Principles and practice of Spectrophotometer By the end of this topic, the student will be able to: 1-Identify the photometer with its main parts and uses. 2- Practice and recognize the principles of photometry and the related laws (Beer-Lambert’s laws).

ILO of the current topic: By the end of this topic the student will be able to: - Identify photometer and its main parts. - Learn the purpose and proper use of a spectrophotometer. - Recognize the principles of photometry and the related laws (Beer-Lambert’s laws).

Spectrophotometer Spectrophotometer - an instrument which can measure the amount of the light absorbed by the sample at any selected wavelength.

What does the word spectrophotometer means? A spectrophotometer consists of two instruments: 1) a spectrometer for producing light of any selected color (wavelength) 2) a photometer for measuring the intensity of light. Spectrometer =light production Photometer = for measuring light intensity Spectrophotometer

Absorption ,transmission principle Construction Application Background What is light Spetrophotometeric laws Absorption ,transmission spectrophotometer principle Construction Application

Background 1. What is light? 2.Electromagnetic spectrum 3. What is Color?

Light is a mixture of different electromagnetic wave lengths What is light Light is a mixture of different electromagnetic wave lengths Light consist of particles (photons) and behaves like a wave

Different Wavelengths Lead To: <380 nm (UV) ~380-750 nm (Visible light) >750 nm (IR) Visible light is only a very small portion of the electromagnetic spectrum.

Any solution containing a compound that absorb light in range of 380-750 nm will appear colored to the eye Incident light (I0) Transmitted light (I) Eye Absorbed light

Incident light (I0) Transmitted light (I) Absorbed light Eye 10

White light All colors Polychromatic light When white (polychromatic) light passes through a coloured solution some of the light is absorbed by the substances in the solution, and the rest passes through. Green solution absorbs light other then green

Monochromatic light Light of one color Red light is absorbed If white light is made to pass through a red filter, all light except red is filtered out and absorbed. Therefore, only red light hits the solution. Red light is absorbed by the green solution

Why Does Color is “deeper” with more Concentration? More molecules means more light is “absorbed” Examples

Definitions & Symbols Intensity (I) Transmittance (T) light (I) Incident light (I0) Transmitted light (I) Absorbed light Intensity (I) Transmittance (T) It’s also referred to as %T or T x 100 T = I/Io I = the rate at which energy in a beam of radiation arrives at a fixed point. T = the ratio of the intensity in a beam of radiation after it has passed through a sample to the power of the incident beam T = I/ Io Where I is the radiation transmitted by the solution and Io is the radiation transmitted by the pure solvent (blank)

Graphical Relationship % transmission is not linearly related to concentration For a graph to be useful, a straight line is needed ABSORBANCE = log(1/T) = -log(T)

Transmittance, absorbance, and concentration

Laws that regulate absorption and transmission of colored solutions: Beer’s Law Lambert’s Law Beer-Lambert Law

Beer’s Law “The concentration of a substance in a solution is directly proportional to the absorbance (A) of the solution” (the amount of light energy absorbed). A α c

Lambert’s Law A α L Incident light I0 When monochromatic light passes through a solution , the intensity of light transmitted decreases exponentially with increasing path length A α L 100 photons  50  25  12.5  6.25  3.125  1.56 T= 0.5 0.25 0.125 0.062 0.031 0.015 Incident light I0 TransmittedLight I absorbing medium Sample 19

= extinction coefficient Beer-Lambert Law Beer’s Law Lambert’s Law Beer-Lambert A = absorbance = extinction coefficient c = concentration L = pathlength of absorbing solution A = εcL http://www.hellma-worldwide.de/en/default.asp

Construction of Photometer Light Source Slit photo cells Wave Length Selector Meter Sample (Cuvette)

1- LIGHT SOURCES UV Spectrophotometer 1. Hydrogen Deuterium Gas Lamp 2. Mercury Lamp Visible Spectrophotometer 1. Tungsten Lamp

(Filter/ Monochromator) 2-Wave Length Selector (Filter/ Monochromator) Each colored solution needs a to be measured properly by the photometer. This is done by choosing the proper monochromator ( filter). 23

3- Slit It is necessary to adjust the intensity of the incident light (I◦) to form a Slit

4- Sample cuvettes UV Spectrophotometer Quartz (crystalline silica) Visible Spectrophotometer Glass-plastic  

5- Light-Detecting photocells Spectrophotometers detect the transmitted light (I)

6- Scale A= logarithmic numerical scale (non uniform) with no unit. T= percentage numerical scale (uniform) with no unit.

