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4.3: Espectrometría ultravioleta y espectrometría visible

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  • <div id="section_5"> <p style="text-align: justify;">Mientras que la interacci&oacute;n con la luz infrarroja provoca que las mol&eacute;culas experimentan transiciones vibracionales,&nbsp;la longitud de onda m&aacute;s corta,&nbsp;radiaci&oacute;n de energ&iacute;a m&aacute;s alta&nbsp;en el UV&nbsp;(200-400 nm)&nbsp;y el&nbsp;rango&nbsp;visible&nbsp;(400-700 nm)&nbsp;del espectro electromagn&eacute;tico hace que muchas mol&eacute;culas org&aacute;nicas se sometan a <strong>transiciones electr&oacute;nicas</strong>.&nbsp;Lo que esto significa es que cuando la energ&iacute;a de la luz visible o UV es absorbida por una mol&eacute;cula,&nbsp;uno&nbsp;de sus electrones salta de una energ&iacute;a m&aacute;s baja a un&nbsp;orbital molecular de energ&iacute;a superior.</p> <h3>4.3A: Transiciones electr&oacute;nicas</h3> <p style="text-align: justify;">Tomemos como nuestro primer ejemplo el caso sencillo de hidr&oacute;geno molecular, H<sub>2</sub>.&nbsp;Como recordar&aacute;s de la secci&oacute;n 2.1A,&nbsp;la imagen orbital molecular para la mol&eacute;cula de hidr&oacute;geno consiste en una uni&oacute;n MO &sigma;,&nbsp;y una energ&iacute;a mayor antienlazante &sigma;* MO.&nbsp;Cuando la mol&eacute;cula est&aacute; en el estado fundamental,&nbsp;ambos electrones est&aacute;n emparejados en el enlace orbital de la m&aacute;s baja energ&iacute;a&#8203; - &nbsp;este es el Orbital Molecular Ocupado M&aacute;s Alto (HOMO).&nbsp;El orbital antienlazante &sigma;*, a su vez, es el Orbital Molecular Desocupado&nbsp;M&aacute;s Bajo (LUMO).</p> <p style="text-align: center;"><img alt="image024.png" class="internal default" height="212" src="/@api/deki/files/33825/=image023.png" style="" width="535" /></p> <p style="text-align: justify;">Si la mol&eacute;cula se expone a luz de una longitud de onda con una energ&iacute;a igual a&nbsp;&Delta;E,&nbsp;la brecha de energ&iacute;a HOMO-LUMO,&nbsp;esta longitud de onda ser&aacute; absorbida y la energ&iacute;a utilizada para topar uno de los electrones desde el HOMO a LUMO -&nbsp;en otras palabras, del orbital &sigma; a el&nbsp;&sigma;*.&nbsp;Esto se conoce como una&nbsp;<strong>transici&oacute;n&nbsp;&sigma; - &sigma;*</strong>.&nbsp;&Delta;E&nbsp;para esta transici&oacute;n electr&oacute;nica es 258 kcal / mol,&nbsp;correspondiente a la luz con una longitud de onda de 111 nm.</p> <p style="text-align: justify;">Cuando una mol&eacute;cula con doble enlace tal como eteno&nbsp;(su nombre com&uacute;n es etileno)&nbsp;absorbe la luz,&nbsp;se somete a una <strong>transici&oacute;n&nbsp;&nbsp;&pi; - &pi;*.&nbsp;</strong>Debido a que los intervalos de&nbsp;energ&iacute;a&nbsp;&pi;-&nbsp;&pi;*&nbsp;son m&aacute;s estrechos que&nbsp;las&nbsp;brechas&nbsp;&sigma;&nbsp;-&nbsp;&sigma;*,&nbsp;eteno absorbe luz a 165 nm -&nbsp;una longitud de onda m&aacute;s larga que el hidr&oacute;geno molecular.&nbsp;</p> <p style="text-align: center;"><img alt="image026.png" class="internal default" height="162" src="/@api/deki/files/33823/=image025.png" width="597" /></p> <p style="text-align: justify;">Las transiciones electr&oacute;nicas</p> <p style="text-align: justify;">The electronic transitions of both molecular hydrogen and ethene are too energetic to be accurately recorded by standard UV spectrophotometers, which generally have a range of 220 &ndash; 700 nm.&nbsp; Where UV-vis spectroscopy becomes useful to most organic and biological chemists is in the study of molecules with conjugated pi systems.&nbsp; In these groups, the energy gap for <span style="font-family: times new roman,times,serif;">&pi;</span> -<span style="font-family: times new roman,times,serif;">&pi;</span>* transitions is smaller than for isolated double bonds, and thus the wavelength absorbed is longer.&nbsp; Molecules or parts of molecules that absorb light strongly in the UV-vis region are called <strong>chromophores</strong>.