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4.4: Espectrometría de masas

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  • <p style="text-align: justify;">Nuestra t&eacute;cnica anal&iacute;tica para la tercera y &uacute;ltima discusi&oacute;n en este cap&iacute;tulo <em>no </em>corresponde con la definici&oacute;n de la espectroscopia,&nbsp;ya que no involucra la absorbancia de la luz por una mol&eacute;cula.&nbsp;En la espectrometr&iacute;a de masas&nbsp;(EM),&nbsp;estamos interesados en la masa&nbsp;-&nbsp;y por lo tanto en el peso molecular -&nbsp;de nuestro compuesto de inter&eacute;s,&nbsp;y, a menudo la masa de los fragmentos que se producen cuando la mol&eacute;cula es causada a separarse.&nbsp;</p> <p style="text-align: justify;">&nbsp;</p> <div id="section_9"> <h3>4.4A: Los fundamentos de una espectrometr&iacute;a de masas&nbsp;</h3> <p style="text-align: justify;">Hay muchos tipos diferentes de instrumentos EM,&nbsp;pero todos ellos tienen los mismos tres componentes esenciales.&nbsp;Primero,&nbsp;hay una fuente de ionizaci&oacute;n,&nbsp;donde&nbsp;la mol&eacute;cula se le da una carga el&eacute;ctrica positiva,&nbsp;ya sea con&nbsp;extrayendo un electr&oacute;n o al a&ntilde;adir un prot&oacute;n.&nbsp;Dependiendo del m&eacute;todo de ionizaci&oacute;n utilizado,&nbsp;la mol&eacute;cula ionizada podr&iacute;a o no romperse en una poblaci&oacute;n de fragmentos m&aacute;s peque&ntilde;os.&nbsp;En la figura de abajo,&nbsp;algunas de las mol&eacute;culas de la muestra permanecen enteras,&nbsp;mientras que otros se fragmentan en trozos m&aacute;s peque&ntilde;os.</p> <p style="text-align: justify;">Siguiente en la l&iacute;nea hay un analizador de masas,&nbsp;donde los fragmentos cati&oacute;nicos son separados seg&uacute;n su masa.&nbsp;</p> <p style="text-align: center;"><img alt="image054.png" class="internal default" height="223" src="/@api/deki/files/33795/=image053.png" width="735" /></p> <p style="text-align: justify;">Finalmente,&nbsp;hay un detector,&nbsp;que detecta y cuantifica el ion separado.&nbsp;</p> <p style="text-align: justify;">Una de las t&eacute;cnicas de EM&nbsp;m&aacute;s comunes&nbsp;utilizada&nbsp;en el laboratorio org&aacute;nico es<strong> ionizaci&oacute;n electr&oacute;nica</strong>.&nbsp;En la fuente de ionizaci&oacute;n,&nbsp;la mol&eacute;cula de muestra es bombardeada por una haz de electrones de alta energ&iacute;a,&nbsp;que tiene el efecto de golpear un electr&oacute;n de valencia fuera de la mol&eacute;cula para formar un <strong>cati&oacute;n radical</strong>.&nbsp;Debido a que una gran cantidad de energ&iacute;a es transferida por este proceso de bombardeo,&nbsp;el cati&oacute;n radical r&aacute;pidamente comienza a romperse en fragmentos m&aacute;s peque&ntilde;os,&nbsp;algunas de ellas est&aacute;n cargadas positivamente y algunas de ellas son neutrales.&nbsp;Los fragmentos neutros son adsorbidos sobre las paredes de la c&aacute;mara o son eliminado por una fuente de aspiradora.&nbsp;En el componente de analizador de masas,&nbsp;los fragmentos cargados positivamente&nbsp;y cualquier&nbsp;<strong>iones moleculares</strong> no fragmentados&nbsp;restante&nbsp;son acelerados trav&eacute;s de un tubo por un campo el&eacute;ctrico.&nbsp;</p> <p style="text-align: center;"><img alt="image056.png" class="internal default" height="365" src="/@api/deki/files/33793/=image055.png" width="647" /></p> <p style="text-align: justify;">Este tubo est&aacute; curvado,&nbsp;y los iones son desviados por un campo magn&eacute;tico fuerte.&nbsp;Los iones de distinta proporci&oacute;n de masa a carga (m/z) son desviados a un grado diferente,&nbsp;lo que resulta en la &quot;clasificaci&oacute;n&quot; de los iones deacuerdo su masa&nbsp;(pr&aacute;cticamente todos los iones tienen cargas de z = 1&nbsp;por lo que la clasificaci&oacute;n de la masa a carga es la misma cosa que la clasificaci&oacute;n de la&nbsp;masa). Un detector al final del tubo de vuelo curvadao registra y cuantifica los iones ordenados.&nbsp;</p> </div> <div id="section_10"> <h3>4.4B: Mirando a los espectros de masas</h3> <p style="text-align: justify;">A continuaci&oacute;n se muestra un resultado t&iacute;pico de un experimento EM de ionizaci&oacute;n electr&oacute;nica (datos EM de la secci&oacute;n se derivan de el <a class="external" href="http://riodb01.ibase.aist.go.jp/sdbs/" rel="external nofollow" target="_blank" title="http://riodb01.ibase.aist.go.jp/sdbs/">Spectral Database for Organic Compounds</a>, un servicio&nbsp;por&nbsp;Internet gratuito proporcionado por AIST en Jap&oacute;n).