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Savitribai phule pune UNIVERSITY SEMINAAR ON
Miss. SNEHAL K. DHOBALE
M-PHARMACY – SEMESTER-1
Padm. Dr. D.Y.PATIL COLLEGE OF
PROF. M.T. MOHITE
Factors affecting RI measurment
Calibration of Refractometer
Advances in Refractometry
Refractometry is the method of measuring substances
refractive index (one of their fundamental physical
properties) for example, assess their composition or purity.
A refractometer is the instrument used to measure refractive
index ("RI"). Although refractometers are best known for
measuring liquids, for quick evaluation of concentration of
Measurement of refractive index of unknown substance.
measure substances dissolved in water and certain oils.
Measurement of RI of unknown substance by measuring angle
of refraction made when the substance is brought into contact
with the medium (prism) of a known refractive index
A refractometer measures the extent to which light is bent (i.e.
refracted) when it moves from air into a sample and is typically
used to determine the refractive index (n) of a liquid sample.
The refractive index is a unitless number, between 1.3000 and
1.7000 for most compounds
The refractive index is a quantity which is a constant for a pure
substance under standard conditions of temperature and
The ratio of the speed of light in a vacuum to the speed of light
in another substance is defined as the refractive index for the
The speed of light in a vacuum is always the same, but when
light moves through any other medium it travels more slowly
since it is constantly being absorbed and re-emitted by the
atoms in the material.
Samples with different refractive indexes will produce different
angles of refraction and this will be reflected in a change in the
position of the borderline between the light and dark regions.
REFRACTIVE INDEX (CONTI…)
Absolute index of refraction (N) is the relation of speed widening of light in the
vacuum to its speed in the present medium
Relative index of refraction (n) is the relation of speed widening of light in the
air to its speed in the present medium
In the case shown, the speed of
light in medium A is greater than
the speed of light in medium B.
The relationship between light's
speed in the two mediums (vA
and vB), the angles of incidence
( A) and refraction ( B) and the
refractive indexes of the two
mediums (nA and nB) is shown:
REFRACTIVE INDEX (conti…)
It is also commonly used to:
Help identify or confirm the identity of a sample by
comparing its refractive index to known values.
Assess the purity of a sample by comparing its refractive
index to the value for the pure substance.
Determine the concentration of a solute in a solution by
comparing the solution's refractive index to a standard curve.
TYPES OF REFRACTOMETER
IMMERSION OR DIPPING
I.) ABBE REFRACTOMETER
Light refraction through liquids to determine
the amount of dissolved solids in liquids by
passing light through a sample and showing
the refracted angle on a scale.
RI of the prism should be greater than that of
In abbe refractometer, the RI can be read
directly, only a few drop of the liquid are
needed , and either white or monochromatic
light can be used.
This refractometer consist, mainly a telescope
and two matched right angle prisms.
I.) ABBE REFRACTOMETER (conti…)
The liquid is placed in contact with the prisms.
Many refractometers are equipped with a thermometer and a means of
circulating water through the refractometer to maintain a given
Most of the refractive index measurements reported in the literature are
determined at 20 or 25 °C.
Operation consists of placing 1 or 2 drops of the water sample on the
prism, closing a glass plate over the sample, then looking through the
eyepiece for the reading
Analyzing Results Finding Refractive Indexes:
Comparison with the literature to know the identity of the
compound or to asses its purity
The following sources list refractive indexes for a wide variety of
The CRC Handbook of Chemistry and Physics
Lange's Handbook of Chemistry
The Merck Index
Chemical catalogs (e.g., the one from Aldrich
Chemical Co. ) MSDS datasheets
OPERATING THE BAUSCH & LOMB ABBE-3L REFRACTOMETER
Open the prism
remove the tissue
Use a pipette to
apply your liquid
sample to the prism.
Be careful not to let
the glass pipet tip
touch the prism
Close the prism
Turn on the lamp
using the switch on
the left side.
Adjust the lamp so
the light shines on
the prism .
AMICI PRISMS OR COMPENSATING
To prevent the chromatic
Light sources in abbe’s
i. natural light
ii. C - line of hydrogen
iii. D - line of sodium vapor
iv. F - line of hydrogen
v. G – line of mercury
Calibration of refractometer
• Make sure that you are holding the refractometer horizontally, and open the daylight
plate to expose the main prism.
• Using a plastic pipette, place two drops of distilled water on the prism.
• Close the daylight plate and press it lightly so that the water spreads across the
entire surface of the prism without any air bubbles or dry spots. Allow the sample to
remain on the prism for 30 seconds.
• Look through the eyepiece to see a circular field with graduations on either side. The upper
portion should be blue and the lower portion white. If the field is not in focus, twist the eyepiece
until the graduations are clearly distinguishable. The boundary between the blue and white
portions should fall on the zero mark of the graduations. If not, turn the calibration screw on top
of the refractometer until the boundary between the colors reaches the zero mark.
• After calibrating the refractometer, open the daylight plate and pat dry the main
II.) IMMERSION REFRACTOMETER
The immersion or dipping refractometer is basically an Abbé
type refractometer of short range and without an illuminating
The dipping refractometer is used primarily for determining
solution concentrations, such as in the sugar industry,
pharmaceuticals, and milk. It is particularly useful for acids
since only the inert prism dips into the solution.
Immersion refractometers always work at the temperature of
the liquid, so they need built in temperature compensation
II.) IMMERSION REFRACTOMETER
Similar to abbes but lacks illuminating prism. lower surface of
the refracting prism dipped into the sample solution.
