液相各检测器介绍

1、LIQUID CHROMALIQUID CHROMATOGRAPHY DEOGRAPHY DETECECTORSORSAnalAnalytes tes that elute from a column need to be detected tothat elute from a column need to be detected toObtain a chromatogramObtain a chromatogram. Detector is a critical component Detector is a critical componentOf anOf any LC s LC s

2、ystemstem. The following the following types of detectors arepes of detectors are used: used:11. . UVUV-visible detectorvisible detector22. . Fluorescence detectorFluorescence detector33. . Electrical conductivitElectrical conductivity detector detector44. . Refractive index detectorRefractive index

3、 detector5 5. . Mass spectral detectorMass spectral detectorUVUV-VISIBLE DEVISIBLE DETECECTORSORSSINGLE WAVELENGSINGLE WAVELENGTH DEH DETECECTORORSingle wavelength detectors consist of the followingSingle wavelength detectors consist of the followingComponents:Components:11. . A low pressure mercurA

4、 low pressure mercury discharge lamp that emits light discharge lamp that emits lightIn the UV and visible regionsIn the UV and visible regions.22. A focusing quart A focusing quartz lens lens.33. A cut A cut-off filter (broad band or bandoff filter (broad band or band-pass) that allowspass) that al

5、lowsSpecific wavelength ranges to be transmitted to the Specific wavelength ranges to be transmitted to the SampleSample.44. Reference and sample cells of small volume Reference and sample cells of small volume (microliters (microliters) ).5 5. Photomultiplier Photomultiplier or photo cells to monit

6、or the or photo cells to monitor the Absorption of lightAbsorption of light.66. Absorbance is calculated from Beer Absorbance is calculated from Beers law:s law: Log ILog IT/I IOO = = l c = A l c = AVARIABLE WAVELENGVARIABLE WAVELENGTH DEH DETECECTORORVARIABLE WAVELENGVARIABLE WAVELENGTH DEH DETECEC

7、TOR OR (CON(CONTD)D)Variable wavelength detector consists of the followingVariable wavelength detector consists of the following components: components:11. . A deuterium and or tungsten lamp to cover the uv A deuterium and or tungsten lamp to cover the uv andandVisible spectral rangesVisible spectra

8、l ranges.22. Collimating mirrors that focus the source beam on to Collimating mirrors that focus the source beam on toA diffraction gratingA diffraction grating. The diffraction grating resolveshe diffraction grating resolvespolpolychromatic beam into its component wavelengthschromatic beam into its

9、 component wavelengths.The spectral resolution is a function of the number ofhe spectral resolution is a function of the number ofLines in the gratingLines in the grating. This arrangement allows a specifichis arrangement allows a specificWavelength to be chosen for detecting the analWavelength to b

10、e chosen for detecting the analytestes.33. A small volume cell into which the eluting mobile phase A small volume cell into which the eluting mobile phaseIs fedIs fed.44. A lens in the front and back of the cell to focus the given A lens in the front and back of the cell to focus the givenWavelength

11、 light into the sample and the transmitted lightWavelength light into the sample and the transmitted lightInto the photomultiplierInto the photomultiplier.FLUORESCENCE DEFLUORESCENCE DETECECTORORFLUORESCENCE DEFLUORESCENCE DETECECTORORA fluorescence detector has the following components:A fluorescen

12、ce detector has the following components:11. . A polA polychromatic light source such as as a xenon lampchromatic light source such as as a xenon lamp.22. This light is focused onto a diffraction grating whichhis light is focused onto a diffraction grating whichAllows the excitation wavelength to be

13、 chosenAllows the excitation wavelength to be chosen.33. The chosen wavelength light is passed through the he chosen wavelength light is passed through the Sample and reference cellsSample and reference cells.44. The light emitted from the sample cell is collected at he light emitted from the sample

14、 cell is collected at 90o o to the incident light to the incident light.5 5. This is incident on a second grating before being senthis is incident on a second grating before being sentTo the photomultipliero the photomultiplier for detection for detection.66. The exit grating enables detection at a

15、chosen emissionhe exit grating enables detection at a chosen emissionWavelengthWavelength.77. Fluorescence is inherentl Fluorescence is inherently more sensitive than more sensitive than Absorption and has a larger liner dAbsorption and has a larger liner dynamic rangenamic range.ELECELECTRICAL COND

16、UCRICAL CONDUCTIVIIVITY Y DEDETECECTORORElectrical conductivitElectrical conductivity detector is used in ion detector is used in ion ChromatographChromatography. It measures the resistance and hence It measures the resistance and hencethe conductivitthe conductivity of the solution from which a sig

17、nal is of the solution from which a signal isGenerated for detectionGenerated for detection.ELECELECTRICAL CONDUCRICAL CONDUCTIVIIVITY Y DEDETECECTORORConductance of a given solution is C and is given bConductance of a given solution is C and is given by C = 1 C = 1/R = R = a a/l lWhere R = resistan

18、ce in ohmsWhere R = resistance in ohms = specific conductance = specific conductance a = cross a = cross-sectional area of the electrodesectional area of the electrode l = distance between the electrodes l = distance between the electrodesEquivalent conductance Equivalent conductance is defined as i

19、s defined as = 1 = 1000 /c cWhere c is the concentration of the electrolWhere c is the concentration of the electrolyte inte inGram equivalentsGram equivalents/literliter. C = 1 C = 1/R = R = caca/11000 l lResistance is inverselResistance is inversely proportional to the concentration proportional t

20、o the concentration.REFRACREFRACTIVE INDEX DEIVE INDEX DETECECTORORREFRACREFRACTIVE INDEX DEIVE INDEX DETECECTOR OR The refractive index detector is the last choice detectorhe refractive index detector is the last choice detector.It is the least sensitive sensitive detectorIt is the least sensitive

