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salinomvcin and narasin using

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nmr.

3 . 1 Intro d u c tio n .

Nuclear m agnetic resonance is a very po w erfu l and w id e ly used a n a ly tic a l to o l p ro v id in g d ire c t in fo rm atio n ab o ut the chem ical environm ent of a p a rtic u la r atom, as long as the nucleus is m agnetic. As such it can be used to stu d y the s tru c tu re and conform ation o f m any m aterials. M ost chem ists use nm r re g u la rly

to id e n tify w he th e r or n o t th e ir reaction p ro d u c t is th a t w h ic h th e y require.

There are however some lim ita tio n s o f th is technique. One is the relative in se n sitivity. M illig ram q u a ntitie s o f m ate ria l are n o rm a lly re q u ire d to obta in a sp e ctrum in a p ra ctic a l tim escale. A second problem is the em barasssm ent o f riches obtained in a spectrum . If a spectrum is ta ke n o f a com plex m ate ria l the lines w ill tend to overlap one another m a kin g th e ir id e n tific a tio n and assig nm e nt d iffic u lt. T h is is a p a rtic u la r problem in proton spectra, as there is only a sm all spectral w id th and the lines tend to be relatively broad w ith m u ltip le t character. It is possible to resolve th is d iffic u lty by the use o f a second dim ension in the acquisition. If the resonances are separated in tw o dim e nsion s th e n there is less lik e lih o o d o f peak overlap occu rring .

3.1.1 Two dim ensional nm r.

Two dim e nsion al (2D) nm r was firs t proposed in 1971 b y J e e n e ri. The technique w h ich he pioneered used tw o tc/ 2 pulses

and has since become w idely used. It is more com m only kno w n as COSY one of the acronyms beloved by n m r spectroscopists in th is case it is ta ke n from C o rre latio n SpectroscopY.2 The in itia l

m ethod has spawned a large num ber of variants m ost of w h ich are some k in d of correlation spectroscopy. These techniques a ll re ly on w h a t is kno w n as frequency labelling fo r th e ir u tility . The general procedure can be represented schem atically as follows:

A cq u ire S o m eth in g --- ---1 S om ething else|— ^ — -

w he re t% and tg are b oth tim e delays and the n a tu re o f the operations vary. E xactly w h a t these operations are w ill depend on the aim o f the experim ent and examples w ill be given late r. In general the sample is m odulated as a fu n ctio n of t^ and detected as a fu n ctio n of t2- The sample w ill in itia lly evolve a t some frequency

V i d u rin g t^ ie. it is labelled w ith th is frequency. D u rin g t2 the

e volution w ill occur at a different frequency, V2. T h is w ill lead to

peaks in the spectrum at (vi,V2) where These are kno w n

as o ff diagonals or cross peaks and are the object o f a ll 2D nm r

techniques.3

This process is best explained using the sim plest system ie a

single sp in 1 /2 nu cleus. I f the lo n g itu d in a l re la x a tio n T j is ignored for sim p licity's sake the behaviour o f the m agnetisation in the ro ta tin g fram e w ith two n /2 pulses w ill be as show n in Figure 3-1.

X

tl

Msin27W-

Mcos2%Yt%

Acquire

^

Figure 3-1. A two dim ensional nm r pulse sequence.

The a cq u isitio n sam ples the m agnetisation o n ly in the x -y plane where the signal in te n sity is M.sin27C.v.ti. So the signal w ill flu ctu a te sinu soida lly w ith t j and decay exponentially w ith tim e as

M= Moe”t i / T 2 Eqn. 3-1.

If a series o f experim ents are perform ed at in te rva ls o f t^ s ta rtin g from zero to several seconds the am p litu d e o f the peak w ould be seen to fluctu ate sinusoidally w ith frequency v, see Figure 3-2. Observing the top o f th is peak w ill produce a p a tte rn like th a t in Figure 3-3 know n as an interferogram . This can be seen to bear a rem arkable resemblence to a free in d u ctio n decay and it can be F o u rie r tra n sfo rm e d in the same way. I f th is is perform ed fo r every c o lu m n o f p o ints obta in e d from an F ID th e n a tw o dim ensional spectrum can be obtained w ith a peak at (vi.Vg). In th is case vg is the chem ical s h ift v of the peak. In th is case the

Figure 3-2. The result of applying the pulse sequence in Figure 3-1 w ith variable tj.^

Figure 3-3. A slice from the data of Figure 3-2 taken parallel to t j through the tops of the peaks.^

I I i i ' ! - I : : i I i I : 1 I r V I' if

inte rfe ro g ram produced was o scilla tin g w ith frequency v so the fre q u e n cy is also the chem ical sh ift. T his leads to a square spectrum w ith a peak at (v,v) ie on the diagonal. T his spectrum in its e lf is n o t p a rtic u la rly useful b ut if the same pulse technique is applied to a system w ith J co u p lin g th e n th is is an e n tire ly d iffe re n t m atter.

3.1.2 Coherence transfer.

It is possible to id e n tify adjacent groups in a spectrum using h om o n u cle a r de co u p lin g .^ For a com plex spectrum th is w ill be made more d iffic u lt as the peaks w ill tend to overlap. T his m akes it hard to decouple only one specific resonance and also d iffic u lt to id e n tify the changes in the spectrum . If it were possible to spread the data o u t over two dim ensions the n peaks are less lik e ly to overlap and cross peaks off the m ain diagonal w ill become obvious. T h is s h o u ld enable th e ch em ist to id e n tify e a sily co u p lin g s

betw een ad jacen t centres and so assign the sp e ctrum . The problem is how to produce these off diagonal peaks.

