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1、检测器的种类及选择方法,简介,种类：紫外检测器(UV)荧光检测器(FD)电化学检测器(ECD)蒸发光散射检测器(ELSD)示差折光检测器(RID)质谱检测器(MSD)氢火焰检测器(FID)热导检测器(TCD)氮磷检测器(NPD)火焰光度检测器(FPD)其它检测器：质谱仪、付立叶变换红外光谱仪、AED、SCD、ELCD、PID、HID等,检测器性能评价指标：响应值（或灵敏度）S：定义 S=R/Q 在一定范围内，信号R与进入检测器的量Q呈线性关系：R=S Q S=R/Q 单位：mV/（mg/cm3）；（浓度型检测器）mV/（mg/s）；（质量型检测器）S 表示单位量的物质通过检测器时，产生的响应信
2、号的大小。S值越大，检测器（也即色谱仪）的灵敏度也就越高。,简介,紫外检测器(UV),紫外检测器是液相色谱中使用最广泛的检测器，几乎所有的液相色谱仪都配此类检测器，是一种选择性检测器。原理：朗伯比尔(LambertBeer)定律，即当一束单色光透过样品池时，若流动相不吸收光，则吸光度(A)与吸光组分的浓度(C)和样品池的光径长度(L)成正比。分类：紫外检测器包括固定波长检测器，可变波长检测器和光电二极管阵列检测器三类。缺点：只能检测有紫外吸收的物质，流动相的选择有 一定限制，流动相的截止波长必须小于检测波长。适用范围：大多数有紫外吸收的化合物。,紫外检测器(UV),固定波长检测器：波长一般为2
3、54nm，以低压汞灯为光源，光源单色性好、光强度大、灵敏度高。可变波长检测器：目前配置最多的检测器。光路系统类似分光光度计，一般采用氘灯或卤钨灯为光源，光束经单色器分光后按需要选择组分的最大吸收波长为检测波长，从而提高灵敏度。二极管阵列检测器(DAD)是20世纪80年代出现的一种光学多通道检测器。在晶体硅上紧密排列一系列光电二极管，每一个二极管相当于一个单色器的出口狭缝，二极管越多分辨率越高，一般是一个二极管对应接受光谱上一个纳米谱带宽的单色光。原理：复色光通过样品池被组分选择性吸收后再进入单色器，照射在二极管阵列装置上，使每个纳米波长的光强度转变为相应的电信号强度，即获得组分的吸收光谱，从而
4、获得特定组分的结构信息，有助于未知组分或复杂组分的结构确定。,紫外检测器(UV),Identification of the Position of Mono-O-glucuronide of Flavones and Flavonols by Analyzing Shift in Online UV Spectrum(max)Generated from an Online Diode Array Detector(1),In this paper,glucuronide(s)of 36 flavones and flavonols were generated using an in vi
5、tro glucuronidation reaction.UPLC/MS/MS was used to confirm the degree(mono-or di-)of glucuronidation in flavonoids with up to four hydroxyl groups.UV spectra of flavonoids and their respective mono-O-glucuronides were generated using UPLC with an online diode array detector.Analysis of the extent o
6、f shift in spectra of glucuronides in band I(300-385 nm)and band II(240-280 nm)regions as reflected by changes in max value was used to identify the position of glucuronidation.,(1)SAOWAPA CHUMANEE,SOMYOTE SUTTHIVAIYAKIT,AND PAKAWADEE SUTTHIVAIYAKIT.Identification of the Position of Mono-O-glucuroni
7、de of Flavones and Flavonols by Analyzing Shift in Online UV Spectrum(max)Generated from an Online Diode Array DetectorJ.Agric.Food Chem.2009,57,17521759.,紫外检测器(UV),The data showed that glucuronidation of the 3-and 40-hydroxyls resulted in band I max hypsochromic shifts(or blue shift)of 13-30 and 5-
8、10 nm,respectively.Glucuronidation of the 5-hydroxyl group caused a band II max hypsochromic shift of 5-10 nm.In contrast,glucuronidation of the 7-hydroxyl group did not cause any max change in band I or II max,whereas glucuronidation of the 6-hydroxyl group did not cause predictable changes in max
