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1、1,第二章,Chapter Two,Ultraviolet and Visible Absorption SpectrumUltraviolet and Visible SpectrophotometryFor Short：UV-VIS,紫外-可见吸收光谱法,2,1.1 吸光光度法的基本原理,特点 灵敏度高：测定下限可达105106mol/L,10-4%10-5%准确度能够满足微量组分的测定要求：相对误差25（12）操作简便快速应用广泛,吸光光度法是基于被测物质的分子对光具有选择性吸收的特性而建立起来的分析方法。,3,1.光的基本性质 电磁波的波粒二象性,c真空中光速 2.9979245810
2、8m/s3.0 108m/s波长，单位：m,cm,mm,m,nm,1m=10-6m,1nm=10-9m,1=10-10m频率，单位：赫芝（周）Hz 次/秒 n折射率，真空中为1,光的传播速度:,波动性,4,磁场向量,电场向量,传播方向,Y,Z,X,与物质作用,5,微粒性,h普朗克(Planck)常数 6.62610-34Js频率E光量子具有的能量 单位：J(焦耳)，eV(电子伏特),光量子，具有能量。,6,波粒二象性,结论:一定波长的光具有一定的能量，波长越 长(频率越低)，光量子的能量越低。单色光：具有相同能量(相同波长)的光。混合光：具有不同能量(不同波长)的光复合在 一起。,真空中:,7
3、,光学光谱区,10nm200nm,200nm 380nm,380nm 780nm,780 nm 2.5 m,2.5 m 50 m,50 m 300 m,8,9,3.溶液中溶质分子对光的吸收与吸收光谱,不同颜色的可见光波长及其互补光,10,Complementary of light:if two kinds of mono lights mixed with each other according to the certain proportion we can gain white light,then we call the two kinds of mono light as comp
4、lementary color light mutually.and this phenomena is called complementary of light.,11,紫外-可见分光光度计,一.主要组成及部件的功能,1.工作原理基仪器结构框图,光源,碘钨灯,氘灯,单色器,测量池,参比池,样品池,光电倍增管,数据处理和仪器控制,12,二。光源（辐射源）,1.光源的要求：,发射强度足够且稳定的连续光谱;光辐射强度随波长的变化小;有足够的使用寿命,钨灯（钨的熔点为3680K）；波长范围：3202500nm；工作温度：3000K；Ihv V34,2.白炽光源,常用类型：白炽光源与气体放电光源,卤
5、钨灯：在钨灯中加入卤化物提高白炽灯的使用寿命,3.气体放电光源,氢弧灯（氢灯）：波长范围：165350nm；氢气压力：0.25mmHg。,氘灯：内充气为氘辐射强度比起氢灯达35倍。,13,三.单色器:平面衍射光栅,四.检测器：PMT-前面已经介绍,光源,碘钨灯,氘灯,单色器,测量池,CCD检测器,数据处理和仪器控制,CCD 光二极管阵列检测器,14,五.测量池：液池、样品池、比色皿。,1.吸收池的材料,玻璃,360 nm,2.25 mm,15,石英,200 nm,2.5 mm,16,2.吸收池的形状,波长范围,3.使用注意事项,容易破碎,17,18,300,400,500,600,700,/n
6、m,350,525 545,Cr2O72-,MnO4-,Cr2O72-、MnO4-的吸收光谱,350,19,苯和甲苯在环己烷中的吸收光谱,20,In the visible light,KMnO4 solution has the strongest absorption to the green light with wave length nearby 525 nm,but the absorption is very weak to the purple and the red light.max=525 nm.When concentration are different,the c
7、urve shape of the light absorption is same,max invariable,absorbance are different.,Looking at the absorption light and the color relations from the absorption curve,21,4.光吸收基本定律:Lambert-Beer定律,A=lg(I0/It)=kbc,朗伯定律(1760)A=lg(I0/It)=k1b,比尔定律(1852)A=lg(I0/It)=k2c,22,Lambert-Beers law,Lambert J H and B
8、eer A have studied the quota relations of light absorption and solution level thickness and the solution concentration in 1760 and in 1852 separately,the two unions is called Lambert-the Bill law,also is called the light absorption law.When a bunch of parallel monochromatic light I0 illumination to
9、fluid level whose length are the b,and to the solutions whose concentration is c,because of the absorption by the absorb particle(molecular or ions)in solution,after through the solution the light intensity is weaken to I:,23,Lambert-Beers law indicates that,after a bunch of monochromatic lights thr
10、ough the solution which include the extinction matter,the absorbance of the solution and the extinction matter concentration and the thickness of the absorption layer are proportional.This is the theory foundation to carry on the quantitative analysis.Proportionality constant K is concerned with mat