This can in fact be achieved using the above pulse sequence.2 The reasons fo r th is become clear if one considers a system w ith h om o n u cle a r coupling. In a coupled system the second pulse causes the m agnetisation in one tra n sitio n w hich arose d u rin g t% to

be shared am ongst a ll its associated tra n sitio n s. The reason fo r th is lies in the concept o f coherence. C onsidering a sa tu ra te d tra n s itio n and one w h ich has ju s t undergone a t i/2 pulse it can be

seen th a t ne ith e r have a z com ponent of the m agnetisation. The p o p u la tio n s o f the a and P states m us t therefore be the same. However in the form er case there is no net m agnetisation, w h ils t

in the la tte r there is a m agnetisation com ponent precessing in the x -y plane. T his arises from the x -y com ponents o f the n u c le i precessing w ith the same phase, w h ich th e y derived from the pulse. In the satu rated sam ple the n u cle i are precessing w ith random phase o r incoherently. Thus experiencing a pulse creates a phase coherence between the a and p states.^

A coherence like th is across a tra n s itio n is called a single q u a n tum coherence. T his is in fact the ro o t cause o f the nm r

signal. This phase coherence is useful as once created it can be

transferred to other states. In an A X system (Figure 3-4) if a single q u a n tum coherence is created across say the A^ tra n s itio n , w h a t are its possible fates? A k pulse w ill excite a ll the p o p u la tio n

excess and tra nsfe r a ll the phase data to the receiving level. So i f such a pulse were to be applied to the tra n s itio n a phase coherence between aa and pp w ould be created. T his w ould be a double q u a ntum coherence as there is a difference o f tw o in the q u a n tum levels o f the tw o states. As the selection ru le fo r a tra n s itio n to be seen is AM = ±1 these ca n n o t be observed. However the y are used in ce rta in nm r experim ents as an e xtra pulse can convert them back to a single qu antum coherence w h ich is observable. The evo lution o f the in visib le m u ltip le q u a n tum coherence w ill proceed along d iffe re nt pathw ays to those o f the observable spectrum and so m ay be of practical use.

I f we re tu rn to o u r single q u a n tum coherence between the

aa and ap states we can consider w h a t happens if a pulse o f other th a n K is adm inistered. This w ill lead to only p a rtia l tra n sfe r of coherence so there w ill now be three phase coherences aa - ap, ap - pp, both single qu antum coherences and the double q u a ntum

pp

a a

Figure 3-4. The energy levels in an AX system

coherence, aa - pp. It is the second o f these, the single q u a n tum coherence across the tra n s itio n w h ich gives rise to the COSY cross peak. This com ponent should be called coherence tra nsfe r. In a real COSY experim ent the pulses are n o t applied selectively to a single tra n s itio n , instead a non-selective pulse is applied across the w hole spe ctrum .^ This pulse can be seen as a sequence, or cascade, o f selective pulses in q u ick succession. T h is m eans th a t w h ile the phase in fo rm atio n in ap is being p a rtia lly transfered to pp the same is happening for a ll the other pairs o f states. T h us d u rin g th is second pulse phase coherence is spread o u t am ongst a ll the possible coherences ( in the AX case there are 6, 4 single, 1

zero and 1 double q u a ntum coherence). E x a ctly how m u ch coherence is tra nsfe rred to each state depends on t j , J and the d u ra tio n of the second pulse.

3.1.3 D ata handling and presentation.

One problem in 2D n m r is the am o u nt o f da ta w h ic h is accum ulated. C onsider a lO ppm iH spectrum w ith 0.2 H z /pt re solutio n at 500MHz. In the one dim ensional spectrum th is gives an acq uisition tim e of 5s and uses 50,000 w ords of m em ory space. T h is is w ell w ith in the capacity o f any m odern spectrom eter. The e q u iva le n t 2D e xp e rim e nt w ith the same res o lu tio n in each dim e nsion requires 50,000 w ords for each tg spe ctrum and 2 x 25 ,00 0 fo r each t^. T h us leading to a to ta l o f 2 ,50 0 ,0 0 0 ,0 0 0 w ords o f com pute r m em ory w h ich is ro u g h ly e q u iva le n t to the

m em ory o f 60 personal com puters. The o th e r p rob le m is the a cq uisition tim e. This is 7.5s for each spectrum (mean t^ + tg) so fo r 25,000 spectra w ith eight scans each to allow fo r phase cycling the to ta l a cq uisition tim e is aroud 17 days. So some decrease in the d ig ita l re solutio n a n d /o r sweep w id th is required to m ake th is a viable operation.^

The sp e ctrum w h ic h res u lts from th is p u lse sequence con tains cross peaks show ing w h ich resonances are coupled to each other. There are also peaks on the m a in diagonal w h ich are

those due to un tran sferred coherence. The diagonal is in fact the ID spectrum . The other fact to note is th a t a coupling from A to X

m us t also m ean a coupling from X to A of the same m agnitude. T h is m eans th a t the spe ctrum is sym m etrical ab o ut the m a in diagonal. T his factor can be used in the processing to elim in ate sp u rio u s signals b y the process o f s ym m etris a tio n .^ T h is is a process w h ich rejects any data w h ich is no t sym m etrical about the

m a in diagonal. In th is m anner it removes a lo t o f noise and