拉曼光谱技术

拉曼光谱

OVERVIEW

1. Raman spectra give information on molecular vibrations and are obtained from changes in the frequency of light observed in a scattering experiment (inelastic scattering).

2. The physical picture arises from considering changes in polarizability (induced dipole moment) that arise if a vibration occurs during the time the electrons are oscillating in response to the applied radiation.

3. The gross selection rule is that the vibrational motion must produce a change in the polarizability of the molecule.

4. The anisotropy of the polarization of the scattering can be measured. Comparison of the spectra polarized perpendicular and parallel to the incident radiation gives information on the symmetry of the vibrational motions.

5. Raman spectra can be obtained in water. This is a major advantage over infrared spectra.

6. Resonance Raman spectra result when the wavelength of the exciting light falls within an electronic absorption band of a chromophore in the molecule. Some vibrations associated with such a chromophore may be enhanced by factors of 1000 or more.

7. The experimental parameters of a band in a spectrum are its position ( ) (which is independent of the frequency of the exciting light), its intensity (which is directly proportional to concentration), and its polarization.

8. The main biological applications of conventional Raman are very similar to those for infrared. Resonance Raman affords a means of probing selective sites in molecules. For example, in metalloproteins, Raman can give information on the nature of the ligand directly attached to the metal.

6.1 引言

拉曼光谱和红外光谱都反映了分子振动的信息，但其原理却有很大差别：红外光谱是吸收光谱，而拉曼光谱是散射光谱。红外光谱的信息是从分子对入射电磁波的吸收得到的，而拉曼光谱的信息是从入射光与散射光频率的差别得到的。拉曼光谱的突出优点是可以很容易地测量含水的样品，而且拉曼散射光可以在紫外和可见光波段量测。由于紫外光和可见光能量很强，因此其量测比红外波段要容易和优越得多。

拉曼光谱得名于印度物理学家拉曼(Raman)。1928年，拉曼首先从实验观察到单色的入射光投射到物质中后产生的散射，通过对散射光进行谱分析，首先发现散射光除了含有与入射光相同频率的光外，还包含有与入射光频率不同的光。以后人们将这种散射光与入射光频率不同的现象称为拉曼散射。拉曼因此获得诺贝尔奖。

当一束入射光通过样品时，在各个方向上都发生散射。拉曼光谱仪收集和检测与入射光成直角的散射光。由于收集和检测的散射光强度非常低，因此拉曼光谱的应用和发展受到很大限制。六十年代激光开始广泛应用，拉曼光谱仪以激光作光源，光的单色性和强度都大大提高，拉曼散射仪的信号强度因而大大提高，拉曼光谱技术得以迅速发展，应用领域遍及物理，材料，化学，生物等学科，并已成为光谱学的一个分支?拉曼光谱学。

6.2拉曼光谱原理

6.2.1光的散射：

入射光通过样品后，除了被吸收的光之外，大部分沿入射方向穿过样品，一小部分光则改变方向，发生散射。一部分散射光的波长与入射光波长相同，这种散射称为瑞利散射(Rayleigh scattering)。1899年，瑞利从实验中得出结论：晴天时天空呈兰色的原因是大气分子对阳光的散射。瑞利还证实：散射光的强度与波长的四次方成反比。这就是瑞利散射定律。由于组成白光的各种颜色的光中，兰光的波长最短，因而散射光强度最大。天空因而呈现兰色。

瑞利当时并没有考虑到散射光的频率变化。他认为散射光与入射光的频率是相同的。所以后来把与入射光波长相同的散射称为瑞利散射，而把波长与入射光不同的散射称为拉曼散射。

6.2.2拉曼散射的产生

6.2.2.1 机械力学的解释

光由光子组成，这是光的微粒性。光子与样品分子间的相互作用，可以用光子与样品分子之间的碰撞来解释。

光照射样品时，光子和样品分子之间发生碰撞。如果碰撞时只是运动方向改变而未发生能量交换即发生了弹性碰撞，则光子的能量不变。由E=hν，能量不变频率也就不变。这就是瑞利散射产生的原因。如果光子和样品分子间发生非弹性碰撞，即光子除改变运动方向外还有能量的改变，一部分能量碰撞时在光子和样品之间发生交换，光子的能量有所增减，则光的频率发生改变。

6.2.2.2 从能级之间的跃迁来分析

光子和样品分子之间的作用也可以从能级之间的跃迁来分析。

Figure 9.1 Processes leading to normal, preresonance, and resonance Raman scattering. (For comparison, the processes involved in IR and fluorescence are shown.) The horizontal lines represent different vibrational energy levels in the two electronic states. The Raman scattering spectrum is also indicated. Note that the intensity of the Stokes lines is greater than that of the anti-Stokes

P240 Fig 9.1

样品分子处于电子能级和振动能级的基态，入射光子的能量远大于振动能级跃迁所需要的能量，但又不足以将分子激发到电子能级激发态。这样，样品分子吸收光子后到达一种准激发状态，又称为虚能态。样品分子在准激发态时是不稳定的，它将回到电子能级的基态。若分子回到电子能级基态中的振动能级基态，则光子的能量未发生改变，发生瑞利散射。如果样品分子回到电子能级基态中的较高振动能级即某些振动激发态，则散射的光子能量小于入射光子的能量，其波长大于入射光。这时散射光谱的瑞利散射谱线较低频率侧将出现一根拉曼散射光的谱线，称为Stokes线。如果样品分子在与入射光子作用前的瞬间不是处于电子能级基态的最低振动能级，而是处于电子能级基态中的某个振动能级激发态，则入射光光子作用使之跃迁到准激发态后，该分子退激回到电子能级基态的振动能级基态，这样散射光能量大于入射光子能量，其谱线位于瑞利谱线的高频侧，称为anti-Stokes线。Stokes线和anti-Stokes线位于瑞利谱线两侧，间距相等，如图9.1所示。