9、values.The paper demonstrated for the first time that a rapid and robust analysis method using max changes in online UV spectra can be used to pinpoint region-specific glucuronidation of flavones and flavonols with hydroxyl groups at the 40-,3-,5-,and/or 7-position(s).,a,b,c,Figure 1.LC-DAD chromato
10、grams of blank shrimp(a),shrimp fortified with a mixture of nitrofuran metabolites at 1 g/kg(b),and sample 5(c).,紫外检测器(UV),紫外检测器(UV),Figure 2.UV spectra of quercetin(3,5,7,30,40-pentahydroxyflavone)(solid black line)and its four mono-O-glucuronides generated by UGT 1A8 andUGT 1A9 at an incubation co
11、ncentration of 10 M.,荧光检测器(FD),原理：具有某种特殊结构的化合物受到紫外光激发后能发射出比激发光源波长更长的光，称为荧光。荧光强度(F)与激发光强度(I0)及荧光物质浓度(C)成正比。优点：灵敏度高、选择性好，是微量组分和体内药物分析常用的检测器之一。缺点：只适用于能够产生荧光的物质的检测，适用范围不如紫外检测器。影响因素较多，对溶剂的纯度、pH值、样品浓度、检测温度等需很好地控制。,荧光检测器(FD),Laser-Induced Fluorescence Detector for Capillary-Based Isoelectric Immunoblot Ass
12、ay(2),(2)James E.Knittle,David Roach,Peter B.Vander Horn,and Karl O.Voss.Laser-Induced Fluorescence Detector for Capillary-Based Isoelectric Immunoblot Assay J.Anal.Chem.2007,79,9478-9483.,We describe a whole-capillary,multicolor laser-induced fluorescence scanner for microfluidic protein analysis s
13、ystems.Separation of proteins is achieved by isoelectric focusing in a short length of fused-silica capillary after which the resolved proteins are immobilized to the capillary wall using photochemistry.The capillary is then evacuated,and fluorescently labeled antibodies are flowed through the capil
14、lary to bind to the immobilized proteins.This technique provides high sensitivity,the ability to spatially resolve and quantify proteins,and provides the opportunity for complete automation.Results obtained by fluorescence detection are compared to those obtained by chemiluminescence while offering
15、enhanced resolution and signal stability.,荧光检测器(FD),Figure 3.(a)Schematic representation of fluorescence imager.(b)Detector optics.,荧光检测器(FD),Figure 4.Comparison of fluorescence and chemiluminescence detection of cIEF-separated GFP.(a)GFP imaged using native fluorescence,(b)GFP detected with labeled
16、 antibody based fluorescence,and(c)GFP detected with labeled antibody-based chemiluminescence.,荧光检测器(FD),Figure 5.(a)Native fluorescence and(b)chemiluminescence profiles of HRP-immunolabeled GFP.,电化学检测器(ECD),电化学检测器是测量物质的电信号变化，对具有氧化还原性质的化合物，如含硝基、氨基等有机化合物及无机阴、阳离子等物质可采用电化学检测器。包括极谱、库仑、安培和电导检测器等。前三种统称为伏安
