Principles of Photometry, Laws of Photometry, its Importance - Quantification of Biomolecules in Micro Concentration. Principles Used in Determining Concentration of Molecules with no Known Weight - Preparation of Standard Graph.

UNIT IV

Principles of photometry, Laws of photometry, its importance - quantification of biomolecules in micro concentration. Principles used in determining the concentration of molecules with no known weight - preparation of standard graph.

Definitions

1. Photometry: “The measurement of light brightness to the human eye is called photometry”. OR “Photometry is a measurement of light transmitting power of a solution to determine the concentration of light absorbing materials in the solution.

”Photometry is a science to measure visible light. The basic unit of measurement used in photometry is the lumen.

2. Photometer: A photometer is an instrument to measure the intensity of electromagnetic radiation of visible light.

Sunlight has three types of radiation. These are UV light, visible light, and IR light. The wavelength of visible light is between 380 nm to 760nm, the wavelength of ultraviolet light (UV light) below 380 nm, and the wavelength of infrared violet light (IR light) above 760 nm.

3. Monochromatic light: A light with a single wavelength is called monochromatic light.

4. Photometric units: Photometry uses standardized units to measure light quantities. These include lumens (lm) for luminous flux, lux (lx) for illuminance, and candelas for luminous intensity.

5. Luminous Flux: Luminous flux measures the total amount of visible light emitted by the source. Its unit is lumen (lm). It measures the brightness of a light source that appears to the human eye. More lumens mean more brightness.

6. Illuminance (E): Illuminance is the amount of luminous flux received per unit area on a surface. Illuminance decreases with distance from the source. This decrease is due to the spreading of light.

7. Inverse Square Law: It states that Illuminance (E) at a point on the surface decreases with the square of the distance (r) from the light source. It means E is directly proportional to 1/r square. This explains why light appears dimmer as moving away from the source.

8. Luminance (L): Luminance measures the brightness of the surface and objects. It is expressed in candelas per square meter. It measures the angle at which light emits or reflects from the surface. It is essential to evaluate the visibility and contrast of the object.

9. Spectral sensitivity: Human eyes are sensitive to certain wavelengths of light. The wavelength of visible light ranges from approximately 380 nanometers (nm) to 700 nanometers (nm). This ranges from violet (shortest wavelength) to red (longest wavelength).

10. Solid angles: Solid angles describe how much light is emitted or received in a particular direction. It affects the light distribution from the source of light.

Photometer

The following are the main components of a photometer

1. Monochromator/Filter: Monochromator transmits the monochromatic light. A filter is used as a monochromator in a photometer to produce monochromatic light. This filter does not transmit monochromatic light. It transmits a narrow band of light between 4 and 20nm. Prism is used as a monochromator in a spectrophotometer. It transmits a single wavelength of light.

2. Light source: A tungsten lamp is used to emit radiant energy as a source of light. It emits a visible range of light (400 to 760 nm). A halogen lamp emits radiant energy as a source of light. It emits visible light, UV light, and IR light in bands 200 to 900 nm.

3. Cuvette (Cell): It is made of glass, quartz, silica or a special type of plastic with a width or diameter of 1 cm. It is round or square. It holds a solution through which light is transmitted during photometry.

4. Photocells: These are made of light-sensitive materials such as selenium, cuprous oxide, etc. Photocells are photo receptors to generate electric current when light radiation passes through the solution in the cuvette.

5. Galvanometer: It measures the electric current generated by photocells.

Photometric Procedure:

The following steps are followed in the photometry procedure:

1. Select three tubes labeled as “Standard” “Test” and “Blank”.

2. Add sample into “Test”, standard specimen into “Standard” and Distilled Water into “Blank”.

3. Incubate all tubes in an incubator at a temperature of 37 degrees C.

4. Switch on the light source to focus on a filter,

5. The filter transmits a narrow band of light.

6. Read the intensities of the test and standard against blank using a photometer.

7. Calculate unknown concentration in test sample by using Beer’s Law.Dr Pramila Singh

Principles of Photometry

1. Light emitted from light source of photometer falls on filter through a condense lens.

2. Filter transmit narrow band of light to cuvette containing colored solution.

3. Light absorbed by the colored solution is directly proportional to colored intensities. (Beer’s Law).

4. Light transmitted through cuvette falls on photoreceptor,

5. Photoreceptor converts radiant energy of light into electrical energy.

6. Galvanometer records the reading in percent transmittance (%T) or in optical density (OD).

7. Photometer or spectrophotometer works mainly on two principles Beer’s Law and Lambert’s Law

   1. i.Beer’s Law: Optical density of solution is directly proportional to the solution concentration.

