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Principles of instrumentation. Photometry. Photometry means “the measurement of light” If a substance can be converted to a soluble, colored material, its concentration may be determined by the amount of color present in the solution.
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Principles of instrumentation
Photometry • Photometry means “the measurement of light” • If a substance can be converted to a soluble, colored material, its concentration may be determined by the amount of color present in the solution. • Photometer & Spectrophotometer are instruments used for this type of measutment, in which a photocell or photomultiplier tube is used to detect the amount of light that passes through a colored solution from a light source. • The greatest sensitivity is obtained when the light permitted to pass through the solution is of a particular wavelength.(The wavelength shows the maximum absorbance for the solution color).
Characteristics of Light • Light is a form of electromagnetic energy that travels in waves. • The wavelength of light is the distance between two beaks of the light wave, it is inversely proportional with its energy. • In the visible region the color of light is a function of its wave • Length increasing from violet towards the red color • Objects that appear colored absorb light at particular • Wavelength and reflect the other parts of the visible spectrum Resulting in many shades of color.
example: a substance that absorbs violet light at 400 nm reflects all other light and appears as yellow green. Spectrophotometer takes advantage of the property of colored solutions to absorb light of specific wavelength. To measure the concentration of a blue solution, light is passed through it at about 590 nm. The amount of yellow light absorbed varies directly in proportion to the concentration of the blue substances in the solution.
Table-1(wavelengths of various types of Radiation) Energy Wavelength
When the light of an appropriate wavelength strikes a cuvet that contains a colored sample, some of the light is absorbed and the rest is transmitted through the sample to the detector. % percent transmittance which represents the proportion of light reaches the detector. % T = (It \ Io) x 100 % Where: Io: is the intensity of light striking the sample. It: is the intensity of transmitted light. Beer’s law It Io
Beer’s law • If the concentration of a solution is increased, the It will decrease and then % T is decreased. • The relationship between the concentration and %T is not linear, but if the logarithm of the %T is plotted against the concentration, a straight line is obtained. -The term absorbance is used to represent – log % T A = - log % T = 1/ log % T
Then we can determine the concentration of x substance by measuring both sample and standard absorbance, which can be made by spectrophotometers.
UV – Visible photometry • Typical coloremetric instruments contain five components: • Stable source of radiation energy. • A transparent container for holding the sample. • A device that isolates a restricted region of the spectrum for measurement. • A radiation detector which converts radiant energy to electrical signals. • A signal processor and read out which displays the transudated signals, a meter scale, a digital meter or a recorder chart.
Radiation sources - In UV region: The most commonly used is deuterium lamp or hydrogen lamp. In which a continues spectrum is produced by the excitation of deuterium (D2) or hydrogen at law pressure, and then produced light with (160-375) nm. - In visible region: Tungeston filament lamp is the most commonly used and produces light at (350-2500) nm.
Note: Colorimeters Photometers - Used filters as wavelength selector • Spectrophotometer • - Used monochromators as • Wavelength selector
Sample containers: • Cuvetes that hold the samples must be made of material that passes radiation in the spectral region of intrest. • Quartz or fused silica may be used in the spectral region (350-3000 nm), mean it may be used in the UV, visible and a part of infrared. • Silicated glass used in (350- 2000 nm) region. • Plastic is used in the visible region • Radiation detectors and read out.
Hemoglobin Concentration Determination
Hemopoiesis • Is the process of blood cell formation which takes place during the embryonic life in the yolk sac; mesenchyme and blood vessels; liver; spleen, thymus and lymph nodes; bone marrow. • While in late fetus & adult takes place in bone marrow and lymphtic tissues in normal situation (medullary hemopoiesis). • In pathological conditions hemopoiesis is (extramedullary) in the liver, spleen and lymph nodes.
Hemoglobin (Hb) is the standard abbreviation for hemoglobin, the oxygen-carrying pigment and predominant protein in the red blood cells. • Hemoglobin is the protein that carries oxygen from the lungs to the tissues and carries carbon dioxide from the tissues back to the lungs. • In order to function most efficiently, hemoglobin needs to bind to oxygen tightly in the oxygen-rich atmosphere of the lungs and be able to release oxygen rapidly in the relatively oxygen-poor environment of the tissues. • It does this in a most elegant and intricately coordinated way.
Hemoglobin forms an unstable, reversible bond with oxygen. In the oxygenated state it is called oxyhemoglobin and is bright red. • In the reduced state it is called deoxyhemoglobin and is purple-blue. • A hemoglobin molecule consists of four polypeptide chains: two alpha chains, each with 141 amino acids and two beta chains, each with 146 amino acids. • The protein portion of each of these chains is called "globin". • The α and β globin chains are very similar in structure and each one of them is liked with a heme molecule.
A heme group is a flat ring molecule containing carbon, nitrogen and hydrogen atoms, with a single Fe2+ ion at the center. • Without the iron, the ring is called a porphyrin. • Changes in the amino acid sequence of these chains results in abnormal hemoglobin's. • For example, hemoglobin S is found in sickle-cell disease, a severe type of anemia in which the red cells become sickle-shaped when oxygen is in short supply.
Normal Human Hemoglobin's • Adult hemoglobin's • Hemoglobin A {2, 2} >95% • hemoglobin A2 {2, 2} <3.5% • hemoglobin F {2, 2} 1-2 % • In the very common laboratory test for hemoglobin (Hb), it is measured as total hemoglobin and the result is expressed as the amount of hemoglobin in grams (gm) per deciliter (dl) of whole blood, a deciliter being 100 milliliters. • Adult men 14-18 gm/dl • Adult women 12-16 gm/dl
polycythemia • Is Above-normal hemoglobin levels • Secondary polycythemia which is may be due to: • Dehydration (sever burns, diarrhea, vomitting, …etc.). • Severe lung or heart disease. • Living at high altitudes. • Heavy smoking. • Primary polycythemia which is due malignant variation in blood cells production in bone marrow.
anemia • Below-normal hemoglobin levels that can be the result of • Iron deficiency or deficiencies in essential vitamins of other elements, such as B12, folate, B6. • Inherited hemoglobin defects, such as sickle cell anemia or Thalassemia. • Other inherited defects affecting the red blood cells. • Excessive bleeding. • Excessive destruction of red blood cells. • Kidney disease. • Bone marrow failure or aplastic anemia. • Cancers that affect the bone marrow.
Measurement of hemoglobin • The Cyanmethemoglobin Method for Hb determination is the reference method. • Specimen • Whole blood, using EDTA as the anticoagulant. Capillary may also be used. • Principle Hb (Fe++) K3Fe (CN)6 methemoglobin (Fe+++ ) Cyanmethemoglobin KCN
Procedure Create a standard curve, using a commercially available cyanmethemoglobin standard which, has constant concentration 25g/dl,
Calculation • Test Hb concentration = Abs. of test /Abs. of standard * conc. of standard OR • Test Hb concentration = Abs. of test * factor obtained by the standard curve
Discussion • Before the test sample is read, the solution should be clear. • A high WBC count: centrifuge specimen and use the supernatant for reading. • Lipemia can also interfere, and a false result can be corrected by adding 0.02 ml of the patient’s plasma to 5 ml of the cyanmethemoglobin reagent, this solution being used as the reagent blank. • Carboxyhemoglobin takes up to 1 hr to convert to cyanmethemoglobin and therefore, theoretically could cause erroneous results in the samples from heavy smokers. However the degree of error is probably not clinically significant.