</p> <p style="text-align: justify;">Let&rsquo;s revisit the MO picture for 1,3-butadiene, the simplest conjugated system (see <a href="Core/Organic_Chemistry/Organic_Chemistry_With_a_Biological_Emphasis/Chapter_02:_Introduction_to_organic_structure_and_bonding_II/Section_2.2:_Molecular_orbital_theory:_conjugation_and_aromaticity" title="Organic Chemistry/Organic Chemistry With a Biological Emphasis/Chapter 2: Introduction to organic structure and bonding II/Section 2.1: Molecular orbital theory: conjugation and aromaticity">section 2.1B</a>).&nbsp; Recall that we can draw a diagram showing the four pi MO&rsquo;s that result from combining the four 2p<sub>z</sub> atomic orbitals. The lower two orbitals are bonding, while the upper two are antibonding.</p> <p style="text-align: center;"><img alt="image028.png" class="internal default" height="183" src="/@api/deki/files/33821/=image027.png" width="553" /></p> <p style="text-align: justify;">Comparing this MO picture to that of ethene, our isolated pi-bond example, we see that the HOMO-LUMO energy gap is indeed smaller for the conjugated system. 1,3-butadiene absorbs UV light with a wavelength of 217 nm.</p> <p style="text-align: justify;">As conjugated pi systems become larger, the&nbsp; energy gap for a <span style="font-family: times new roman,times,serif;">&pi;</span> - <span style="font-family: times new roman,times,serif;">&pi;</span>* transition becomes increasingly narrow, and the wavelength of light absorbed correspondingly becomes longer.&nbsp;&nbsp; The absorbance due to the <span style="font-family: times new roman,times,serif;">&pi;</span> - <span style="font-family: times new roman,times,serif;">&pi;</span>* transition in 1,3,5-hexatriene, for example, occurs at 258 nm, corresponding to a <span style="font-family: times new roman,times,serif;"><strong>&Delta;</strong></span>E of 111 kcal/mol.</p> <p style="text-align: center;"><img alt="image030.png" class="internal default" height="221" src="/@api/deki/files/33819/=image029.png" width="605" /></p> <p style="text-align: justify;">In molecules with extended pi systems, the HOMO-LUMO energy gap becomes so small that absorption occurs in the visible rather then the UV region of the electromagnetic spectrum.&nbsp; Beta-carotene, with its system of 11 conjugated double bonds,&nbsp; absorbs light with wavelengths in the blue region of the visible spectrum while allowing other visible wavelengths &ndash; mainly those in the red-yellow region - to be transmitted. This is why carrots are orange.</p> <p style="text-align: center;"><img alt="image032.png" class="internal default" height="136" src="/@api/deki/files/33817/=image031.png" width="572" /></p> <p style="text-align: justify;">The conjugated pi system in 4-methyl-3-penten-2-one gives rise to a strong UV absorbance at 236 nm due to a <span style="font-family: times new roman,times,serif;">&pi;</span> - <span style="font-family: times new roman,times,serif;">&pi;</span>* transition.&nbsp; However, this molecule also absorbs at 314 nm.&nbsp; This second absorbance is due to the transition of a non-bonding (lone pair) electron on the oxygen up to a <span style="font-family: times new roman,times,serif;">&pi;</span>* antibonding MO:</p> <p style="text-align: center;"><img alt="image034.png" class="internal default" height="203" src="/@api/deki/files/33815/=image033.png" width="654" /></p> <p style="text-align: justify;">This is referred to as an <strong>n</strong><strong> - <span style="font-family: times new roman,times,serif;">&pi;</span></strong><strong>* transition</strong>.&nbsp; The nonbonding (n) MO&rsquo;s are higher in energy than the highest bonding p orbitals, so the energy gap for an n - <span style="font-family: times new roman,times,serif;">&pi;</span>* transition is smaller that that of a <span style="font-family: times new roman,times,serif;">&pi;</span> - <span style="font-family: times new roman,times,serif;">&pi;</span>* transition &ndash; and thus the n - <span style="font-family: times new roman,times,serif;">&pi;</span>* peak is at a longer wavelength.