1、Cary Eclipse荧 光 分 光 光 度 计,2,荧光分析法的历史,荧光的发现历史16世纪 Monardes “极可爱的天蓝色”17世纪 Boyle&Newton 荧光现象19世纪 Stokes 是一种发射光，萤石 荧光 20世纪 荧光分析法发展成为一种高效、灵敏的光谱分析手段 第一台荧光计 1928年 第一台光电荧光计1952年 第一台商品化的荧光分光光度计,3,光源,样品,光检测器,谱图,Abs,单色器,紫外-可见分光光度计,4,激发,发射,光源,样品,检测器,荧光分光光度计,单色器,单色器,5,荧光测量的意义：,灵敏度高 比UV-Vis方法灵敏 100 - 1000 倍 UV-Vi
2、s方法测量的是一定波长的光透过样品后的衰减，是高背景上的相对值 荧光是样品被激发后发出的光，测量的是低背景上的绝对发光,6,荧光测量的意义：,选择性好 能吸收光辐射的分子不一定都能发出荧光共轭键，刚性平面结构，给电子取代基 能发出荧光的分子结构或介质环境不同，荧光光谱也不相同吸收（激发）和发射光谱 混合物的分析测定（同步扫描）,7,Excitation and Emission spectra of Anthracene,Light source,Excitation scan,Emission scan,Int,Int,8,荧光测量的意义： Why do fluorescence measu
3、rements?,分析应用 浓度测量 皮摩尔 材料鉴定研究光物理化学作用的机理测量分子动能,9,荧光分析法的应用领域,生命科学RNA/DNA研究蛋白质、氨基酸和酶的分析细胞内的离子浓度研究：Na+, K+, Ca2+, H+, Mg2+ , Zn2+等,10,荧光分析法的应用领域,临床、法医分析荧光免疫分析蛋白质、氨基酸和酶的分析细胞内的离子浓度研究：Na+, K+, Ca2+, H+, Mg2+ , Zn2+等,11,荧光分析法的应用领域,新药研制和药物分析药物研制： 组合化学药物分析： 可卡因、吗啡、鸦片碱、巴比妥类、酚噻嗪、阿托品、异烟肼、利血平、色胺、阿司匹林、青霉素、四环素等药物的分
4、析,12,荧光分析法的应用领域,环境、自来水和污水水质监测叶绿素、油污染、多环芳烃、表面活性剂等的分析 食品科学食品添加剂、维生素含量和霉素等的分析 化学、化工和材料科学常规分析、新化合物的光化学行为研究等无机离子及其配合物的分析晶体材料的共振荧光,13,Cary Eclipse 设计的设计思路,新颖、现代的设计 荧光、磷光、化学/生物发光三种测量模式 先进的Cary 系列软件 波长范围200nm - 900nm 多种附件可选,14,Cary Eclipse的设计特点,光源 Light Source光学元件 Optics检测器 Detector软件设计 Software,15,对光源的要求,稳
5、定 使用寿命长安装、维护方便 足够的强度UV-Vis中： Abs = bC 荧光分析中：F = kI0 bC I0为激发光强度除了与样品浓度有关外，荧光强度还与激发光强度成正比,16,Cary Eclipse的闪烁Xe灯模块,低功率消耗 仅12 W 输出功率最高 75 KW普通只有几百瓦 灯的电子采用模块设计，安装维护方便,17,Cary Eclipse的闪烁Xe灯模块,闪烁Xe灯与Schwarzschild聚光元件的结合，使得Eclipse获得了最高的输出功率,18,The Power of Xenon!,Eclipse的闪烁Xe灯只在测量时开启避免了连续光源长时间照射造成的荧光衰减和光敏样
6、品的降解使用寿命长可连续闪烁20，000小时，保修两年基本与仪器同寿命，终生不用更换普通Xe灯的寿命2000小时,19,忽略室内光线的干扰，可开放样品室操作 Dark current correction(before sample) Ratio result =(sample PMT-dark)/(ref PMT-dark) x 1000卓越的微池测量性能 40 uL 微池 荧光素的检测限 10 pM,The Power of Xenon!,20,Mercury lines from room light,Raman band,21,22,Cary Eclipse,sample chambe
7、r open,sample chamber closed,Raman band,23,Cary Eclipse 蒽的发射谱图,sample chamber open,sample chamber closed,24,Cary Eclipse的单色器,扫描速度24000nm/min，且消除了波长漂移 标准配置的自动/手动可调的多级滤光片，有效消除了散射光干扰,25,散射的识别和摒除,26,Prescan capabilities,Automates the process of determining the optimum Ex. and Em. wavelengths for your an
8、alysis! Provided in: Advanced Reads Concentration Scan Kinetics Lifetimes,27,Cary Eclipse的单色器,Czerny Turner 设计 狭缝宽度可调 1.5, 2.5, 5, 10, 20, 10 nm 圆形 水平取向 1cm标准液池， 样品量0.5 mL增加信噪比 扩大了检测区域 增强发光信号 灵敏度比垂直狭缝提高530倍,0.5mL,28,2 支R928 光电倍增管 动态范围宽 : 400-1000 Volts 对红区发射敏感 标准配置革命性的新电子设计 数据采集速度达 80 点/秒：对不到1秒的快速反应
9、的测定精度更高 扫描速度达24000nm/min，且消除了波长漂移 磷光寿命测量的数据采集速度达每点/ 微秒（10-6s）,Cary Eclipse 检测器,29,为何使用R928光电倍增管?,R928 PMT检测波长范围更宽:200 to 900 nm全波长范围内具有卓越的灵敏度，而不是仅仅在350nm! 无论在紫外还是可见-近红外光区（UV和Vis/NIR） 内测量低浓度样品时都能获得更高的灵敏度 其他品牌把这一能力作为升级的可选配置,30,光电倍增管电压设置,PMT 电压设置 线性范围 400 - 1000 V 起始设置为“Medium” 灵活性在不牺牲分辨率的条件下，增加信号强度 延长
