Analytical Chemistry Experiment 4 Determination of Riboflavin Medicine Year 1 Group 37 NGOH KEXUAN B101099154

20th November 2010

Aim: To determine the concentration of riboflavin via spectrophotometric method.

Fluorescence emitted by riboflavin 1) When certain wavelength of light with an energy (E= hv), reach the molecules of riboflavin in
the solution, electron of riboflavin will absorb the energy and excited to a higher energy level.

2) Electron which excited will try to get back to their ground stat e. This can be done by electronic transition, vibrational trans ition and rotational transition . 3) When the electron successfully get back to their ground state, the remaining energy is
perform in the way of light. The light is mentioned as fluorescence.

4) In this experiment, beside knowing that the riboflavin can emits fluorescence, we also
investigate the effect on concentration of riboflavin on its Intensity. By using the Intensity value recorded from spectrophotometer, we can determine the concentration of riboflavin in the solution.

5) According to F = kC, we know that fluorescence intensity is in linear relation with concentration of the solution. F: Fluorescence intensity k: Constant (Quantum Yield + Molar extinction coefficient) C: Concentration of solution One-to-one correspondence can be applied for any intensi ty to the certain concentration, after we have drawn the linear line.
About Riboflavin Structural formula of riboflavin:

Detector : To detect the fluorescence intensity. ¨§  Riboflavin is a water-soluble vitamin (Vitamin B2 which is involved in vital metabolic processes in the body. and green vegetables. 10-mL transfer pipette 5. enriched cereals/grains. growth. " $ #! ! "!   Light Sou % ! luo s n ilt ad ut & "! !# !# !" ! !    Sp t ophoto   t ©  Especially good dietary sources of riboflavin are milk (and other dairy products . 2-mL pipette 2. Sample in cell : The solution to be determined. liver. 200-mL volumetric flask 8. eggs. £ ¢¡   . 5-mL pipette 3. Safety pipette filler 6. Read out : To provide the readable data. Test-tubes x 10   2 © 1 Light source : To provide light. Filter : To make sure the output beam is of the chosen wavelength. Beakers x 2 9. ¦ ¥ ¤  donating groups of riboflavin suc as -NH2or .H enhance fluorescence Elec That is why riboflavin is fluorescent. 50-mL volumetric flask 7. The data shown in the spectrophotometer is the light intensity. and energy produc tion. Amplifier : To enlarge the information. meats. 10-mL graduated pipette 4. ilt Sampl In C ll t to App tus: 1. Necessary for normal cell function. Fluorescence : The fluorescence emitted by molecules in solution.

We have decided to prepare the following concentration of riboflavin: 1. Calculation as below: V1 x (1x10-5M) = 100ml x (1x10-6M) V1 = 10ml 5. V1 x (1x10-5M) = 100ml x (1. Preparation of Standard solution 4 ( 1. It was then diluted quantitatively by adding sufficient distilled water.5 x 10-6M) 15mL of 1x 10-5 M riboflavin solution was added into a 100-mL volumetric flask. Preparation of riboflavin solution (1 x 10 -5M) 20mL of 1 x 10-4M riboflavin solution was added into a 200 -mL volumetric flask. Preparation of Standard solution 2 (5 x 10-7M) 5mL of 1 x 10-5M riboflavin solution was added into a 100-mL volumetric flask. It was then diluted quantitatively by adding sufficient distilled water. Calculation as below: V1 x (1x10-5M) = 100ml x (5x10-7M) V1 = 5ml 4.10. 3.5x10-6M) V1 = 15ml . Calculation as below: V1 x (1x10-4M) = 200ml x (1x10-5M) V1 = 20ml 2. It was then diluted quantitatively by adding sufficient distilled water. Preparation of Standard solution 1 ( 5 x 10 -8 M) 1mL of 1 x 10-5M riboflavin solution was added into a 200 -mL volumetric flask. Cell 11. It was then diluted quantitatively by adding sufficient distilled water. It was then diluted quantitatively by adding sufficient distilled water. Spectrophotometer Material: 1. Riboflavin solution (2 x 10 -6M) Procedure: We were required to prepare 5 different concentration of riboflavin solution with the range of 2› 10-6~ 5×10 -8M. Preparation of Standard solution 3 (1 x 10-6M) 10mL of 1x 10-5 M riboflavin solution was added into a 100-mL volumetric flask. Calculation as below: V1 x (1x10-5 M) = 200ml x (5x10-8 M) V1 = 1ml We use micropipette to get 1ml of 1 x 10-5 M riboflavin solution.