&nbsp; In general, n - <span style="font-family: times new roman,times,serif;">&pi;</span>* transitions are weaker (less light absorbed) than those due to <span style="font-family: times new roman,times,serif;">&pi; - </span><span style="font-family: times new roman,times,serif;">&pi;</span>* transitions.</p> <div>&nbsp;</div> <div> <div> <div class="exercise"> <p class="boxtitle" >Exercise 4.3</p> <p style="text-align: justify; visibility: visible;">How large is the <span style="font-family: times new roman,times,serif;">&pi; - </span><span style="font-family: times new roman,times,serif;">&pi;</span>* transition in 4-methyl-3-penten-2-one?</p> <p style="text-align: justify; visibility: visible;"><a href="Core/Organic_Chemistry/Organic_Chemistry_With_a_Biological_Emphasis/Solution_Manual/Chapter_04_Solutions" title="Organic Chemistry/Organic Chemistry With a Biological Emphasis/Solution Manual/Chapter 4 Solutions">Solution</a></p> </div> </div> </div> <div> <div class="exercise"> <p class="boxtitle" >Exercise 4.4</p> <p style="text-align: justify;">Which of the following molecules would you expect absorb at a longer wavelength in the UV region of the electromagnetic spectrum? Explain your answer.</p> <p style="text-align: center;"><img alt="image036.png" class="internal default" height="142" src="/@api/deki/files/33813/=image035.png" width="280" /></p> <div>&nbsp;</div> <div>&nbsp;</div> <div><a href="Core/Organic_Chemistry/Organic_Chemistry_With_a_Biological_Emphasis/Solution_Manual/Chapter_04_Solutions" title="Organic Chemistry/Organic Chemistry With a Biological Emphasis/Solution Manual/Chapter 4 Solutions">Solution</a></div> </div> </div> </div> <div id="section_6"> <h3>4.3B: Looking at UV-vis spectra</h3> <p style="text-align: justify;">We have been talking in general terms about how molecules absorb UV and visible light &ndash; now let's look at some actual examples of data from a UV-vis absorbance spectrophotometer. The basic setup is the same as for IR spectroscopy: radiation with a range of wavelengths is directed through a sample of interest, and a detector records which wavelengths were absorbed and to what extent the absorption occurred.&nbsp; Below is the absorbance spectrum of an important biological molecule called nicotinamide adenine dinucleotide, abbreviated NAD<sup>+ </sup>(we'll learn what it does in <a href="Core/Organic_Chemistry/Organic_Chemistry_With_a_Biological_Emphasis/Chapter_16:_Oxidation_and_reduction_reactions/Section_16.04:_Hydrogenation////dehydrogenation_reactions_of_carbonyls,_imines,_and_alcohols" title="Organic Chemistry/Organic Chemistry With a Biological Emphasis/Chapter 16: Oxidation and reduction reactions/Section 16.4: Hydrogenation//dehydrogenation reactions of carbonyls, imines, and alcohols">section 16.4</a>)&nbsp; This compound absorbs light in the UV range due to the presence of conjugated pi-bonding systems.</p> <p style="text-align: center;"><img alt="image038.png" class="internal default" height="405" src="/@api/deki/files/33811/=image037.png" width="700" /></p> <p style="text-align: justify;">You&rsquo;ll notice that this UV spectrum is much simpler than the IR spectra we saw earlier: this one has only one peak, although many molecules have more than one.&nbsp; Notice also that the convention in&nbsp; UV-vis spectroscopy is to show the&nbsp; baseline at the bottom of the graph with the peaks pointing up.&nbsp; Wavelength values on the x-axis are generally measured in nanometers (nm) rather than in cm<sup>-1</sup> as is the convention in IR spectroscopy.&nbsp;</p> <p style="text-align: justify;">Peaks in UV spectra tend to be quite broad, often spanning well over 20 nm at half-maximal height.&nbsp; Typically, there are two things that we look for and record from a UV-Vis spectrum..