10、PMT寿命,31,标准的Windows传统的软件,多任务 图标操作Start applications from icons Eclipse方法的快捷键 复制、剪帖和添加注解 等等 and so on.,32,Cary Eclipse 软件包功能,扫描激发、发射和同步扫描 浓度测量 动力学80 点/秒-极快的动力学数据采集速度 时间分辨 认证等,33,Scan Application,34,数据采集模式,荧光 (寿命 10-11 to 10-7 sec)利用光源产生发射在光源处于“on”的状态下，测量发射 磷光 (寿命 10-3 to 102 sec)利用光源产生发射在光源处于“off”的状态
11、下，测量发射 化学/生物发光在光源处于“off”的状态下，产生发射在光源处于“off”的状态下，测量发射,35,荧光 VS 磷光 - 萘,36,荧光 VS 生物/化学发光 - 钌的配合物,化学发光,荧光,37,所有的方法参数在一个窗口设置完成!,Cary Tab Instrument params. Options Tab Lamp, PMT voltage, filter options, data display options Accessories Tab Analyze Tab Setup Calculations Reports Tab Report format Auto sto
12、re Tab Data Storage options,38,所有应用的控制界面,39,新的数学计算能力,运行完后的计算 Polarization calculations Polarization Anisotropy Sum Function Mean Deriv 1, 2, 3, 4 Integrate e.g Quantum Yield calculations Interpolate,40,3D graphics - 3D Grams software,GRAMS 3D software Very powerful/fast 3D data manipulation Zooming
13、in, fly mode, rotating spectra Variety of data displays Industry acceptance Save As *.SPC file Saves visible traces into Grams format If multiple files are visible, data is saved automatically as a GRAMS meta file Open GRAMS 3D and directly import Eclipse data,41,3D-Graphics,At customer shipments 3D
14、 Grams will be integrated into the Eclipse software Icon will appear on graphics toolbar Automatically startup GRAMS 3D and load visible data,42,等高线图,43,New Graphics capabilities,Advanced tick mark options Auto tick mark options Manual tick mark options # ticks for X and Y axes # decimal places X an
15、d Y axes,44,Peak Labels,45,Copy and Paste Graphs,No more Grey borders!,46,New Auto Store capabilities - Email,Auto convert Auto store Tab Email your stored data to colleagues Setup system to automatically email your data at the end of a collect Startup run and go back to your office. Obtain an email
16、 when your data has been collected!,47,Cary Eclipse ADLs,ADL provided as standard, offering unlimited software flexibility! Control third party accessories via software Perform specialized calculations with ease! Create dedicated analyzers to enhance ease of useWe will provide free ADLs on our Web p
17、age Same as UV-Vis ADL programs,48,Cary Eclipse Validation,The most complete Validation documentation (IQ/OQ/PQ etc.) in the market! An aid for pharmaceutical labs, that are consistently audited Installation/Operation/Performance QualificationThe most comprehensive suite of instrument Validation tes
18、ts included as standard An aid for pharmaceutical labs, that are consistently audited. Automation of tests will save time and will guarantee the tests are performed reproducibly day after day,49,Cary Eclipse,Factory tests Includes all tests performed at Final Acceptance stage, prior to shipment Inst
