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Principles of Fluorescence Spectroscopy. Chemistry Department XMU. Chapter Two. Instrumentation for Fluorescence Spectroscopy. Instrumentation. 2.1 General Description 2.2 light Sources 2.3 Monochromators 2.4 Sample Cuvette 2.5 Detectors
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Principles of Fluorescence Spectroscopy Chemistry Department XMU XIAMEN UNIVERSITY
Chapter Two Instrumentation for Fluorescence Spectroscopy XIAMEN UNIVERSITY
Instrumentation 2.1 General Description 2.2 light Sources 2.3 Monochromators 2.4 Sample Cuvette 2.5 Detectors 2.6 Correction of Fluorescence Spectra 2.7 Sensitivity of spectrofluorometer 2.8 Commercial spectrofluorometer XIAMEN UNIVERSITY
General description Excitation mono- chromator Sample cuvette Light source Emission mono- chromator PMT XIAMEN UNIVERSITY
Excitation Monochromator Grating Emission Monochromator Grating PMT 150 W Xenon Source Sample cuvette F-4500 Schematic diagram XIAMEN UNIVERSITY
Dual grating excitation monochromtor Xenon Lamp Emission monochromtor PMT PMT Ref cell Sample cuvette SLM Schematic diagram XIAMEN UNIVERSITY
PMT Emi Mono SC Exci Mono XL PMT FLR XIAMEN UNIVERSITY
I p S Ideal Spectrofluorometer • Intensity of light source as a function of wavelength • Efficiency of mono-chromator • Sensitivity of PMT to photons • Property of monochromator independent of anisotropy of light XIAMEN UNIVERSITY
光谱的失真 真实的光谱应是物质的性质决定,即由和决定。但测得的光谱与I 0,有关,故光谱失真; F 由检测器所检测,检测的失真也会导致光谱失真。 S —检测灵敏度 XIAMEN UNIVERSITY
激发光谱的失真 • 光源的强度相对于波长的分布随光源的不同而不同,对特定光源而言,光源的强度随波长的变化而变化。 At a em, san ex Apparent excitation Light profile Excitation XIAMEN UNIVERSITY
光源的波长特性 氙-汞灯在紫外区的光源强度比氙灯强得多,但光谱输出不如氙灯平滑。 XIAMEN UNIVERSITY
365 nm 光源的波长特性 XIAMEN UNIVERSITY
单色器的波长特性和偏振特性引起的光谱失真 激发单色器对不同波长的光子和不同偏振角度的光子具有不同的透过率,引起进一步的光谱失真。 XIAMEN UNIVERSITY
全息光栅 机刻光栅 E┷ E┷ EII EII 单色器的波长特性与偏振特性 XIAMEN UNIVERSITY
发射光谱的失真 At a ex, san em • 发射单色器的波长特性 • 检测器的波长特性, S • 某些波长的光强度超过线性范围(对激发和发射光谱均引起失真) XIAMEN UNIVERSITY
2.2 light source 2.2.1 气体放电灯 氙灯 xenon • 波长特征 扫描水的锐利光谱可获得 • 工作条件 高压启动, 20 – 40 Kv; 工作电流大; 内充氙气,室温气压 5 atm, 工作气压 20 atm. 能量分布 • 使用的安全措施 XIAMEN UNIVERSITY
Corrected and uncorrected spectra XIAMEN UNIVERSITY
气体放电灯 高压汞灯 能量分布 常用365 nm的光激发荧光物质 (国产960型) XIAMEN UNIVERSITY
气体放电灯 氙-汞灯 紫外区的强度比氙灯强的多,但光谱输出不如氙灯平滑。 XIAMEN UNIVERSITY
气体放电灯 低压汞灯 产生尖锐的汞线光谱,不适宜作光源,但可用于作波长校正,其中253.7 nm 的光线最强。 XIAMEN UNIVERSITY
2.2.2 激光光源 气体激光器 固体激光器 可调谐染料激光器 半导体激光器 Reference E. W. Small, “Laser source and micro channel plate detectors for pluse fluorometry” in “Topics in fluorescence spectroscopy”, V1. Techniques, pp.99-145, ed. By J. R. Lakowicz, Plenum Press, NY, 1991. XIAMEN UNIVERSITY
激光光源的基本特性 激光光源是一种受激辐射的光源,具有单色性好、谱线强度大、方向性好、相干性好的光源 在普通光源中,单色性最好的氪灯,在低温条件下,谱线宽度 = 0.047Å。激光的谱线宽度可小于10-7Å 激励发射光与激励光子具有相同的偏振方向, 激光是偏振光,有利于荧光偏振测定。 XIAMEN UNIVERSITY
Spontaneous emission Stimulated emission Induced absorption 激光工作物质 光学共振腔 受激发射 Stimulated emission 激励源(泵浦源,光源和电源) 反射镜>99% 反射镜≈90% XIAMEN UNIVERSITY
