Photometry

Photometry is defined as the measurement of the luminous intensity of light or the amount of luminous light falling on a surface from such a source. Filter photometers use filters to obtain required wavelengths Spectrophotometers use prisms or grating to obtain required wavelengths

There are six types of Photometry 1- Spectrophotometry 2- Flame Emission Spectrophotometry 3- Atomic absorption Spectrophotometry 4- UV- Visible Spectrophotometry 5- Fluorometry 6- Nephlometry and Turbidimetry

Spectrophotometry Nature of light The term “light” is used to describe the visible form of electromagnetic radiation as well as the ultraviolet(UV)and infrared (IR) form of electromagnetic radiation,which are invisible.

The wavelength of light is defined as the distance between two peaks as the light travels in a wave like manner. This distance is expressed in nanometers (nm) for wavelengths commonly used in photometry. 1nm = 10 Aº

* The human eye responds to radiant energy with wavelengths between about 380 and 750 nm. * Modern instrumentation (spectrophotometers) permits measurements at both shorter-wavelengths (UV) and longer- wavelength (IR) portion of the spectrum

Beerś- lambertś law * The base of colorimetric analysis is the measurement of the concentration of a colored substance. When a ray of a monochromatic light of certain wavelength (λ) of initial intensity (Io) passes through a colored solution, the solution will absorb some of the light and some will be transmitted with the intensity of (Is). The intensity of the transmitted light beam (Is) will be less than (Io).

Transmittance (T) of light is defined as T=Is/ Io. When the concentration of the colored compound is increased, the transmittance (T) is decreased. The relation is inverse and logarithmic. % Transmittance, %T = 100 T

Consequently, it is more convenient to define a new term, absorbance (A) that will be directly proportional to concentration hence: 1 100% A = - log T = log = log T % T = log100 – log % T = 2 – log % T Or Absorbance, A = log10 I0 / Is A = log10 1 / T  A = log10 100 / %T A = 2 - log10 %T 

As the light passes through a solution, the intensity of light transmitted decrease, exponentially with increasing path length (Lambert's law) and with increasing concentration of the absorbing substance (Beer's-law). The combination is often termed the Beer's - Lambert's law. The overall equation expressed as: A= a b c Where A = the absorbance a = Constant defined as moler absorptivity b = light path in centimeters c = concentration of the absorbing compound

Calibration & Absorbance Curves The variations in chemical reaction condition are: 1-Time 2-Temperature 3-Concentration & age of the reagent 4-Instruments behavior All these combine to cause variability in the absorbance (A) of the sample.

Calibration curve . A calibration curve is a method used in chemistry to determine the concentration of an unknown sample solution. It is a graph generated by experimental means, with the concentration of solution plotted on the x-axis and the observable variable — for example, the solution’s absorbance — plotted on the y-axis. The curve is constructed by measuring the concentration and absorbance of several prepared solutions, called calibration standards. Once the curve has been plotted, the concentration of the unknown solution can be determined by placing it on the curve based on its absorbance or other observable variable.

Absorption curve Used to find the wavelength of maximum absorbance for standard of known concentration.

Measurement of Concentration of the Unknown (test) We can compare the concentration of the test solution with a standard solution measured in the same way: A test C test = ----------------- x C std A std C std/ A std= factor

Reference cell (blank): Identical to the sample cell, exept that the compound of interest is omitted from the solvent. This will correct for the effect of : Absorbance of light by the cell (cuvette) Reflectance of light by the cell wall Absorbance of light by reagent Test solution: Made from serum or other unknown specimen. Standard solution: Made from a known concentration of a substance to be measured. Quality control: Made from pooled serum of a known chemical constituent.

Spectrophotoeters Components The major components of a single-beam spectrophotometer 1- Light source (e.g. tungsten, deuterium or hydrogen) lamps 2- Monochromator, for selection of desired color or wavelength 3- Slit for isolation of narrow beam 4- Absorption cell (cuvette), where a portion of the radian energy is absorbed 5- Detector (photocell) converts the light energy to electrical energy 6- Meter to register the electrical energy

Home work 1-Which of the following relationships between absorbance and %transmittance is incorrect? A). A = log10 100 / %T B). A = 2 - log10 %T C).A = log10 1 / T D).All are correct 2- Why is it generally preferable to use absorbance as a measure of absorption rather than % transmittance? A).Because %T cannot be measured as accurately as absorbance B).Because %T is dependant on the power of the incident radiation C).Because absorbance is proportional to the concentration of the analyte, whereas %T is not D.)none of the above