Spectrofuorometer lab. 1

Fluorescence spectroscopy or (spectrofluorometry) Is a type of electromagnetic spectroscopy which analyzes fluorescence from a sample. It involves using a beam of light, that excites the electrons in molecules of certain compounds and causes them to emit light.

fluorophore A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation.fluorescent Fluorophores typically contain several combined aromatic groups, or plane or cyclic molecules with several π bonds. Fluorochromes absorb light of a particular wavelength, they are transiently excited and then emit light of a longer wavelength as they return to their unexcited state. The emitted light is referred to as fluorescent light (fluorescence). The intensity of the emitted light is directly proportional to the amount of fluorochrome in the sample. Not all the light energy that is absorbed by the fluorochrome is emitted as fluorescence because some of the energy is quickly dissipated as heat or vibrational energy.

Instrument design The instrument design must provide some mechanism by which different excitation and emission wavelengths of light are selected. This is achieved by use wavelength selectors which are monochromators. Monochromators are device made of several components, one component disperses the spectrum of visible light, while the other component is adjustable, and is used to select the particular wavelength of light that will pass through the monochromator.

Instrument design A spectrofluorometer must have an excitation monochromator and emission monochromator because The absorption and emission spectrum of one compound differs from that of another compound. It should always be kept in mind that the wavelength selector must be adjusted for every compound tested.

Principle of spectrofuorometer function The excitation light signals is visible light consisting of many wavelengths radiating in all directions. Xenon arc lamp is often used as an alternative source because A device known as excitation monochromators is used to focus the light onto a prism or grating, which cause dispersion of the spectrum of light. Computer driven exit slits determine the wavelength of the light that exits the monochromators and enters the cuvette. it is crucial that all extraneous wavelengths of the light be blocked before entering the cuvette because this would contribute to the measured emission.

Principle of spectrofuorometer function The excitation light penetrates the cuvette and is absorbed by the fluorochrome, when the fluorochrome absorbs light energy it, it is excited momentarily. As the fluorochrome reverts back to its more stable state, light of longer wavelength is emitted. Fluorescence is emitted in all directions. A slit in the emission monochromator allow light to focused onto the wave selector. The emission monochromator is set to the wavelength of light emitted as the fluorochrome returned to its stable state.

Principle of spectrofuorometer function The emission monochromator is spatially arranged at 90º to the excitation monochromators to minimize the amount of excitation light that reaches the detector. The detector is often a photomultiplier tube (electron tube) that absorbs the emitted light and ejects electron in proportion to the amount of light absorbed.

A schematic of spectrofluorometer v v v

Determination of unknown quinine sulphate concentration using calibration curve

In five 50 ml volumetric flasks, prepare a serial dilutions of quinine sulphate by pipette 2,4,6,8,10 ml from the stock solution of Q.S (5 g/L). Complete to volume with 0.2N H2SO4 and mix well. Set the λ excitation at 350 nm and λ emission at 450 nm. Record the relative fluorescence intensity for each dilution, and for the unknown sample. Plot the calibration curve by plotting relative fluorescence intensity versus concentration. Determine the unknown conc. of QS from the graph. 0 procedure

Relative Fluorescence intensity Calculate conc. of Q.S for each diluted flask Stock Q.S concentration (5 g / L) ml that you pipette from quinine sulphate stock ? from the instrument C X V = C ̄ X V 5 (g / L) X 2 ml = C ̄ X 50 ml 2ml ? from the instrument C X V = C ̄ X V 5 (g / L) X 4 ml = C ̄ X 50 ml 4ml ? from the instrument C X V = C ̄ X V 5 (g / L) X 6 ml = C ̄ X 50 ml 6ml ? from the instrument C X V = C ̄ X V 5 (g / L) X 8 ml = C ̄ X 50 ml 8ml ? from the instrument C X V = C ̄ X V 5 (g / L) X 10 ml = C ̄ X 50 ml 10ml ? from the instrument Unknown sample conc. From the calibration graph calculation

Unknown concentration How to determine unknown concentration from the calibration curve

Determination of unknown quinine sulphate concentration using linear equation

YX press AC …... Mode ….. (choose 3: STAT)…... (Choose 2: A +B X) 2- Fill the schedule with the calculated concentration in X column, and with fluorescence intensity obtained in Y column. As shown. After each number you add press = to transfer to the next row 3- press AC….. Shift 1 ….. Choose 7: Reg ……. (Then choose 1 : A) …. Press = ……. You will have intercept value. 4- press AC….. Shift 1 ….. Choose 7: Reg ……. (Then choose 2 : B) …. Press = ……. You will have slope value. Y = a + bx Unknown Conc. slope Unknown fluorescence intensity intercept

Several factors affect the amount of fluorescence that is measured and so not all emitted light is measured. The most common factor is quenching. Quenching refers to a decrease in fluorescent intensity. Quenching by the solvent may occur when the fluorochrome interacts with molecules in the solution. A variety of processes can result in quenching such as : energy transfer, complex-formation and collisional quenching. The most common chemical quenchers are: Oxygen, iodide ions, chloride ions and acrylamide. Factors that affect fluorescence measurements

processes can result in quenching

Determination of the quenching effect of KI on the fluorescence of quinine sulphate

procedure In eight 100 ml volumetric flasks, add 16 ml of Q.S. Add 0,1,1.5,2.5,5,7,10,15 ml of KI ( stock conc. 0.1M) to each flask. Complete to volume with distilled water. Read and record relative fluorescence intensity for each flask then plot intensity versus conc. of KI Comment on the curve

calculation IntensityCalculate conc. of KI for each diluted flask ((Stock conc. 0.1M ml of KIml of Q.S. ? from the instrument C X V = C ̄ X V 0.1 M X 0 ml = C ̄ X 100 ml 016 ? from the instrument C X V = C ̄ X V 0.1 M X 1 ml = C ̄ X 100 ml 116 ? from the instrument C X V = C ̄ X V 0.1 M X 1.5ml = C ̄ X 100 ml ? from the instrument C X V = C ̄ X V 0.1 M X 2.5ml = C ̄ X 100 ml ? from the instrument C X V = C ̄ X V 0.1 M X 5 ml = C ̄ X 100 ml 516 ? from the instrument C X V = C ̄ X V 0.1 M X 7 ml = C ̄ X 100 ml 716 ? from the instrument C X V = C ̄ X V 0.1 M X 10 ml = C ̄ X 100 ml 1016 ? from the instrument C X V = C ̄ X V 0.1 M X 15ml = C ̄ X 100 ml 1516

Effect of of KI on the fluorescence of quinine sulphate

What is the difference between Fluorescence spectroscopy and spectrophotometer ?