적외선 분광기
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개요
출처: 비 M. 동과 지웨이 첸, 캘리포니아 대학교 어바인, 캘리포니아 화학학과
본 실험에서는 현재 기능성 군을 식별하여 알 수 없는 화합물의 정체를 해명하기 위해 적외선(IR) 분광법(진동 분광법이라고도 함)의 사용을 시연할 것이다. IR 분광은 미지의 깔끔한 샘플과 함께 감쇠된 총 반사(ATR) 샘플링 기술을 사용하여 IR 분광계에서 얻을 것이다.
Principles
두 원자 사이의 공유 결합은 스프링과 연결된 질량 m1과 m2를 가진 두 개의 물체로 생각할 수 있습니다. 당연히 이 결합은 특정 진동 주파수로 늘어나고 압축됩니다. 이 
주파수는 k가 스프링의 힘 상수인 방정식 1에의해 주어지며, c는 빛의 속도이며, μ 감소된 질량(수학식2)이다. 주파수는 전형적으로 반전 센티미터(cm-1)로표현되는 파수로 측정됩니다.
수학식 1에서주파수는 스프링의 강도에 비례하며 개체의 질량에 반비례합니다. 따라서 C-H, N-H 및 O-H 결합은 수소가 가벼운 원자이기 때문에 C-C 및 C-O 결합보다 더 높은 스트레칭 주파수를 가지고 있습니다. 이중 및 삼중 채권은 더 강한 스프링으로 간주 될 수 있으므로 C-O 이중 채권은 C-O 단일 채권보다 더 높은 스트레칭 빈도를 가지고 있습니다. 적외선은 상대 결합 강도와 일치하는 700 nm에서 1mm에 이르는 파장을 가진 전자기 방사선입니다. 분자가 공유 결합의 자연 진동 주파수와 동일한 주파수로 적외선을 흡수하면 방사선으로부터의 에너지는 결합 진동의 진폭이 증가합니다. 두 원자의 전자성(전자를 유치하는 경향)이 매우 다른 경우, 전하 분리가 발생하여 이폴 순간을 초래한다. 예를 들어, C-O 이중 결합(carbonyl group)에서 전자는 탄소보다 전기음이 더 높기 때문에 탄소 원자보다 산소 원자 주위로 더 많은 시간을 보낸다. 따라서, 산소에 부분적인 음전하와 탄소에 부분적인 양전하의 결과로 그물 이폴 순간이 있다. 한편, 대칭 알키네는 양쪽의 두 개의 개별 이폴 순간이 서로 를 취소하기 때문에 그물 이폴 순간이 없습니다. 적외선 흡수의 강도는 결합이 늘어나거나 압축될 때 이폴 모멘절의 변화에 비례합니다. 따라서, 카보닐 그룹 스트레치는 IR에서 강렬한 밴드를 표시하고, 대칭 내부 알키네는 C-C 트리플 본드(그림 1)의스트레칭을위한 작은, 보이지 않는 경우, 밴드를 표시합니다. 표 1은 몇 가지 특성 흡수 주파수를 보여줍니다. 도 2는 한츠쉬 에스테르의 IR 스펙트럼을 나타낸다. N-H 단일 결합의 경우 3,343cm-1의 피크와 카보닐 그룹의 경우 1,695cm-1의 피크를 확인합니다. 이 실험에서 ATR 샘플링 기술이 사용되며 적외선이 ATR 결정과 여러 번 접촉하는 시료를 반사합니다. 일반적으로 굴절률이 높은 재료는 게르마늄 및 아연 셀레니드와 같은 사용됩니다. 이 방법을 사용하면 추가 준비 없이 고체 또는 액체 분석을 직접 검사할 수 있습니다.
그림 1. C–O 더블 및 C–C 트리플 본드스트레칭과이폴 순간의 결과로 변경을 보여주는 다이어그램.
표 1. 유기 분자에 존재하는 공유 결합의 특징적인 IR 주파수.
그림 2. 한츠쉬 에스테르의 IR 스펙트럼.
Procedure
Results
Table 2: Appearance and observed IR frequencies of the compounds listed in Figure 3.
| Compound Number | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Appearance | clear liquid | white solid | clear liquid | clear liquid | clear liquid | clear liquid | yellow liquid | white solid | white solid | clear liquid |
| Observed frequencies (cm-1) | 1691, 1601, 1450, 1368, 1266 |
2773, 2730, 1713, 1591, 1576 |
2940, 2867, 1717, 1422, 1347 |
3026, 2948, 2920, 1605, 1496 |
2928, 2853, 1450, 904, 852 |
3926, 3315, 2959, 2120, 1461 |
3623, 3429, 3354, 2904, 1601 |
3408, 3384, 3087, 1596, 1496 |
3226, 2966, 1598, 1474, 1238 |
3340, 2959, 2861, 1468, 1460 |
Applications and Summary
In this experiment, we have demonstrated how to identify an unknown sample based on its characteristic IR spectrum. Different functional groups give different stretching frequencies, which allow the identification of the functional groups present.
As shown in this experiment, IR spectroscopy is a useful tool for the organic chemist to identify and characterize a molecule. In addition to organic chemistry, IR spectroscopy has useful applications in other areas. In the pharmaceutical industry, this technique is used for quantitative and qualitative analysis of drugs. In food science, IR spectroscopy is used to study fats and oils. Lastly, IR spectroscopy is used to measure the composition of greenhouse gases, i.e., CO2, CO, CH4, and N2O in efforts to understand global climate changes.