Stokes线和anti-Stokes线统称为拉曼谱线。由于振动能级间距还是比较大的，因此，根据波尔兹曼定律，在室温下，分子绝大多数处于振动能级基态，所以Stokes线的强度远远强于anti-Stokes线。拉曼光谱仪一般记录的都只是Stokes线。

6.2.2.3 从光的波动性来分析

由于光同时具有波动性，因此也可以从光的波动性分析拉曼散射的产生：电磁波的交变电场可以用E=E0cos(2πν't)表示，其中E是任意时刻t的电场强度，E0为交变电场的振幅，ν'为频率。样品分子的电荷分布在交变电场的作用下会发生变形，其正电荷和负电荷的中心会发生位置上的相对移动或分离，产生诱导偶极矩μ，μ=αE，其中E为入射光的交变电场强度，α是分子的极化率(polarizability)。分子极化率是衡量分子在电场作用下电荷分布发生改变的难易程度或诱导偶极矩(induced dipole moment)的大小，也就是单位电场强度诱导产生的偶极矩的大小。

如果分子的振动引起分子极化率的改变，则分子具有拉曼活性。

以双原子分子为例，设分子极化率α随分子振动而变化，则α可按台劳级数展开。忽略高次项，可得到：

α=α0+(dα/dq)0q

式中α0是分子在平衡位置时的极化率，q=r-r e，是双原子分子核间距r与平衡位置时核间距r0的差。(dα/dq)0表示平衡位置上α对q的导数。

由μ=αE=[α0+(dα/dq)0q]E0cos(2πν't)

根据前面红外原理中所推得的方程d2q/dt2= -kq/μ的解q=q0cos2πνt

可以有

μ=[α0+(dα/dq)0q0cos2απνt]E0cos2πν't

=α0E0cos2πν't+q0E0(dα/dq)0cos2πνtcos2πν't

=α0E0cos2πν't

+(1/2)q0E0(dα/dq)0[cos2π(ν'+ν)t+cos2π(ν'-ν)t]

式中前一项α0E0cos2πν't对应于样品分子产生的波长未变化的散射即瑞利散射，第二项反映分子极化率随分子振动而改变(即(dα/dq)0不为零)时分子产生的与入射光频率不同的散射光。散射光与入射光频率的差值即分子的振动频率，这就是拉曼散射。

红外吸收的光频率是分子的振动频率，拉曼散射光与入射光的频率差也反映了分子振动能级之间的差。

6.2.3 拉曼散射的选择定则（参考书P242）

外加交变电磁场作用于分子内的原子核和核外电子，可以使分子电荷分布的形状发生畸变，产生诱导偶极矩。极化率是分子在外加交变电磁场作用下产生诱导偶极矩大小的一种度量。极化率高，表明分子电荷分布容易发生变化。

μ= αE

如果分子的振动过程中分子极化率也发生变化，则分子能对电磁波产生拉曼散射，称分子有拉曼活性。有红外活性的分子振动过程中有偶极矩的变化，而有拉曼活性的分子振动时伴随着分子极化率的改变。因此，具有固有偶极矩的极化基团，一般有明显的红外活性，而非极化基团没有明显的红外活性。拉曼光谱恰恰与红外光谱具有互补性。凡是具有对称中心的分子或基团，如果有红外活性，则没有拉曼活性;反之，如果没有红外活性，则拉曼活性比较明显。(This is another important principle of vibrational spectroscopy, the rule of mutual exclusion. This rule states that for any molecule containing a true inversion center of symmetry, the infrared active vibrations are Raman inactive and vice versa.)

一般分子或基团多数是没有对称中心的，因而很多基团常常同时具有红外和拉曼活性。当然，具体到某个基团的某个振动，红外活性和拉曼活性强弱可能有所不同。有的基团如乙烯分子的扭曲振动，则既无红外活性又无拉曼活性。

下图显示了甲苯的红外谱和拉曼谱。可以看到：在某些频率处两者是吻合的，而在另

一些频率上，只有一种谱上有峰。

FIGURE 1.12. Comparison of IR (top) and Raman (bottom) spectra of toluene. Some lines appear at the same frequency in both the IR and the Raman spectrum. However, some lines show in the IR but not in the Raman spectrum. The intensities of the IR lines arc different from those of the Raman lines, although many of them appear at the same frequency. This reflects the difference in selectivity of two fundamentally different processes.

例. 包含两个相同原子的双原子分子的红外活性和拉曼活性如何? (参考书P242例9.2)

答：具有红外活性的分子振动必须引起分子固有偶极矩的变化。对于含两个相同原子的分子来说，由于它没有固有偶极矩，因此这个振动不可能发生固有偶极矩的变化，这个振动没有红外活性。但是在分子振动过程中，分子会变形，这就会引起电子相对于核的分布的变化。分子的极化率会反映这种变形，因而在分子振动中极化率会发生变化，这种振动因此有拉曼活性。

例. CO2的拉曼光谱(参考书P242例9.3)。

Figure 9.2

CO2有四个基本的振动模式，但其中只有一个有拉曼活性，为什麽?