</p> <p style="text-align: center;"><img alt="image058.png" class="internal default" height="335" src="/@api/deki/files/33791/=image057.png" width="635" /></p> <p style="text-align: justify;">La muestra es acetona.&nbsp;En el eje horizontal esta el valor de m/z (como hemos dicho anteriormente,&nbsp;la carga z es casi siempre 1,&nbsp;por lo que en la pr&aacute;ctica, esto es lo mismo que la masa).&nbsp;En el eje vertical es la abundancia relativa de cada ion detectado.&nbsp;En esta escala,&nbsp;el ion m&aacute;s abundante,&nbsp;llamado el <strong>pico de base</strong>,&nbsp;est&aacute; establecido en 100%,&nbsp;y todos los otros picos se registran en relaci&oacute;n con este valor.&nbsp;Para acetona,&nbsp;el pico de base est&aacute; en m/z = 43 -&nbsp;vamos a discutir la formaci&oacute;n de este fragmento un poco m&aacute;s tarde.&nbsp;El peso molecular de la acetona es 58,&nbsp;por lo que podemos identificar el pico a m/z = 58 como la correspondiente al pico del ion molecular, o pico principal<span class="rangySelectionBoundary" id="selectionBoundary_1461728649225_6821270920340012" style="line-height: 0; display: none;">&#65279;</span><span class="rangySelectionBoundary" id="selectionBoundary_1461728643143_33700495491622484" style="line-height: 0; display: none;">&#65279;</span></p> <p style="text-align: justify;">The sample is acetone.&nbsp; On the horizontal axis is the value for m/z (as we stated above, the charge z is almost always +1, so in practice this is the same as mass). &nbsp;On the vertical axis is the relative abundance of each ion detected.&nbsp; On this scale, the most abundant ion, called the <strong>base peak</strong>, is set to 100%, and all other peaks are recorded relative to this value. For acetone, the base peak is at m/z = 43 - we will discuss the formation of this fragment a bit later.&nbsp; The molecular weight of acetone is 58, so we can identify the peak at m/z = 58 as that corresponding to the <strong>molecular ion peak</strong>, or <strong>parent peak</strong>.&nbsp; Notice that there is a small peak at m/z = 59: this is referred to as the <strong>M+1 peak</strong>.&nbsp; How can there be an ion that has a greater mass than the molecular ion?&nbsp; Simple: a small fraction - about 1.1% - of all carbon atoms in nature are actually the <sup>13</sup>C rather than the <sup>12</sup>C isotope. The <sup>13</sup>C isotope is, of course, heavier than <sup>12</sup>C&nbsp; by 1 mass unit.&nbsp; In addition, about 0.015% of all hydrogen atoms are actually deuterium, the <sup>2</sup>H isotope.&nbsp;&nbsp; So the M+1 peak represents those few acetone molecules in the sample which contained either a <sup>13</sup>C or <sup>2</sup>H.&nbsp; &nbsp;</p> <p style="text-align: justify;">Molecules with lots of oxygen atoms sometimes show a small <strong>M+2 peak</strong> (2 m/z units greater than the parent peak) in their mass spectra, due to the presence of a small amount of <sup>18</sup>O (the most abundant isotope of oxygen is <sup>16</sup>O). Because&nbsp; there are two abundant isotopes of both chlorine (about 75% <sup>35</sup>Cl and 25% <sup>37</sup>Cl) and bromine (about 50% <sup>79</sup>Br and 50% <sup>81</sup>Br), chlorinated and brominated compounds have very large and recognizable&nbsp; M+2 peaks. Fragments containing both isotopes of Br can be seen in the mass spectrum of ethyl bromide:&nbsp;</p> <p style="text-align: center;"><img alt="image060.png" class="internal default" height="307" src="/@api/deki/files/33789/=image059.png" width="647" /></p> <p style="text-align: justify;">Much of the utility in electron-ionization MS comes from the fact that the radical cations generated in the electron-bombardment process tend to fragment in predictable ways.&nbsp; Detailed analysis of the typical fragmentation patterns of different functional groups is beyond the scope of this text, but it is worthwhile to see a few representative examples, even if we don&rsquo;t attempt to understand the exact process by which the fragmentation occurs.&nbsp; We saw, for example, that the base peak in the mass spectrum of acetone is m/z = 43.&nbsp; This is the result of cleavage at the &lsquo;alpha&rsquo; position - in other words, at the carbon-carbon bond adjacent to the carbonyl.&nbsp; Alpha cleavage results in the formation of an acylium ion&nbsp; (which accounts for the base peak at m/z = 43) and a methyl radical, which is neutral and therefore not detected.