17、检测器，用于具有氧化还原性质的化合物的检测，电导检测器主要用于离子检测。其中安培检测器(AD)应用较广泛，更以脉冲式安培检测器最为常用。原理：在两电极之间施加一恒定电位，当电活性组分经过电极表面时发生氧化还原反应(电极反应)，电量(Q)的大小符合法拉第定律QnFN。因此，反应的电流(I)为：InFdNdt，式中n为每摩尔物质在氧化还原过程中转移的电子数，F为法拉第常数，N为物质的摩尔数，t为时间。当流动相的流速一定时，dNdt与组分在流动相中的浓度有关。,电化学检测器(ECD),优点：灵敏度很高，尤其适用于痕量组分分析。缺点：干扰比较多，如生物样品或流动相中的杂质、流动相中溶解的氧气及温度的变
18、化等都会对其产生较大的影响。电极寿命有限，对温度和流速的变化比较敏感。适用范围：应用范围广，凡具氧化还原活性的物质都能进行检测，本身没有氧化还原活性的物质经过衍生化后也能进行检测。,(3)Ju Qiu,Nozomi Saito,Mai Noguchi,Keiichi Fukui,Kayo Yoshiyama,Kazusato Matsugano,Norihiko Terehara,and Toshiro Matsui.Absorption of 6-O-Caffeoylsophorose and Its Metabolites in Sprague Dawley Rats Detected b
19、y Electrochemical DetectorHigh-Performance Liquid Chromatography and Electrospray Ionization Time-of-FlightMass Spectrometry MethodsJ.Agric.Food Chem.2011,59,62996304,(3)Absorption andmetabolism of a natural compound,6-O caffeoylsophorose(CS)from acylated anthocyanins in a red vinegar fermented with
20、 purple sweetpotato,were clarified.The absorption of CS and conjugated CS in blood from orally administrated SpragueDawley rats at a dose of 400 mg/kg was investigated by electrochemical detectionhigh performance liquid chromatography.As a result,CS was successfully detected in rat plasma(AUC06h,108
21、.6(8.1 nmol h/mL)and was found to be an intact absorbable polyphenol.In addition,half of the absorbed CS was detected as its conjugates(AUC06h,50.7(5.7 nmol h/mL)as well as caffeic and ferulic acids from CS.,电化学检测器(ECD),电化学检测器(ECD),Figure 6.Typical ECD-HPLC chromatograms of standard(A)and ratplasma
22、before(B)and 30 min after(C)a single oral administration of400 mg/kg CS to SD rat.Peaks 1,2,and 3 denote CS,caffeic acid,andferulic acid,respectively.HPLC and ECD conditions are described in the Materials and Methods.,Microfabricated Electrochemical Detector for High-Performance Liquid Chromatograph
23、y(4),(4)Evan T.Ogburn,Michael Dziewatkoski,Don Moles,Jay M.Johnson,and William R.Heineman.Microfabricated Electrochemical Detector for High-Performance Liquid ChromatographyJ.Anal.Chem.2011,83,69636970,A microfabricated electrochemical cell has been developed as a disposable detector for flow inject
24、ion analysis(FIA)and high-performance liquid chromatography(HPLC).The simplicity of the fabrication process allows this detector to be used as a low-cost,disposable device that can be replaced easily if its performance degrades rather than disassembling the detector and polishing the electrode surfa