Why you must use a blank solution ? To eliminate Reflection and Light scattering that may decrease the intensity of transmitted light and affect measurement. So we use a BLANK Reference=Blank Test Known concentration=Standard *Is similar to the sample in every thing except the measured compound is absent from the solvent *It is used to adjust the zero of the spectrophotometer and eliminate any interference .

STEPS IN DEVELOPING A SPECTROPHOTOMETRIC ANALYTICAL METHOD

The instrument must be warmed for 15 min.

Set a monochromatic wavelength for the maximum absorption wavelength.

4-place it into sample holder . 3-Wipe the tube containing the blank solution with a lab wipe 4-place it into sample holder . Close cover

6-Remove blank tube, wipe off the cuvette of sample and insert it 5-set Zero absorbance 6-Remove blank tube, wipe off the cuvette of sample and insert it 7-read and record the absorbance. 8- Calculate the concentration of The sample using Beer Lambert Equation: A = εCL

Spectrophotometer Applications Qualitative identifying unknown compounds by their absorption spec Quantitative 1-Absolute Method 2- Comparative method A = εcL Atest Ctest= ________ x cStand AStand compare the absorbance of the test solution to that of the standard solution of same wave length.

Qualitative Spectrophotometric assays What is meant by absorption Spectra? An absorption spectrum can be generated by measuring the absorbance of the pure compound in solution at variety of wavelength

1- Heme proteins or Cytochromes show a distinct absorbance in the visible range from 500-600 nm 2- The nitrogenous bases that comprises nucleic acids are known to absorb strongly at 260 nm

3- The aromatic rings on tryptophan and tyrosine are known to absorb strongly at 280 nm.

Quantitative Spectrophotometric assays

Comparative method Test Reference=Blank Standard = Known concentration 39

A Standard = εC Standard L A test = εC test L A Standard = εC Standard L A test εC test L C test ______ = _______ = ______ A Standard εC Standard L C Standard A test C test= ________ x c Standard A Standard

Students will be divided into groups Write their comments Identify photometer with its main parts and uses Recognize the principles of photometry and the related laws (Beer-Lambert’s laws) by demonstration Write their comments

Identify 1-6 in the given illustration 1) Light source 2 1 3 4 5 6 Identify 1-6 in the given illustration 1) Light source 2) Wavelength Selector 3) Slit 4) Cuvette 5) Light-Detecting Photocells 6) A scale or a meter

In the given light spectrum: Region 1 represents: infrared light Region 2 represents: Visible light Region 3 represents: Ultraviolet light

According to the Beer-Lambert Law, Absorbance is dependent on : A) Distance traveled by the light through the sample. B) Extinction coefficient of the sample. C) Solution concentration. D) A and C. E) A, B and c The Beer-Lambert law (or Beer's law) is the linear relationship between absorbance and concentration of an absorbing species. The general Beer-Lambert law is usually written as: A = a(lambda) * b * c where A is the measured absorbance, a(lambda) is a wavelength-dependent absorptivity coefficient, b is the path length, and c is the analyte concentration. When working in concentration units of molarity, the Beer-Lambert law is written as: A = epsilon * b * c where epsilon is the wavelength-dependent molar absorptivity coefficient with units of M -1 cm -1 .

You have three readings given by the opposite test tubes… match each reading with its test tube : a) 0.66 b) 0.31 c) 0.88

How would you determine the concentration of an unknown protein sample? Explain in details. Use a set of standard proteins with known concentration. Find out their absorbance by using a spectrophotometer. Plot absorbance on y-axis vs. protein concentration on x-axis and plot the results. Draw a best fit line and use it to determine the concentration of the unknown protein after determining their absorbance using the same spectrophotometer

Given the following standard curve, answer the following question 1 1.5 2.0 2.5 3.0 3.5 An unknown protein has an absorbance of 0.45, what would its concentration be in mg?