</p> <p style="text-align: center;"><img alt="image062.png" class="internal default" height="153" src="/@api/deki/files/33787/=image061.png" width="608" /></p> <p style="text-align: justify;">After the parent peak and the base peak, the next largest peak, at a relative abundance of 23%, is at m/z = 15.&nbsp; This, as you might expect, is the result of formation of a methyl cation, in addition to an acyl radical (which is neutral and not detected).&nbsp;</p> <p style="text-align: center;"><img alt="image064.png" class="internal default" height="116" src="/@api/deki/files/33785/=image063.png" style="" width="431" /></p> <p style="text-align: justify;">A common fragmentation pattern for larger carbonyl compounds is called the <strong>McLafferty rearrangement</strong>:</p> <p style="text-align: center;"><img alt="image066.png" class="internal default" height="142" src="/@api/deki/files/33783/=image065.png" style="" width="720" /></p> <p style="text-align: justify;">The mass spectrum of 2-hexanone shows a 'McLafferty fragment' at m/z = 58, while the propene fragment is not observed because it is a neutral species (remember, only cationic fragments are observed in MS). The base peak in this spectrum is again an acylium ion.</p> <p style="text-align: center;"><img alt="image068.png" class="internal default" height="318" src="/@api/deki/files/33781/=image067.png" width="654" /></p> <p style="text-align: justify;">When alcohols are subjected to electron ionization MS, the molecular ion is highly unstable and thus a parent peak is often not detected.&nbsp; Often the base peak is from an &lsquo;oxonium&rsquo; ion.</p> <p style="text-align: center;"><img alt="image070.png" class="internal default" height="150" src="/@api/deki/files/33779/=image069.png" width="620" /></p> <p style="text-align: center;"><img alt="image072.png" class="internal default" height="290" src="/@api/deki/files/33777/=image071.png" width="638" /></p> <p style="text-align: justify;">Other functional groups have predictable fragmentation patterns as well. By carefully analyzing the fragmentation information that a mass spectrum provides, a knowledgeable spectrometrist can often &lsquo;put the puzzle together&rsquo; and make some very confident predictions about the structure of the starting sample.</p> <div align="center">&nbsp;</div> <div><span class="script">template.ExampleStart()</span></div> <div id="section_11"> <p style="text-align: justify;"><u>Exercise 4.8</u>: The mass spectrum of an aldehyde gives prominent peaks at <em>m/z</em> = 59 (12%, highest value of <em>m/z</em> in the spectrum),&nbsp; 58 (85%), and 29 (100%), as well as others.&nbsp; Propose a structure, and identify the three species whose <em>m/z</em> values were listed.</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> <p><span class="script">template.ExampleEnd()</span></p> <p>&nbsp;</p> <h3>4.4C: Gas Chromatography - Mass Spectrometry</h3> <p style="text-align: justify;">Quite often, mass spectrometry is used in conjunction with a separation technique called gas chromatography (GC).&nbsp; The combined GC-MS procedure is very useful when dealing with a sample that is a mixture of two or more different compounds, because the various compounds are separated from one another before being subjected individually to MS analysis.&nbsp; We will not go into the details of gas chromatography here, although if you are taking an organic laboratory course you might well get a chance to try your hand at GC, and you will almost certainly be exposed to the conceptually analogous techniques of thin layer and column chromatography.&nbsp; Suffice it to say that in GC, a very small amount of a liquid sample is vaporized, injected into a long, coiled metal column, and pushed though the column by helium gas.&nbsp; Along the way, different compounds in the sample stick to the walls of the column to different extents, and thus travel at different speeds and emerge separately from the end of the column.&nbsp; In GC-MS, each purified compound is sent directly from the end of GC column into the MS instrument, so in the end we get a separate mass spectrum for each of the compounds in the original mixed sample.&nbsp; Because a compound's MS spectrum is a very reliable and reproducible&nbsp; 'fingerprint', we can instruct the instrument to search an MS database and identify each compound in the sample.</p> <p style="text-align: justify;">The extremely high sensitivity of modern GC-MS instrumentation makes it possible to detect and identify very small trace amounts of organic compounds.