelength. least volatile.
➢ It is independent of flame ➢ A large number of
temperature. interferences are possible,
➢ High sample throughput such as the formation of non-
➢ Easy to use volatile compounds, smoke
formation which will absorb
➢ High precision
light, contamination etc.
➢ Inexpensive technique
 Ultraviolet-visible Spectrophotometry
 Involves the spectroscopy of photons in the UV-visible region.
 It uses light in the visible and adjacent (near ultraviolet (UV) and near
infrared (NIR)) ranges.
 UV radiation is often used in visible spectrophotometry to determine the
existence of fluorescence in a given sample.
UV and Visible spectrophotometers:
 The most common spectrophotometers are used in the UV and visible
regions of the spectrum.
 Some of these instruments also operate into the near-infrared region as
well.
 Visible region 400-700 nm spectrophotometry is used extensively in
colorimetry science.
 Ink manufacturers, printing companies, textiles vendors, and many more,
need the data provided through colorimetry.
 IR Spectrophotometry
 Spectrophotometers designed for the main infrared region are quite different
because of the technical requirements of measurement in that region.
 One major factor is the type of photosensors that are available for different
spectral regions.
 But infrared measurement is also challenging because virtually everything
emits IR light as thermal radiation, especially at wavelengths beyond about 5
μm.
 Another complication is that quite a few materials such as glass and plastic
absorb infrared light, making it incompatible as an optical medium.
 Ideal optical materials are salts, which do not absorb strongly.
 Samples for IR spectrophotometry may be smeared between two discs of
potassium bromide or ground with potassium bromide and pressed into a
pellet.
 Where aqueous solutions are to be measured, insoluble silver chloride is used
to construct the cell.
 IR Spectrophotometry
The sequence of events in a spectrophotometer:
 The light source shines into a monochromator.
 A particular output wavelength is selected and beamed at the sample.
 The sample absorbs light.
 The photodetector behind the sample responds to the light stimulus and
outputs an analog electronic current which is converted to a usable format.
Applications:
 The most common application of spectrophotometers is the measurement
of light absorption.
 The use of spectrophotometers is not limited to studies in physics.
 They are also commonly used in other scientific fields such as chemistry,
biochemistry, and molecular biology.
 They are widely used in many industries including printing and forensic
examination.
 COLORIMETRY
 A filter isolates a rather broad band of wavelengths at which the
substances to be estimated absorb maximally.
Principle:
 The isolated beam of light then passes through the sample placed in a
cuvette of fixed path length where a part of the incident energy is
absorbed so that the transmitted beam of light is reduced in intensity
in proportion to the concentration of the sample in solution.
 The transmitted beam of light then falls on a photocell or a
phototube, which converts the light signal into an electrical signal.
 This is finally read out as transmittance or optical density on a
suitable meter.
 COLORIMETRY
Development of color:
 Development of color is linked to the concentration of a
substance in solution.
 Concentration can be measured by determining the extent of

absorption of light at the appropriate wavelength.

