Simulation of Scanning Fluorescence Spectrometer.doc

Simulation of Scanning Fluorescence Spectrometer This is a simulation of a scanning spectrofluorometer. The simulation displays excitation spectra, emission spectra, and synchronous spectra, relative fluorescence intensity, and absorbance at the excitation wavelength. Operating instructions are contained in the scrolling text field in the upper right of the screen.Answer the following questions.1. Set A=1 ppm and B=0. Determine the wavelengths of maximum excitation and emission for component A. What is its Stokes shift?2. Dose Vavilovs Law*seem to hold for compound A, that is, is the shape of the emission spectra independent of the excitation wavelength, and vice versa, except for the scatter peaks?3. Is there any sign of Rayleigh or Raman scatter? How could you distinguish these from genuine fluorescence?4. Check the blank (click on “Insert Blank”).Increase the sensitivity setting as necessary. Is there any sign of dark current or background fluorescence? What are the main features of the excitation and emission spectra of the blank? Estimate the spectral bandpass of the monochromators.5. Dose the wavelength separation between the Rayleigh and Raman scatter peaks in the emission spectrum vary with excitation wavelength? What is the frequency, in cm-1, of the vibration causing the Raman peak? What vibration is most likely the cause?6. Find the combination of excitation and emission wavelength that gives the best precision of measurement of low concentrations of component A. Estimate the detection limit of component A in ppm. Is the detection limit lower by fluorescence or by absorption measurement? By approximately what factor?7. Over most of the concentration range, what is the source of noise in the intensity readings and in the spectra? How could you prove this?8. Is there evidence of non-linearity in the relationship between concentration and intensity at high concentrations? What is the most likely source of the non-linearity?9. Vary the wavelength offset and observe the synchronous spectrum. What offset gives the largest peak height? Explain the effect of Rayleigh and Raman scatter on the synchronous spectrum. Note: this is a constant wavelength synchronous spectrum.10. Set A=0 and B=1 ppm. Determine the wavelengths of maximum excitation and emission for component B. What is its Strokes shift? Can mixtures of these two components be determined by fluorescence measurement?*Vavilovs Law states that the shape of the fluorescence emission spectrum of a single fluorophor is independent of the excitation wavelength, and vice versa. This holds only for the fluorescence peaks, not Raman or scatter peaks.扫描荧光光谱仪的模拟这是模仿一个扫描分光荧光计。该模拟显示激发光谱，发射光谱和同步光谱,相对的荧光强度,在激发波长下的吸光度。操作说明书被包含在屏幕右上方的滚动文本区域。回答下列问题：1. 设定A= 1 ppm和B = 0。确定A的最大激发和发射波长。它的斯托克斯位移是多少？2. 瓦维洛夫定律是否适用于化合物A？就是说，发射光谱的形状不依赖于激发波长，反之亦然，除了散射波峰。3. 是否有瑞利和拉曼散射的标志？你怎么能从真正的荧光区分出这些？4. 检查空白（点击“插入空白”）。必要时提高敏感度的设定。是否有任何暗电流或背景荧光的标志?空白的激发和发射光谱的主要特点是什么？估计单色仪的频谱带通。5. 瑞利和拉曼散射中的波长分离在发射光谱中随激发波长变化吗?造成拉曼峰的振动频率是多少？振动是最有可能是什么原因引起的?6. 找出能最好地精密测量低浓度的成分A的激发和发射波长的结合。用ppm估计成分的检测限度.用荧光或吸光度测量检出限是否会降低?大约什么因素?7. 在大多数的浓度范围内, 在强度读数和光谱中什么是噪音的来源?如何证明?8. 是否有证据证明在高浓度范围浓度和强度不成线性关系？造成这种非线性最可能的原因是什么？9. 改变波长的偏移，观察同步光谱。峰值最大时偏移量是多