答：二氧化碳的对称伸缩振动改变了核和电子的相对位置，因而分子的极化率也发生改变，分子具有拉曼活性。在不对称伸缩振动中，一个键的伸展效果被另一个键的收缩抵消了，因此极化率总体上没有变化。另外两种振动模式也是如此。这里要注意的是：具有拉曼活性的振动是无红外活性的。事实上，如果一个分子具有对称中心，则具有红外活性的振动没有拉曼活性。反之，具有拉曼活性的振动没有红外活性。

6.2拉曼谱的量测及参数

RAMAN INSTRUMENTATION

6.2.1拉曼谱仪：

Types of Instrumentation and Trends in the 1990s

Raman instrumentation has an interesting heritage, and in some ways the technique has seen more changes, and more diversity than any other analytical technique. This may be due to two aspects of its evolution?one technical and the other perception versus acceptance. The Raman effect is extremely weak, and Raman spectroscopy has obviously gained over the years by technology that improves signal and detectability. Not all these technological "breakthroughs" follow the same developmental trail. Secondly, the technique is in some ways too closely-related to infrared spectroscopy, and is treated as a poor relative.

Raman spectroscopy, in practical terms and for specific applications, can be demonstrated to have considerable advantages over infrared spectroscopy. However, as a general analytical technique, it is difficult to demonstrate that it offers any net advantages. And, it has been seen to have some clear disadvantages, in particular the common interference from broad-band sample fluorescence, which can totally mask the spectrum. Consequently, when people justify the purchase of new laboratory instrumentation, the safe decision tends to be infrared spectroscopy, which is well established, and has evolved consistently over the past 40+ years. With Raman, there is less practical history, and the instrument platforms are constantly changing. Today, there are a couple of technology choices which are not necessarily mutually exclusive?there are pros and cons to the selection. When it comes to a specific application, however, it can be easy to demonstrate whether Raman or infrared is the better choice.

There are currently two main technological approaches to the design of Raman instrumentation ? a monochromator or spectrograph-based CCD system and FT-Raman. As mentioned above, because of practical and technical constraints, these two approaches do not necessarily produce exactly the same end result. Qualitatively, both generate the same fundamental spectrum for a given material, however, the overall appearance of the spectrum or the impact of the sample, may be different. This section will provide an overview of instrumentation as it exists in the 1990s, and it will provide a general discussion of the trends and applications.

The greatest technological difficulty for Raman spectroscopy has been the weakness of the Raman spectral signal, compared to the magnitude of the main excitation wavelength. The intensity of a particular Raman line can be in the range 10-6 to 10-10 of the main excitation line (possibly even as low as 10-12). The issues are: how to measure such a low signal in the presence of a dominant signal, how to remove effectively the interference from the dominant signal, and, if possible how to enhance the weaker signal. One approach to increasing the absolute intensity of the signal is to increase the power of the source ? the laser power. While this may be possible, it does not remove the fundamental dynamic range problem, or the interference problem, which only becomes worse with increased laser power. For years, the traditional Raman instruments featured scanning monochromators. The important criteria for the monochromator design were to minimize straylight, to enable the very weak signals to be measured, and to design for maximum Rayleigh line rejection. The original solution was to use more than one monochromator, with commercial systems being based on double and triple monochromators. While these had the desired optical properties, the use of up to three monochromators made these instruments mechanically complex, and severely limited the optical throughput of the instrument (down to 5% or less overall efficiency).

典型的拉曼谱仪装置图如下图所示。

(A.T.Tu, FIGURE 2.5.) Essential parts of a Raman spectrometer. Normally the scattered light at 90? is examined.(instfig.pcx)

由于散射效率大约为10-6~10-7，所以通常使用激光器作为光源，通过滤片和聚焦镜投

射到样品上。这时光向各个方向散射。散射光包括瑞利散射(弹性散射)和拉曼散射(非弹性散射)。弹性光散射强度比拉曼散射高出103以上，所以色散系统必须精心设计，消除种种杂散光。为了达到高分辨率，一般采用双联或三联单色仪作为分光系统。一般在与入射光成90

度的方向上接收散射光，采用光电倍增管作为接收器，然后经过信号处理电子系统和计算机，从显示屏或记录仪输出。输出参数为各个波数上的散射光绝对值，散射光波数的绝对值和拉曼波数(即入射光与散射光的波数差)，可以直接显示在屏幕上。

With the introduction of laser line rejection filters, and in particular the holographic notch filters, the need for the second and third monochromators was effectively eliminated. A side benefit of the use of these rejection filters and the elimination of the additional monochromators, was the associated reduction in instrument size.

The next significant technology boost was provided by the move towards detector arrays?initially with intensified photodiode arrays, and more recently with the cooled CCD (charge-coupled device) arrays. With such devices, the need to scan the monochromator mechanically is removed.The result is a high efficiency spectrographic system with no moving parts. Today, the limitations of the technology are the cost of high-performance spectroscopic CCD array cameras, and the overhead associated with cooling. However, with major advances in imaging technologies in the 1990s there has been a corresponding expansion in CCD technology. As a result, it is anticipated that CCD devices with improved performance and lower costs will continue to become available. The trades that have to be made with a CCD device are spectral range versus spectral band width, and the impact of the signal cut-off between 1000 and 1100 nm for silicon. These issues will be discussed in greater detail, later.

