Spectrophotometry & Colorimetry

Spectrophotometry
Spectrophotometry is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength It involves with the use of a spectrophotometer. A spectrophotometer is a photometer that can measure intensity as a function of the light source wavelength.

 A spectrophotometer is commonly used for the measurement of transmittance or reflectance of solutions, transparent or opaque solids, such as polished glass, or gases.

Principles
A spectrophotometer is employed to measure the amount of light that a sample absorbs. The instrument operates by passing a beam of light through a sample and measuring the intensity of light reaching a detector.

Classes

Single Beam Spectrophotometer

 Light from the source lamp is passed through a monochromator, which diffracts the light into a "rainbow" of wavelengths and outputs narrow bandwidths of this diffracted spectrum. Discrete frequencies are transmitted through the test sample. Then the photon flux density (watts per metre squared usually) of the transmitted or reflected light is measured by the sensor. The transmittance or reflectance value for each wavelength of the test sample is then compared with the transmission (or reflectance) values from the reference sample.

Types

Double Beam Spectrophotometer

It is to determine the amount of light of a specific wavelength absorbed by an analyte in a sample. A double beam instrument compares the light intensity between two light paths by splitting the light source into two separate beams. The splitting of the beam is accomplished either statically using a partially transmitting mirror or through attenuation of the beams optical devices.

Kinds
UV-Visible Spectrophotometer uses light over the ultraviolet range (185 400 nm) and visible range (400 - 700 nm) of electromagnetic radiation spectrum a solution sample that absorbs light over all visible ranges appears black in theory

Kinds
IR Spectrophotometer uses light over the infrared range (700 15000 nm) of electromagnetic radiation spectrum if all visible wavelengths are transmitted the solution sample appears white

Components and Functions

Light source- Most commonly, a spectrophotometer uses white light as its light source. White light is a combination of all visible wavelengths. Ultraviolet and infrared wavelengths may also be used. Monochromator- is an optical device that transmits a mechanically selectable narrow band of wavelengths of light or other radiation chosen from a wider range of wavelengths available at the input. Cuvette- is a small tube of circular or square cross section, sealed at one end, made of plastic, glass, or fused quartz (for UV light) and designed to hold samples for spectroscopic experiments. Detector- transduces light energy into electrical energy

Colors and their Wavelength

Color Wavelength (nm) Frequency

Violet
Blue Green Yellow

420
470 530 580

7.1x1014
6.4 x 1014 5.7 x 1014 5.2 x 1014

Orange
Red

620
700

4.8 x 1014
4.3 x 1014 Red

Orange
Yellow Green Blue Violet

Specificity of the color reactions

Very few reactions are specific for a particular substance, but may give colors for a small group of related substances only. This may be achieved by isolating the substance by the normal methods of inorganic analysis. But these separation methods are often tedious and time consuming. .

Beers Law

Uses
A spectrophotometer has important uses, mainly being in the forensic sciences i.e. chemistry, biology, biochemistry, physics, etc. and medical field and the purpose being research. Research conducted is for varied reasons, laboratory as well as business or industrial purposes. We will learn about the uses of spectrophotometer in both these fields in detail in the following section along with some information on how to use a spectrophotometer.

Advantages
cheap and fast It also works for a wide variety of samples and can detect things very strongly

Disadvantages
very weak emission of ultraviolet light; very great variation of light intensity in the useful spectrum; the intensity of light at 290 nm is about 900 times less than at 700 nm; the proportion of interfering light is considerable, which means that expensive blocking filters need to be used; the system for compensating variations in light intensity with wavelength must have a wide dynamic range; the service life is relatively short; the light output is relatively low; the dissipated power is considerable; and the electric supply means for the lamp is relatively heavy and bulky

Applications
Spectrophotometry is widely used for quantitative analysis in various areas . (In biochemistry) used to determine enzyme-catalyzed reactions. (In clinical applications),used to examine blood or tissues for clinical diagnosis. There are also several variations of the spectrophotometry such as atomic absorption spectrophotometry and atomic emission spectrophotometry. A blank is used in order to cancel out or zero the absorbance of all the other components in the sample except the component whose absorbance is to be measured.

Colorimetry
Colorimetry or colourimetry is a technique "used to determine the concentration of colored compounds in solution." In colorimetry, the color of the sample is compared with a color of a standard in which the color is known.

 A colorimeter is a device used to test the concentration of a solution by measuring its absorbance of a specific wavelength of light.

Classes
Colorimeter A colorimeter makes a rough estimate of the color by taking three wideband readings. Spectrophotometer A spectrophotometer is commonly used for the measurement of transmittance or reflectance of solutions, transparent or opaque solids, such as polished glass, or gases.

Classes
Densitometer A densitometer is a type of colorimeter that helps control color by measuring the darkness and lightness of a printed material during the printing process. Spectroradiometer A spectroradiometer is a type of colorimeter that measures the intensity and irradiance of color.

Types
Visual Colorimetry A procedure for the determination of the color of an unknown solution by visual comparison to color standards (solutions or color-tinted disks).

Types
Photoelectric Colorimetry Measurement of the colorant concentration in a solution by means of the tristimulus values of three primary light filter-photocell combinations.

Components and Functions

a light source, which is usually an ordinary filament lamp an aperture is a hole or an opening through which light travels a detector which measures the light which has passed through the solution a set of filters in different colors that are used to select the wavelength of light which the solution absorbs the most.

Uses
Uses of Colorimeters Besides being valuable for basic research in chemistry laboratories, colorimeters have many practical applications. used to test for water quality, by screening for chemicals such as chlorine, fluoride, cyanide, dissolved oxygen, iron, molybdenum, zinc and hydrazine. used to determine the concentrations of plant nutrients (such as phosphorus, nitrate and ammonia) in the soil or hemoglobin in the blood and to identify substandard and counterfeit drugs

Advantages
Simplicity of Design Material Costs Time Consumed Learning Curve

Differences
What is the difference between Colorimeter and Spectrophotometer? A colorimeter quantifies color by measuring three primary color components of light (red, green, blue), whereas spectrophotometer measures the precise color in the human visible light wavelengths. . Colorimetry uses fixed wavelengths, which are in the visible range only, but spectrophotometry can use wavelengths in a wider range (UV and IR also). Colorimeter measures the absorbance of light, whereas the spectrophotometer measures the amount of light that passes through the sample.