Name: how dyes absorb visible light
Uploaded: 2020-03-26T07:22:29.000Z
Duration: 4 min 55 s
Channel: JoVE

当光到达某种物质时，一部分被它吸收，而其余部分则通过它被反射或透射。我们感知到的物质的颜色取决于它最有可能反射的波长。例如，一块我们看到的蓝色织物含有一种染料，这种染料会强烈反射蓝光并强烈吸收橙色和红色光。
染料通常是共轭化合物，这意味着它们具有交替的双键和单键。电子可以在共轭系统内自由移动。不同颜色的染料吸收的光波长必须不同。当我们看几个例子时，我们可以看到吸收的波长随着共轭量的增加而增加。
那么，波长与共轭程度有什么关系呢？让我们考虑一下分子能级。我们可以将离域电子视为占据分子轨道 （MO）。分子吸收光的能量正好是将电子激发到更高能量的分子轨道。最可能的转变是从最高占据的分子轨道（称为 HOMO）到最低的未占据分子轨道（LUMO）。因此，我们预计吸收最多的波长与 HOMO - LUMO 能隙相匹配。
很少或没有偶联的分子通常具有较大的 HOMO-LUMO 间隙。它们吸收紫外线并反射所有可见光，因此呈现白色或无色。共轭键通过降低分子的能级来稳定分子，尤其是在高能下。共轭程度越高，HOMO - LUMO 间隙越小，吸收波长越大。金属和替代品也会影响间隙。
让我们看一个例子。视黄醇有一个小的共轭系统，而叶绿素 a 有一个包含氮和镁的大系统。视黄醇在 325 nm 处吸收，而叶绿素 a 在 430 和 662 nm 处吸收。正如预期的那样，视黄醇的能量差距更大。
我们可以使用紫外和可见光或紫外-可见分光光度计来研究吸收。分光光度计由光源、一种控制样品接收波长的方法和一个光检测器组成。样品通常是透明溶液。吸光度可以在特定波长下测量，也可以在某个波长范围内测量，因为化合物通常在多个波长处吸收。此外，我们看到每个跃迁的波长范围，因为分子处于不同的方向和振动状态。
在测量过程中，光要么被吸收，要么在不接触任何分子的情况下穿过，要么从溶剂或化合物分子上反射。我们忽略了向后反射的少量光线。有时，可能被分子吸收的光反而会从分子上反射回来。我们描述了一种物质在特定波长下传输具有独特摩尔衰减系数的能力。虽然吸光度随浓度而变化，但摩尔衰减系数不会。
测量后，分光光度计以称为透射率的比率比较接收光和原始光。吸光度是透射率的负基 10 对数。如果分光光度计具有溶剂的吸光度，则会减去它以仅显示化合物。结果通常显示为吸光度与波长的关系。化合物吸收最多的波长称为 lambda max。如果我们计算每个波长的摩尔衰减系数，它在 lambda max 时最高。
摩尔衰减系数、吸光度、样品浓度和光程长度（即光穿过样品的距离）与 Beer-Lambert 定律相关。如果我们知道任何三个变量，我们就可以计算第四个变量。
在本实验中，您将使用紫外-可见分光光度计分析荧光素、β-胡萝卜素和靛蓝染料的吸收特性。然后，您将使用 Beer-Lambert 定律创建 β-胡萝卜素校准曲线，然后确定 β-胡萝卜素溶液的浓度。