With the gains in performance experienced with FTIR instrumentation compared to dispersive infrared instruments, there has been a natural desire to determine whether or not the same level of performance can be achieved for Raman spectroscopy. Originally, this experiment was considered to be impractical because of noise considerations and the extraordinarily large dynamic range involved between the excitation (Rayleigh) line and the Raman signals. However, with the advent of the laser line rejection filters mentioned above, Hirschfeld and Chase demonstrated the feasibility of FT-Raman spectroscopy. Original experiments were performed with the 647.1 nm line of a Krypton gas laser, and with a silicon detector. However, the real justification for the move to FT-Raman spectroscopy was the ability to use near-infrared lasers. Moving from visible to NIR excitation helped to remove one of the major interferences encountered with Raman spectroscopy?the occurrence of broadband fluorescence.This, plus the expected gain in performance from the multiplex advantage, offered by Fourier spectroscopy, made the technique of FT-Raman an attractive alternative to the conventional dispersive-based methods of measurement.A second, practical advantage for a user is that Raman can be performed on an existing FTIR instrument, without significant redesign.In fact, most of the major FTIR vendors offer Raman as an accessory for their high-end instruments. In most cases, the 1064 nm line of the Nd:YAG laser, operating with powers of up to 4 W, are used for excitation. Following the success of the FT-Raman accessories, some dedicated FT-Ramans instruments were produced, with notable gains in performance linked to the optimization of the optical system.In particular, gains

were experienced by the use of high reflectivity optics,the reduction in the number of optical elements, and the use of high sensitivity detectors, matched to the laser image.

Figure 22 Schematic diagram of a FT-Raman Spectrometer

One of the major applications of Raman spectroscopy has been microscopy, with the benefits of the spatial resolution of the laser light source.Raman microscopy gained popularity in the mid 1970s from the pioneering work of Delhaye, et al. and by the introduction of the MOLE by Instruments SA. Later, following the gain in popularity of infrared microscopy, with microscope accessories optimized for commercial FTIR instruments, a parallel implementation was made for FT-Raman. Likewise, commercial Raman microscopes are offered either as accessories or as dedicated systems for use with CCD-based technology. An important recent development is in the area of Raman spectroscopic imaging microscopy, a two-dimensional experiment, where the Raman spectrum is scanned with an AOTF device, and the main image is generated by a CCD array. This technology is expected to have significant impact on studies in materials science, in polymer chemistry, and in the area of biological and medical research.

One of the major benefits of Raman spectroscopy is the fact that the primary measurement involves visible or NIR radiation. This permits the use of conventional glass or quartz optics for imaging. Furthermore, it opens up the opportunity to use optical fibers for remote sampling. In such an arrangement, a single fiber is used for the transmission of laser radiation to the sample, and second fiber, or a series of fibers (Figure 19), transmits the Raman scattered radiation back to the spectrometer.

Figure 19 Schematic diagram of a Raman fiber-optic sampling probe featuring a fiber-optic

Bundle

The construction of the sample-light interface in this case is very important to maximize the coupling between the laser and the sample, and the subsequent collection of the scattered radiation. Silica fibers are normally used, which can transmit visible or near infrared radiation over relatively long distances without significant light loss. Usually, the Raman spectrum from silica is very weak, however, over the distance covered by optical fibers, the contribution can be significant. To overcome this problem, sampling probes featuring optical filtering in the measurement head are utilized. An example of such a probe head is shown in Figure 20.

Figure 20 Schematic diagram of a Raman fiber-optic sampling probe featuring filter elements in the measurement head (Courtesy of Kaiser Optical Systems, Inc.)

As noted earlier, there are several important areas of application where Raman excels over infrared. Often, these are based on practical issues, such as the ability to use glass in the optical system,the lower Raman scattering of water, which permits the study of aqueous media, and the opportunity to have noncontact sampling. A secondary issue is that unlike mid-infrared spectroscopy, there is no interference from atmospheric water vapor or carbon dioxide.This, coupled to the scaling down in instrument size, the ability to perform remote measurements, and the availability of mechanically simple instruments, has made Raman a practical tool for process applications. Applications have ranged from raw material screening to reaction monitoring, with a major focus on the analysis of polymeric products.

6.2.2. 拉曼谱参数：(参考书P243－245)

拉曼谱的参数主要是谱峰的位置和强度。

6.2.2.1 谱峰(谱线)位置：

峰位是样品分子电子能级基态的振动态性质的一种反映。它是用入射光与散射光的波数差来表示的。峰位的移动与激发光的频率无关

6.2.2.2 强度：

Unlike the traditional infrared measurement, the Raman effect is an emission phenomenon, and is not constrained by the laws of absorption. The intensity of a recorded spectral feature is a linear function of the contribution of the Raman scattering center, and the intensity of the incident light source. The measured intensity function is not constant across a spectrum, and is constrained by the response of the detector at the absolute frequency (wavelength) of the point of measurement. Also, Raman scattering is not a linear effect, and varies as a function

of λ4, where λ is the absolute wavelength of the scattered radiation. While Raman intensity may be used analytically to measure the concentration of an analyte, it is necessary to standardize the output in terms of the incident light intensity, the detector response and the Raman scattering term, both as a function of the absolute measured wavelength.

拉曼散射强度与产生谱线的特定物质的浓度有关，成正比例关系。而在红外谱中，谱的强度与样品浓度成指数关系。)样品分子量也与拉曼散射有关，样品分子量增加，拉曼散射强度一般也会增加。对于一定的样品，强度I与入射光强度I0、散射光频率νs、分子极化率α有如下关系：

I=CI0νs4α2

这里C是一个常数。

在共振拉曼谱中，谱的加强是由于极化率α的增加引起的。

6.2.2.3 退偏比(depolarization ratio)

如参考书P242中所说，一个分子的电荷在某一个方向上可能比在另一个方向上容易变形，称这种分子的极化率各向异性；相反，则称为各向同性。把一个朝向与偏振光平行的偏振片放在偏振光的路径上，则偏振光可以通过偏振片。若偏振片转动90度，则偏振光不能通过偏振片。