내레이션 대본
Infrared, or IR, spectroscopy is a technique used to characterize covalent bonds.
Molecules with certain types of covalent bonds can absorb IR radiation, causing the bonds to vibrate. An IR spectrophotometer can measure which frequencies are absorbed. This is generally represented with a spectrum of percent IR radiation transmitted through the sample at a given frequency in wavenumbers. In this type of spectrum, the peaks are inverted, as they represent a decrease in transmitted light at that frequency.
The absorbed frequencies depend on the identity and electronic environment of the bonds, giving each molecule a characteristic spectrum. However, each type of bond will absorb IR radiation within a specific frequency range, and will have a common peak shape and absorption strength. Peaks can therefore be assigned to specific bonds, allowing identification of an unknown compound from the IR spectrum.
This video will illustrate the characterization of an unknown organic compound with IR spectroscopy and will introduce a few other applications of IR spectroscopy in organic chemistry.
A covalent bond between two atoms can be modeled as a spring connecting two bodies with masses m1 and m2. This “spring” has a resonance frequency, which, in this case, is the frequency of light corresponding to the quantum of energy needed to excite an oscillation in the bond at that same frequency, but with even greater amplitude.
The resonance frequency of a bond depends on the bond strength and length, the identity of the involved atoms, and the environment. For instance, a conjugated bond will vibrate in a different frequency range than a non-conjugated bond.
The resonance frequency also depends on the vibrational mode, which is the oscillation pattern of the atoms within a molecule. The most common vibrational modes observed by IR spectroscopy are stretching and bending. Linear molecules have 3N minus 5 vibrational modes, where N is the number of atoms, and non-linear molecules have 3N minus 6 vibrational modes.
IR spectrophotometry is primarily performed by shining a broad-spectrum light source through an interferometer, which blocks all but a few wavelengths of light at any given time, onto the sample. An IR detector measures the light intensities for each interferometer setting. Once data has been collected over the desired frequency range, it is processed into a recognizable spectrum by Fourier transform.
The sample can be gaseous, liquid, or solid, depending on the construction of the instrument. For a standard detector, gases and liquids are placed in a cell with IR-transparent windows, and solids are suspended in oil or pressed into a transparent pellet with potassium bromide. The IR light is then directed through the sample to the detector.
An alternate method for solid and liquid samples is attenuated total reflectance, or ATR. In this method, the pure sample is placed in contact with a crystal surface. IR light is then reflected off the underside of the crystal into a detector, with the absorbed frequencies reflecting more weakly. The sample doesn’t need to be processed first, as the light does not travel through it.
Now that you understand the principles of IR spectroscopy, let’s go through a procedure for identifying an unknown organic compound using the ATR sampling technique on an FTIR instrument.
To begin the characterization procedure, turn on the FTIR spectrometer and allow the lamp to warm up to operating temperature.
Ensure that the ATR crystal is clean. Then, with no sample in place, use the spectrometer software to record a background spectrum.
Next, obtain a solid sample of an unknown organic compound and note its appearance. Using a clean metal spatula, carefully place the sample on the crystal surface. Alternatively, for liquid samples, a pipette is used to transfer samples to crystal surface.
Carefully screw down the probe until it locks into place to fix the sample against the crystal surface.
Then, collect at least one IR spectrum of the unknown sample. After data collection has finished and the background has been subtracted, use the analysis tools in the software to identify the wavenumbers of the peaks.
When finished with the spectrometer, remove the sample and clean the probe with acetone. Save the spectra, close the software, and turn off the spectrometer.
In this experiment, the unknown sample may be one of ten organic compounds, each with five characteristic IR peaks. Based on the phase and visual appearance of the unknown, 8 of the possibilities may be eliminated.
The spectrum from the unknown compound shows a wide peak near the 3,300 wavenumber region, indicative of either an -OH or -NH stretching absorption. The peaks to right indicate the presence of carbon-carbon double bonds and carbon oxygen bonds. Of the two remaining compounds, only one has an -OH group so the compound is phenol.
IR spectrophotometry is a widely used characterization tool in biology and chemistry. Let’s look at a few examples.
In this procedure, FTIR spectroscopy performed with the ATR method was used to obtain IR absorbance images of tissue by introducing a microscopy component into the instrument. Each pixel in the image had a corresponding IR spectrum, allowing determination of the molecular composition of the tissue with excellent spatial resolution. The tissue image could also be displayed at different frequencies to visualize the distribution of molecule types throughout the tissue.
The molecular vibrations of peptide groups in a protein are affected by protein conformational changes. By monitoring a protein sample with step-scan FTIR, which has a temporal resolution on the order of tens of nanoseconds, protein dynamics can be monitored via the changes in their absorbance spectra. The data can be presented as individual spectra or as 3D plots of intensity, frequency, and time for peak identification and further analysis.
You’ve just watched JoVE’s introduction to IR spectroscopy. You should now be familiar with the underlying principles of IR spectroscopy, the procedure for IR spectroscopy of organic compounds, and a few examples of how IR spectroscopy is used in organic chemistry. Thanks for watching!