# Colorimetry Extinction coefficient (ε) Lambda max (λ max ) Qualitative vs. quantitative analysis

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1 Lab Week 2 - Spectrophotometry Purpose: Introduce students to the use of spectrophotometry for qualitative (what is it) and quantitative (how much is there of it) analysis of biological samples and molecules. Key Concepts/Terms: Absorbance (A) Absorbance spectrum Beer-Lambert Law Blank vs. sample cuvette Calibration curve Laboratory Skills: Colorimetry Extinction coefficient (ε) Lambda max (λ max ) Qualitative vs. quantitative analysis Transmittance (T) UV vs. visible radiation Wavelength (λ) (i) (ii) (iii) (iv) (v) How to determine absorbance spectrum of unknown compounds (qualitative analysis). How to determine the λ max of a compound. How to use an absorbance spectrum to identify a pure compound. How to use the Beer-Lambert Law to determine the concentration of compounds in solution. How to prepare and graph standard or calibration curves for quantitative analysis using colorimetry. Background Basic Laws of Light Absorption. For a uniform absorbing medium (solution: solvent and solute molecules that absorb light) the proportion of light radiation passing through it is called the transmittance, T, and the proportion of light absorbed by molecules in the medium is absorbance, Abs. Transmittance is defined as: T = I/I o where: I o = intensity of the incident radiation entering the medium. I = intensity of the transmitted radiation leaving the medium. T is usually expressed as percent transmittance, %T: %T = I/I o x 100 The relationship between percent transmittance (%T) and absorbance (A) is given by the following equation: A = 2-log (%T) On most spectrophotometers two scales are present, %T and Abs. Absorbance has no units (Why?) and varies from 0 to 2 (linear region for most substances is from 0.05 to 0.7). The Beer- Lambert Law states that Abs is proportional to the concentration (c) of the absorbing molecules, the length of light-path through the medium and the molar extinction coefficient: A = εcl where: ε = molar extinction coefficient for the absorbing material at wavelength in units of 1/(mol x cm) c = concentration of the absorbing solution (molar) l = light path in the absorbing material (l=1 cm for our purposes)

2 The Beer-Lambert Law may not be applicable to all solutions since solutions can ionize/polymerize at higher concentrations, or precipitate to give a turbid suspension that may increase or decrease the apparent absorbance. Further, the Beer-Lambert Law is most accurate between Abs of 0.05 to Above 0.70, the measured Abs tends to underestimate the real Abs. Below 0.02 Abs many instruments are not accurate (remember that Abs is the log of a ratio). For example, a protein solution had an Abs of 1.42, but when it was diluted 1:4 it had an Abs of 0.45 meaning that the original, undiluted solution had a read Abs of 4 x 0.45 = 1.80 Abs. UV/Visible Spectroscopy. The absorption spectrum (plural, spectra), or more correctly the absolute absorption spectrum, of a compound may be shown as a plot of the light absorbed by that compound against wavelength. Such a plot for a colored compound will have one or more absorption maxima (λ max s ) in the visible region of the spectrum (400 to 700 nm). Absorption spectra in the ultraviolet (200 to 400 nm) and visible regions are due to energy transitions of both bonding and nonbonding outer electrons of the molecule. Usually delocalized electrons are involved such as the B bonding electrons of C=C and the lone pairs of nitrogen and oxygen. Since most of the electrons in a molecule are in the ground state at room temperature, spectra in this region give information about this state and the next higher one. As the wavelengths of light absorbed are determined by the actual transitions occurring, specific absorption peaks may be recorded and related to known molecular substructures. The term chromophore is given to that part of a molecule that gives rise independently to distinct parts of an absorption spectrum, for example the carbonyl group. Conjugated double bonds lowers the energy required for electronic transitions and results in an increase in the wavelength at which a chromophore absorbs. This is referred to as a bathochromic shift, whereas a decrease in conjugation, caused for example by protonating a ring nitrogen atom, causes a hypochromic shift which leads to a decrease in wavelength. Hyperchromic and hypochromic effects refer to an increase and a decrease in absorbance respectively. Instrumentation. To obtain an absorption spectrum, the absorbance of a substance must be measured at a series of wavelengths. Absorption in the visible and ultraviolet regions can be measured by a UV/visible spectrophotometer. UV/Vis spectrometers consist of three basic components, (i) a light source and a mechanism to select a specific wavelength of light in the UV/visible region of the spectrum, (ii) a chamber where a cuvette containing a test solution can be introduced into the light path, and (iii) a photocell that can determine the amount of light absorbed by the sample (or the intensity of light transmitted through the sample). The light source is usually a tungsten lamp for the visible region of the spectrum, and either a hydrogen or deuterium lamp for ultraviolet wavelengths. Cuvettes are optically transparent cells that hold the material(s) under study and are used to introduce samples into the light path. A reference cuvette optically identical to, and containing the same solvent (and impurities) as the test cuvette is always required for setting the spectrophotometer to read zero absorbance at each wavelength used. For accurate work, the optical matching of the two cuvettes should always be checked. Glass and plastic absorb strongly below 310 nm and are not useful for measuring absorbance below that wavelength. Quartz or silica cells are used when measuring absorption of ultraviolet wavelengths by a solution since they are transparent to wavelengths greater than 180 nm. When a cuvette is positioned in the light path it becomes an integral part of the instrument's optical system, so it should be treated with the same care given to other optical components. The use of scratched or contaminated cuvettes should be avoided since they reflect and/or absorb radiation that