如果一个分子位于原点0，对入射光进行散射。下图只表示了沿Y方向散射的射向观测者的光。对于高度对称的分子如CH4和SF6而言，极化率是各向同性的。当这类分子的完全对称的振动模式(例如CH4中C-H键的伸缩振动)与在XZ平面上偏振的入射光相互作用时，散射光将在YZ平面上偏振。这样，当一个偏振片平行于YZ平面放置时，只有这个平面上的散射光能够通过。这部分光的强度称为I∥。若偏振片转动90度，则YZ平面上的光将无法通过。偏振片处于这个位置时测得的散射光强度称为I⊥。

ρ=I⊥/I∥

定义为退偏比。对于CH4的对称C-H伸缩振动来说，退偏比ρ=0。

(A.T. Tu FIGURE 1.32.) The intensity of scattered tight can be measured two different ways. (A) Light comes through a parallel-oriented polarizer and is parallel to the incident light (I//). (B) Light comes through a perpendicularly oriented polarizer and is perpendicular to the incident light (I⊥). The ratio of I// to I⊥ is called the depolarization ratio, and it is related to symmetry of vibrational modes.

大多数分子的对称程度比CH4或SF6小，因此，极化率是各向异性的。一般来说，分子散射的光在XZ和ZY平面上都有偏振。在平行于入射光偏振方向的方向上(如图中的Z方向)，测到的散射光强度与垂直于入射光偏振方向的方向(如图中所示的X方向)上测得的强度是不同的，但其比值一般不为零。

如果入射光是平面偏振光，则退偏比

ρ= I⊥/I∥=3α2a/(45α i +4α2a)

其中α i是极化率的各向同性部分，αa是极化率的各向异性部分，I⊥是垂直于入射光的方向上偏振的散射光强度，I∥是平行于入射光的方向上偏振的散射光强度。

对于平面偏振光来说，退偏比与振动的不对称程度有关，其值在0到3/4之间。任何分子的不完全对称的振动，其退偏比ρ为3/4(αi =0)。对于完全对称的振动，ρ≤3/4。上面提到的CH4的例子中，退偏比为。但一般来说，ρ值在0到3/4之间，其大小取决于分子极化性质的变化和分子键的对称性。一个完全对称的振动在进行任何对称操作后不变，这些对称操作只是交换了分子中对等原子的平衡位置。因此，退偏比的量测可以提供有关分子对称性的信息，而且有助于拉曼谱线的指认。

6.2.3增强拉曼光谱：

There are some enhanced Raman methods, which for some compounds produce significantly intensified Raman spectra, and which overcome some aspects of this dichotomy. Two such techniques are resonance Raman（参考书P243）and surface-enhanced Raman spectroscopy (SERS). One other Raman-based technique worthy of mention is coherent anti-Stokes Raman spectroscopy (CARS)further discussion of this technique is beyond the scope of this book.

Resonance Raman is particularly interesting because it can turn Raman into a highly specific probe for certain functional groups or chemical sites. Resonance Raman occurs when the laser excitation frequency coincides with an electronic absorption band. In this case, the vibrations associated with the absorbing chromophore are enhanced by as much as 103 to 106 times the normal Raman intensity. These intensified Raman lines are linked to the specific chromophore site and functional groups or sites, within the molecule, that interact with the chromophoric group. The early resonance Raman experiments were with the visible lines of the argon ion laser, and this obviously constrained the technique to a limited set of colored compounds. Of these, the work with the heme chromophore of the hemeglobin molecule were the most significant. It is possible to observe the influence of external molecular ligands, such as oxygen, carbon monoxide and cyanide, on the critical heme site, free of interference from the remaining of the protein structures. Moving to shorter wavelengths from the visible towards, and into the ultraviolet regions might, at first site, seem to be impractical because one normally equates high levels of native fluorescence with the use of UV excitation. However, many compounds absorb in the ultraviolet spectral region, and so there is a high probability for the resonance Raman effect to occur. Also, it has been observed that below 260 nm excitation that there is virtually no interference from fluorescence. One of the main issues here has been the appropriate selection of a laser operating in the UV range. One approach is to use a dye laser pumped by a Nd:YAG or a XeCI excimer laser, coupled to frequency doubling and tripling crystals to provide a wavelength selectable range of 200 to 750 nm (Nd:YAG) and 206-950 nm (excimer). Both these laser systems provide a pulsed laser output. A practical

alternative, where continuous wavelength tuning is not a requirement, is an

intracavity frequency doubled argon ion laser which provides continuous output of five excitation lines in range 230 nm to 260 nm.

当入射光引起分子中电荷的平移时，则发生散射。电荷的平移通过分子极化率反映出来。

散射的强度与极化率的平方成正比。

当激发光的频率接近且小于两个电子能级之间的频率差时，则产生所谓的preresonance，

当激发光的频率等于两个电子能级之间的频率差时，则会发生共振，这时产生很大的电子电

荷频移或分子变形的概率很高，这时就会产生对光的吸收。这也说明对于某些振动，当入射

光接近或等于某个吸收跃迁频率时，极化率会变得比较大。这种振动称为共振加强的

(resonance enhanced)振动。这种加强取决于电子跃迁的强度及振动的对称性。如果只有一个单一的电子态，则振动加强必须是对称的。即这种振动不能改变分子的对称性。如果某个被激发的生色团有不止一个电子跃迁，则振动的对称性就不那么重要了。

