關聯性磁振頻譜

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關聯性磁振頻譜(Correlation spectroscopy, COSY)是二維核磁共振(NMR)頻譜學中的一種技術，其他二維NMR技術包括有J頻譜(J-spectroscopy)、交換譜(Exchange spectroscopy, EXSY)及核歐佛豪瑟效應頻譜(Nuclear Overhauser effect spectroscopy, NOESY)。二維NMR頻譜對於分子提供了比一維NMR頻譜更多的資訊，在對分子結構的決定上極為有用，尤其當分子極為複雜，難以用一維NMR來研究之時。歷史上第一項二維NMR實驗方法——COSY是由Jean Jeener於1971年首先提出，其為布魯塞爾自由大學(Université Libre de Bruxelles)教授。真正的實驗由Walter P. Aue、Enrico Bartholdi及理查·恩斯特(Richard R. Ernst)三人首先完成，於1976年發表。^[1]

[编辑] 原理

二維NMR實驗牽涉到一連串的一維實驗組。每單項實驗包含了一組射頻脈衝序列，脈衝間存有些延遲時間(delay periods)。脈衝的時機、頻率以及強度決定了每個NMR實驗的異同點。在延遲時間期間，核自旋被允許自由旋進，此段時間稱作演化時間(evolution time)，長度是可以預先決定好。核頻率在最後的脈衝之後才被偵測。在相續的單項實驗中，演化時間逐步增加；每個單項實驗所收到的資料形成了一組二維資料組。^[2]

其中一例二維NMR實驗為同核種(homonuclear)COSY脈衝序列，包括了第一個脈衝(p1)，接著是演化時間(t1)，再接著是第二個脈衝(p2)，最後是測量時間(t2)。電腦會將所有的頻譜組集成演化時間(t1)的函數。最後，透過傅利葉轉換將時間相依的訊號轉為二維頻譜。

The two-dimensional spectrum that results from the COSY experiment shows the frequencies for a single 同位素 (usually hydrogen, ¹H) along both axes. (Techniques have also been devised for generating heteronuclear correlation spectra, in which the two axes correspond to different isotopes, such as ¹³C and ¹H.) The intensities of the peaks in the spectrum can be represented using a third dimension. More commonly, intensity is indicated using contours or different colors. The spectrum is interpreted starting from the diagonal, which consists of a series of peaks. The peaks that appear off of the diagonal are called cross-peaks. The cross-peaks are symmetrical (both above and below) the diagonal and indicate which hydrogen atoms are spin-spin coupled to each other. One can determine which atoms are connected to one another by only a few chemical bonds by matching the center of a cross-peak with the center of each of two corresponding diagonal peaks. The peaks on the diagonal when matched with cross-peaks are coupled to each other.

For example: a CH₃CH₂COCH₃ molecule 2-丁酮(2-butanone)would show three peaks on the diagonal, due to the three distinct hydrogen groups. By drawing a line straight down from a cross-peak to the point on the diagonal directly above or below it, and then drawing a line from the cross-peak directly across to another peak on the diagonal, one can determine which peaks are coupled. This is done in such a way that the lines from the cross-peak form a 90° angle between the two peaks on the diagonal. The matching peaks, as determined by using the cross-peaks, indicate which hydrogens are coupled, giving a clearer understanding of the structure of the molecule under examination.

右圖是COSY is an example of a COSY NMR spectrum of 黃體素(progesterone)in DMSO-d6. The spectrum that appears along both the x- and y-axes is a regular one dimensional ¹H NMR spectrum. The COSY is read along the diagonal - where the bulk of the peaks appear. Cross-peaks appear symmetrically above and below the diagonal.

[编辑] COSY

COSY-90 is the most common COSY experiment. In COSY-90, the sample is irradiated with a radio frequency pulse, p1, which tilts the nuclear spin by 90°. After p1, the sample is allowed to freely precess during an evolution period (t1). A second 90° pulse, p2, is then applied, after which the experimental data are acquired. This is done repeatedly using a series of different evolution periods (t1). At the conclusion of data acquisition the data is Fourier transformed in each dimension to generate the two dimensional spectrum. It is only because the evolution period is varied that cross-peaks appear in the spectrum.

Cross-peaks result from a phenomenon called 磁化轉移(magnetization transfer). Depending on the experiment, this transfer can be achieved through space or bonds, or even through chemical or physical means. In COSY, magnetization transfer occurs through the bonds.

Another member of the COSY family is COSY-45. In COSY-45 a 45° pulse is used instead of a 90° pulse for the first pulse, p1. The advantage of a COSY-45 is that the diagonal-peaks are less pronounced, making it simpler to match cross-peaks near the diagonal in a large molecule. Additionally, the relative signs of the coupling constants can be elucidated from a COSY-45 spectrum. This is not possible using COSY-90.^[3] Overall, the COSY-45 offers a cleaner spectrum while the COSY-90 is more sensitive. Related COSY techniques include double quantum filtered COSY and multiple quantum filtered COSY.

COSY NMR has useful applications. Organic chemists often use COSY to elucidate structural data on molecules that are not satisfactorily represented in a one-dimensional NMR spectrum. Using cross-peaks, along with the diagonal spectrum, one can often discover much about the structure of an unknown molecule.

[编辑] NOESY

NOESY is 2D NMR technique which takes advantage of the 核歐佛豪瑟效應(Nuclear Overhauser effect) and 磁偶極交互作用 to create a "through-space" 2D NMR spectrum. NOESY spectra can be assigned using a technique called Sequential Walking.