25、ce,which is the usual procedure.The detector consists of thin film-metal electrodesplatinum working electrode,platinum auxiliary electrode,and silver/silver chloride coated on Pt reference electrodedeposited on a polyimide substrate with a locking layer of chromium in between.A microfluidic cover ma
26、de of polyimide directs the solution flow across the electrodes.,电化学检测器(ECD),Figure 7.Schematic diagrams of the microfabricated electrochemical detector:electrode platform(A)and microfluidic cover layer(B).,电化学检测器(ECD),蒸发光散射检测器(ELSD),20世纪80年代出现的通用性质量型检测器。不论化合物是否存在紫外、荧光或电负性基团，均可用ELSD检测。原理：将洗脱液引入雾化器与气
27、体混合形成均匀的微小液滴，蒸发除去流动相而样品组分形成气溶胶，进入检测室，用强光或激光照射气溶胶产生光散射，用光电二极管检测散射光。散射光的强度(I)与组分的质量(m)通常具有下述关系：IKmb或lgIblgm+lgK，式中K和b为与漂移管温度、雾化气体压力及流动相性质等实验条件有关的常数。因此，散射光的对数响应值与组分质量的对数呈线性关系。优点：通用型检测器，对各种物质有几乎相同的响应。缺点:灵敏度相对较低，流动相必须是挥发性的，不能用非挥发性的缓冲盐及表面活性剂。适用范围:适用于挥发性低于流动相的组分，主要用于糖类、高级脂肪酸、磷脂、维生素、氨基酸、甘油三酯、皂苷及甾体等等无紫外吸收或紫外
28、末端吸收的化合物的检测。,蒸发光散射检测器(ELSD),(5)Xianfeng Li,Fred Monsuur,Bart Denoulet,Agnieszka Dobrak,Pieter Vandezande,and Ivo F.J.Vankelecom.Evaporative Light Scattering Detector:Toward a General Molecular Weight Cutoff Characterization of Nanofiltration MembranesJ.Anal.Chem.2009,81,18011809,Evaporative Light Sc
29、attering Detector:Toward a General Molecular Weight Cutoff Characterization of Nanofiltration Membranes(5),An evaporative light scattering detector(ELSD)coupled with HPLC was used for the first time to characterize membranes.Polydispersed PEG-200,600,and 1000 were selected as probe molecules to stud
30、y the dependence of membrane retention on molecular weight via a gradient eluted HPLC separation coupled to ELSD detection.The results show that HPLC/ELSD is a really general and powerful technique to study the nanofiltration(NF)process since it does not require any special properties for the solute
31、s(chromospheres or fluorophores)and possesses the required sensitivity.Especially in solvent resistant NF(SRNF),where a wide range of organic solvents is used,the ELSD detector was not affected by the interaction between solvent and solutes,which is a critical issue compared to other more common det
32、ectors.,Figure 8.HPLC/ELSD results of PEG-600 Performed at different column temperatures.,Figure 9.HPLC/ELSD results of PEG-600 with different injection volumes.,蒸发光散射检测器(ELSD),Figure10.HPLC/ELSD results of 1000 ppm PEG-200 in methanol.,Figure 11.PEG-200 chromatograms obtained with the two different