共振拉曼谱典型的增强倍数是102~103，因此共振拉曼谱在10-4 mol?dm-3或更低的样品

浓度下即可测得。这样，共振拉曼谱就提供了一种以接近紫外光谱的灵敏度选择性地探测生

色团振动频率的手段。

共振拉曼在研究生物大分子的结构和功能时很有用，多生物分子都含有能给出共振拉曼

谱的基团，如类胡萝卜素、黄素、视紫红质、各种含铜与铁的化合物、叶绿素等。共振拉曼

谱仪的使用范围主要受到激光光源频率有限这一现实情况的限制。

As noted, another technique that provides an enhanced Raman spectral output is SERS ? surface-enhanced Raman spectroscopy. Unlike resonance Raman, the laser wavelength is not important, unless aqueous-based measurements are made with near-infrared excitation. As the name implies, the measurement is specific in nature, and somewhat limited in application to surfaces or interfaces. Most studies of SERS have been performed on electrode surfaces. An signal enhancement, in the order of 106is observed for adsorbed species on certain metallic electrode surfaces, notably metals such as gold, silver, platinum and to some extent copper.The original experiments involved calomel (Hg2C12) on a mercury surface, and later work involved organics, such as pyridine adsorbed on roughened silver electrode surfaces. The enhancement phenomenon is not restricted to electrodes, and similar effects have been observed for other substrates involving metals, such as colloidal suspensions of metals and metals deposited or embedded in oxides. A critical factor in all the experiments is the surface roughness, which is nominally at the atomic scale. The origin of the enhancement is believed to be associated with an increased electric field in the region of the molecule under study. Although experiments involving charged metal surfaces might appear to be limited, the phenomenon does open up interesting applications in the area of metallic corrosion, the study of batteries materials, and new electrolytic studies in the contested area of cold fusion.

6.3拉曼光谱的特点和应用

6.3.1. 优缺点：

Advantages and Disadvantages of Vibrational Spectroscopy

Raman and infrared spectroscopy for protein and nucleic acid structure analysis have the following notable advantages:

1. Raman and infrared spectroscopy are nondestructive techniques. Ordinarily the sample may be recovered and assayed for biological activity after spectroscopic examination.

2. Raman and infrared methods are applicable to samples of virtually any morphological form. For proteins and nucleic acids, this includes solutions (aqueous and nonaqueous), suspensions, precipitates, gels, films, fibers, single crystals, and polycrystalline and amorphous solids. Data obtained from a given sample in one morphological state are generally transferable to another morphological state of the same sample. This has important practical benefits-for example, in comparing the molecular structure of a protein in the crystal with that prevailing in solution.

3. A small sample volume is required for these methods. Approximately 1μl is sufficient for conventional Raman spectroscopy and approximately 10 μl for Fourier transform infrared spectroscopy. This represents an advantage over many other structural methods, including X-ray crystallography and magnetic resonance spectroscopy.

4. Raman scattering and infrared absorption processes occur on a time scale that is very short (≈10-15sec) in comparison to the time scales of fluorescence (> 10-9sec) and nuclear magnetic resonance phenomena ((≈ 10-6 sec). Thus, vibrational spectroscopy is suitable for time-resolved studies of biological processes that are inaccessible by fluorescence and magnetic resonance methods.

5. There exists a large database of infrared and Raman spectra of proteins, nucleic acids, and their constituents, for which reliable band assignments, normal mode analyses, and spectra-structure correlations have been made. This facilitates interpretation of the often complex vibrational spectra obtained from proteins, nucleic acids, and their complexes.

The following advantages are specific to Raman spectroscopy:

1. Both H2O and D2O generate very weak Raman scattering, thus producing relatively little interference with the Raman spectrum of the dissolved solute. This constitutes a significant advantage over infrared absorption spectroscopy, where both H2O and D2O are highly problematic solvents. The innocuous Raman characteristics of H2O and D2O likewise facilitate the exploitation of Raman spectroscopy for monitoring hydrogen isotope exchange processes in proteins, nucleic acids, and their assemblies. Similarly, hydrogen isotope exchanges can be employed in the Raman effect for purposes of measuring isotope shifts to establish or confirm definitive vibrational spectroscopic assignments.

2. The fundamental selection rule for Raman spectroscopy, i.e., that relatively high Raman intensity accrues from molecular vibrations with which there is associated a large change in molecular polarizability, favors the electron-rich substituents of proteins and nucleic acids. Thus, the Raman spectrum of a typical protein is dominated by spectral bands assignable to main-chain peptide groups, aromatic side chains (Trp, Phe, Tyr), sulfur-containing side chains (Met, Cys), and side-chain carboxyls (Asp, Glu, Asn, Gln). Raman intensities associated with saturated hydrocarbon groups are intrinsically rather weak. However, the large numbers of such groups typically present in a protein result in a few relatively intense Raman bands associated with groups

frequencies of the methylene and methyl substituents. In the case of nucleic acids, the Raman spectra are dominated by bands attributable to vibrations of the backbone phosphate groups and of the purine and pyrimidine rings, especially in-plane skeletal stretching and exocyclic carbonyl stretching modes of the latter.

3. Raman intensities are enhanced dramatically (by several orders of magnitude) when the energy of the incident photon is selected in resonance with a molecular electronic transition of a protein or nucleic acid chromophore. This constitutes the resonance Raman (RR) effect. Therefore, structural information about the chromophore can be obtained by use of relatively dilute protein or nucleic acid solutions through the RR mechanism. This technique is particularly valuable for proteins containing chromophores that absorb in the visible (metalloproteins, retinal proteins, etc.) or near-ultraviolet (nucleotide-binding proteins, nucleoprotein complexes, etc.). Ultraviolet resonance Raman (UVRR) spectroscopy has found more extended use in applications to nucleic acids and nucleoprotein assemblies.

Vibrational spectroscopy for protein and nucleic acid structure analysis has the following notable disadvantages:

1. Although band resolution in vibrational spectra is superior to that achievable in electronic spectra of condensed phases, the spectral resolution of vibrational spectroscopy is still inferior to that of high-field magnetic resonance spectroscopy. Inadequate resolution is especially problematic in infrared spectra of proteins and nucleic acids and can severely limit the usefulness of the data. To overcome this limitation, the experimentalist may employ a strategy based on chemical or biological modification, such as isotope editing or site-directed mutagenesis.

