Spectrophotometry Practical Lesson on Medical Chemistry and Biochemistry

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1 Spectrophotometry Practical Lesson on Medical Chemistry and Biochemistry General Medicine Jiřina Crkovská (translated by Jan Pláteník) 2010/2011 1

2 Spectrophotometry is one of the most widely used instrumental techniques in analytical practice. It is an optical method based on estimation of absorption of light either in the UV range having wavelength nm, or in the visible light (VIS) range of wavelength nm by molecules of the analyte in solutions. The principle of spectrophotometry is interaction of electrons placed in the bonding or non-bonding orbitals with the photons of UV-VIS radiation. The energy of radiation excites the electrons in orbitals to a higher excited level; it is associated with absorption of certain quantum of radiation, which has definite energy contents, hence also a definite wavelength. The method is able to estimate a particular analyte if at least part of the analyte molecule absorbs radiation. It means that it must contain a configuration of atoms or functional groups that are responsible for this absorption these structural elements are called chromophores. The chromophores can consist of functional groups, or certain types of chemical bonds, such as the unsaturated bonds. Such chromophores are found especially in organic compounds. If a substance contains chromophores we can estimate absorption of radiation by the substance and in this way directly determine its concentration. The absorption of light is quantitatively described by the Lambert-Beer law A = ε.c.l [λ] A absorbance ε molar absorption decadic coefficient, at the wavelength λ, unit is l. mol -1. cm -1 c concentration of the analyte in mol/l l path length of the light (i.e. width of the cuvette containing the measured solution, through which the ray of UV-VIS radiation passes). Absorbance tells how much of light is consumed by the measured solution. It is directly related to the absorption of radiation. If the absorption of light is zero, the absorbance is zero as well. The Lambert-Beer law is valid for diluted solutions to concentration about 10-2 mol/l. Food dyes Various substances are added to foodstuffs to improve their taste, flavor, appearance or stability in storage. These food additives are denoted by E numbers. The codes E 100 to E 182 are reserved for food dyes. The Act No. 110/1997 Sb. defines dyes as substances that provide the foodstuffs with a color that would be absent without using the dye, or restore a color that was altered or attenuated during technological processing. The dyes can be classified to two groups natural and synthetic dyes. In the practical lesson we will work with synthetic dyes. The color of drinks is often caused by addition of synthetic dyes soluble in water. These substances can in sensitive persons induce allergic reactions and can be harmful if consumed in a higher quantity. That is why for various kinds of food and beverages limits have been set, to which the manufacturers are obliged to adhere. In flavored non-alcoholic beverages the maximal concentration of a dye should not exceed 100 mg/kg. 2

3 E-102, food yellow 4, C 16 H 9 N 4 Na 3 O 9 S 2 Trisodium -hydroxy-l-(4-sulfonatophenyl)-4-[(4-sulfonatophenyl)diazenyl]-pyrazole-3- carboxylate It is used in bakery and diary products, mustard, candy, non-alcoholic and even alcoholic beverages. The substance can induce allergic reactions and asthmatic attacks in sensitive individuals. It has been associated with hyperactivity in infancy. E-110, food yellow 3, C 16 H 10 N 2 Na 2 O 7 S 2 Disodium 6-hydroxy-5-[(4-sulfonatophenyl)diazenyl]-naphthalene-2-sulfonate It is used in bakery and diary products, mustard, candy, non-alcoholic and even alcoholic beverages. The substance can induce allergic reactions and asthmatic attacks in sensitive individuals. This substance has been also linked to the hyperactivity in children. E-123, food red 9, C 20 H 11 N 2 Na 3 O 10 S 3 Trisodium -3-hydroxy-4-[(4-sulfonato-l-naphthyl)diazenyl]-naphthalene-2,7- disulfonate In Czech Republic this dye is approved only for coloring of the following foodstuffs: aperitif wines, alcoholic liquors, fish eggs and milt. In 1976 FDA (The Food and Drug Administration) banned this dye as a possible carcinogen. In addition, this substance causes allergic reactions in sensitive individuals. 3