3 will give you inaccurate measurements. Also, bubbles, turbidity, fingerprints, or condensation on, or inside, cuvettes should be avoided since they will diminish the accuracy of readings. Note: The cuvettes commonly used for accurate work have an optical path length of 1 cm and require 2.5 to 3.0 ml of sample for all accurate reading. This applies to the Spec 20 tubes that you will be using in this laboratory. Colorimetry. Many substances that do not absorb or have a very low absorbance in the visible region of the spectrum will react quantitatively with specific reagents to give a colored product that can be measured in the spectrophotometer. The absorbance (A) of such solutions can be plotted against the quantity or concentration (c) of the test substance producing the color. This graph is known as the calibration curve (or standard curve). Since it is often possible to produce comparatively high absorbances with relatively small amounts of material, colorimetry is widely used in biochemistry to assay a wide range of biologically important molecules. Some important points to bear in mind when carrying out colorimetry are as follows: (i) unlike straightforward spectrophotometry, colorimetry is a destructive technique; i.e., once reacted, the sample cannot be recovered; (ii) a chromophore reflects the complementary color(s) that it absorbs, i.e. a yellow compound appears yellow because it absorbs blue light and therefore it must be estimated in the blue region of the spectrum; (iii) colorimetric assays are usually most sensitive at the λ max of the chromophore produced. If this is not known, the spectrum needs to be determined before commencing the assay; (iv) reference cuvettes (i.e., blanks) should contain everything except the substance being assayed i.e., all the reagents in the same concentrations as in the test cuvettes; (v) the reference cuvette and its contents require, if anything, more care because any error in these will be reflected in all of the values obtained; (vi) assays should normally be performed in duplicate or triplicate, and individual values, not means, should be plotted. This procedure allows the experimenter to justifiably omit erroneous values from the calibration curve; (vii) the line of best fit should be drawn through the data points, not necessarily the line that passes through the origin and the other points. (viii) calibration curves may vary as batches of reagents and standards vary. Therefore, new calibration curves should be prepared each time an assay is run; (ix) calibration curves should never be extrapolated beyond the highest absorbance value measured. It is always more accurate to repeat an assay at a concentration which falls within the most accurate region of the calibration curve (i.e., you may need to dilute a solution to accomplish this). This is usually from 0.05 to 0.70.