2. The Raman process of inelastic light scattering is inherently weak compared to other light absorption and emission processes. Thus, considerable effort in sample purification and care in sample handling are necessary to avoid even minute traces of fluorescent impurities or other chromophores that may interfere with detection of the Raman scattering.

3. The inherent weakness of the Raman scattering mechanism in comparison to other mechanisms of interaction of radiation with matter also imposes a requirement for sophisticated and relatively costly instrumentation.

4. Although small sample volumes are sufficient for Raman and infrared analyses, relatively high solute concentrations are required ≈10-100μg/μl).

和红外谱相比，拉曼光谱有以下优点：

(1)一些在红外光谱中为弱吸收或强度变化的谱带，在拉曼光谱中可能成为强谱带，例如基团S-S、C=C、N=N、C≡C、C≡N、C=S、S-H、X=Y=Z、C=N=C、O=C=O等。环状化合物对称伸缩振动具有很强的拉曼谱线，用拉曼光谱来研究比较方便。

(2)拉曼光谱在低波数方向的测量范围较宽，常规测量范围为40~4000cm-1，有利于重原子的振动信息研究。

在低波数范围内，红外光谱的测量有困难。但拉曼光谱所测定的是?ν，因此可以选择

适当的激发光(ν')来把振动光谱移到便于测定的紫外、可见光区域。

(3)拉曼光谱样品大小形状可以多样，不必粉碎、研磨，不必透明。而且样品池或样品杯也可以采用玻璃材料，因为玻璃是弱拉曼散射体。

(4)拉曼光谱比较适于测试生物样品。生物样品一般含水，而水对红外光有很强的吸收，因此用红外光谱测试生物样品技术上要求较高。但拉曼光谱中水的吸收比较弱，因此是含水生物样品的理想检测手段。许多情况下，可以用拉曼光谱来检测活体中的生物物质。只要样品对激光能量吸收不强，测量时就不至造成样品结构上的变化。现在，一些拉曼谱仪上还有专门的显微附件，可以检测样品上微小区域内的物质组分和结构。

(5)拉曼光谱中没有倍频和组合频等红外谱中常见的干扰，因此拉曼光谱比红外光谱简单，容易分析。

(6)拉曼光谱的激发光和拉曼散射光在紫外-可见波段能量较红外光高，因此检测起来比红外光谱容易。

但拉曼光谱也有一些缺点：

(1)有些样品本身的发光本底较强，这样就使得拉曼光谱的信噪比受到影响。此外，样品分子量增加时拉曼光谱的信噪比也会降低。

(2)激光束焦点上能量集中，可能对样品造成损伤。

付力叶拉曼光谱仪的出现，在一定程度上克服了上述缺点，为拉曼光谱更广泛的应用提供了更有力的手段。

6.3.2 数据分析Analysis of Data

The successful application of vibrational spectroscopy in protein and nucleic acid analysis presumes definitive band assignments and requires a combination of experimental and theoretical approaches. Tactics employed toward this objective include

(1)comparison of Raman frequencies and intensities with corresponding infrared data when

available;

(2)determination of vibrational frequency shifts accompanying stable-isotope substitutions,

such as 1H→2H(D), 12C→13C, 14N→15N, and 16O→18O;

(3)evaluation of the effects of pH, temperature, and other environmental factors on the spectra;

(4)detailed and well-analyzed Raman and infrared spectra of smaller, more symmetrical

molecules that are structurally related to the biomolecular constituents;

(5)normal coordinate calculations;

(6)measurements of depolarization ratios of the Raman bands in isotropic solutions of the

molecules;

(7)collection and analysis of polarized spectra of oriented samples when feasible;

(8)measurement of Raman excitation profiles that correlate Raman intensities with laser

excitation wavelengths.

Digitally computed difference spectra can facilitate the visualization of changes in vibrational

band intensities or frequencies accompanying changes in sample temperature, molecular environment, or the like. However, because the Raman spectrum is not an absorption spectrum, its intensities are not governed by the Beer-Lambert law, and the comparison of two independently recorded Raman spectra by subtraction can be subject to considerable uncertainty. This is dealt with by comparing intensities in the two spectra only after normalization of both the minuend and subtrahend to a reliable internal intensity standard. A normalization procedure should be deemed reliable only after careful consideration of the origin of the band in question, and preferably after validation on a suitable model system. In the absence of reliable internal normalization, a process of trial and error may be the only recourse for computation of a Raman difference spectrum. Similar concerns apply to the procedure of solvent correcting a vibrational spectrum (infrared or Raman). It should be kept in mind that the protein or nucleic acid solute may influence the structure of the aqueous solvent, and therefore its vibrational spectrum, as much as the water molecules may influence the structure of the dissolved biomolecule. Accordingly, it is virtually impossible to compensate completely a solution spectrum for contributions of the aqueous solvent by simply subtracting therefrom the spectrum of the pure solvent.

The computational power of microcomputers has ushered in an era of easy manipulation of all types of experimental data, including infrared and Raman spectra. A particularly popular procedure is the method of Fourier deconvolution, which is applied to enhance the resolution of overlapping bands. Also commonly employed is the method of least-squares curve fitting, which can be used to fit a complex bandshape to an arbitrary number of simpler band components. While the utility of such procedures cannot be questioned, considerable caution must be exercised in their application. The vibrational spectroscopist is well advised to avoid these computational approaches whenever the collection of additional experimental data can serve the same objective.

