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Exercise 1. Determination of the Anthocyanin Concentration in Table Wines and Fruit Juices Using Visible Light Spectrophotometry

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Exercise 1. Determination of the Anthocyanin Concentration in Table Wines and Fruit Juices Using Visible Light Spectrophotometry Reprinted from Gallik S., Cell Biology OLM P a g e | 1 Exercise 1. Determination of the Anthocyanin Concentration in Table Wines and Fruit Juices Using Visible Light Spectrophotometry A. Introduction Anthocyanins are natural plant pigments that give various fruits, vegetables and flowers red, blue and purple color. Blueberries, blackberries, raspberries and grapes, and wines and fruit juices made of these fruits are relatively rich in anthocyanins. Unlike chlorophylls and carotenoids, anthocyanins are water soluble; they are considered one of the largest and most important group of water-soluble pigments in the plant kingdom. YakimaBlueberries.com Anthocyanins are relatively strong natural antioxidants. They have recently caught the attention of scientists and the public because of their possible use in fighting the effects of aging and reducing the risk of cancer and cardiovascular disease through their antioxidant power. In recent years, research into the cellular effects of anthocyanins has intensified, and many research groups are working to determine the concentration of anthocyanins in fruits, vegetables, fruit juices, and wines. The specific objective of the experiment to be performed in this exercise is to determine the concentration of anthocyanin pigment in various wines and fruit juices. To accomplish this objective, students will use a variety of pipets to prepare various standards and samples. Once these samples are prepared, students will employ visible-light spectrophotometry to measure the amount of anthocyanin pigment in samples. In the process, students will gain a working understanding of pipeting and spectrophotometry, two tools commonly used in cell biology, biochemistry and molecular biology laboratories. Copyright © 2011, 2012 by Stephen Gallik, Ph. D. Licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. All text falls under this copyright and license. The only figures that fall under this copyright and license are those sourced to Stephen Gallik, Ph. D. Other figures may be copyrighted by others. Go to the on-line lab manual for image attribution & copyright information. Contact author at [email protected] . Reprinted from Gallik S., Cell Biology OLM P a g e | 2 B. Spectrophotometry Introduction Spectrophotometry is the science of measuring the light-absorbing and light- transmitting characteristics of a substance. Spectrophotometry is the science of measuring the light-absorbing and light- transmitting characteristics of a substance. Many substances absorb light and transmit light of specific wavelengths within the ultraviolet (200 - 400 nm), visible (400 - 700 nm) and near-infrared (700 - 1000 nm) regions of the electromagnetic spectrum. These light-absorbing / light-transmitting characteristics of a substance are useful in determining the presence and concentration of that substance in a sample. ADInstruments.com The Spectrophotometer The instrument used to measure the amount of light of a specific wavelength absorbed or transmitted by a substance is called a spectrophotometer. In a spectrophotometer, a sample of the substance is placed across the path of a light beam of a chosen specific wavelength. The spectrophotometer determines the intensity of the light entering the sample and the intensity of the light leaving the sample, then calculates the amount of light transmitted and absorbed by the http://faculty.uca.edu/march/bio1/scimethod/spectro.htm substance. A diagram of the light path through a visible-light spectrophotometer is shown to the right. A beam of light emerges from its source and passes through a prism, which dissects the light into a continuous spectrum of wavelengths. The user can select which single specific wavelength of Copyright © 2011, 2012 by Stephen Gallik, Ph. D. Licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. All text falls under this copyright and license. The only figures that fall under this copyright and license are those sourced to Stephen Gallik, Ph. D. Other figures may be copyrighted by others. Go to the on-line lab manual for image attribution & copyright information. Contact author at [email protected] . Reprinted from Gallik S., Cell Biology OLM P a g e | 3 light passes to the sample with the monochromator. Light of a single wavelength is called monochromatic light. The monochromatic light that passes to the sample is the known as incident light, its intensity is represented the value Io. As the incident light passes through the sample, a certain amount of the light will be absorbed by the sample. The monochromatic light that is not absorbed emerges from the sample; it is called transmitted