高灵敏度荧光检测圆二色性（FDCD）测量

荧光检测的圆二色性（FDCD）可用于测量光活性样品被圆偏振光激发时荧光强度的差异。FDCD提供了优越的选择性，因为它允许用户在一组非荧光的手性分子中测量一个特定的荧光发色团的圆二色性。在FDCD测量过程中，摩尔可以根据FDCD结果和以下方程计算出圆二色性：Δε = εl  - εr  = (3.032 ∙ 10-5) ∙ S ∙ (1 - 10-A) / c ∙ l ∙ 10-A)具有高粘度的溶剂也会产生各向异性效应，从而产生伪影。为了进一步减少这些影响，FDCD-465包含了一个平衡掩模。如果F-y方向的荧光由于样品本身的堵塞而不能到达检测器，则F+x + F-x的荧光强度会略大于F+y + F-y，这可能会造成伪影。通过使用平衡掩模，这些荧光信号的强度可以被平衡，以消除伪影，即使在使用高粘度的溶剂。该应用说明表明，如果一个样品可修正为FDCD测量，与标准CD测量相比，可以获得更高的选择性和灵敏度。Application NoteCDSSpectroscopy High-sensitivity FDCD measurement2/4Application Note High-sensitivity fluorescence-detected circula r dichroism (FDCD) measuremen t F luore s ce nc e -d e te ct e d c i r c u lar d i c h r o i sm (FDCD) c a n b e u s e d t o m e as u r e th e d iffe renc e in fl u ore s c enc e in ten sit ies w hen an o p t i cally ac ti ve sa m p l e has bee n excite d wit h c irc u l a rly p o l a r iz e d lig h t. FDCD o f f e rs su p e ri o r s e l ec ti vi t y in th a t i t all o ws th e u ser t o measu re th e c irc u l a r d ichr o i s m of a specific fl u o resce n t c h r o mophore in a grou p of no n -f luo r es c e nt ， c h iral m o le cul e s . D u ring an F D C D me a s ure me n t，the mol a r c ir cu l a r d i c h r o i s m c a n be c al culat ed fr o m the F DCD r esu l t s and th e fo ll owi n g eq u ation ： where e -8 is th e d i ffe r e n ce betwee n l ef t a n d r ight circ u lar dich r o i sm， S i s the me a s ur ed FD C D v a lu e， A is th e a b sorbance of th e sample ， c is the con c e n tra ti o n a n d l i s the cell 's path l en g th. Th e J A SCO F D CD-465 i s a h ighly sensiti v e FDCD me a s ur e m e n t a c ce s so r y w h i c h co mb i n es b oth a c y lindr ical cel l a n d a n elliptica l cylin de r mi r r o r (Figu r e 1). T h is design all ow s f o r th e col l ec t ion o f a l l ra d i a te d f lu o r e sce n c e l i g h t w h ich is e m i tt ed i n a c ir cum f eren t ia l d i rectio n f rom th e ce ll Th e r esu lt not only improves th e l i g h t co l lection e f ficiency，bu t a l s o th e ab i l ity t o e l iminate th e ar t i f a ct s e f fect s c a u s e d b y f l u o r es c en c e a ni s ot ro py.23 S o lv en t s with hig h v iscos i t ie s c an a l s o co n t r i bu te t o a n isot r opy effects ， ind u ci n g art if ac t s . To fur th er minimize the s e e ff e c t s ， the F DCD-465 in clu de s a b a l anci n g m a s k . I f th e f luor e s c e n c e ligh t in th e F -y d ir ecti o n d o es n ot re ac h the d etec t o r due to a blockage f rom th e ce l l i t self ， the f lu or es c e n c e i nten s i t y of Fx +F _ ca n b e c om e s l i g h tly gr e a t e r than F +F， wh i c h coul d ca u se an a r ti f a c t. By us in g the b a l an c ing mask ， the intens i ties of th ese fluorescence signals c an b e balan c ed t o e l i min a t e th e a r tifacts ， e v e n w h e n u sin g so lv e nt s with h igh vi sc o si t i es. I n th i s applica ti o n n ote ， th e J-1500 CD spe c t r om e t er a n d FCDC-465 accessory we r e u sed t o meas u re ammo n ium d -10-ca mpho rs u lf o ni c a c i d in w a t er. JASCO INC. 28600 Mary's Court ， Ea s t on， MD 21601 U S A FDCD-465 Exterior view Interior view Figure 1. Exterior and interior view of the FDCD-465 and its components. Keywords J -1500， J -820， c ir cu l ar d i c h r o i s m，f lu o rescen c e-de t ected c i r c u l a r d i c h r o i s m， F D CD -465， flu o r escen c e， bi oc hem i str y Experimental FDCD CD Spectral bandwidth 4nm Spectral bandwidth 2nm Accumulations 16 times Accumulations 8 times Results S i nce the FDC D is an excitation spec t rum ， t h e excitation and emissio n wavelengt h s need to be obtained u s ing a f luo r e sc enc e sp e c t r om e t e r pr i or t o F D C D me asur e