# CLS Spectrophotometry Review questions winter 2011

Flashcard maker : Richard Molina
 Define: beers law
 The mathematical relationship that connects Absorbance of energy, concentration of a solultion and PATH LENGTH. The equation is:   Absorbance=(a)absorbancy of the solution molecule X(times) *(b)the path length X(times) (c) the concentration of the solute in solution. A=a*b*c
 Beers Law what are the components?
 A=Absorbance   a=the absorbancy of the solution molecule b= the path length c=the concentration of the solute in solution
 What is the mathematical relationship between % T and Absorbance?
 P=measured transmitted light Po= incident light %T=(P/Po)(100%)  The relationship is inverse and logarithmic: if the path lenght is unchanged then linear increases in concentration are measured as expotential decreases in transmittance. Since an inverse logarithmic term is cumbersome a new term, ABSORBANCE can be derived to be used in place of %T. this formula is used A=LOG 100% -LOG%T or 2–LOG%T
 What is the relationship of Absorbance to Concentration?
 Absorbance is directly proportional to concentration of the absorbing species if beers law is followed.
 What is the appearance of the relationship of absorbance and concentration when plotted on a linear graph?     A perfect positive slope.   When a graph of absorbance vs. concentration is plotted for the standard solutions, a direct relationship should result,
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 What is the appearance of the relationship of absorbance to concentration when plotted on semi log paper?
 A graph of absorbance versus concentration will result in a straight line relationship when plotted on coordinate graph paper. A graph of % T  versus concentration  results in a straight line relationship when plotted on semi log graph paper. Absorbance plotted on coordinate scale is usually preferred to %T plotted on semi log paper However,  both result in a straight line and either can be used to determine concentration
 What happens to Absorbance and %T when concentration increases?
 This is a direct relationship when concentration increases the absorbance increases An indirect relationship when concentration increases %T  decreases.
 What happens to Absorbance and %T when concentration decreases?
 A direct relationship when concentration decreases Absorbance decreses An indirect relationship when concentrationd decreases %T increases.
 What happens to Absorbance and %T when the light path increases?
 Absorbance is directly proportional to the light path and the concentration, doubling the path length results in incident light contacting twice the number of molecules in solution. this causes absorbance to double.   %T is indirectly proportional to the light path and the concentration, doubling the path length results in incident light contacting twice the number of molecules in solution. this causes %T to to be reduced by 50%.
 What happens to Absorbance and %T when the light path decreses?
 Absorbance is directly proportional to the light path and the concentration, reducing  the path length results in incident light contacting less  molecules in solution. this causes absorbance to decrease.   %T is indirectly proportional to the light path and the concentration, reducing the light path length results in incident light contacting less molecules in solution. this causes %T to to be increased.
 What condition is necessary for obeying beers law?
 YOU MUST MAINTAIN STAY IN THE LINEAR RANGE The graph will flatten out and you will not be able to maintain a linear relationship Because transmittance inherently decreases logarithmically, linearity breaks down at higher concentrations . The same applies for lower end limits.
 What formula for calculating concentrations of unknowns using absorbance values [standard concentrations are known]
 C = (Au/As)Cs   C= concentration of the unknown Cs=the concentrationof the standard Au=the absorbance of the unknown As=the absorbance of the standard
 What is stray light and stray energy?
 Stray light (stray energy) is caused by the presence of any light other than the wave length of measurement reaching the detector.
 How can high levels of stray light influence absorbance values?
 Stray light is the most common cause of loss of linearity at high analyte concentrations. Transmitted incident light is lowest when absorbtion is the highest. Therefore, stray light is a greater percentage of the detector response when sample concentration is high. Stray light is usually most significant when measurements are made at the extreams of the visable spectrum because lamp output and/or detector response is low.
 How can one measure the level of stray light empiricaly?
 By using A sharp cut-off filter. Sharp cut-off filters transmit almost all incident light until the cut-off wave-length is reached. At that point, they cease to transmit light. Because they give an all or none effect, only stray light reaches the detector when the selected wavelength is beyond the cut-off.
 What concepts are used to govern the selection of appropriate wavelength from a spectral curve? .
 1) record the absorption spectrum of the material that is being measured.                                                2). This spectrum will help to determine the best wavelength for the spectrophotometric analysis. The optimum wavelength for a specific analysis depends on: 1  the absorption maxima of the chromogen..2 the slope of the absorbance peak.3.the absorption spectrum of interfereing chromogens. If the absorption peak is narrow any small error in the setting of the spectrophotophotometer  at this wave length results in a large change in absorbance. because spectrophotometers use manually set wave lengths, this can cause large run to run imprecision and analytical error. This can be avoided using a wider absorption peak with this peak small changes in wavelength adjustment result in only small changes in absorbtivity and precision and accracy are high. 1.) choose an absorption peak with the greatest possible molor avsorptivity. 2.) Choose a relatively broad peak. 3.) choose a peak that is as far as possible from the absorption peaks  of commonly interfereing chromogens.
 What is meant by nominal wave?
 the nominal wavelength of the bandpass light beam is the wave length at which the peak intensity of light occurs. The wavelength of maximum transmittance.