11、ter property,insert light wave length and temperature and so on.The additive property of the absorbance:When any wave length monochromatic light through solution including many kinds of extinction matter,the total absorbance of the solution should be equal to the sum of various matter Absorbance.Thi
12、s rule is called the Absorbance additive property.Using this property,we may carry on the multi-component determination and the determination of certain chemical reaction equilibrium constant.,24,T透光率（透射比）（Transmittance）,A=lg(I0/It)=lg(1/T)=-lgT=kbc,25,吸光度A、透射比T与浓度c的关系,26,K 吸光系数 Absorptivity,当c的单位用g
13、100mL-1表示时，用 表示，A bc，叫做比消光系数,27,吸光度与光程的关系 A=bc,28,吸光度与浓度的关系 A=bc,29,朗伯-比尔定律的适用条件,单色光 应选用max处或肩峰处测定2.吸光质点形式不变 离解、络合、缔合会破坏线性关系 应控制条件(酸度、浓度、介质等)3.稀溶液 浓度增大，分子之间作用增强,30,亚甲蓝阳离子水溶液的吸收光谱a.6.3610-6 mol/Lb.1.2710-4 mol/Lc.5.9710-4 mol/L,亚甲蓝阳离子单体 max=660 nm二聚体 max=610 nm,二聚体的生成破坏了A与c的线性关系,31,溶液浓度的测定,A bc,工作曲线法
14、(校准曲线),朗伯-比尔定律的分析应用,32,6.吸光度的加和性与吸光度的测量,用参比溶液调T=100%（A=0），再测样品溶液的吸光度，即消除了吸收池对光的吸收、反射，溶剂、试剂对光的吸收等。,33,2.2 光度分析的方法和仪器2.2.1 光度分析的几种方法,方便、灵敏，准确度差。常用于限界分析。,1.目视比色法,34,colorimeter,35,2.光电比色法,通过滤光片得一窄范围的光(几十nm),36,3.吸光光度法和分光光度计,通过棱镜或光栅得到一束近似的单色光.波长可调,故选择性好,准确度高.,37,Working process,light source,monochromato
15、r,Manifest record system,Photoelectricity transformation,colorithrough,38,722型分光光度计结构方框图,光源,吸收池,检测系统,分光系统,39,分光光度计的主要部件,光源：发出所需波长范围内的连续光谱，有足够 的光强度,稳定。可见光区：钨灯，碘钨灯(3202500nm)紫外区：氢灯，氘灯(180375nm)单色器：将光源发出的连续光谱分解为单色光的 装置。棱镜:玻璃3503200nm,石英1854000nm 光栅:波长范围宽,色散均匀,分辨性能好,使用方便,40,吸收池：(比色皿)用于盛待测及参比溶液。可见光区：光学玻璃
16、池 紫外区：石英池检测器：利用光电效应，将光能转换成 电流讯号。光电池，光电管，光电倍增管指示器：低档仪器：刻度显示 中高档仪器：数字显示，自动扫描记录,41,棱镜：依据不同波长光通过棱镜时折射率不同,单色器,42,光栅：在镀铝的玻璃表面刻有数量很大的等宽度等间距条痕(600、1200、2400条/mm)。,原理：利用光通过光栅时发生衍射和干涉现象而分光.,43,检测器,硒光电池,Ag、Au,44,光电管,红敏管 625-1000 nm蓝敏管 200-625 nm,45,光电倍增管,待扫描,160-900 nm,1个光电子可产生106107个电子,46,Light source:Using 6
17、 12 V tungsten lamps as the light source of the visible light area,it can launch continuous spectrum at the range of 360 800 nm.Still have deuterium lamp as light source,be used to launch ultraviolet light.,47,monochromator：It is a device that decomposition the continuous spectrum which is sent out
18、from the light source into mono-color.There have prism and grating two kinds。,48,The coloritrough also called absorbing pool.Be used for contain test solution.they are fabricated by glass and quartz material.Be marked according to thickness:0.5 cm，l cm，2 cm，3 cm，5 cm，10 cm,49,Detector:Accepting the
19、transmission that launched from the colorithrough and changing into an electric signal that carrying out measurement.It can divided into phototube and photomultiplier,the sensitivity of the photomultiplier is more than 200 times higher than the phototube.Apply to wavelength range is 160 700 nm.photo
