罗丹明B相对量子产率的测定

不同的分子和环境条件不仅可以影响一个分子是否会产生荧光，而且还可以决定所发射的荧光辐射的强度。分子的量子产量来描述，定义为吸收的光子数与样品发射的光子数的比值。测量荧光量子产量有两种方法：绝对法和相对法。绝对方法通过使用积分球检测所有样品的荧光，直接得到量子产率。该相对方法将标准样品的荧光强度与未知样品的荧光强度进行比较，以计算出未知样品的量子产率。因此，所得到的结果取决于标准样品的量子产率值的准确性。Application NoteFP-0009 2/4Determination of the Relative Quantum YieldApplication Noteof Rhodamine B Determination of the Relative Quantum Yield of Rhodamine B Introd u ction Differe n t molecula r and envi r onmen t al condit i ons not only effect whether a molecule will f l uoresce o r n ot， but can a l so determine the i n te n sity of the emi t ted fl uoresce n ce radiat i on . A molecu l e’s e f ficiency to f l uoresc e is descr i b e d by its quantum yie l d an d i s d e fin e d as t he ratio o f t h e numb er of photons a b sorb e d to t h e numb er of photons emitted by t h e samp l e. There are two methods F-8500for measur i n g the fluorescence quantum yield： t h e absol u te method a nd the r elat i ve m ethod. Th e abso l u t e metho d di r ectlv obtains th e q u an t um yi e l d by de t e cti n g all sampl e f luor e sc e nce through t h e use of FP-8500an i n tegrating sphere. The r elative met h od compares the f luorescence inte n sity of a standard S p ect r o fl uorometer sa m ple with t h e f l uoresce n ce i n tensity of a n u nk nown sample to calculate the quantum y i eld of the u n known sam p le. T h erefore， th e obtai n ed results depe n d on t h e accuracy of the sta n dard sam p le's quantum yield v a lue. This applicatio n not e will d e monstrate how to obtain the rela ti v e q u a n t u m yie l d of Rhoda m i n e B us i ng fl u oresce i n as a sta n dard sample. Keyword s FP-8500， FUV-803， F l uorescence， Quantum Y i e l d ， Rela ti ve Met h od E xper i me n tal In ord er to calculate th e re l at i ve qua n t u m yield of an u nknown sampl e ， th e following information is required： the quantum yie l d of the standard sample， the absorpt i on spectra of the samples， and the a r ea of the samples' emission spectra after spectral correct i on. Additionally， whe n t h e solvent of the standa r d sample is d i fferent from that of the unknown sample，t h e average r efr a c t ive i n dex v a l u e i s n eeded. A dil u tion rat i o is also r equi r ed i f t he standard and/o r unk n own s a mples are dilut e d. By o b tai n ing the aforement i oned parameters， th e re l ative q u antum yie l d of unk n own sample， D ， can be calcu l ated using the f ollowing equation： whe r e D i s t h e qu a ntum yield of t he st a ndard sam p le，A an d A a re th e absorba n ce of t he st an dard a nd unk n own sample s ， r e sp e ctively， Fand F are t h e areas of the s t andard and unknown emission spect r a， r e spectiv e ly， nsand nar e the r efract i ve indices of t he standard and unknown samples， r espec ti vely， and D/D i s the di l u ti on r atio. Pr i or to obta i ning t he relative quantum yield calculat i ons， r hodam i ne B was used to co rr ect the excitation spectrum a n d a cal i brated h alog en lig h t sou r ce was measured f rom 450 t o 700 nm to correct the em i ss i on spect rum . Absorbance Emission Excitation Wavelength 500nm Excitation Bandwidth 2.5nm 5nm Emission Bandwidth 10 nm 5nm Scanning Speed 200 nm/min 200 nm/min Data Interval 1nm 0.5nm Response Time 0.5 sec 0.5 sec PMT Voltage 230V 430V Fo r absorbance measurements， 5400 pg/L and 72 p g/L of fluorescei n and r h odamine B were dissolved i n ethanol，respectively. For fluo r escence measu r emen t s， 7200 ug/L a n d 36 pg/L of fluorescein and r hodamine B were d i ssolved i n ethanol ， respect i ve l y. Res u lts T h e absorpt i on spect r a of f luorescein and rh odami n e B were measu r ed using the [Absorbance Measureme n t] program a n d FUV-803 A b sorba n ce measureme n t cel l b l ock. T h e results a re illustr a ted i n Figure 1 a n d i n d i cate tha t the a bsor b ance of f luoresc ei n at 450 nm was 0.490 a n d th e absorbanc e of r hoda m i n e B at 500 nm was 0.225. JASCO INC. Wave l engt h [nm] Figure 1. Absor p t i on s p ect r a of f l uorescei n (b lu e) an d rh odamine B (p u r p le). Int. Figure 2. Fluorescerce emission spectra of fluorescein (blue) ard rhodamire B (purple). T h e e mission sp e ctra of r hodamin e B and f l uor e sc e in we r e then measured and are s h own i n Figure 2. I n order to preve n t i nner fi l ter ef f ects or r eabsorption of the em i tted f luorescence radiation， the sample so l u ti ons were diluted so that the sample absorbance was less t han 0.02. T h e a r ea unde r f l uo r escein's emissio n spectrum was 727204 and th e a r ea un d e r rh odami n e B's emission spect r um was d e te rmin e d t o b e 243513. Sinc e e thanol is u s e d as t h e so l ve n t for bot h t h e sta n dard and unk n own solut i on s ， th e average refractive i ndex of the solven t i s not r equi r ed . However， when the solvents used are d i fferent ， the publ i s h ed value is necessary. T h e standard sa m ple solution was di l uted by 75 t i m es and t he unknown sample 100 t imes， which makes the dilutio n r atio 1.33. T h e r e l at i ve quantum yield of Rhodam i ne B was t h en calculated by applying the obtai n ed p aramete r s to th e equ a tion ab ove. The calcula t ed r elat i ve quant u m yield of r h odami n e B i s 92% and wit h i n range of t h e p u b lished valu e s of 69-97%. Con c l u sio n T hi s a p plica t io n n ot e d e mon s t r at e s t h at the FP -8500 can be us e d to e asily d e termi n e a sam p le 's r el a t i v e qu an tum ef f iciency f r om a standard sample. References 1. Kazuhiko Kinos hi ta and Koshi n Mihas h i . Fluor e sc en c e measur e me n ts -Appl i cations for Biochemical Scien ce s. Th e Spectrosco p ic Society of Japan， Measu r ement Met h od Series 3， Japan Scienti f ic Societies Press， 1983.