&nbsp; The first is <span style="font-size: 16px;"><span style="font-family: times new roman,times,serif;"><strong>&lambda;</strong></span></span><sub>max,</sub> which is the wavelength at maximal light absorbance.&nbsp; As you can see, NAD<sup>+</sup> has <span style="font-size: 16px;"><span style="font-family: times new roman,times,serif;"><strong>&lambda;</strong></span></span><sub>max</sub><sub>,</sub> = 260 nm.&nbsp; We also want to record how much light is absorbed at <span style="font-size: 16px;"><span style="font-family: times new roman,times,serif;"><strong>&lambda;</strong></span></span><sub>max</sub><sub>. </sub>&nbsp;Here we use a unitless number called <strong>absorbance</strong>, abbreviated 'A'.&nbsp; This contains the same information as the 'percent transmittance' number used in IR spectroscopy, just expressed in slightly different terms.&nbsp; To calculate absorbance at a given wavelength, the computer in the spectrophotometer simply takes the intensity of light at that wavelength <em>before</em> it passes through the sample (I<sub>0</sub>), divides this value by the intensity of the same wavelength <em>after</em> it passes through the sample (I), then takes the log<sub>10</sub> of that number:</p> <p style="text-align: center;">\[A = log \dfrac{I_0}{I}\]</p> <p style="text-align: justify;">You can see that the absorbance value at 260 nm (A<sub>260</sub>) is about 1.0 in this spectrum.&nbsp;</p> <div> <div class="exercise"> <p class="boxtitle" >Exercise 4.5</p> <p style="text-align: justify;">Express A = 1.0 in terms of percent transmittance (%T, the unit usually used in IR spectroscopy (and sometimes in UV-vis as well).</p> <p style="text-align: justify;"><a href="Core/Organic_Chemistry/Organic_Chemistry_With_a_Biological_Emphasis/Solution_Manual/Chapter_04_Solutions" title="Organic Chemistry/Organic Chemistry With a Biological Emphasis/Solution Manual/Chapter 4 Solutions">Solution</a></p> </div> </div> <p style="text-align: justify;">Here is the absorbance spectrum of the common food coloring Red #3:</p> <p style="text-align: justify;">&nbsp;</p> <p style="text-align: center;"><img alt="image040.png" class="internal default" height="426" src="/@api/deki/files/33809/=image039.png" width="682" /></p> <p style="text-align: justify;">Here, we see that the extended system of conjugated pi bonds causes the molecule to absorb light in the visible range.&nbsp; Because the&nbsp; <span style="font-size: 16px;"><span style="font-family: times new roman,times,serif;"><strong>&lambda;</strong></span></span><sub>max </sub>of 524 nm falls within the green region of the spectrum, the compound appears red to our eyes.</p> <p style="text-align: justify;">Now, take a look at the spectrum of another food coloring, Blue #1:</p> <p style="text-align: justify;">&nbsp;</p> <p style="text-align: center;"><img alt="image042.png" class="internal default" height="437" src="/@api/deki/files/33807/=image041.png" width="680" /></p> <p style="text-align: justify;">Here, maximum absorbance is at 630 nm, in the orange range of the visible spectrum, and the compound appears blue.&nbsp;</p> </div> <div id="section_7"> <h3>4.3C: Applications of UV spectroscopy in organic and biological chemistry</h3> <p style="text-align: justify;">UV-vis spectroscopy has many different applications in organic and biological chemistry.&nbsp; One of the most basic of these applications is the use of the <strong>Beer - Lambert Law</strong> to determine the concentration of a chromophore.&nbsp; You most likely have performed a Beer &ndash; Lambert experiment in a previous chemistry lab.&nbsp; The law is simply an application of the observation that, within certain ranges, the absorbance of a chromophore at a given wavelength varies in a linear fashion with its concentration: the higher the concentration of the molecule, the greater its absorbance. If we divide the observed value of A at <span style="font-size: 16px;"><span style="font-family: times new roman,times,serif;">&lambda;</span></span><sub>max</sub> by the concentration of the sample (<em>c</em>, in mol/L), we obtain the <strong>molar absorptivity</strong>, or <strong>extinction coefficient</strong> (<strong>&epsilon;</strong>), which is a characteristic value for a given compound.