&nbsp; GC-MS is being used increasingly by environmental chemists to detect the presence of harmful organic contaminants in food and water samples.&nbsp;&nbsp; Airport security screeners&nbsp; also use high-speed GC-MS instruments to look for residue from bomb-making chemicals on checked luggage.&nbsp;</p> <p style="text-align: justify;">&nbsp;</p> </div> </div> <div id="section_12"> <h3>4.4D: Mass spectrometry of proteins - applications in proteomics</h3> <p style="text-align: justify;">Mass spectrometry has become in recent years an increasingly important tool in the field of <strong>proteomics</strong>.&nbsp; Traditionally, protein biochemists tend to study the structure and function of individual proteins.&nbsp; Proteomics researchers, in contrast, want to learn more about how large numbers of proteins in a living system interact with each other, and how they respond to changes in the state of the organism.&nbsp; One very important subfield of proteomics is the search for protein <strong>biomarkers</strong> for human disease.&nbsp; These can be proteins which are present in greater quantities in a sick person than in a healthy person, and their detection and identification can provide medical researchers with valuable information about possible causes or treatments.&nbsp;&nbsp; Detection in a healthy person of a known biomarker for a disease such as diabetes or cancer could also provide doctors with an early warning that the patient may be especially susceptible, so that preventive measures could be taken to prevent or delay onset of the disease.</p> <p style="text-align: justify;">New developments in MS technology have made it easier to detect and identify proteins that are present in very small quantities in biological samples.&nbsp; Mass spectrometrists who study proteins often use instrumentation that is somewhat different from the electron-ionization, magnetic deflection system described earlier.&nbsp; When proteins are being analyzed, the object is often to ionize the proteins <em>without</em> causing fragmentation, so 'softer' ionization methods are required.&nbsp; In one such method, called <strong>electrospray ionization</strong>, the protein sample, in solution, is sprayed into a tube and the molecules are induced by an electric field to pick up extra protons from the solvent.&nbsp; Another common 'soft ionization' method is 'matrix-assisted laser desorption ionization' (<strong>MALDI</strong>).&nbsp; Here, the protein sample is adsorbed onto a solid matrix, and protonation&nbsp; is achieved with a laser.</p> <p style="text-align: justify;">Typically, both electrospray ionization and MALDI are used in conjunction with a time-of-flight (TOF) mass analyzer component.&nbsp;</p> <p style="text-align: center;"><img alt="image074.png" class="internal default" height="198" src="/@api/deki/files/33775/=image073.png" width="750" /></p> <p style="text-align: justify;">The ionized proteins are accelerated by an electrode through a column, and separation is achieved because lighter ions travel at greater velocity than heavier ions with the same overall charge.&nbsp; In this way, the many proteins in a complex biological sample (such as blood plasma, urine, etc.) can be separated and their individual masses determined very accurately.&nbsp; Modern protein MS is extremely sensitive &ndash; very recently, scientists were even able to obtain a mass spectrum of <em>Tyrannosaurus rex</em> protein from fossilized bone! (<a href="http://www.sciencemag.org/content/31...2/277.abstract" title="http://www.sciencemag.org/content/31...2/277.abstract">Science <strong>2007</strong>, 316, 277</a>).</p> <p style="text-align: justify;">In one recent study,&nbsp; MALDI-TOF mass spectrometry was used to compare fluid samples from lung transplant recipients who had suffered from tissue rejection to control samples from recipients who had not suffered rejection.&nbsp; Three peptides (short proteins) were found to be present at elevated levels specifically in the tissue rejection samples.&nbsp; It is hoped that these peptides might serve as biomarkers to identify patients who are at increased risk of rejecting their transplanted lungs.&nbsp; (<a href="http://onlinelibrary.wiley.com/doi/1...01036/abstract" title="http://onlinelibrary.wiley.com/doi/1...01036/abstract">Proteomics <strong>2005</strong>, 5, 1705</a>).</p> </div> <p>&nbsp;</p> <p><span class="script"> Template.Soderberg() </span></p>