   2. ii. Lambert’s Law: Optical density of colored solution is directly proportional to the path of light (diameter/width of the cuvette).

Laws of photometry

Photometry working is based on Beer’s Law and Lambert’s law. Beer's Law is also known as the Beer-Lambert Law. It is a fundamental principle in photometry and spectroscopy. It states that the absorbance (A) of a solution is directly proportional to the concentration (C) of the solution and the path length of the light through the solution.

Beer's Law states a linear relationship between absorbance and concentration of substance in solution at a specific wavelength.

The mathematical expression of Beer's Law is:

A = εlc

• A is the absorbance of the solution.

• ε is the molar absorptivity or extinction coefficient, a constant that characterizes the absorbing species and the wavelength of light used. It has units of L mol⁻¹ cm⁻¹.

• l is the path length of the light through the solution, it is measured in centimeters (cm). It remains constant in photometry or spectroscopy.

• c is the concentration of the solution. It is expressed in moles per liter (mol/L).

Limitations: Beer's Law is applicable only for dilute solutions and monochromatic light. Deviations may occur at higher concentrations due to interactions between molecules or changes in the solution's properties.

Importance of Photometry

Photometry is used in medical science for accurate diagnosis, treatment, and research. It is used in

1. Blood Tests: Photometry is used to measure the concentration of various substances in blood, such as glucose, cholesterol, proteins, and electrolytes.

2. Urine Analysis: Photometry is used to analyze urine samples. It detects abnormalities like proteinuria, hematuria, and changes in pH.

3. Spectrophotometry: It measures the intensity of light absorbed or transmitted by a sample at specific wavelengths. It is used to identify and quantify substances in biological fluids, tissues, and cells.

4. Pulse Oximetry: It is non-invasive method to measure the oxygen saturation level in blood using photometric principles.

Quantification of biomolecules in micro concentration

Quantification is a process to determine or measure the quantity of object. Quantification of biomolecules in micro concentration helps to diagnose diseases such as cancer, diabetes, infection, etc in early stage. Photometry is helpful to quantify biomelecules in micro concentration in several ways.

1. Sensitivity: Photometric techniques are highly sensitive to detect small amounts of biomolecules.

2. Quantification: Photometry allows quantitative measurement of biomolecules to determine their concentration based on the absorption or emission of light.

3. Specificity: Photometric method can measure specific biomolecules by selecting appropriate wavelength of light. It is especially useful while dealing with complex mixture of biomolecules in a sample.

4. Speed and precision: Photometric method to quantify is rapid and provides accurate result.

5. Cost effective: Photometric method is relatively cost effective.

6. Non destructive: Photometric method is a non destructive method. It preserves integrity of the sample.

Dr. Pramila Singh

Principles used in determining the concentration of molecules with no known weight - preparation of standard graph.

• Principle: The concentration of molecules with no known weight can be determined by using photometry. Beer-Lambert Law is followed in this determination. Beer’s Law states concentration of a colored substance is directly proportional to the optical density (OD). The optical density (OD) of the test solution (Concentration of molecules with unknown weight) is compared with the optical density (OD) of the standard solution (of known concentration). The concentration of the test solution can be determined by using a formula derived from Beer’s Law.

• The concentration of test solution = OD Test/OD Standard X concentration of standard.

Procedure:

• 1. Test solution: Prepare potassium dichromate solution as test solution.

• 2. Standard solution: Prepare a standard solution of potassium dichromate. Dissolve 50mg potassium dichromate in 100 ml of distilled water. The strength of the standard solution shall be 50mg/dl.

• 3. Add 5 ml of test solution into the cuvette. Note down its optical density (OD) at 420 mm (violet filter) using a photometer.

• 4. Add 5 ml of the standard solution into the cuvette. Note down its optical density (OD) at 420 mm (violet filter) using a photometer.

• 5. Determine test solution optical density by using Beer’s Law formula

Concentration of test solution = OD Test/OD Standard X 50.

Preparation of a standard graph to determine the  concentration of molecules with no known weight (Unknown weight)

• The standard graph is prepared by using any one of two graphs. Both graphs follow Beer’s Law. These are

• 1. % Transmittance (%T) of a series of standard solution concentrations at a particular wavelength in the photometer. %T (on the Y axis of the graph) and concentration (on the X axis of the graph).

• 2. OD of a series of standard solution concentrations at a particular wavelength in a photometer. OD (on the Y axis of the graph) and concentration (on the X axis of the graph).

• Graph for % T (Y-axis) and concentration (X-axis) shall be a curve. The graph for OD (Y-axis) and concentration (X-axis) shall be a straight line. OD density graph gives accurate results in the determination of molecule concentration solution with unknown weight. Beers Law states OD of solution is directly proportional to concentration.