Theme 4- REFRACTOMETRICAL METHOD OF THE DETERMINATIONS OF THE CONCENTRATIONS OF THE SOLUTIONS AND OF THE BLOOD SAMPLES

Question 1

What are refractometers used for in medicine

Answer 1

-Used to determine protein in urine, blood
serum, and other biological media.
-With the help of these devices, the sugar content in the blood and urine of patients is analyzed.

Question 2

What is the refraction of light

Answer 2

-When a light ray passes from a less dense medium to a more dense medium (e.g., from air to glass), it bends towards the normal and when it passes from a denser medium to a less dense
medium (e.g., from glass to air) it bends away from the normal.
-This phenomenon of deviation of light rays from their original path, when they pass from one medium to another, is called refraction of light

Question 3

What happens to the speed of light as it travels from one medium to another

Answer 3

-The speed of light changes.
-A ray of light from a rarer medium to a denser medium slows down and bends towards the normal.
-On the other hand the ray of light going from a denser medium to a rarer medium is sped up and bends away from the normal due to the interactions between the bound electrons of
the medium and the electric field of the radiation.

Question 4

What is the index of refraction/ refractive index

Answer 4

It is the bending ability of a material

Question 5

What is the refractive index (n) of a material

Answer 5

-It is defined as the ratio of the speed of light in vacuum to that in the material medium.
-Therefore refractive index n of a medium;
n = c / v
-Where v is the velocity of the propagation of the radiation in the medium and c is the velocity in a vacuum

Question 6

What does the extent to which a ray bends depend on

Answer 6

-The refractive index of a material
-The angle of incidence

Question 7

The 2 laws of refraction

Answer 7

-First law of refraction: The incident ray, the
refracted ray and the normal at the point of
incidence all lie in the same plane.
-Second law of refraction: This law is called as the Snell’s law of refraction

Question 8

Snell’s law of refraction

Answer 8

When medium 1 is a vacuum, n1 =1 because v1 becomes equal to c. then n2 becomes n
which is called absolute refractive index of medium 2

n1/n2 = v2/v1

Question 9

Variables that Affect Refractive Index Measurements

Answer 9

1) Temperature
2) Wavelength
3) Pressure

Question 10

How does temperature affect refractive index measurements

Answer 10

-Temperature influences the refractive index of a medium primarily because of the accompanying change in density.
-Since the density of a liquid usually decreases with temperature, it is not surprising that the speed of light in a liquid will normally increase as the temperature increases.
-Thus, the index of refraction normally decreases as the temperature increases for a liquid.

Question 11

How does wavelength affect refractive index measurements

Answer 11

-In most liquids and solids the speed of light, and hence the index of refraction, varies significantly with wavelength.
-This variation is referred to as dispersion, and it is what causes white light moving through a prism to be refracted into a rainbow.
-Shorter wavelengths are normally refracted more than longer ones.
-Thus, for the most accurate measurements it is necessary to use monochromatic light.

Question 12

How does pressure affect refractive index measurements

Answer 12

-The refractive index of a substance increases with pressure because of the accompanying rise in density.
-The effect is most pronounced in gas rather than solids and liquids.

Question 13

Instruments for Measuring Refractive Index

Answer 13

-Critical Angle Refractometers; the most widely used for the measurement of the refractive index are of the critical angle type.
-Critical Angle; critical angle is the incident angle at which light (instead of getting to the other side of phase boundary) gets refracted in such a way that it becomes parallel to the phase boundary surface. Critical angle can be easily calculated if we know refractive indices of both media

Question 14

Amici Prism

Answer 14

-It is a Tri prism constructed from different varieties of glass and is so designed that it does not deviate a ray of light corresponding to the sodium D line.
-Rays of other wavelengths are deviated, however, and by rotating the Amici prism about the axis of the instrument it is possible to counteract the dispersion of light caused by refraction at the liquid interface.
-The important feature enables white light to be used for illumination

Question 15

Abbe refractometer

Answer 15

-Abbé refractometer working principle is based on critical angle.
-Sample is put between two prisms - measuring and illuminating.
-Light enters sample from the illuminating prism, gets refracted at critical angle at the bottom surface of measuring prism, and then the telescope is used to measure position of the border between bright and light areas.
-Telescope reverts the image, so the dark area is at the bottom, even if we expect it to be in the upper part of the field of view.
-Knowing the angle and refractive index of the measuring prism it is not difficult to calculate refractive index of the sample.
-Surface of the illuminating prism is matted, so that the light enters the sample at all possible
angles, including those almost parallel to the surface.

