Spectrphotometry Dr M M Mir

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M Muzaffar Mir... Basic concepts of spectrophotometry

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Spectrophotometry :

Spectrophotometry بسم الله الرحمن الرحيم

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Concentration: The concentration of a chemical solution refers to the amount of solute that is dissolved in a solvent . تركيز المحلول الكيميائي يشير إلى كمية المذاب أن يذوب في المذيبات Units of Concentration Concentration may be expressed several different ways, using percent composition by mass , volume percent , mole fraction , molarity , molality , or normality . BASIC CONCEPTS

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Percent Composition by Mass (%) This is the mass of the solute divided by the mass of the solution (mass of solute plus mass of solvent), multiplied by 100. Example: Determine the percent composition by mass of a 100 g salt solution which contains 20 g salt. Solution: 20 g NaCl / 100 g solution x 100 = 20% NaCl solution . Volume Percent (% v/v) : Volume percent or volume/volume percent most often is used when preparing solutions of liquids. Volume percent is defined as: v/v % = [(volume of solute)/(volume of solution)] x 100% Note that volume percent is relative to volume of solution, not volume of solvent . BASIC CONCEPTS

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Molarity (M) Molarity is the number of moles of solute dissolved per liter of solution . It is probably the most commonly used unit of concentration. Example : 1M solution of Na OH contains 40 g of NaOH [ gm mol weight of NaOH] in one litre of NaOH solution. Molality (m) Molality is the number of moles of solute per kilogram of solvent. Example : I molal solution of NaOH contains 40 g NaOH in dissolved in 1000 g of water. BASIC CONCEPTS

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Normality (N) Normality is equal to the gram equivalent weight of a solute per liter of solution. Normality is the only concentration unit that is reaction dependent. Normality = molarity x n (where n = the number of protons exchanged in a reaction). Examples : For HCl -- Normality is equal to molarity For H 2 SO 4 -- Normality = Molarity x 2 Why ???????? BASIC CONCEPTS

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Dilutions We can make a dilute solution by adding solvent to a solution or a part of it. Adding solvent results in a solution of lower concentration. We calculate the concentration of a solution following a dilution by applying this equation: M i V i = M f V f where M is molarity, V is volume, and the subscripts i and f refer to the initial and final values. Example: How many milliliters of 5.0 M NaOH are needed to prepare 300 mL of 1.0 M NaOH ? Solution: 5.0 M x V i = 1.0 M x 300 V i = 1.0 M x 300 / 5.0 M V i = 60 mL BASIC CONCEPTS

Terminology used in Spectrophotometery :

Terminology used in Spectrophotometery Photometery : defined as measurement of intensity of light. A photometer is used for this purpose. Spectrphotometery: defined as measurement of intensity of light at selected wavelength. A spectrophotometer is used for this purpose. Absorbance: The amount of light energy absorbed by a medium. It is given by 2-log %T Transmittance, %T : is the portion of light energy that passes through a medium.

Terminology used in spectrophotometry :

Terminology used in spectrophotometry Absorption spectrum: When the absorbance of a substance is plotted as a function of wavelength, the graph is called as absorption spectrum. Wavelength: The linear distance travelled by one complete cycle of electromagnetic energy. Visible light: The visible light to human eye in the approximate wavelength range of 390 to 780 nm. Ultraviolet light : The region of electromagnetic radiation from 180 to 390 nm.

Principle of Spectrophotometery :

Principle of Spectrophotometery I o I s Transmittance ,T = I s / I o % T = I s / I o x 100% As the concentration of solute increases , more light is absorbed and less light is transmitted. %T varies inversely and logarithmically with concentration.

Principle of Spectrophotometery :

Principle of Spectrophotometery Absorbance is more convenient to use and is directly proportional to concentration. A = - log I s / I o = - log T = log 1/T let us convert T to %T A = log 1/T x 100%/100% = log 100%/%T .. Rearranging means = log 100%- log %T So, A = 2- log %T

Principle of Spectrophotometery :

Principle of Spectrophotometery Beer’s Law : states that absorbance of radiant light energy is directly proportional to the concentration of a substance . امتصاص الطاقة من الضوء يتناسب طرديا مع تركيز من مادة Lambert Law : states that absorbance of radiant light energy is directly proportional to the path length through the cell. امتصاص الطاقة من الضوء المنبعث يتناسب طرديا مع طول مسار Combined together, the Beer Lambert law [ referred simply as Beer’s Law ] provides a mathematical relation ship between absorbance of radiant light energy , conc. of solution and path length. A = abc A is absorbance ; a, absorptivity ; b, light path of the solution in cm ; and c, the concentration of the substance in interest . Absorptivity is a constant related to the chemical nature of the substance. It is also related to the units of b and c. Molar absorptivity with symbol, ε [Epsilon] is used when c is expressed as moles per liter and b in centimeters.

The main Components of a Spectrophotometer:

The main Components of a Spectrophotometer

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monochromator sample cell detector light source slit diffraction grating The main Components of a Spectrophotometer

The Components of a Spectrophotometer:

Light source : A variety of light sources are used. A tungsten lamp is used for visible region Hydrogen and deuterium lamps are used for UV region Filters/ Monochromators: A range of wavelengths or a specific wavelength is obtained by use of a filter or a monochromator. A filter absorbs a set of wavelengths and selectively transmits desired wavelengths. In a monochromator a prism or a grating disperses radiant light into a spectrum. A desired wavelength is isolated by using a slit. Cuvette: A small device in which a sample is placed for spectrophtmetric measurement is called a cuvette or a cell. Glass cells are used for visible region Quartz [silica] cells are used for UV region. The Components of a Spectrophotometer

The Components of a Spectrophotometer-cont’d:

The Components of a Spectrophotometer-cont’d Detectors: These are devices which help in the measurement of emitted light energy [or absorbance]. The absorbance is related to the concentration of the substance by using a standard. They are of different types Charge coupled devices , CCD Photodiodes Photomultiplier tubes

The Components of a Spectrophotometer cont’d:

The Components of a Spectrophotometer cont’d Other components Readout devices: To display readings Analog Digital Microprocessors: Help in storing and analyzing data. Recorders: Help in recording absorbance as a function of wavelength or time.

Spectral characteristics:

Spectral characteristics Wavelength [nm] Region name Color observed <380 Ultraviolet , UV Invisible 380-440 Visible Violet 440-500 Visible Blue 500-580 Visible Green 580-600 Visible Yellow 600-620 Visible Orange 620-750 Visible Red >800 Infrared Not visible The wavelength intervals are approximate. The UV and Infrared regions are further subdivided .

Quality control checks of spectrophotometer performance.:

Quality control checks of spectrophotometer performance. Several quality control checks should be performed to make sure that spectrophotometer is performing well. These checks include: wavelength accuracy linearity of detector response stray light photometer accuracy

Applications of Spectrophotometry :

Applications of Spectrophotometry Chemical analysis Analysis of biomolecules like proteins,carbohydrates, vitamins etc. Analysis of enzymes Drug Assays Others.

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