&nbsp;</p> <p style="text-align: center;">\[ &epsilon; = \dfrac{A}{c}\]</p> <p style="text-align: justify;">The absorbance will also depend, of course, on the <strong>path length</strong> - in other words, the distance that the beam of light travels though the sample.&nbsp; In most cases, sample holders are designed so that the path length is equal to 1 cm, so the units for molar absorptivity are&nbsp; mol * L<sup>-1</sup>cm<sup>-1</sup>.&nbsp; If we look up the value of e for our compound at <span style="font-size: 16px;"><span style="font-family: times new roman,times,serif;">&lambda;</span></span><sub>max</sub>, and we measure absorbance at this wavelength, we can easily calculate the concentration of our sample.&nbsp;&nbsp; As an example, for NAD<sup>+</sup> the literature value of <span style="font-size: 16px;"><span style="font-family: times new roman,times,serif;">&epsilon;</span></span> at 260 nm is 18,000 mol * L<sup>-1</sup>cm<sup>-1</sup>.&nbsp;&nbsp; In our NAD<sup>+</sup> spectrum we observed A<sub>260</sub> = 1.0, so using equation 4.4 and solving for concentration we find that our sample is 5.6 x 10<sup>-5</sup> M.&nbsp;&nbsp;</p> <div> <div class="exercise"> <p class="boxtitle" >Exercise 4.6</p> <p style="text-align: justify;">The literature value of <span style="font-size: 16px;"><span style="font-family: times new roman,times,serif;">&epsilon;</span></span> for 1,3-pentadiene in hexane is 26,000 mol * L<sup>-1</sup>cm<sup>-1</sup> at its maximum absorbance at 224 nm.&nbsp; You prepare a sample and take a UV spectrum, finding that A<sub>224</sub> = 0.850.&nbsp;&nbsp; What is the concentration of your sample?</p> <p style="text-align: justify;"><a href="Core/Organic_Chemistry/Organic_Chemistry_With_a_Biological_Emphasis/Solution_Manual/Chapter_04_Solutions" title="Organic Chemistry/Organic Chemistry With a Biological Emphasis/Solution Manual/Chapter 4 Solutions">Solution</a></p> </div> </div> The bases of DNA and RNA are good chromophores: <p style="text-align: center;"><img alt="image044.png" class="internal default" height="178" src="/@api/deki/files/33805/=image043.png" width="617" /></p> <p style="text-align: justify;">Biochemists and molecular biologists often determine the concentration of a DNA sample by assuming an average value of&nbsp; <span style="font-size: 16px;"><span style="font-family: times new roman,times,serif;">&epsilon;</span></span> = 0.020 ng<sup>-1</sup>&times;mL&nbsp;for double-stranded DNA at its <span style="font-size: 16px;"><span style="font-family: times new roman,times,serif;">&lambda;</span></span><sub>max</sub> of 260 nm (notice that concentration in this application is expressed in mass/volume rather than molarity:&nbsp; ng/mL is often a convenient unit for DNA concentration when doing molecular biology).</p> <div> <div class="exercise"> <p class="boxtitle" >Exercise 4.7</p> <p style="text-align: justify;">50 mL of an aqueous sample of double stranded DNA is dissolved in 950 mL of water.&nbsp; This diluted solution has a maximal absorbance of 0.326 at 260 nm.&nbsp; What is the concentration of the original (more concentrated) DNA sample, expressed in mg/mL?</p> <p style="text-align: justify;"><a href="Core/Organic_Chemistry/Organic_Chemistry_With_a_Biological_Emphasis/Solution_Manual/Chapter_04_Solutions" title="Organic Chemistry/Organic Chemistry With a Biological Emphasis/Solution Manual/Chapter 4 Solutions">Solution</a></p> </div> </div> <p style="text-align: justify;">Because the extinction coefficient of double stranded DNA is slightly lower than that of single stranded DNA, we can use UV spectroscopy to monitor a process known as DNA melting.