6.3.3.拉曼光谱在生物学中的应用

(ANTHONY T. TU eds.,RAMAN SPECTROSCOPY IN BIOLOGY: Principles and Applications, John WILEY & SONS, New York)

PART II. BIOLOGICAL MOLECULES

CHAPTER 3. PROTEINS

1.Peptide-Bond Vibrations 66

1.1. A mide A and B Bands 67

1.2. A mide I, II, and III Bands 68

1.3. A mide V, VI, and VII Bands 71

1.4. A mide IV Band 71

Secondary Structure (peptide-Backbone

Structure) 72

Conformational Analysis from Amide I and

III Bands 73

Comparison of Amide 1 and III Bands

78

2.3. O ther Structurally Sensitive Lines 80 The D- and L-Amino Acid Copolymers

81

2.5. R ight and Left-Handed α-Helices 82

2.6. D egree of polymerization 82

2.7. S olid and Aqueous Phases 83

2.8. E ffect of Protein on Water 84 Quantitative Estimation of Secondary

Structure 84

3. Side Chains 86

3.1. T yrosine 87

3.2. T ryptophan 89

3.3. P henylalamne 90

3.4. H istidine 91

3.5.Disulfide Bond 91

3.6. C-S 94

3.7. Sulfhydryl Group (-SH) 96

4. Preresonance Raman 97

5. IR and Raman 97

6. Low-Frequency Vibrations 99

6.1. Internal Vibrations 100

6.2. Intermolecular Vibrations 101

7. Raman-Spectra Background 103

8. Application 103

8.1. D enaturation 103

8.2. C hemical Modification 106

8.3. C omparison of Related Proteins 107

8.4. G lycoproteins 108

8.5. B lood Coagulation 108

References 109

CHAPTER 4. ENZYMES AND IMMUNOGLOBULINS

1. Enzyme Action 117

1.1. P apain 117

1.2. C hymotrypsin 120

1.3. C arboxypeptidase A 121

1.4. P eroxidase 123

1.5. T hymidylate Synthetase 123

1.6. O thers 124

2. Enzyme- Inhibitor Complexes 125

2.1. C hymotrypsin 125

2.2. C arbonic Anhydrase 125

2.3. T rypsin 127

2.4. O ther Enzymes 127

2.5. E nzyme-Drug Interaction 128

3. Isozymes 128

4. Immunology 129

4.1. A ntigen-Antibody Reactions 129

4.2. H apten-Antibody Interactions 129

4.3. C ryoglobulin 130

References 131

CHAPTER 5. NUCLEIC ACIDS

1. Background 134

1.1. T automerism 135

1.2. H ydrogen-Deuterium Exchange 135

1.3. O rigin of Base Vibrations 135

2. Principal Raman Lines 137

2.1. Aqueous Solution 13 8

2.2. In D20 141

3. Structurally Sensitive Lines 141

3.1. H eat Treatment 141

3.2. p H Treatment 141

3.3.P hosphodiester-Bond Vibrations 141

4. Conformational Analysis 145 ,

4.1.Double-Stranded Polynucleotides, DNA,

and RNA 145

4.2. U se of Phosphodiester Bands 145

4.3. M elting of Nucleic Acids 147

4.4.Base Stacking and Hydrogen Bonding

150

4.5. S ingle-Stranded Polynucleotides 151

4.6. G el Formation 152

5. Native RNA 153

5.1. Transfer RNA 153

5.2.Rjbosomal RNA 155

5.3.

6. Reactions of Nucleic Acids 156

6.1. A lkylation 156

6.2. P olypeptides 157

6.3. M etal Ions 158

6.4. D rugs 163

7. Special Techmques 165

7.1. Preresonance and Resonance Raman

Spectroscopies 165

7.2. Others 167

References 167

CHAPTER 6. NUCLEOPROTEINS--VIRUS AND

CHROMOSOME

1. Viruses 174

1.1. Plant Viruses 175

1.2. Bacteriophages 180

2. Chromosome 182

2.1. Histones 182

2.2.DNA-Histone Interactions in Nucleosomes

183

2.3.References 185

CHAPTER 7. LIPIDS AND BIOLOGICAL MEMBRANES

1. Brief Review of Lipids and Membranes 187

2.Vibrations of Fatty Acids and Phospholipids

190

2.1. L ow-Frequency Vibrations 191

2.2. S tructurally Sensitive Raman Bands 191

2.3. U nsaturated Fatty Acids 199

2.4. P hase Transition (Melting Behavior) 201

2.5.Quantitative Estimation of Conformation

206

3. Interaction of Lipids 207

3.1. Lipid-Lipid Interactions 207

3.2. Lipid-Protein Interactions 209

3.3. Lipid-Ion Interactions 213

3.4. Lipid-Antibiotic Interactions 213

3.4.I nteractions with Other Compounds

218

4. Biological Membranes 219

4.1. E D, throcyte Membranes 219

4.2. S arcoplasmic Reticulum Membranes 223

4.3. S treplococcus faecalis Membrane 224

4.4. N erves 224

4.5. O ther Plasma Membranes 225

References 226

CHAPTER 8. CARBOHYDRATES

1. Assignment of Raman Bands 235

2. Glycosidic Linkage 235

2.1. IR Absorption Spectroscopy 235

2.2. Raman Spectroscopy 241

3. Functional Groups 242

3.1. O-H Vibrations 242

3.2. C-H Vibrations 244

3.3. N-H Vibrations 245

3.4. C arboxyl and Acetyl Groups 245

3.5. S ulfates 245

https://www.360docs.net/doc/2d2210643.html,parison of Solid and Aqueous Phases

248

5. Conformation 249