染料的紫外-可见光谱

吸光度
当光与物质相互作用时，一部分光被吸收，而其余部分则通过它被反射或透射。我们认为具有某种颜色的物质会反射可见光范围内的光。我们能够看到的物质的颜色取决于反射的光的波长。我们认为是蓝色的物质会反射可见光谱蓝色范围 （430 – 480 nm） 的光。根据色轮，相同的物质吸收与反射光互补的光。因此，蓝色物质吸收可见光谱橙色区域 （590 – 630 nm） 中的光。并非所有化合物都能在可见区域吸收，因此，它们在人眼中看起来是无色的。
光由其能量 E 和波长 λ 定义。其中，h 是普朗克常数，c 是光速。
<img src="/CDNSource/lm/labs/70/70_Concepts_1.jpg" alt="能量方程 \（E = \frac{hc}{\lambda}\）、公式、波长、光子能量计算" data-altupdatedat="1754907201000">
光的波长与其能量成反比。因此，高能量光具有较短的波长。
<h4>染料</h4>
不同颜色的染料吸收的光波长不同。大多数染料是具有交替双键和单键的偶联化合物，通常在可见光区域吸收光。
染料分子的共轭部分可以很短，这意味着共轭程度低，双键和单键交替很少，或者长，意味着有许多交替的双键和单键高度共轭。这些交替的双键不一定只在两个碳之间。这些共轭键可以包括羰基以及碳和氧之间的双键。共轭程度决定了化合物吸收的光的波长。例如，具有高度共轭的化合物比具有较低共轭度的化合物吸收更长的波长。
基于分子轨道理论，离域电子占据分子轨道。最高占据的分子轨道或 HOMO 是具有电子的最高能量轨道。最低的未占据分子轨道 （LUMO） 是没有电子的最低能量轨道。很少或没有偶联的分子通常在 HOMO 和 LUMO 之间具有较大的能隙。然而，共轭分子在 HOMO 和 LUMO 之间的能隙较小。
为了将电子从较低能级激发到较高能级，或从 HOMO 激发到 LUMO，分子必须吸收能量等于两个轨道之间能隙的光。因此，具有较大能隙的分子需要更高能量的光（例如紫外光）来激发电子。然而，染料具有较小的能隙，并且需要较低能量的光（例如可见光）来激发电子。
<img src="/CDNSource/lm/labs/70/70_Concepts_2.jpg" alt="能隙方程ΔE（HOMO-LUMO）= hc/λ;分子光谱学，量子化学。" data-altupdatedat="1754907201000">
因此，具有较大能隙的分子需要更高能量的光（例如紫外光）来激发电子。然而，染料具有较小的能隙，并且需要较低能量的光（例如可见光）来激发电子。
回想一下，光的能量与波长成反比。因此，高能量光的波长较短，而低能量光的波长较长。
<h4>分光光度计</h4>
在实验中，使用 UV-Visible （UV-Vis） 分光光度计测量吸光度。该仪器利用光源，该光源由光栅转换为特定波长的光，这些光将穿过样品并进入另一端的检测器。样品必须在液体中，因此如果有机化合物是固体，则需要溶剂。该溶液保存在称为比色皿的样品架中。根据样品的不同，比色皿可能由石英晶体、玻璃或塑料制成，并具有特定的光程。此 pathlength 是光线必须穿过样本的距离。由于溶剂也会吸收光，因此需要单独的溶剂样品空白。因此，当仪器捕获样品化合物的吸光度光谱时，它可以减去溶剂的背景光谱，以显示仅由样品引起的吸光度。透射率 （T） 是穿过样品的原始光的分数。
<img src="/CDNSource/lm/labs/70/70_Concepts_3.jpg" alt="透射率方程T=P/P₀公式，光谱分析和光学研究的关键。" data-altupdatedat="1754907201000">
其中，P0 是光束在照射样品之前的辐照度或每单位面积每秒的能量。P 是照射到探测器的光束的辐照度。P 通常低于 P0，因为一些光被样品吸收。
吸光度 A 定义为透射率的负对数。
<img src="/CDNSource/lm/labs/70/70_Concepts_4.jpg" alt="吸光度公式：A=log（P₀/P）=-logT;光谱分析中的光学原理。" data-altupdatedat="1754907201000">
吸光度的值范围介于 0（无吸收）和 2（99% 吸收）之间。当没有光被吸收时，P0 等于 P，透射率等于 1。因此，吸光度为零。如果 90% 的光被吸收，则透射 10%，T 等于 0.1。这导致吸光度等于 1。如果 99% 的光被吸收，则 1% 的光被透射 （T= 0.01），吸光度等于 2。
获得的光谱是吸光度与波长的关系图。对于紫外-可见分光光度计，此范围在 200 至 800 nm 之间。
<h4>比尔-兰伯定律</h4>
特定化合物的透射率和吸光度与化合物在溶液中的浓度有关。这种关系由 Beer-Lambert 定律描述。
<img src="/CDNSource/lm/labs/70/70_Concepts_5.jpg" alt="比尔-朗伯定律方程; A=εcl;光谱学中的吸收图;光学研究。" data-altupdatedat="1754907201000">
样品的吸光度等于化合物浓度、光程和摩尔衰减系数的乘积。该系数对于每种化合物都是唯一的，并且会因波长而异。但是，如果波长保持不变，则无论浓度如何变化，摩尔衰减系数都将相同。对应于样品最高吸光度的波长（称为 λmax）也将具有最大的摩尔衰减系数。
<h4>引用</h4>
<ol>
	<li style="font-size:17px">Silberberg， M.S. （2012）.化学:物质和变化的分子性质。马萨诸塞州波士顿:麦格劳·希尔。</li>
	<li style="font-size:17px">哈里斯，哥伦比亚特区（2015 年）。 定量化学分析。 纽约州纽约市:W.H. Freeman and Company。</li>
</ol>