Question 16

What Abbe did to prevent dispersion

Answer 16

-To prevent dispersion Abbé added two compensating Amici prisms into his design.
-Not only telescope position can be changed to measure the angle, also position of Amici prisms can be adjusted, to correct the dispersion.
-In effect edge of the shadow is well defined
and easy to locate.

Question 17

Human blood and its functions

Answer 17

-Human blood is a fluid connective tissue that performs multiple essential body functions, including transport, defense, and regulation.
-Blood transports gases, nutrients, proteins, and waste products.
-It defends the body against pathogens and forms clots to prevent blood loss.
-Blood also helps regulate body temperature, maintains the osmotic pressure that keeps fluid
in the intravascular space, and stabilizes the body’s acid-base balance

Question 18

Components of human blood

Answer 18

-Blood consists of approximately 45% formed elements (red blood cells, white blood cells, and platelets) by volume suspended in 55% liquid plasma.
-Plasma carries formed elements, proteins, and other substances throughout the body.
-The cardiovascular, endocrine, digestive, skeletal, and urinary systems all regulate blood composition.
-The average adult has about 5 liters of blood, or about 7-8% of body weight.

Question 19

What is serum

Answer 19

Serum is the liquid fraction of whole blood that is collected after the blood is allowed to clot.

Question 20

Separation of serum and plasma from blood

Answer 20

-The clot is removed by centrifugation and the resulting supernatant, designated serum, is
carefully removed using a Pasteur pipette.
-Plasma is produced when whole blood is collected in tubes that are treated with an anticoagulant.
-The blood does not clot in the plasma tube.
-The cells are removed by centrifugation.
-The supernatant, designated plasma is carefully
removed from the cell pellet using a Pasteur pipette

Question 21

Serum preparation

Answer 21

-Collect whole blood in a covered test tube.
-If commercially available tubes are to be used, the researcher should use the red topped tubes. -These are available from Becton Dickinson (BD).
-After collection of the whole blood, allow the blood to clot by leaving it undisturbed at room temperature.
-This usually takes 15–30 minutes.
-Remove the clot by centrifuging at 1,000–2,000 g for 10 minutes in a refrigerated centrifuge.
-The resulting supernatant is designated serum.
-Following centrifugation, it is important to
immediately transfer the liquid component (serum) into a clean polypropylene tube using a
Pasteur pipette.
-The samples should be maintained at 2–8°C while handling.
-If the serum is not analyzed immediately, the serum should be apportioned into 0.5 ml aliquots, stored, and transported at –20°C or lower.
-It is important to avoid freeze-thaw cycles because this is detrimental to many serum components.
-Samples which are hemolyzed, icteric or lipemic can invalidate certain tests

Question 22

Plasma preparation

Answer 22

-Collect whole blood into commercially available anticoagulant-treated tubes e.g., EDTA-treated (lavender tops) or citrate-treated (light blue tops).
-Heparinized tubes (green tops) are indicated for some applications; however, heparin can often be contaminated with endotoxin, which can stimulate white blood cells to release cytokines.
-Cells are removed from plasma by centrifugation for 10 minutes at 1,000–2,000 x g using a refrigerated centrifuge.
-Centrifugation for 15 minutes at 2,000 x g depletes platelets in the plasma sample.
-The resulting supernatant is designated plasma. -Following centrifugation, it is important to
immediately transfer the liquid component (plasma) into a clean polypropylene tube using a
Pasteur pipette.
-The samples should be maintained at 2–8°C while handling.
-If the plasma is not analyzed immediately, the plasma should be apportioned into 0.5 ml aliquots, stored, and transported at –20°C or lower.
-It is important to avoid freeze-thaw cycles.
-Samples which are hemolyzed, icteric, or lipemic can invalidate certain tests