light, and its intensity is represented by the value I (or I1). The intensity of the transmitted might is detected by a photodetector. Once the transmitted light is detected, the instrument calculates the fraction of the incident light transmitted by the sample, a value known as transmittance (T), T = I1 / I0. From the transmittance value, the instrument will calculate the amount of the monochromatic light absorbed by the sample, a value known as absorbance (A), using the formula A = - log10 T. Both the transmittance and absorbance are displayed on the display screen of the instrument. Transmittance & Absorbance The amount of monochromatic light absorbed by a sample is determined by comparing the intensities of the incident light (I0) and transmitted light (I1). The ratio of the intensity of the transmitted light (I1) to the intensity if the incident light (I0) is called transmittance (T) . T = I1 / I0 Because the intensity of the transmitted light (I1) is never greater than the intensity of the incident light (I0), transmittance (T) is always less than 1. http://en.wikipedia.org/wiki/File:Beer_lambert.png In practice, one usually multiplies T by 100 to obtain the percent transmittance (%T), which ranges from 0 to 100%. %T = T * 100 If the T of a sample is 0.40, the %T of the sample is 40%. This means that 40% of the photons in the incident light emerge from the sample as transmitted light and reach the photodetector. If 40% of the photons are transmitted, 60% of the photons were absorbed by the sample. From the transmittance or % transmittance, one can calculate the quantity known as absorbance (A). Absorbance is the amount of light absorbed by a sample. It is calculated from T or %T using the following equations: Copyright © 2011, 2012 by Stephen Gallik, Ph. D. Licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. All text falls under this copyright and license. The only figures that fall under this copyright and license are those sourced to Stephen Gallik, Ph. D. Other figures may be copyrighted by others. Go to the on-line lab manual for image attribution & copyright information. Contact author at [email protected] . Reprinted from Gallik S., Cell Biology OLM P a g e | 4 A = - log10 T or A = log10 (1/T) A = 2 - log10 %T These equations reveal that transmittance and absorbance are inversely related. That is, the more a particular wavelength of light is absorbed by a substance, the less it is transmitted. Moreover, the inverse relationship between A and T is not linear, it is logarithmic. Therefore, if 50% of the photons of monochromatic light are transmitted by a sample, and 50% of the photons are absorbed, T = 0.5, but A is not 0.5, A is 0.3, due to the inverse logarithmic relationship beween T and A. If 10% of the photons of monochromatic light are transmitted by a sample, and 90% of the photons are absorbed, T = 0.1, but A is not 0.9, A = 1.0. When A is 2.0, 99% of the photons of monochromatic light are absorbed, and when A is 3.0, 99.9% of the photons of monochromatic light are absorbed. The inverse logarithmic relationship between absorbance and transmittance and between absorbance and %T are clearly shown in the graphs below. In these graphs, as transmittance (top graph) and %T (bottom graph) increase from 0 to 1.0 and 0% to 99%, respectively, absorbance decreases logarithmically from 2.0 to 0. Stephen Gallik, Ph. D. Sample Calculations Three Sample Calculations of Absorbance from T and %T Calculation #1: if T = I1 / I0 = 0.999 then %T = T * 100 = 99.9 Copyright © 2011, 2012 by Stephen Gallik, Ph. D. Licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. All text falls under this copyright and license. The only figures that fall under this copyright and license are those sourced to Stephen Gallik, Ph. D. Other figures may be copyrighted by others. Go to the on-line lab manual for image attribution & copyright information. Contact author at [email protected] . Reprinted from Gallik S., Cell Biology OLM P a g e | 5 and A = 2 - log10 %T = 2 - log10 99.9 = 2 - 1.9995 = 0.0005 Calculation #2: if T = I1 / I0 = 0.50 then %T = T * 100 = 50 and A = 2 - log10 %T = 2 - log10 50 = 2 - 1.69897 = 0.301 Calculation #3: if T = I1 / I0 = 0.20 then %T = T * 100 = 20 and A = 2 - log10 %T = 2 - log10 20 = 2 - 1.301 = 0.699 Beer's Law The amount of light absorbed by a sample is dependent on the concentration of the pigment in the sample (c), path length (l), and the extinction coefficient of the pigment (Ε). Determining the amount of monochromtic light absorbed by a substance is most- commonly used to determine the concentration of that substance in a sample. The concentration (c) of a substance in a sample is one of three factors that affect the amount of light absorbed by a sample. The other two are path length (l), that is the distance the light http://en.wikipedia.org/wiki/File:Beer_lambert.png travels through the sample, and the extinction coefficient of the absorbing substance (Ε).
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