4 1. Spectrophotometric estimation of molar concentration of food dyes 1.1 Absorption maximum of stock solution of food dye. Demonstration Reagents: 1. stock solution of food dye E-102 of c = 55 µmol/l 2. stock solution of food dye E-110 of c = 55 µmol/l 3. stock solution of food dye E-123 of c = 62 µmol/l 4. deionised water Principle: The absorption curve is obtained by continuous measurement of absorbance at various wavelengths and plotting these data to a graph, where the x axis contains the wavelength (independent variable) and the y axis is used for the absorbance (dependent variable). Regions with the highest absorbance values are called the absorption maxima. Procedure: a. According to the instructions for use of the spectrophotometer Lightwave II + set the wavescan range nm (Lightwave II+ - short instructions). b. Set the spectrophotometer reading to zero; use deionised water as the blank. c. Into a cuvette put about 1 ml of stock solution of food dye and in the range of preset wavelengths measure the absorption curve. Record the wavelength of the absorption maximum Estimation of molar concentration of food dyes in given sample. Reagents: 1. stock solution of food dye E-102 of c = 55 µmol/l 2. stock solution of food dye E-110 of c = 55 µmol/l 3. stock solution of food dye E-123 of c = 62 µmol/l 4. deionised water Principle: Estimation of concentration of given substance requires a blind sample (reference sample, blank) and usually one or more standards. The blind sample contains all components used in the assay except for the substance that is measured. The standard is a solution of the measured substance of known concentration. 4

5 When the absorbances of the sample (A sa ) and standards (A st ) are measured, the value of concentration of the measured substance can be obtained in several ways: 1. Calculation from the Lambert-Beer law, if the molar absorption coefficient is known. The most commonly used unit for the molar absorption coefficient is l. mol -1. cm -1. This unit is convenient because usually cuvettes with the path length 1 cm are used. Then a simple calculation according to the formula A = ε. c gives molar concentration of the solution (mol/l). Another used unit is cm 2. mol -1. This unit is 1000-times smaller than the unit l. mol -1. cm Calibration graph method. For construction of calibration graph we use the measured absorbances of the standards, i.e. solutions of known concentrations of the estimated substance. On the x axis we plot the standard concentration (independent variable) and on the y axis the standard absorbance (dependent variable). The graphical expression of the relationship is a straight line with slope k and passing through the origin. Calibration graph for the photometric estimation of NO 3 - A 540 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0, [µmol/l] 3. Calibration factor method Calibration factor (f) is the reciprocal value of the straight line slope (k). 1 f = k The factor can be calculated from the values obtained for the calibration line: by dividing the value of concentration c α with the corresponding measured value of absorbance A α. cα f α = kde α = 1...n A where α 5

6 From the values f 1 f n the arithmetic mean is calculated that gives the value of the calibration factor f. The absorbance of the sample whose concentration is unknown is then multiplied with the factor. In practice it is possible to store the factor in the spectrophotometer memory and read directly concentrations of the analyzed samples. This method is therefore suitable for routine processing of high number of unknown samples. 4. Standard sample method In addition to the sample of unknown concentration (c sa ) also a standard sample whose concentration is known is processed. The standard concentration is chosen so that it would be in the middle of the calibration line, or at the upper limit of the physiological range for the given analyte. The unknown sample concentration is calculated from the absorbances of both samples and the known concentration. The absorbance is directly proportional to the concentration of the absorbing substance: c sa c st c st = then c sa =. A sa A sa A st A st A st standard absorbance c st standard concentration A sa sample absorbance c sa sample concentration Procedure: a. Prepare and mark 6 test tubes (1-6). b. The stock solution of one of the food dyes (E-102, E-110, E-123 of given concentrations) is to be successively diluted with distilled water, always to the concentration that is one half of the previous solution. Repeat 5-times in total (it is geometrical dilution). Proceed according to the following table: Test tube Deionised Solution of Molar absorbance No. 1 water food dye concentration A (ml) (ml) Note: (µmol/1) - 2 Stock solution From tube From tube From tube From tube From tube 5 6