4 Pump bands Idler transition 3 Meta-stable state hv E 2hv 2 Short – lived state Idler transition 1 Ground state 激光形成机制 XIAMEN UNIVERSITY
4P (3P44P) 4D02/5 514.5 nm 2P3/2 4S (3P44S) Ar + e→(Ar+)*+2e Ar+3P5 Ar+ + e→Ar Ar3P6 氩离子激光器 Meta-stable state Laser transition • 波长514.5 nm, 单波长光源; Short-lived state • 气体放电形成粒子数翻转,耗电量大; • 可调节电流控制激光强度 XIAMEN UNIVERSITY
Argon Laser XIAMEN UNIVERSITY
Nonradiative transition Pump bands 4F3/2 Lifetime 200 s 590 nm 750 nm 800 nm 1064 nm 4F11/2 Nonradiative transition Nd3+ Nd:YAG激光器(掺钕钇 铝石榴石激光器) Meta-stable state 单波长,532 nm Short-lived state 泵浦源:氪弧灯 价廉 XIAMEN UNIVERSITY
Nd:YAG 激光器 XIAMEN UNIVERSITY
T2 S2 S1 nr~1ps Isc~10 ns F ~1ns T1 532 nm 600 nm laser transition P ~100 ms S0 可调谐染料激光器 2d cos = m的光才具有低的损耗,形成振荡,输出激光 XIAMEN UNIVERSITY
Dye laser XIAMEN UNIVERSITY
Laser dye 波长可调谐(可选用不同的荧光染料作为工作物质,产生不同波长的激光) XIAMEN UNIVERSITY
Ec Conduction band hv P Bandgap, Eg 2hv Ev Valence band N e Semiconductor laser diode 半导体激光二极管 Diode Laser 二极管激光器 通过PN结电注入泵浦的方式实现受激发射的半导体器件。 体积小,结构简单,效率高,能直接调制,但输出功率、单色性和方向性不如其他激光器好。 XIAMEN UNIVERSITY
Reference • T. Imasaka, “Diode laser in analytical chemistry”, Talanta, 48 (1999),305-320 • D. J. S. Birch and G. Hungerford, “Instrumentation for red / Near-infrared fluorescence” in “Topics in fluorescence spectroscopy”, V4, probe design and chemical sensing, ed. By J. R. Lakowicz, Plenum press, UY,1994 XIAMEN UNIVERSITY
2.2.3 Semiconductor light emitting diode LED, light-emitting diode 工作原理 LED特点 在低压(2v)、小电流(几十毫安至200毫安)下工作,功率小,体积小,稳定,可靠,寿命长(105-106小时),调制方便,通过调制LED电流来调制光输出。 XIAMEN UNIVERSITY
I I t 2.2.4 Pulse source 脉冲光源 Flash lamp 闪光灯 Light source for steady – state fluorescence measurement Pulse source Light source for time-resolved fluorescence measurement 气体放电灯 典型的脉冲宽度为2 ns 锁模氩离子激光器 脉冲宽度 100 ps, 脉冲频率 76 MHz 锁模离子激光器泵浦的脉冲染料激光器 脉冲宽度可达 ps 级,频率一般小于1000 Hz XIAMEN UNIVERSITY
Time-intensity profile of nitrogen flash lamp From JRL’s PFS XIAMEN UNIVERSITY
Wavelength Characteristics of Nitrogen flash lamp 337nm 358nm 316 nm From JRL’s PFS XIAMEN UNIVERSITY
Wavelength profile of deuterium flash lamp 典型脉冲宽度为2 nm 氘闪光灯 From JRL’s PFS XIAMEN UNIVERSITY
混合 F = 10 ns 3ns 1ns 10ps 脉冲宽度 脉冲宽度对时间分辨荧光测定的影响 XIAMEN UNIVERSITY
Flash xenon lamp FLR Varian XIAMEN UNIVERSITY
凹面光栅 Concave grating Plane grating 2.3 Monochromators 单色器 Be used to disperse polychromatic or white light into the various colors or wavelengths. Diffraction gratings 衍射光栅 XIAMEN UNIVERSITY
Consideration in selecting grating Dispersion 色散力 High Efficiency The stray light levels 杂散光水平 The most important factor in selecting a grating XIAMEN UNIVERSITY
Efficiency distribution XIAMEN UNIVERSITY
ᅩ II Efficiency distribution average XIAMEN UNIVERSITY
NATA and glycogen in buffer Polarizer condition VV VH Polarization characteristics of monochromators NATA: N-乙基-L-色氨酰胺 From JRL’s PFS XIAMEN UNIVERSITY
Polarization characteristics of monochromators From JRL’s PFS XIAMEN UNIVERSITY