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Savitribai phule pune UNIVERSITY SEMINAAR ON 
Miss. SNEHAL K. DHOBALE 
M-PHARMACY – SEMESTER-1 
YEAR 2014-15 
Padm. Dr. D....
CONTENT 
Refractometry 
Principle 
Refractometer 
Refractive index 
Instrumentation 
Factors affecting RI measurment...
REFRACTOMETRY 
 Refractometry is the method of measuring substances 
refractive index (one of their fundamental physical ...
PRINCIPLE 
Measurement of RI of unknown substance by measuring angle 
of refraction made when the substance is brought int...
REFRACTOMETER 
A refractometer measures the extent to which light is bent (i.e. 
refracted) when it moves from air into a...
REFRACTIVE INDEX 
 The ratio of the speed of light in a vacuum to the speed of light 
in another substance is defined as ...
REFRACTIVE INDEX (CONTI…) 
Absolute index of refraction (N) is the relation of speed widening of light in the 
vacuum to ...
REFRACTIVE INDEX (conti…) 
It is also commonly used to: 
 Help identify or confirm the identity of a sample by 
comparing...
INSTRUMENTATION 
TYPES OF REFRACTOMETER 
ABBE’sREFRACTOMETER 
IMMERSION OR DIPPING 
REFRACTOMETER 
PULFRICH REFRACTOMETER ...
I.) ABBE REFRACTOMETER 
 Light refraction through liquids to determine 
the amount of dissolved solids in liquids by 
pas...
I.) ABBE REFRACTOMETER (conti…) 
 The liquid is placed in contact with the prisms. 
 Many refractometers are equipped wi...
Analyzing Results Finding Refractive Indexes: 
Comparison with the literature to know the identity of the 
compound or to...
OPERATING THE BAUSCH & LOMB ABBE-3L REFRACTOMETER 
Open the prism 
assembly and 
remove the tissue 
Use a pipette to 
appl...
AMICI PRISMS OR COMPENSATING 
To prevent the chromatic 
aberration 
Light sources in abbe’s 
refractometer : 
i. natural l...
Calibration of refractometer 
1. 
• Make sure that you are holding the refractometer horizontally, and open the daylight 
...
II.) IMMERSION REFRACTOMETER 
 The immersion or dipping refractometer is basically an Abbé 
type refractometer of short r...
II.) IMMERSION REFRACTOMETER 
 Similar to abbes but lacks illuminating prism. lower surface of 
the refracting prism dipp...
II.) IMMERSION METHOD 
 The immersion method relies on having immersion oils of 
known refractive index and comparing the...
1.) Relief 
Relief can be defined as the degree to which a mineral grain or 
grains appear to stand out from the mounting ...
2.) BECKE LINE 
 Movement of the Becke line as the stage is lowered. The becke line may be 
considered to consist of a co...
III.) PULFRICH REFRACTOMETER 
 Pulfrich refractometer is the design 
(after Abbé refractometer) that 
became commercially...
III.) PULFRICH REFRACTOMETER 
Right angled prism attached 
to a small glass block 
containing the sample 
light is incid...
23 
ADVANCES IN REFRACTOMETRY
Colloidal refractometry: meaning and measurement of refractive index 
for dispersions. Mansur Mohammadi 
Abstract 
 This ...
Refractometry as a measure of the immunoglobulin status of the 
newborn dairy calf: comparison with the zinc sulfate turbi...
APPLICATIONS & USES 
1) It is used in the examination of organic compounds (oils, solvents, etc.), 
solutions, food produc...
REFERENCES 
1. Gurdeep.R. Chatwal, Sham k.Anand, INSTRUMENTAL METHOD OF 
CHEMICAL ANALYSIS, Himalaya publishing house, 200...