 COLORIMETRY

Applications:
 Used in determination of amount of many substances in blood,
urine and saliva. Example,
1. Determination of blood glucose
2. Blood urea
3. Serum creatinine
4. Serum proteins
5. Serum cholesterol
6. Urine creatinine
7. Serum inorganic phosphate
 Color Analysis
 Even if two colors look the same to one person, slight differences may
be found when evaluated with a color measurement instrument. If the
color of a sample does not match the standard, customer satisfaction is
compromised and the amount of rework and costs increase. Because of
this, identifying color differences between a sample and the standard as
early in the production process as possible is important.
 Color difference can be defined as the numerical comparison of a
sample’s color to the standard. It indicates the differences in absolute
color coordinates and is referred to as Delta (Δ). These formulas
calculate the difference between two colors to identify inconsistencies
and help users control the color of their products more effectively.
 To begin, the sample color and the standard color should be measured
and the values for each measurement saved. The color differences
between the sample and standard are calculated using the resulting
colorimetric values.
 Identifying Color Differences Using CIE L*a*b*
Coordinates
 The L*a*b* color space was modeled after a color-opponent theory stating that
two colors cannot be red and green at the same time or yellow and blue at the
same time. As shown below, L* indicates lightness, a* is the red/green
coordinate, and b* is the yellow/blue coordinate. Deltas for L* (ΔL*), a* (Δa*)
and b* (Δb*) may be positive (+) or negative ( -). The total difference, Delta E
(ΔE*), however, is always positive.
 ΔL* (L* sample minus L* standard) = difference in lightness and darkness (+ =
lighter, – = darker)
 Δa* (a* sample minus a* standard) = difference in red and green (+ = redder, –
= greener)
 Δb* (b* sample minus b* standard) = difference in yellow and blue (+ =
yellower, – = bluer)
 Identifying Color Differences Using CIE L*a*b*
Coordinates
 Looking at the L*a*b* values for each apple in Figure, we can objectively
determine that the apples don’t match in color. These values tell us that Apple
2 (sample) is lighter, less red, and more yellow in color than Apple 1
(standard). If we put the values of ΔL*=+4.03, Δa*=-3.05, and Δb*=+1.04 into
the color difference equation, it can be determined that the total color
difference between the two apples is 5.16.
5.16 = [4.03^2 + -3.05^2 + 1.04^2] ^1/2
 Identifying Color Differences Using CIE L*C*H*
Coordinates
 The L*C*h color space is similar to L*a*b*, but it describes color
differently using cylindrical coordinates instead of rectangular
coordinates. In this color space, L* indicates lightness, C* represents
chroma, and h is the hue angle. Chroma and hue are calculated from the
a* and b* coordinates in L*a*b*. Deltas for lightness (ΔL*), chroma
(ΔC*), and hue (ΔH*) may be positive (+) or negative ( -). These are
expressed as:
 ΔL* (L* sample minus L* standard) = difference in lightness and
darkness (+ = lighter, – = darker)
 ΔC* (C* sample minus C* standard) = difference in chroma (+ =
brighter, – = duller)
 ΔH* (H* sample minus H* standard) = difference in hue
Identifying Color Differences Using CIE L*C*H*
Coordinates
 Identifying Color Differences Using CIE L*C*H*
Coordinates
 Looking at the L*C*h values for each apple in Figure, we can objectively
determine that the apples don’t match in color. Like the L*a*b* values, these
values tell us that Apple 2 (sample) is lighter and duller in appearance than
Apple 1 (standard). The positive ΔH* value of +1.92 indicates Apple 2 falls
counterclockwise to Apple 1 in the L*C*h color space. This tells us that Apple
2 is less red than Apple 1.
 ELECTROPHORESIS

 Electrophoresis is the study of the movement of charged molecules

in an electric field.
Principle:
 The charged biomolecules are separated according to their mass or
charge by the use of an electric field.
Types:
➢ Paper Electrophoresis
➢ Agarose Gel Electrophoresis
➢ Polyacrylamide Gel Electrophoresis
➢ SDS-PAGE
➢ Horizontal Electrophoresis
➢ Vertical Electrophoresis
 Gel Electrophoresis
Principle:
 •Gel electrophoresis involves use of a “gel” or “gelatinous material” which is
electrically transparent and acts as a porous sieve and thus forming a suitable
support medium for electrophoresis of charged molecules.
 Separation is based on size, shape and charge.
 Small sized molecules with net high charge will move faster and greater distance
in the gel towards the electrodes than large sized charged molecules. A suitable
staining dye is used to locate the position of separated molecules across the gel.
 Gel Electrophoresis
SEPERATION:
 By placing molecules in wells in gel and applying an electric
current, molecules will move through matrix at different rates
➢ Usually determined by mass
➢ Toward the positive anode if negatively charged
➢ Toward the negative cathode if positively charged
Applications:
➢ Estimation of DNA molecule
➢ Analysis of PCR product
➢ Determining molecular weight of protein
➢ Diagnose various diseases of kidney, liver and CVS
➢ Separation of organic acids, alkaloids, carbohydrates, amino acids,
alcohols etc.
Chromatography
 Chromatography
Chromatography is a technique for separating
mixtures into their components in order to analyze,
identify, purify, and/or quantify the mixture or
components.

• Analyze
Separate
• Identify
• Purify

Mixture Components • Quantify

 Uses for Chromatography
 Chromatography is used by scientists to:

• Analyze – examine a mixture, its components, and their

relations to one another
• Identify – determine the identity of a mixture or
components based on known components
• Purify – separate components in order to isolate one of
interest for further study

• Quantify – determine the amount of the a mixture

and/or the components present in the sample
 Uses for Chromatography
 Real-life examples of uses for chromatography:
Pharmaceutical Company – determine amount of each chemical
found in new product
Hospital – detect blood or alcohol levels in a patient’s blood
stream
Law Enforcement – to compare a sample found at a crime scene to
samples from suspects

Environmental Agency – determine the level of pollutants in the

water supply

Manufacturing Plant – to purify a chemical needed to make a

product
Chromatography- Principle
 Few Terminology
Detailed Definition:
Chromatography is a laboratory technique that
separates components within a mixture by using the
differential affinities of the components for a mobile
medium and for a stationary adsorbing medium through
which they pass.