吸光度
当光与物质相互作用时，一部分光被吸收，而其余部分则通过它被反射或透射。...

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UV-Vis Spectroscopy of Dyes

how dyes absorb visible light

Dyes absorb visible light because their molecular structure lets them interact with specific wavelengths. When light hits a substance, some light is absorbed and the rest is reflected or transmitted. The color we see depends on the wavelength that is reflected. A blue substance reflects blue light, around 430 to 480 nm, and absorbs the complementary orange region, around 590 to 630 nm. Compounds that do not absorb in the visible range appear colorless to the human eye. Light also has energy and wavelength, and higher-energy light has a shorter wavelength.

Most dyes are conjugated compounds. This means they contain alternating double and single bonds, and in some cases carbonyl groups and carbon-oxygen double bonds also take part in the conjugated system. The length of the conjugated region affects the wavelength the dye absorbs. A dye with more conjugation absorbs a longer wavelength than a dye with less conjugation. This happens because delocalized electrons occupy molecular orbitals. The highest occupied molecular orbital, or HOMO, is the highest-energy orbital with an electron, and the lowest unoccupied molecular orbital, or LUMO, is the next available empty orbital.

Molecules with little or no conjugation usually have a large energy gap between the HOMO and LUMO. Conjugated molecules have a smaller gap. An electron must absorb light with the same energy as that gap to move from the HOMO to the LUMO. Because of this, molecules with a large gap need higher-energy UV light, while dyes with a smaller gap can be excited by lower-energy visible light. Since light energy and wavelength are inversely related, higher-energy light has a shorter wavelength.

Absorbance is measured experimentally with a UV-Visible, or UV-Vis, spectrophotometer. The instrument uses a light source and a monochromator to select specific wavelengths. That light passes through a liquid sample and then reaches a detector. If the compound is a solid, it must first be dissolved in a solvent. The solution is placed in a cuvette, which may be made of quartz crystal, glass, or plastic. The cuvette has a pathlength, which is the distance the light travels through the sample. Because the solvent also absorbs light, a blank made of solvent alone is used to subtract background absorbance.

The instrument measures transmittance, which is the fraction of the original light that passes through the sample. P0 is the irradiance before the sample, and P is the irradiance that reaches the detector. Absorbance is the negative log of transmittance. It ranges from 0, which means no absorption, to 2, which means 99% absorption. If 90% of the light is absorbed, transmittance is 0.1 and absorbance is 1. If 99% is absorbed, transmittance is 0.01 and absorbance is 2. The spectrum is a plot of absorbance versus wavelength, and for UV-Vis spectroscopy, the range is 200 to 800 nm.

The amount of light absorbed by a dye also depends on its concentration in solution. This relationship is described by the Beer-Lambert law. Absorbance equals the concentration of the compound multiplied by the path length and the molar attenuation coefficient. The molar attenuation coefficient is unique to each compound and changes with wavelength. If the wavelength is kept constant, it does not change with concentration. The wavelength with the highest absorbance is called lambda max, and it also has the largest molar attenuation coefficient.

