신부경, 안광일, 이윤형, 장태완

From www.cyworld.com/yschem

▶ Objectives

• Understand the basic principles of spectroscopy.

• Determine “Fluorescence Quantum Yield” using dilution method.

▶ Theory

• from www.colourtherapyhealing.com

RotElecVib

Etot = Eelec+Evib+Erot

From “Principles of Instrumental Analysis”,Skoog.., 5th

From “Principles of Instrumental Analysis”,Skoog.., 5th

<Jablonski Diagram>

From “Physical Chemistry”, Donald McQuarri..,

Kasha’s Rule

▶ Apparatus• UV/VIS Spectrophotometer : UV-160A• Fluorometer (Spectrofluorophotometer)

: RF-5301 PC• Cell• Volumetric flask• Beaker• Pipette

간단한 형광분석계 구성요소

1) Light Source

2) Monochromator

3) Detecter

From www.gmi-inc.com/categories/cary5000.htm

Deuterium tungsten lamp (D2 : UV190~350nm) (WI : IR, VIS :320~1900nm) Continuous spectrum

UV/VIS spectrophotometer Fluorometry

From curie.umd.umich.edu/srsmith/uvvis.html

Xenon arc : (250~1400nm)Continuous spectrum

FluorometryPMT(Photomultiplier tube)

UV/VIS spectrophotometerSi photo-diode detector

From edic.hu-berlin.de/…/html/chapter2.html

From www.daeilgaebal.co.kr/web_daeil_eng/main_d_3.htm

From www.shimadzu.co.uk/fluorescence/rf-1501.html

▶ Reagent• Ethanol (Solvent)• Rhodamine 6G• Cresyl violet perchlorate• Nile blue A perchlorate

1) Aromatic compound

2) Ring에 붙은 치환기가 EDG일 경우

3) Rigid, Planar한 분자

SynonymBasic Red 1

Molecular FormulaC28H31N2O3Cl

Molecular Weight479.01

고리수 : 4 축합수 : 3Rigid, Planar 분자

From omlc.ogi.edu/spectra/PhotochemCAD/html/rhodamine6G.html

SynonymOxazine 9 perchlorate

Molecular FormulaC16H11N3O · HClO4

Molecular Weight361.74

고리수 : 4 축합수 :4Rigid, Planar 분자

From omlc.ogi.edu/spectra/PhotochemCAD/html/cresylviolet.html

SynonymBasic Red 1

Molecular FormulaC20H20ClN3O5

Molecular Weight417.84

고리수 : 4 축합수 :4Rigid, Planar 분자

From www.sigmaaldrich.com/catalog/search/TablePage/9541118

▶ Procedure

• 시료 준비.(Analyte1ml + Ethanol 9ml)

• Absorption을 측정.• Solvent(Ethanol)을 찍어서 바탕값 보정.• (530nm : Rhodamine 6G, C.V.P ,Nile blue)

• 시료 희석.• 각각의 형광양자수율과 오차율 계산.

*주의사항1. 에탄올을 일정량 넣고 형광물질을 넣어준 다음 다시

에탄올을 넣어 희석.2. 형광물질은 극히 소량을 사용하여도 된다.

(Rhodamine 6G:발암물질)3. 에탄올 및 각 시약에 다른 Pipet을 사용.4. 기기에서 형광과 흡광을 측정할 때에는 Cuvet표면에

이물질이 없도록 처리 후 측정.

▶ Results

300 400 500 600 700 8000

20000

40000

60000

80000

100000

120000

Extin

ctio

n Co

effic

ient

(M-1cm

-1)

Wavelength (nm)

R6G Cresyl Nile Blue

πConjugation Expansion

Absorption Red Shift

500 550 600 650 700 750 8000

20

40

60

80

100

Corre

ctio

n Fa

ctor

Wavelength (nm)Correction Factor (Red Line)

PMT Sensitivity Curve

300 400 500 600 700 8000

20000400006000080000

100000120000

wavelength(nm)

Fluorescence Intensity (a.u.)Extin

ctio

n Co

effic

ient

(M-1cm

-1)

300 400 500 600 700 8000

20000400006000080000

300 400 500 600 700 8000

1000020000300004000050000600007000080000

R6G

Cresyl Violet

Nile Blue

▶ Fluorescence Quantum Yield

• Φ =

• D = Area of fluorescence spectrumA = Optical Density

• ΦX : ΦR = :

Number of photon emitted(D)Number of photon absorbed(A)

DX DR

AX AR

• ΦX = ΦR

= ΦR

AR(λR)×IR(λR)×nX×DX

AX(λX)×IX(λX)×nR×DR

AR(λR)×DX

AX(λX)×DR

• I : Intensity of lightn : Refractive index

▶ ResultUV-VIS Fluorescence

λ(nm) O.D Area Φ

Rhodamine 6G

530 0.149 28727.1 0.95(ref)

Cresyl violet perchlorate

530 0.068 6644.7 0.48(-11%)

Nile Blue A perchlorate

530 0.074 2390.5 0.16(-33%)

▶ Discussion & Further study• 형광에 영향을 미치는 요인

- Rigid한 구조- 온도와 점도의 영향- pH의 효과- 용액중의 산소의 영향

형광수율(기기분석책)

▶ References• “Fluorescence and Phosphorescence”, David Rendell, 1987,

Wiley• “Introduction to Fluorescence spectroscopy”, Ashutosh Shama,

Stephen G.Schulman, 1999, University Science Books• “Physical Chemistry; A Molecular Approach”, Donald A.

McQuarrie, John D. Simon, 1997, University Science Books• “Principles of instrumental Analysis” 5th.ed, Skoog,

Holler,Nieman, 1999, Freedom Academy Publishing Co.,• “Spectrophotometry and Spectrofluorimetry”, Michael. G. Gore,

2000, Oxford University