5 2. Calculate the extinction coefficient (ε) of the standards. The extinction coefficient (ε) in the Beer-Lambert equation is a measure of how strongly a compound in solution absorbs light at a particular wavelength. In other words, at equal molar concentrations in solution, a compound that has an ε = 35,000 at the λ max for that compound, absorbs light more strongly than another compound with ε = 5,000 at its λ max. Extinction coefficients are useful numbers that allow researchers to calculate the concentration of a compound in solution (quantitative analysis). You will be using spectrophotometers to calculate the concentration of compounds in solution throughout the term, so the investment of time in this lab will save you time in the future. The Beer-Lambert equation states that A=εcl at the λ max of a compound. Therefore, if you know A at the λ max, c, and l (always equal to 1 cm for Spec 20 s), you can calculate ε by rearranging the Beer-Lambert equation as follows: ε=a/cl By the same reasoning, if you know A, ε and l, you will be able to calculate the molar concentration (c) of a compound in solution. a. Calculate the molar concentration (c) of your undiluted standard compound. i) The concentration of each of the three standards is 10 mg/ml. ii) The molecular weights of the three colored compounds are: b. Calculate the molar concentration of the diluted standard. Determine the molar concentration of standard that gave an A 0.6 at the λ max. You will need to use the initial concentration of your standard (expressed in mol/l) and the dilution data from Part 1. c. Substitute your calculated values for A at λ max, c and l and solve for ε. Note: Don't forget all the good stuff you learned about significant figures and error in previous labs. Report your estimate of ε with the number of significant figures you can justify. Where would you expect the greatest amount of error to occur? During the dilution? During calculation of the molar concentration? During the absorbance measurement? Be sure to finish your calculations of the molar extinction coefficient BEFORE going on to the next part of the laboratory. 3. Determine the concentration of proteins in solution using a colorimetry. The Bradford assay can be used to detect and quantify the level of proteins in solution. Cytochrome c is a red colored protein that you analysed earlier by determining its spectrum in the visible range. It is red because it binds a porphyrin group similar to the heme in hemoglobin that makes your blood red. The protein portion of cytochrome c is colorless because it does not absorb in the visible range. Proteins containing tryptophan, phenylalanine and tyrosine absorb at about 280 nm due to the π electrons characteristic of the conjugated double bonds in their R groups. One way to estimate the concentration of proteins in solution is to measure absorbance at 280 nm. Unfortunately, this technique is extremely inaccurate for proteins because of the varying levels of tryptophan, phenylalanine and tyrosine in many proteins. Because of such inaccuracies, a variety of assays have

7 APPENDIX Spectra and their Biochemical Usefulness. Molecules interact differentially with radiation of different wavelengths (λ s) i.e., they absorb more strongly at specific λ s and less at others. An absorbance spectrum may be represented as a graph of the amount of energy absorbed by a system plotted against λ. Different types of instruments are required for the study of different regions of the spectrum. Some of these regions yield information of great use to the biochemist and are used routinely, whereas other regions requiring more sophisticated instrumentation and expertise are used only in detailed research of biological macromolecules. Visible and UV spectra arise due to the outer electrons of atoms changing between major electronic energy levels. When molecules absorb visible and ultraviolet light they change their rotational and vibrational energy levels (electrons are raised to unoccupied higher orbitals). These spectra are used routinely in biochemistry. Fluorescence spectra may also arise from when electrons that went to higher orbitals return to their ground state (lower) orbital, they release some of the energy as light. The rest is released as vibrational energy (heat). Vibration rotation spectra are caused by changes in the vibration energy levels. They occur in the near infrared region and may be accompanied by changes in the rotational energy levels. Such spectra are sometimes used in studies of the detailed structure of biological macromolecules in nonaqueous environments. Electron spin resonance spectra and nuclear magnetic resonance spectra arise due to changes in the direction of the spins of electrons and nuclei respectively in an intense magnetic field. These two types of spectra are valuable for studying the structure of biological molecules. Revised 09/02 krd and jcm

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