19、allation Wavelength Accuracy (Xe) SBW Sensitivity Stray light ASTM limit of detection using Quinine Bisulphate,Validation tests and performance specifications,50,Cary Eclipse,GLP privileges ON by default Factory set instrument parameters not accessible Most customers will not want to change these pa
20、rameters For those who do, they need to have Administrator privileges,51,Cary Eclipse具有各种荧光分光光度计中最大的样品室 可以容纳如N2超低温装置和多孔板读数器等多种附件，也为各种自制装置提供了最大的扩展性,Cary Eclipse 样品室,52,Eclipse Accessories - Temperature,Single cell thermostatting Single Cell Peltier holder Monitor Block, Probes 1, 2,3,4 Water thermost
21、atted cell holder PCB 150 Monitor: PCB 150, Probes 1,2,3,4 (None): Other Water baths Monitor: Probes 1,2,3,4,53,Cary Eclipse Accessories - Multicell/Temperature,4 cell Multicell holder peltier thermostatted temperature controller Monitor: Block, Probe 1,2,3,4 UNIQUE Nobody offers multicell Peltier c
22、ontrol,Temperature controls all on one page! Communication ports, Temp. monitor, Temp. display etc.,54,Accessories - Multicell/Temperature,4 cell Multicell holder water thermostatted PCB 150 water bath Monitor: PCB 150, Probe 1,2,3,4 Water bath (other) Monitor: Probe 1,2,3,4,55,Cary Eclipse 多孔板读数器,5
23、6,Sensitivity Cary Eclipse VS PE LS-50B,96 well microplate 384 well microplate,21pM 17pM,140pM -,Cary Eclipse LOD,LS-50B LOD,384 well Eclipse LOD is an order of magnitude better than PEs 96 well LOD!,57,Cary Eclipse 多孔板读数器,具备荧光分光光度计软件的所有功能扫描、浓度等 不存在光纤传输的光强损失问题，灵敏度同类中最高96孔 21 皮摩尔384孔 17 皮摩尔 测量速度快96孔的
24、浓度测量 50秒即可完成394孔的浓度测量 90秒内完成,58,多孔板 定位,59,Accessories,固体样品支架 光纤耦合器 荧光探头Fluorescence dip probe 快速过滤附件 马达驱动的起偏器 自动进样器 Sipper,60,Cary Eclipse performance specifications,Wavelength range 200-900 nm zero order selectable Wavelength accuracy 0.5 nm, 541.9 nm 1.0 nm (200-900nm) Wavelength reproducibility50
25、0:1 RMS, 350nm excitation, 10/10nm slits,61,Cary Eclipse performance specifications,Limit of detection (LOD) 1.0pM fluorescein using standard cell 10pM fluorescein using 40ul microcell 25pM Quinine Bisulfate (QBS) in standard cell Minimum sample volume 0.5ml using standard cell SBW (Excitation and E
26、mission) 1.5, 2.5, 5, 10, 20nm limiting resolution 1.5nm,62,Cary Eclipse performance specifications,Signal averaging 0.0125 to 999 sec (Fluorescence) 1 us to 10 sec Gate time (Phosphorescence) 40us to 10 sec Gate time (Bio/Chemi-luminescence) Maximum scan rate 24,000 nm/min, 1,839,080 cm-1/min, 240,000 /min, 228 eV/min Data collection rate (kinetics) points/min/cell 4800pts (1 cell) 6pts (4cells) 55pts (4 cells with Dwell time=0.013sec),