33、 ELSD detectors.,蒸发光散射检测器(ELSD),蒸发光散射检测器(ELSD),Figure 12.Typical HPLC/ELSD results of PEG-600 before(feed)and after filtration(permeate)through a PDMS membrane in different solvents,示差折光检测器(RID),工作原理；利用组分与流动相的折光率的不同，其响应信号(R)与组分的浓度(Ci 成正比：R=ZCi(ni-n0)，式中Z为仪器常数，ni为i组分的折光率，n0为流动相的折光率。优点：通用型检测器，只要组分的折光率
34、与流动相的折光率有足够的差别就能检测。缺点：灵敏度低、受环境温度、流量及流动相组成等波动的影响大，一般不能用于梯度洗脱。适用范围：RID为通用型检测器，适用于无紫外吸收化合物的分析，如糖类分析。,质谱检测器(MSD),与液相色谱仪联用的质谱检测器是将色谱系统流出的组分经过液质联用接口在一定条件下离子化后，进入质量数分析器(如四极杆质谱、离子阱质谱、二级或多级串联质谱等)，按照离子的质荷比大小分离，记录响应信号并列成谱图。一般地，采用全离子扫描模式可得到特定组分的质谱图，从而得到组分的化学结构信息;采用选择离子监测(SIM)模式可得到特定质量数碎片离子的色谱图，可用于定量测定。优点：灵敏度高，选
35、择性好，能同时给出组分的结构信息。缺点：响应信号受离子化效率限制，仪器较为昂贵，通常需专人使用与维护。适用范围：组分的结构鉴别，微量及痕量组分的分析，药物代谢分析等。,氢火焰检测器(FID),特点:(1)典型的质量型检测器;(2)对有机化合物具有很高的灵敏度;(3)无机气体、水、四氯化碳等含氢少或不含氢的物质灵敏度低或不响应;(4)氢焰检测器具有结构简单、稳定性好、灵敏度高、响应迅速等特点;(5)比热导检测器的灵敏度高出近3个数量级，检测下限可达10-12gg-1。应用：FID是多用途的破坏性质量型检测器。广泛应用于有机物的常量和微量检测。,氢火焰检测器(FID),A区：预热区B层：点燃火焰C
36、层：热裂解区：温度最高D层：反应区,原理：（1）CnHm CH，含 CnHm的载气由喷嘴喷出进入火焰时，在C层发生裂解反应（2）CH+O CHO+e，自由基在D层火焰中与激发态原子氧或分子氧反应（3）CHO+H2O H3O+CO，正离子CHO+与火焰中大量水分子碰撞而发生分子离子反应（4）化学电离产生的正离子和电子在外加恒定直流电场的作用下分别向两极定向运动而产生微电流（约10-610-14A）；（5）组分在氢焰中的电离效率很低，大约五十万分之一的碳原子被电离。（6）离子电流信号输出到记录仪，得到峰面积与组分质量成正比的色谱流出曲线,热导检测器(TCD),结构：热敏元件装入检测池池体中，制成热
37、导池，再将热导池与电阻组成惠斯顿电桥。原理：不同的气体有不同的热导系数。钨丝通电，加热与散热达到平衡后，两臂电阻值：R参=R测;R1=R2 则：R参R2=R测R1 无电压信号输出；记录仪走直线（基线）。,进样后，载气携带试样组分流过测量臂而这时参考臂流过的仍是纯载气，使测量臂的温度改变，引起电阻的变化，测量臂和参考臂的电阻值不等，产生电阻差，R参R测 则：R参R2R测R1，这时电桥失去平衡，a、b两端存在着电位差，有电压信号输出。信号与组分浓度相关。记录仪记录下组分浓度随时间变化的峰状图形。,特点：热导检测器是一种通用型检测器。被测物质与载气的热导系数相差愈大，灵敏度也就愈高。此外，载气流量和
38、热丝温度对灵敏度也有较大的影响。热丝工作电流增加倍可使灵敏度提高37倍，但是热丝电流过高会造成基线不稳和缩短热丝的寿命。热导检测器结构简单、稳定性好，对有机物和无 机气体都能进行分析，其缺点是灵敏度低。应用：TCD对所有物质均有响应，结构简单、性能可靠，定量准确，经久耐用。广泛用于各种气体分析。它是一种通用的非破坏性浓度型检测器，理论上可应用于任何组分的检测，但因其灵敏度较低，故一般用于常量分析。,热导检测器(TCD),氮磷检测器(NPD),NPD 对含N、P 的有机物的检测肯有灵敏度高，选择性强，线性范围宽的优点，它已成为目前测定含N 有机物最理想的气相色谱检测器；对含P 的有机物，其灵敏度
39、也高于火焰光度检测器，而且结构简单，使用方便；广泛用于环境、临床、食品、药物、香料、刑事法医等分析领域，成为最常用的气相色谱检测器，目前几乎所以的商品色谱仪都装备这种检测器。,火焰光度检测器(FPD),FPD是分析S、P 化合物的高灵敏度、高选择性的气相色谱检测器。当含S、P 的化合物进入检测器，在富氢焰（H2 与O2 体积比）中燃烧时，从基态到激发态发出特征光谱，分别发射出（350-480）nm 和(480-600)nm 的一系列特征波长光，其中394nm 和526nm 分别为含S 和含P化合物的特征波长。其特征光透过特征光单色滤光片直接投射在光电倍增管上，通过光电倍增管将光信号转换成电信号，经微电流放大器放大传输给色谱工作站的数据采集卡，数据采集卡将其模拟信号转换成数字信号，便可得到相应的谱峰。广泛用于环境、食品中S、P 农药残留物的检测。以前一直将FPD 作为S 和P 化合物的专用检测器，后由于氮磷检测对P 的灵敏度高于FPD，而且更可靠，因此FPD 现今多只作为S 化合物的专用检测器。,Thank You!,