21、sensitive detector. It is sensitive It is sensitiveTo changes in ambient temperature and pressure ando changes in ambient temperature and pressure andFlow rateFlow rate. It can onl It can only be used with isocratic elutions be used with isocratic elutions And is unsuitable for gradient elutionsAnd

22、is unsuitable for gradient elutions.It consists of the following components:It consists of the following components:11. . A light source which is usuallA light source which is usually tungsten lamp tungsten lamp.22. An optical mask confines the beam to the region of the An optical mask confines the

23、beam to the region of theCellCell.33. A lens collimates the light beam which then passes A lens collimates the light beam which then passes through both the sample and reference cells to a planethrough both the sample and reference cells to a planeMirrorMirror.44. The mirror reflects the beam back t

24、hrough the samplehe mirror reflects the beam back through the sampleREFRACREFRACTIVE INDEX DEIVE INDEX DETECECTOR (CONOR (CONTD)D)5 5. The location of the beam rather than its intensithe location of the beam rather than its intensity is isDetermined bDetermined by the angular deflection of the beam

25、caused the angular deflection of the beam causedB By the refractive index difference between the contents the refractive index difference between the contentsOf the two cellsOf the two cells.66. As the beam changes the position of focus on the As the beam changes the position of focus on the Photoel

26、ectric cellPhotoelectric cell, the output changes and the resulting the output changes and the resulting Difference signal is electronicallDifference signal is electronically modified to provide a modified to provide a Signal proportional to the concentration of solute in theSignal proportional to t

27、he concentration of solute in theSample cellSample cell.REFRACREFRACTIVE INDEX DEIVE INDEX DETECECTOR (CONOR (CONTD)D)ION CHROMAION CHROMATOGRAPHYOGRAPHYIon ChromatographIon Chromatography is based on the exchange of ions of is based on the exchange of ions ofLike charge between the solution and the

28、 stationarLike charge between the solution and the stationary phase phase.ION CHROMAION CHROMATOGRAPHY (CONOGRAPHY (CONTD)D)The equilibria involved in cation he equilibria involved in cation and anion exchange and anion exchange chromatographchromatography are: are:ION CHROMAION CHROMATOGRAPHY (CONO

29、GRAPHY (CONTD)D)When When ION CHROMAION CHROMATOGRAPHY (CONOGRAPHY (CONTD)D)K decreases in the order for MK decreases in the order for M+ ions ionsTl l+AgAg+CsCs+RbRb+KK+NHNH44+NaNa+LiLi+The order for anions is he order for anions is These orders are based on the ionic radii of the varioushese order

30、s are based on the ionic radii of the various ions ions.ION CHROMAION CHROMATOGRAPHY (CONOGRAPHY (CONTD)D)OSO-OONa+Sodium Dodecyl SulfateCH3CH3CH3NCH3+Br- Octyltrimethylammonium BromideION CHROMAION CHROMATOGRAPHY (CONOGRAPHY (CONTD)D)H3CSO4-CH3SiCH3CH3H3CSO4-CH3SiCH3CH3+2+MM2+K0SiCH3CH3CH3H3CSO4+ H

31、L+ ML+H+SiCH3CH3CH3H3CSO4-M+K1SIZE EXCLUSION CHROMASIZE EXCLUSION CHROMATOGRAPHYOGRAPHYIt is also known as gel permeation or gel filtrationIt is also known as gel permeation or gel filtrationChromatographChromatography. As the name implies the separation As the name implies the separationIs based on

32、 the siIs based on the size of the anale of the analyte moleculeste molecules. TheheStationarStationary phase contains fairl phase contains fairly large 1 large 10 m uniformm uniformSi Si or polor polymer particlesmer particles. Anal Analyte and solvent molecules te and solvent molecules Diffuse in

33、and out of the pores of these particlesDiffuse in and out of the pores of these particles.Retention of the analRetention of the analyte depends on the pore site depends on the pore size and thee and theSiSize of the moleculee of the molecule. It is thus important that the pore It is thus important t

34、hat the poreSiSize be in the range of diameters of the moleculese be in the range of diameters of the moleculesBeing separatedBeing separated. If the molecular diameter is significantl If the molecular diameter is significantlySmaller than the pore diameter theSmaller than the pore diameter they wil

35、l simpl will simply be be Washed out unretainedWashed out unretained.SIZE EXCLUSION CHROMASIZE EXCLUSION CHROMATOGRAPHY OGRAPHY (CON(CONTD)D)SiSize exclusion is often applied to large molecules suche exclusion is often applied to large molecules suchAs polAs polymersmers, proteins etc proteins etc.S

36、IZE EXCLUSION CHROMASIZE EXCLUSION CHROMATOGRAPHY OGRAPHY (CON(CONTD)D)The total volume of a packed sihe total volume of a packed size exclusion column ise exclusion column is V Vt t = V= Vg g + V Vi i + V Vo o V Vt t = total volume; V= total volume; Vg g = volume occupied b = volume occupied by the

37、 solid the solid matrix of the gel (stationar matrix of the gel (stationary phase) phase) V Vi i = volume of the solvent held in the pores = volume of the solvent held in the pores V Vo o = free volume outside the gel particles = free volume outside the gel particlesThe retention volume for molecule

38、s too large to enter thehe retention volume for molecules too large to enter thePores = VPores = Vo o Retention volume for molecules too small to be retainedRetention volume for molecules too small to be retainedIn the pores = VIn the pores = Vi i + V Vo o SIZE EXCLUSION CHROMASIZE EXCLUSION CHROMATOGRAPHY OGRAPHY (CON(CONTD)D)Molecules of intermediate siMolecules of intermediate size that are retai