20、multiplier is widely used in modern spectrophotometry.In the early age it also use photocell.,50,Manifest record system:Its function is to demonstrate or record the enlarging single by using absorbance A or transmission T.The usually used manifest equipment of the spectrophotometry is galvanometer,t
21、iny safety form,digital display record instrument.,51,2.2.2 分光光度计的类型,52,多通道仪器（Multichannel Instruments）,光电二极管阵列(通常具有316个硅二极管)photodiode arrays(PDAs)同时测量200820nm范围内的整个光谱,比单个检测器快316倍,信噪比增加 316 1/2 倍.,其他类型分光光度计,将光度计放入样品中,原位测量.对环境和过程监测非常重要.,纤维光度计,53,镀铝反射镜,纤维光度计示意图,54,纤维光度计,55,56,2.3 吸光光度法的灵敏度与准确度,一.灵敏度的
22、表示方法:摩尔吸光系数(),A=b c=A/bc(Lmol-1cm-1),越大,灵敏度越高:1045104 为中等灵敏度;105为高灵敏度;104为低灵敏度.,57,吸光物质的特征常数()；在最大吸收波长max处，常以max表示。在温度和介质条件一定时，仅与吸光物质的结构与性质有关，可作为定性鉴定的参数；不随浓度c 和光程长度b 的改变而改变：b c/A。吸光能力与测定灵敏度的度量；max越大表明该物质的吸光能力越强，测定的灵敏度越高。105：超高灵敏；C=A/b=0.01/105=10-7 mol/L=(610)104：高灵敏；C=A/b=0.01/5 104=210-7 mol/L 104
23、：不灵敏。C=A/b=0.01/104=10-6 mol/L 在数值上等于浓度为1 mol/L、液层厚度为1cm时该溶液在某一波长下的吸光度。,58,例1 邻二氮菲光度法测铁(Fe)=1.0mg/L,b=2cm,A=0.38 计算 和,解：c(Fe)=1.0 mg/L=1.010-3/55.85=1.810-5mol/L,59,c=1.0mg/L=1.010-3 g/1000mL=1.010-4g/100mL,60,二.准确度仪器测量误差,光度计的读数误差一般为0.22(T),由于T与浓度c不是线性关系，故不同浓度时的T引起的误差不同。,61,浓度测量的相对误差与T(或A)的关系,实际工作中，
24、应控制T在1070,A在0.151.0之间(调c,b,),62,最佳的吸光度测量范围,-lgT bC,dlgT-b d C,0.4343 d T/T-b d C,d C/C=0.4343 d T/(T lgT),A=-lgT 0.4343,T 0.368,C/C 100=0.4343 T/(T lgT)100=0.4343(0.01)/0.368(-0.4343)100=2.7%,最佳的吸光度范围：A0.20.8,当 T=0.01，即透射率的测量误差为1%时：,63,2.4 显色反应与分析条件的选择,2.4.1 显色剂与显色反应,无机显色剂:SCN-Fe(SCN)2+H2O2 TiOH2O22
25、+,有机显色剂:,64,有机显色剂,OO型：,ON型：,PAR,S型：,双硫腙,NN型：,丁二酮肟,邻二氮菲,磺基水杨酸,65,显色反应的选择,*灵敏度高，一般104*选择性好*显色剂在测定波长处无明显吸收。对照性好,max60 nm.*反应生成的有色化合物组成恒定，稳定。*显色条件易于控制，重现性好。,66,Request to the color reaction,(1)Have a good selectivity(2)Have a high sensitivity(3)Contrast ratios should be big.namely if the chromogenic age
26、nt have color,so the difference of the maximal absorb wavelength between color compound and chromogenic agent must be big,general demand above 60 nm.(4)The composition of the colored compound must be constant,chemical property is going to stabilize.(5)The condition of the color reaction needs easily
27、 control.,67,2.4.2 显色条件的确定,c(R),c(R),c(R),1.显色剂用量（c(M)、pH一定）,Mo(SCN)32+浅红Mo(SCN)5 橙红Mo(SCN)6-浅红,Fe(SCN)n3-n,68,2.显色反应酸度（c(M)、c(R)一定）,pH1pHpH2,pH,69,邻二氮菲亚铁反应完全度与pH的关系,Fe2+3R,c(R)R=10-4molL-1,31021.3,70,pH38为适宜的酸度范围,71,3.显色温度及显色时间,另外，还有介质条件、有机溶剂、表面活性剂等,T1(),T2(),t(min),A,72,3.Time and temperature,The
28、speed of the color reaction sometimes fast sometimes slow.most of the color reaction need a certain time before the color of the solution reach to stability.The color of some color compound may decrease because of the oxide of the air,reagents decompose or be volatized.The proper coloration time and
29、 the stabilize degree of the colored solution,must ascertain by an experiment.Draw the A-t curve,ascertain the proper time according to the curve.Different color reaction needs different temperature,general color reaction may be completed under the room temperature.But some color reaction needs to h