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Snehal refractometry

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Snehal refractometry

  1. 1. Savitribai phule pune UNIVERSITY SEMINAAR ON Miss. SNEHAL K. DHOBALE M-PHARMACY – SEMESTER-1 YEAR 2014-15 Padm. Dr. D.Y.PATIL COLLEGE OF PHARMACY,AKURDI, PUNE. GUIDED BY, PROF. M.T. MOHITE
  2. 2. CONTENT Refractometry Principle Refractometer Refractive index Instrumentation Factors affecting RI measurment Calibration of Refractometer Advances in Refractometry Uses 2
  3. 3. 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 dissolved substances.  Measurement of refractive index of unknown substance. measure substances dissolved in water and certain oils. 3
  4. 4. PRINCIPLE 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 4
  5. 5. REFRACTOMETER 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 pressure. 5
  6. 6. REFRACTIVE INDEX  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 substance  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. 6
  7. 7. 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: 7
  8. 8. 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. 8
  9. 9. INSTRUMENTATION TYPES OF REFRACTOMETER ABBE’sREFRACTOMETER IMMERSION OR DIPPING REFRACTOMETER PULFRICH REFRACTOMETER 9
  10. 10. 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 the sample  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. 10
  11. 11. 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 temperature.  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 11
  12. 12. 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 substances: 12 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
  13. 13. OPERATING THE BAUSCH & LOMB ABBE-3L REFRACTOMETER Open the prism assembly and 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 assembly. Turn on the lamp using the switch on the left side. Adjust the lamp so the light shines on the prism . 13
  14. 14. AMICI PRISMS OR COMPENSATING To prevent the chromatic aberration Light sources in abbe’s refractometer : i. natural light ii. C - line of hydrogen lamp(653.6nm) iii. D - line of sodium vapor lamp(589.3nm) – iv. F - line of hydrogen lamp(486.1nm) v. G – line of mercury vapor lamp(435.8nm) PRISMS 14
  15. 15. Calibration of refractometer 1. • Make sure that you are holding the refractometer horizontally, and open the daylight plate to expose the main prism. 2. • Using a plastic pipette, place two drops of distilled water on the prism. 3. • 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. 4. • 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. 5. • After calibrating the refractometer, open the daylight plate and pat dry the main prism.. 15
  16. 16. II.) IMMERSION REFRACTOMETER  The immersion or dipping refractometer is basically an Abbé type refractometer of short range and without an illuminating prism.  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 16
  17. 17. 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. 17
  18. 18. II.) IMMERSION METHOD  The immersion method relies on having immersion oils of known refractive index and comparing the unknown mineral to the oil.  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 I. Relief II. Becke Line 18
  19. 19. 1.) Relief 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 mineral. 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 stand out. If noil ≠ nmineral then the light travelling though the oil-mineral boundary is refracted and the mineral grain appears to stand out. 19
  20. 20. 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 mineral grain.  If nmin < noil, the cone diverges upwards and if nmin > noil the cone converges upwards.  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. 20
  21. 21. 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 dispersion determinations. 21
  22. 22. 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 22
  23. 23. 23 ADVANCES IN REFRACTOMETRY
  24. 24. Colloidal refractometry: meaning and measurement of refractive index for dispersions. Mansur Mohammadi Abstract  This paper introduces a new way of investigating the microstructure of high phase volume dispersions.  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 edge.  It can cope with a wide range of concentrations and size distributions. The sizing results for polystyrene latices and liposome dispersions compare well with conventional methods. 24
  25. 25. 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 of life.  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
  26. 26. 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 26
  27. 27. REFERENCES 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 3. http://www.refractometer.pl/hand-held-refractometer 4. www.refractometer.pl/ 5. "Microscope Objectives: Immersion Media" by Mortimer Abramowitz and Michael W. Davidson, Olympus Microscopy Resource Center (website), 2002 6. www.colorado.edu/physics/phys1230/phys1230.../6 7. Journal of dairy science, Evaluation of the Brix refractometer to estimate immunoglobulin G concentration in bovine colostrum . J.D. Quigley1, A. Lago2 27
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