How Dyes Absorb Visible Light

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hydrolysis of an ester

Soap Formation from Triglycerides

Triglycerides are lipids made of a glycerol backbone linked to three fatty acids. They are common in vegetable oils and animal fats. Plant lipids are the basis of vegetable oils, while animal lipids are used as lard or general sources of fats. A fatty acid has a long carbon chain, usually between 12 and 20 carbons, with a weak organic acid at one end. Some fatty acids contain double bonds. These are unsaturated fats. The double bond usually gives the chain a bent, cis shape. As a result,...

synthesis of luminol

Luminol Chemiluminescence in Forensics

Luminol chemiluminescence shows how a chemical reaction can produce light and help with blood detection. Chemical reactions may be endothermic or exothermic, depending on whether they absorb or release heat as reactants become products. The enthalpy of a reaction is the energy difference between reactants and products, or ΔH. A positive ΔH means the reaction is endothermic. A negative ΔH means it is exothermic. Many reactions do not happen in a single step. They can pass through intermediates...

esterification

Ester Formation and Everyday Fragrances

Esters are organic compounds with a carbonyl group attached to an oxygen that connects to another alkyl or aryl group. Their general formula is RCOOR'. They are often made by reacting a carboxylic acid with an alcohol in an esterification reaction. In this process, the hydroxyl group of the acid is replaced by an alkyl or aryl group. Many simple esters have familiar fruit and flower smells because their structures are small and less complex. Formic acid and methanol can combine to form methyl...

identification of unknown aldehydes and ketones

identification-of-unknown-aldehydes-and-ketones

Carbonyl Tests for Aldehydes and Ketones

Aldehydes and ketones are carbonyl compounds, which means they contain a C=O functional group. A ketone has two alkyl or aryl groups attached to the carbonyl carbon. Acetone is the simplest ketone, with two methyl groups. An aldehyde is similar, but it has at least one hydrogen attached to the carbonyl carbon. Formaldehyde is the simplest aldehyde, and acetaldehyde has one hydrogen and one methyl group. The carbonyl carbon in both aldehydes and ketones is electrophilic because the oxygen atom...

uv-vis spectroscopy of dyes

Dyes absorb visible light because their molecular structure lets them interact with specific wavelengths. When light hits a substance, some light is absorbed and the rest is reflected or transmitted. The color we see depends on the wavelength that is reflected. A blue substance reflects blue light, around 430 to 480 nm, and absorbs the complementary orange region, around 590 to 630 nm. Compounds that do not absorb in the visible range appear colorless to the human eye. Light also has energy and...

identifying alcohols

Alcohol Tests for Primary, Secondary, and Tertiary

Alcohol tests can help sort alcohols by structure and reactivity. Alcohols are organic molecules with a hydroxyl group attached to an alkyl or aryl group, written as ROH. If the carbon with the hydroxyl group has one R group, the alcohol is primary. If it has two R groups, it is secondary. If it has three R groups, it is tertiary. Alcohols can also be aromatic when they contain a benzene ring. Phenol is the simplest aromatic alcohol. To identify an alcohol, chemists can use its chemical...

chemistry

Comprehensive, curriculum-focused videos for both instructors and students in introductory chemistry lab courses.

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Learn how dye structure affects visible light absorption, how UV-Vis spectrophotometers measure absorbance, and how Beer-Lambert law links absorbance to concentration.

How Dyes Absorb Visible Light | JoVE K12

通过这段 4 分钟的教师指南，准备教授 染料的紫外-可见光谱。涵盖 JoVE Lab Manual 上 Chemistry 实验的关键准备步骤。

Chemistry 中的 染料的紫外-可见光谱 教师准备 - 8.8K+ 观看次数 | JoVE Lab Manual

在这段 10 分钟的学生实验流程视频中，逐步学习 染料的紫外-可见光谱 的操作步骤。属于 JoVE Lab Manual 上的 Chemistry 系列。

Chemistry 中的 染料的紫外-可见光谱 学生实验流程 - 37K+ 观看次数 | JoVE Lab Manual

How Dyes Absorb Visible Light

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