Simplest design is a refractometer that you just immerse
partially into solution to make a measurement.
In most cases dip refractometer is just a liquid proof Abbé
refractometer mounted in a tube, with an additional
immersible light source, or mirror that helps direct natural light
on the measuring prism surface.
II.) IMMERSION METHOD
The immersion method relies on having immersion oils of
known refractive index and comparing the unknown mineral to
Immersion oil is available with refractive indices that cover the
range found for most minerals.
Typically the immersion oil will have indices of refraction in
the range 1.4 to 1.8 in increments of 0.002, 0.004 or 0.005.
There are two components to comparing the index of refraction
of the mineral and the immersion oil
II. Becke Line 18
Relief can be defined as the degree to which a mineral grain or
grains appear to stand out from the mounting material, whether it is
an immersion oil, Canada balsam or other medium, or another
If the indices of refraction of the
oil and mineral are the same, light
passes through the oil-mineral
boundary un-refracted and the
mineral grains do not appear to
If noil ≠ nmineral then the light
travelling though the oil-mineral
boundary is refracted and the
mineral grain appears to stand out.
2.) BECKE LINE
Movement of the Becke line as the stage is lowered. The becke line may be
considered to consist of a cone of light that extends upwards from the
If nmin < noil, the cone diverges upwards and if nmin > noil the cone
If the stage is lowered, the plane of focus goes from F1 to F2 and the Becke
Line appears to move towards the material of the higher refractive index.
III.) PULFRICH REFRACTOMETER
Pulfrich refractometer is the design
(after Abbé refractometer) that
became commercially avilable in
19th century. It is another type
of critical angle refractometer
The Pulfrich refractometer is actually
only as accurate as a standard Abbé
(one unit in the fourth decimal place)
in absolute determinations.
However, it can give measurements to
2-3 units in the fifth-place for
differential measurements and for
III.) PULFRICH REFRACTOMETER
Right angled prism attached
to a small glass block
containing the sample
light is incident at grazing
incidence(at right angle)
Angle of refraction is
measured with the help of
telescope n = RI of sample =
RI of glass block
n= RI OF SAMPLE
nG= RI OF GLASS BLOCK
Colloidal refractometry: meaning and measurement of refractive index
for dispersions. Mansur Mohammadi
This paper introduces a new way of investigating the microstructure of high phase
It is a light scattering method where, instead of scattering intensities, one obtains
refractive index, n, of a dispersion The measured values of n on undiluted
dispersions can provide a measure of the volume-weighted average particle size.
The accurate measurement of n for turbid fluids is not trivial and total internal
reflection refractometers that rely on the visual identification of the degraded cut-off,
or critical, edge of a dark-light border (or even most of modern automatic ones)
cannot cope with this task.
Here a total internal reflection refractometer is described that relies
on differentiating the light profile for accurate identification of the degraded cut-off
It can cope with a wide range of concentrations and size distributions. The sizing
results for polystyrene latices and liposome dispersions compare well with
Refractometry as a measure of the immunoglobulin status of the
newborn dairy calf: comparison with the zinc sulfate turbidity test
Naylor JM, Kronfeld DS. American Journal of Veterinary Research
Immunoglobulins were quantitated by single radial immunodiffusion in 34 female
Holstein-Friesian calves which had been kept with their dams for the first 24 hours
The mean immunoglobulin G1 (IgG1) concentration was 1.063 g/dl; IgG2, 0.093
g/dl; IgM, 0.171 g/dl; and IgA, 0.125 g/dl
Both serum total protein as measured by refractometer and zinc sulfate turbidity
measured at 498 nm gave good correlations with total protein, which were
significant, P less than 0.001.
Plasma total protein had a slightly poorer correlation with total immunoglobulins,
presumably due to variable fibrinogen content.
Plasma total protein gave a better correlation with total immunoglobulins than did
any of the immunoglobulin classes individually.
Total protein by refractometer underestimated naturally occurring or added
immunoglobulins by one-third 25
APPLICATIONS & USES
1) It is used in the examination of organic compounds (oils, solvents, etc.),
solutions, food products, serum protein concentration.
2) In veterinary medicine, a refractometer is used to measure the total
plasma protein in a blood sample and urine specific gravity.
3) In gemmology, a refractometer is used to help identify gem materials by
measuring their refractive index.
4) Since the index of refraction of a pure substance is constant at constant
temperature and pressure, it can be used as a means of identification.
5) It is used to determine the purity of oils, fats, and waxes.
6) It is used to determine the amount of sugar in sugar solutions and in
general, for determining total solids in fruit juices, tomato products,
honey, syrups and soda water
1. Gurdeep.R. Chatwal, Sham k.Anand, INSTRUMENTAL METHOD OF
CHEMICAL ANALYSIS, Himalaya publishing house, 2008, p.no.2.382-2.597.
2. Skoog, Douglas A., F. James Holler and Timothy Nieman. PRINCIPLES OF
INSTRUMENTAL ANALYSIS. Fifth Edition. New York, 1998.p.no.156-178
5. "Microscope Objectives: Immersion Media" by Mortimer Abramowitz and
Michael W. Davidson, Olympus Microscopy Resource Center (website), 2002
7. Journal of dairy science, Evaluation of the Brix refractometer to estimate
immunoglobulin G concentration in bovine colostrum . J.D. Quigley1, A. Lago2