30、eat till certain temperature can completed;Some colored complex compounds also decompose easily under the higher temperature.Therefore,we should choose the appropriate temperature according to different condition carrying out a coloration.Temperature have an impact on the absorption of the light and
31、 the color depth,therefore the temperature of the standard sample and the reference sample should be the same one.,73,2.4.3 干扰及消除,测Co2(含Fe3+):(掩蔽法),1.化学法,74,测Co2:(生成络合物性质不同),如不能通过控制酸度和掩蔽的办法消除干扰，则需采取分离法。,测Fe3:(控制pH),75,2.物理法,选择适当的测定波长,76,1.仅络合物有吸收，溶剂作参比。如 phenFe2+标准曲线2.待测液也有吸收，被测液作参比。如 测汽水中的 Fe3.显色剂或
32、其他试剂也有吸收，空白溶液作参比 例:邻二氮菲光度法测Li2CO3中的 Fe，参比溶液为不含Li2CO3样品的所有试剂。4.干扰组分与显色剂有反应,又无法掩蔽消除时：1）掩蔽被测组分，再加入显色剂，作参比.2）加入等量干扰组分到空白溶液中,作参比.,-选择适当的参比溶液,77,3.The selective of the reference solution(1)When the test solution,regent,color-developing regents are all colorless,we can use water as the reference solution.(
33、2)When reagent and color-developing regents are all colorless,but other ions in sample solution have color,we should take the sample solution that not adding a color-developing agent as the reference.(3)When reagent and color-developing agents are all have color,we may add appropriate masking agent
34、in one test solution to sheltering the measured components,will make that no longer act with the color-developing agent,then adding the color-developing agent,reagent according to handling procedures,used the above solution as reference solution,so it can eliminate the disturbance of some coexist co
35、mponents.,78,2.5 吸光光度法的应用,1.单一组分测定,1)金属离子:Fe-phen,Ni-丁二酮肟,Co-钴试剂,2)磷的测定:DNA中含P9.2%,RNA中含P9.5%,可得核酸量.,H3PO4+12(NH4)2MoO4+21HNO3=(NH4)3PO412MoO3+12NH4NO3+12H2O,磷钼黄(小),磷钼(V)蓝(大),79,The application of the absorption photometry The absorption photometry not only widely used to determine the micro ingredi
36、ent,but also can used to measure the constant component and the multi-components.At the same time,it also can used to study the chemical equilibrium,the determination of complex compound composition.Quantitative analysis(1).Single component determination1.General method:A-c standard curve method2.Di
37、fferential method:It is not use reagent blank whose c=0 as comparison,but use a standard solution cs whose concentration is slightly minor than test solution cx,then determine the absorbance of the test solution.Af=Ax-As=b(cx-cs)=bc cx=cs+c,80,3)蛋白质测定溴甲酚绿、考马司亮蓝等4)氨基酸测定茚三酮(紫色化合物)5)水质检测:NH4+、NO2-、Mn2+
38、、Fe2+、SO42-、Hg2+-6)药物含量测定比吸光系数定量；荷移光谱法测定.7)紫外吸收(UV):NO2-、NO3-、SO42-、SO32-、CO32-、SCN-、酪氨酸、色氨酸、苯丙氨酸、蛋白质等。,81,2.多组分的测定,xl1,eyl1,exl2,eyl2由x,y标液 在1,2处分别测得,在1处测组分x,在2处测组分y,b)在1处测组分x;在2处测总吸收,扣除x吸收,可求y,c)x,y组分不能直接测定A1=exl1bcx+eyl1bcy(在1处测得A1)A2=exl2bcx+eyl2bcy(在2处测得A2),82,.Measuring many kind of components
39、 at the some time The foundation of measuring many kinds of components in one kind of test specimen simultaneously is the addition of the absorbance,namely the sum total absorbance is equal to the absorbance of each components.,83,3.光度滴定,84,.Determine the composition of the compound and acid-base di