1 139. V1 x (1x10-5M) = 100ml x (2x10-6M) V1 = 20ml 7.8 ( 2x10 5383 5371. It was then diluted quantitatively by adding sufficient distilled water. (As there is no mark on cell) The intensity data of 5 standard solutions and 1 unknown concentration solution were readily collected.B. The cell should be wiped clean and dry only outside with tissue. Put the cells back into a 200-mL beaker with water to avoid sudden collision with beaker base.2 5x10-8 151.5x10 4282 4270. 2.4 889. . Caution: The solution must be prepared from low concentration to hi h concentration. 1. Cell with distilled water was placed in the slot of the spectrophotometer.6.6 . 3. . Preparation of blank solution A tube of distilled water was prepared.2 1400.2 -6 Riboflavin 1x10-6 5x10-7 2742 1412 2730. The cell with about 2 cm high (the effective height is considered as 1 cm) ' should be handled only by the upper edge.3 sample 901. Spectrophotometer operation Zero error calibration would be done as following : a.2 -6 1. RESULT: ( Solution Blank Concentration(M) Intensity Deduction blank 11. B. 8. Arrange the cells in order. All solution is free of bubbles. 4. Preparation of unknown concentration Fe 2+ solution A cell of unknown concentration riboflavin solution was given by instructor. 1. Preparation of Standard solution 5 ( 2 x 10-6M) 20 mL of 1 x 10-5 M riboflavin solution was added into a 100-mL volumetric flask.

467 X = 3. So. However.205X + 34. The unstable electrons will try to get back to ground state by different ways. The excited electron will soon fall back to it ground state. The phenomena is called quenching effect.20x + 34. 9 @ @ 578 78 7B CB 7 7 A5 9 76 5 1 2 h th o of fluo s n hen certain wavelength of light with quantized energy reaches the molecules in riboflavin solution. The energy left are displayed in the form of light (fluorescence . there are still molecules which haven t excited yet.467 Substitute the intensity of unknown.46 R² = 0. such as electronic D 3 .998 4000 nte nsit y 2000 1000 E 3000 0 0 50 100 150 200 -8 Concentration of riboflavin (x 10 ) 250 Con nt ation of un no n: Calculation: Y = 27. electrons will be excited to higher energylevel.14 x 10-17 M Con lusion: The experimental result for unknown concentration is 3. there is not enough of energy for absorption of the molecules causing some of it became excited and some of it remain on ground state.14 x 10-17M is ussion: 5 87 7 8 7 ) ) 4 2 1 0) finition of qu n hing ff t hen the concentration of fluorescent solution if too high.6 = 27. get.Plot of Int nsit against on nt ation of iboflavin 6000 5000 y = 27.205X + 34. results a less intensity. the energy which supposed to be displayed in the form of light is absorbed by the remaining molecules. when a light with certain energy (E=hv reach the molecule. 889. e should claim that the intensity is higher if the concentration is higher.

F G REFERENCE: Teacher s pdf file wikipedia http: en. vibrational transition and rotational transition. The concentration of fluorescent solution is the major factor. ormally.05.org wiki Riboflavin H H HH . 3. When the electrons get back to ground state from singlet exciton (S 1) in the way of electronic transition. It is due to the intermolecular collision (both fluorescent molecule and non -fluorescent molecule). Factors that affect the fluorescence intensity in the experiment. Besides.wikipedia. transition into triplet exciton and electronic transition. presence of oxidizing species and exposure to light.transition. we control the concentration of solution for the absorbance become smaller than 0. fluorescence is emitted. There are also some other form of energy such as heat energy . When the concentration is too high.Riboflavin fluorescence is very sensitive to p environment. the plot of intensity against concentration will not be a linear.

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