me n t s . A n exc i t a ti o n w a vel e n g t h of 285 nm w a s ob t a in e d f r o m t h e f lu ore s c e n ce e x p eri me n ts al o ng w ith a f lu o res c en c e em i s si o n m ax ima a t 427 nm. The s e w a ve len g ths we re th en u s e d to meas u re the F D C D of 0.0024 M ammon iu m d -10-campho r su lf on i c ac i d (d-10-ACS)， wh i c h i s s h own in Fi g u re 2. Figure 2. FDCD (top)， CD (middle)， and absorption (bottom) spectra of 0.0024 M d-10-ACS in water was obtained us i ng a L38 exc i tation light cut-off f i lter and a 1 cm pathlength cell. 28600 Mary's Cour t ， E asto n ， MD 21601US A Based o n the obt a ined FDCDC，CD ， a n d a bsor p t i o n spect r a ， th e CD spec tru m wa s conver t ed from FDCD u sing equ ati on 1. A co mp a ri so n o f a m easured CD s p ectru m a n d C D sp e ctr u m calculated f rom F DCD meas u remen t s i s s h own in Fi g u re 2and d epic t s th at th e spec tr a a r e i n good agreem e n t with eac h oth e r . Figure 3. Comparison of the measured CD spectrum (lilac) and CD spectrum calculated from FDCD data (magenta). F i g ur e 4 s h o w s th e FDCD a n d CD spec tr a of (1S ，2S)-t r ans-c y clohexanediol bi s (6-met h oxy-2-n a ph thoa t e) a n d acetoni tril e s o lu ti o n a t v a r y in g c o n c e n tra t ion s， r e sp ective l y . Fo r th e F D CD data (left)， t he d iff e r e n c e in the f l uo r es c ence in t e n sit y e xc i t ed by circ u larly p ola ri z e d lig h t i s n ormal i z ed a g a in st the inte n s i ty o f the t otal f lu o r e sc en ce . Th e re f o r e ， th e F D C D sig n al (m d eg) show n i n Figu r e 4 d i splays a con s tant valu e regard l ess o f sample con c e n trat i on . In th e CD m ea s urements， a CD s i gn al co u ld o n l y b e o b se r ved w ith a co n cen tr a t io n o f 2 ·10M . Th e co n cent r atio n s a t 2·108 M an d 2· 10° M d i d not sh ow u s a bl e CD s i gn al s . H oweve r ， u sin g th e F D C D-465 a c cessor y fo r spe c t r a l measu r eme n ts， a sig n al w as c l ear l y obse r ved with a conce n tra ti o n two orders of mag n itu d e lower t h an 2·10M. Figure 4. FDCD of (1S，2S)-trans-cyclohexanediol bis (6-methoxy-2-naphthoate) (left) and CD of acetoni t rile (right) spectra at varying low concentrations. We kindly thank Tatsuo Nehira (Hiroshima University) and Katsunori Tanaka (Osaka University)for providing us with information regarding (1S， 2S)-trans-cyclohexanediol bis (6-methoxy-2-naphthoate). 28600 Mary's Cour t ， E asto n ， MD 21601US A Conclusion Th i s ap p l i ca ti o n n ote d emonstr a tes t hat if a sample i s ame n dab l e t o FDCD measurement ， mu ch h igher sele c tiv it y and sen si tiv ity can be ach i e v ed c om p ared to standard CD mea s u r e m e n ts. References 1. Tur ner， D.H.， T in o co Jr ， l .， a n d M. Ma estre ， (1974) JACS， 96，4340-4342. 2. Nehir a ， T .， tan a ka ， K .， T ak ak u w a ， T .， O h s hima， C .， M a s a go， H.， P e s c ite l li， G.， Wad a ， A .， an d N . B e rova，(2005) Appli ed Spectrosc o py， 59，121-125. 3. T a n a ka ， K .， P escitelli， G.， N ak anis h i， K .， a n d N . Be r ova ， (2005) Monats h e f te f u r Chemie /C h e m ical Mo n th l y， 136， 367-395 4. T .Nehira ，(2005) Mo n ats h efte fur C h emie /Chem i c al Monthl y ， 136， 447-487. 5. D on g， J.G.， Wa da ， A.， T ak a k uw a， T .， N aka n i s h i， K.， a nd N. Be r o v a ， (1997) J ACS， 119， 12024-12025. 6. M u t o， K.， Mo c h iz u k i， H.， Yo s h ida ， R.， Is h i i， T.， and T . H a n da，(1986) J A CS， 108， 6416-6417. 7.N e h ira， T .， Pa ri sh ， C. A .， Joc h usc h ， S.， T u rro， N . J .， N a k an i s h i， K.， an d N. Be rov a，(1999) JACS， 121， 8681-8691 28600 Mary's Cour t ， E asto n ， MD 21601US A