40、ssociation constant,The determination of the composition of the compound 1.Mole ratio method We can determine the composition of the compound(complex ratio)and stability constant Suppose the complex reaction is:M+nRMRn Usually we fix the concentration of the metallic ion M,increase the concentration
41、 of the complexing agent gradually.ligand is R,obviously,R should be colorless or not remarkably absorbs in the designation wavelength range.Then dilute to the identical volume,obtains a series of solutions whose R/M is 1,2,3.,preparing the corresponding reagent blank,under certain wave length,measu
42、ring its luminosity,then draw the curve,we can obtain the complexing ratio from the curve.,85,典型的光度滴定曲线,依据滴定过程中溶液吸光度变化来确定终点的滴定分析方法。,86,4.络合物组成的测定,(1)摩尔比法:固定cM,改变cR,87,(2)等摩尔连续变化法:,88,表观形成常数的测定(设M、R均无吸收),c(M)=c(R)=c,MmRn型?,89,5.一元弱酸离解常数的测定,HL HL,KaH+L/HL,高酸度下，几乎全部以HL存在，可测得AHLHLc(HL)；低酸度下，几乎全部以L存在，可测得
43、AL Lc(HL).代入整理：,或,配制一系列c相同,pH不同的溶液,测A.,90,Measuring the acid-base dissociation constant If there has monohydric weak acid,dissociation according to the following equations:HB=H+B-Ka=B-H+/HB Preparing three kinds of solution whose analytical concentration c=HB+B-is equal,but its pH is different.pH of
44、 the first solution is near its pKa,then in the solution HB and B-are coexist,measure its absorbance in 1cm absorption cell under some certain wavelength:,91,MO吸收曲线,由每份溶液的一对pH、A，可求得一个Ka,取平均值即可.,92,The second kind of solution is the acid solution whose pH is above two units lower than its pKa,then th
45、e existence body of the weak acid almostly is HB,determine the absorption under the above wave length.The third kind of solution is the base solution whose pH is above two units higher than its pKa,then existence body of the weak acid almostly is B-,determine the absorption under the above wave leng
46、th.,93,MO离解常数的测定作图法,94,Reorganize the overall equations:,95,双波长分光光度法消除浑浊背景干扰,1 2,A 1-A 2=A Ac,例 牛奶中微量Fe的测定,96,1.A compound had a molar absorptivity of 6.74x103 L/cmmol.what concentration of the compound would be required to produce a solution having a transmittance of 7.77%in a 2.50-cm-cell?2.The fo
47、llowing data were abtained for solution of substance X and Y(1.2-cm-cells)C(mol/L)A at 320nm A at 460nmX 4.6x10-3 0.965 0.106Y 5.1x10-3 0.094 1.213a.Calculate the molar absorptivity of X and Y at each wavelengthb.A solution containing both X and Y was found to have an absorbance of 0.431 at 320 nm a
48、nd 0.686 at 460nm when measured in 0.5-cm-cells.Calculate the molar concentration of each species in the solution assuming both species obey L-Bs Law,97,Sandell Sensitivity or Index of sensitivity is indicated with S.S refers that when instrument detection limit A=0.001,in the unit cross sectional area optical path can examine the smallest content of the extinction matter t,its unit is gcm-2,