7 c. Calculate the resulting molar concentrations of the diluted solutions (round off to two significant figures) and fill in the table above. From the provided graph of the food dyes absorption spectra choose a wavelength suitable for absorbance measurement of the given food dye. d. Measure the absorbances of the diluted solutions against distilled water (blank sample) and fill in the table. e. Plot the results to the calibration graph and calculate the calibration factor. f. Against distilled water measure the absorbance of an unknown sample (obtained from your instructor) and estimate concentration of this sample using Calculation with the standard Calculation with the calibration factor Reading from the calibration graph. g. Compare precision of all the used methods. 2. Estimation of time dependent change in concentration of NADH Nicotinamide adenine dinucleotide, NAD in short, is a coenzyme consisting of nicotinamide, adenine, two ribose molecules and two phosphates, connected together in the same way as nucleotides are (adenosine diphosphate, then ribose and then nicotinamide attached). 7

8 In the cell NAD participates in redox reactions, i.e. it is a coenzyme of oxidoreductases. The coenzyme occurs in two forms: NAD + is the oxidized form that accepts electrons from other molecules and is itself reduced. In this way NADH + H + originates, which provides electrons and itself gets oxidized. This electron transfer is the major function of NAD +. NAD + 2 H NADH H + H H H N + C O H 2 H 2 H N + C O H H + Reversible hydrogenation of nicotinamide adenine dinucleotide, which occurs on the pyridine ring of nicotinamide, leads to the reduced form and is associated with a distinct change in the absorption spectrum. The oxidized form (NAD + ) has an absorption maximum at wavelength 260 nm. Reduction cancels the aromatic character of the pyridine ring and its transition to a quinoid form (NADH + H + ) is associated with emergence of another absorption maximum at 340 nm. This maximum is utilized for estimation of concentration of the coenzyme in a reaction mixture. The used molar absorption coefficients are: at 340 nm 6.22 x 10 3 l. mol -1. cm -1 at 365 nm 3.41 x 10 3 l. mol -1. cm -1. Absorption Absorpční spectrum spektrum of NAD NAD + + a and NADH NADH A 1,5 NAD + 1,0 NADH 0,5 0, nm 8

9 We can prepare a reaction mixture form the estimated enzyme, e.g. lactate dehydrogenase, its coenzyme (NADH) and corresponding substrate (pyruvate) and under optimal condition follow the reaction rate by measuring changes in absorbance of NADH at the wavelength 340 nm. CH 3 -CHOH-COOH + NAD + CH 3 -CO-COOH + NADH + H + lactate pyruvate Reagents: 1. Reagent 1: Tris buffer, ph 7.2, 127 mmol/l pyruvate 2.5 mmol/l 2. Reagent 2: NADH in Tris buffer ph 7.2, 127 mmol/l, 3. Serum 4. Reagent 3: Tris buffer, ph 7.2, 127 mmol/l Procedure: a. In a cuvette prepare a blank sample: 800 µl reagent 1 (Tris buffer and pyruvate) 200 µl reagent 3 (Tris buffer) 20 µl serum. b. Mix. c. According to the instructions for use of the spectrophotometer Lightwave II + set the wavescan range nm (Lightwave II+ - short instructions). d. Into another cuvette measure: 800 µl reagent 1 (Tris buffer and pyruvate) 200 µl reagent 2 (NADH) 20 µl serum. e. Mix well and after exactly 2 minutes record the spectrum. f. Read A for wavelengths 281, 340 and 365 nm and write the values to the table. g. Repeat the measurement 4-times more in exactly one minute intervals; write down the absorbances for the given wavelengths to the table. h. From the measured absorbances calculate the NADH concentration in all measurements and plot to a graph as function of concentration on time. Calculate the (difference) of concentration between each two particular measurements and plot to a graph as function of on time. 9

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