&nbsp;&nbsp; If&nbsp; a short stretch of double stranded DNA is gradually heated up, it will begin to &lsquo;melt&rsquo;, or break apart, as the temperature increases (recall that two strands of DNA are held together by a specific pattern of hydrogen bonds formed by &lsquo;base-pairing&rsquo;).</p> <p style="text-align: justify;">&nbsp;</p> <p style="text-align: center;"><img alt="image046.png" class="internal default" height="357" src="/@api/deki/files/33803/=image045.png" width="557" /></p> <p style="text-align: justify;">As melting proceeds, the absorbance value for the sample increases, eventually reaching a high plateau as all of the double-stranded DNA breaks apart, or &lsquo;melts&rsquo;.&nbsp; The mid-point of this process, called the &lsquo;melting temperature&rsquo;, provides a good indication of how tightly the two strands of DNA are able to bind to each other.&nbsp;</p> <p style="text-align: justify;">In <a href="Core/Organic_Chemistry/Organic_Chemistry_With_a_Biological_Emphasis/Chapter_16:_Oxidation_and_reduction_reactions/Section_16.08:_Observing_the_progress_of_hydrogenation_and_dehydrogenation_reactions_by_UV_spectroscopy" title="Organic Chemistry/Organic Chemistry With a Biological Emphasis/Chapter 16: Oxidation and reduction reactions/Section 16.8: Observing the progress of hydrogenation and dehydrogenation reactions by UV spectroscopy">section 16.8</a> we will see how the Beer - Lambert Law and UV spectroscopy provides us with a convenient way to follow the progress of many different enzymatic redox (oxidation-reduction) reactions.&nbsp; In biochemistry, oxidation of an organic molecule often occurs concurrently with reduction of nicotinamide adenine dinucleotide (NAD<sup>+</sup>, the compound whose spectrum we saw earlier in this section) to NADH:</p> <p style="text-align: justify;">&nbsp;</p> <p style="text-align: center;"><img alt="image048.png" class="internal default" height="190" src="/@api/deki/files/33801/=image047.png" width="703" /></p> <p style="text-align: justify;">Both NAD<sup>+</sup> and NADH absorb at 260 nm.&nbsp; However NADH, unlike NAD<sup>+</sup>, has a second absorbance band with <span style="font-size: 16px;"><span style="font-family: times new roman,times,serif;">&lambda;</span></span><sub>max</sub> = 340 nm and <span style="font-size: 16px;"><span style="font-family: times new roman,times,serif;">&epsilon;</span></span> = 6290 mol L<sup>-1</sup>cm<sup>-1</sup>.&nbsp; The figure below shows the spectra of both compounds superimposed, with the NADH spectrum offset slightly on the y-axis:</p> <p style="text-align: center;"><img alt="image050.png" class="internal default" height="397" src="/@api/deki/files/33799/=image049.png" width="610" /></p> <p style="text-align: justify;">By monitoring the absorbance of a reaction mixture at 340 nm, we can 'watch' NADH being formed as the reaction proceeds, and calculate the rate of the reaction.</p> <p style="text-align: justify;">UV spectroscopy is also very useful in the study of proteins.&nbsp; Proteins absorb light in the UV range due to the presence of the aromatic amino acids tryptophan, phenylalanine, and tyrosine, all of which are chromophores.&nbsp;</p> <p style="text-align: center;"><img alt="image052.png" class="internal default" height="212" src="/@api/deki/files/33797/=image051.png" width="700" /></p> <p style="text-align: justify;">Biochemists frequently use UV spectroscopy to study conformational changes in proteins - how they change shape in response to different conditions. When a protein undergoes a conformational shift (partial unfolding, for example), the resulting change in the environment around an aromatic amino acid chromophore can cause its UV spectrum to be altered.</p> </div> <div> <h2 style="text-align: justify;">Contributors</h2> <p style="text-align: justify;"><span class="script">template.SpanishSoderberg()</span><span class="title mt-title-edit"><span class="script">template.TransVazquez()</span></span><span class="script">template.HideTOC()</span></p> </div>