Terminology:
• Differential – showing a difference, distinctive
• Affinity – natural attraction or force between things
• Mobile Medium – gas or liquid that carries the components
(mobile phase)
• Stationary Medium – the part of the apparatus that does
not move with the sample (stationary phase)
Chromatography- Classification
 Chromatography- Classification
 There are two classification schemes:
 Mobile phase
◼ gas (GC)
◼ Gas-solid
◼ Gas-liquid
◼ water (LC)
◼ organic solvent (LC)
◼ supercritical fluid (SCFC)

 Attractive forces
Classification based on Attractive Forces
 Adsorption - for polar non-ionic compounds

 Ion Exchange - for ionic compounds

 Anion - analyte is anion; bonded phase has positive charge
 Cation – analyte is cation; bonded phase has negative charge

 Partition - based on the relative solubility of analyte in mobile

and stationary phases
 Normal – analyte is nonpolar organic; stationary phase MORE polar
than the mobile phase
 Reverse – analyte is polar organic; stationary phase LESS polar than the
mobile phase

 Size Exclusion - stationary phase is a porous matrix; sieving

Partition Chromatography
Paper chromatography
Paper chromatography- Procedure
Paper chromatography
Ascending and Descending Paper
Chromatography
Paper Chromatography -Analysis
Two dimensional chromatography
Significance of Paper chromatography
Thin layer chromatography (TLC)
Thin layer chromatography- Procedure
Thin layer chromatography- Procedure
Thin layer chromatography- Advantages
Adsorption chromatography
Adsorption chromatography
Ion – Exchange chromatography
Ion – Exchange chromatography
Ion – Exchange chromatography-Procedure
Types of ion exchanges resins
Ion – Exchange chromatography
Gel filtration chromatography or Molecular
Sieve chromatography
Gel filtration chromatography or Molecular
Sieve chromatography
Gel filtration chromatography or Molecular
Sieve chromatography
Affinity chromatography
Affinity chromatography- Significance
Affinity chromatography- Significance
High performance liquid chromatography
High performance liquid chromatography
High performance liquid chromatography
 High pressure liquid chromatography is a liquid chromatographic
technique in which the solution is pumped through the column at pressures
up to and sometimes exceeding 4000 psi inlet pressure.
Principle:
 The Stationary Phase is a finely divided solid held inside the column. It
may be a solid orliquid, but must have a large surface area. High
performance liquid chromatography is basically a highly improved form of
column chromatography.
 Instead of a solvent being allowed to drip through a column under gravity,
it is forced through, under high pressures of upto 4000 atmospheres, that
makes it much faster.
 It also allows to use a very much smaller particle size for the column
packing material which gives a much greater surface area for interactions
between the stationary phase and the molecules flowing past it.
 This allows a much better separation of the components of the mixture.
High performance liquid chromatography
High performance liquid chromatography

Applications:
 To study metabolic pathways, reproductive mechanisms and
drug metabolism.
 It is useful in kinetic chemotherapeutic studies.

 Useful to spot metabolic or genetic defects.

 Used to study adulteration, contamination and decomposition

of foods.
 Essential oils, terpines, and terpine derivatives can all be

analyzed by HPLC.
 Can handle the separation of alkaloids and vitamins,

especially the water-soluble ones, better than gas

chromatography.
 High performance liquid chromatography

❖ Detectors:
 UV-vis
 Refractive Index (RI)
 Mass spectrometry (MS)
 Electrochemical (EC)
 amperometric

 NMR - novel
High performance liquid chromatography

Chromatogram - Detector signal vs.

retention time or volume

Detector Signal 1 2

time or volume
Gas liquid chromatography-
Gas liquid chromatography
 Instrumentation for GLC

 Carrier gas Oven

 N2, He, H2
 Injector
 Column
 Detector
 Computer
 Gas liquid chromatography

 Capillary (open tubular)

 Inner wall modified with thin (1 m) film of liquid
 0.3 - 0.5 mm ID; 10 - 50 m length

 Packed
 Solid particles either porous or non-porous coated
with thin (1 m) film of liquid
 1 - 8 mm ID; 1 - 10 m length
Gas liquid chromatography-
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APPLICATIONS OF BIOSENSORS
 In food industry, biosensors are used to monitor the freshness of food.
 Drug discovery and evaluation of biological activity of new compounds.
 Potentiometric biosensors are intended primarily for monitoring levels of
carbon dioxide, ammonia, and other gases dissolved in blood and other liquids.
 Environmental applications e.g. the detection of pesticides and river water
contaminants.
 Determination of drug residues in food, such as antibiotics and growth
promoters.
 Glucose monitoring in diabetes patients.
 Analytical measurement of folic acid, biotin, vitamin B12 and pantothenic
acid.
 Enzyme-based biosensors are used for continuous monitoring of compounds
such as methanol, acetonitrile, phenolics in process streams, effluents and
groundwater.
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Thanks