 What is meant by effective bandwidth? wavelength encompassing 1/2 the transmittance.   two types of slits are present inmonochromaters. the first at he entrance.focuses the light on the grating of the prism, where it can be despersed with a minimum of stray light, the second slit at the exit determines the band width of light that will be selected from the dispersed spectrum. The effective bandwidth is the portion of the curve that is most effective it is seen on the curve as the middle 1/3 that is 1/2 the height of the curve,
 What is meant by spectral bandwidth?
 Your UV/Vis Spectrophotometer has a monochromator (grating or prism) that disperses the light from the light source towards a small opening (a slit) that let’s through a narrow spectrum of wavelengths (narrow spectrum, but spectrum nevertheless), centered at the selected wavelength. This spectrum contains a gaussian distribution of wavelengths and the width of this distribution (at half height) is called spectral bandwidth… it is 1/2 of the entire gaussian curve;
 What is the relationship between resolution and instrument spectral bandwidth?
 Bandpass refers to the range of wavelengths passing through a sample. The narrower the bandpass the greater the photometric resolution. Band pass can be made smaller by reducing the width of the exit slit. accurate absorbance measurements require a bandpass less than 1/5 th the natural bandpass of a chromaphore. ; The spectral bandwidth of a spectrophotometer is related to the physical slit-width of the monochromator and, therefore, to the resolution capabilities of the instrument. resolution is defined as the ability of an instrument to separate light into finite, distinct wavelength regions and distinguish these finite regions from each other. Resolution is primarily governed by the physical slit width of the instrument,in combination with the inherent optical dispersion of light from the exit of the monochromator to the detector of the instrument. Reducing the physical slit width decreases the spectral bandwidth and improves the ability of the instrument to resolve closely spaced peaks.
 What is the basic schematic of a single beam spectrophotometer?
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 Name some different types of monochromaters, their advantages and disadvantages, and how they work. ;
 Filters; glass Glass filters: one or more layers of colored glass: They have selective transmittance. They;Transmit more radiant energy in some parts of the spectrum than in others. ;Limited because of wide spectral bandpass.
 Name some different types of monochromaters, their advantages and disadvantages, and how they work. ; calibration filters ; Variable glass filters with specific spectral maxima wavelenghts. DIDIMIUM CRYSTAL
 DIDIMIUM CRYSTAL Has absorbance maxima at 573, 586,685,741,803 nm good for calibrating at these wavelength. some peaks have broadish plateau (you lose detail) but is useful for general lab spectrophotometers.
 What is a wratten filter?
 wratten filters:consist of colored gelatin between clear glass plates. Glass filters:one or more layers of colored glass: They have selective transmittance. They;Transmit more radiant energy in some parts of the spectrum than in others. A wratten filter is a wide band pass filter and is unsuitable for spectrophotometer calibration.
 What is an interference filter? Interference filters work on a different principle which is the same as that underlying the play of colors from a soap film, namely interference. When radiant energy strikes the thin film, some is reflexed from the frount surface, but some of the; radiant energy that penitrates the film is reflexed by the surface on the other side. the latter rays of energy now have traveled farther than the first by a distance two times the film thickness. If the two reflected rays are in phase there resolute intensity id doubled, whereas if they are out of phase they destroy each other.Therefore , when white light strikes the film , some reflected wave lengths will be augmented and some destroyed, resulting in colors.
 Narrow bandpass filters based on constructive and destructive interference. ; Constructive interference allow specific wavelengths through. Destructive interference prevents all other wave lengths from getting through.
 Name some different types of monochromaters, their advantages and disadvantages, and how they work. ;calibration filters Variable glass filters with specific spectral maxima wavelenghts. HOLMIDE OXIDE CRYSTAL ;
 HOLMIDE OXIDE CRYSTAL Has absorbance maxima at 241,279,287,333.361,418,453,536,636 nm The greater range, number of peaks and very narrow peak plateaus, makes this a choice for spectrophotometers at the high end / critical use as well as for checks in the UV range.
 what is a glass blocking filter? also called a CUT-OFF filter. Cut-off filters transmit almost all incident light until the cutoff wavelength is reached. At that point they cease to transmit light;. Because they give an all or none effect, only stray light reaches the detector when the slected wavelength;is beyond the cut-off.;
 A cut off filter a filter added to the interference filter to get rid of unwanted wave lengths, ; LIKE STRAY LIGHT!!!
 MONOCHROMATERS: Diffraction gratings: ; You change the position of the slit to get different bandwidths.
 A grating consist of a large number of parallel equally spaced lines ruled on a surface. Dispersion by a grating is; is linear therefore only two different wavelengths must be checked to certify the wavelength accuracy.
 MONOCHROMATERS: Prisms: dispersion by prism is nonlinear becoming less linear at different wavelengths (over 550 nm). Therefore, in certifing wavelength calibration, three different wavelengths must be checked. Prisms give only one order of emerging spectrum and thus provide higher optical eficiency because the entire incident energy is dispersed over the single emerging spectrum.
 A prism will split up white light because different wavelengths will travel at different velocities through glass.; If the glass is a triangular shape the white light will split up into the spectrum as with grating the exit slit will select the particular bandwidths. it is not as linear as with a grating they can have the narrowest band pass and greatest intensity they are the very best
 Name the different light detection devices in light detection instruments. Their advantages and disadvantages and breifly describe how they function. ; PHOTOVOLAIC CELL (Barrier Layer Cell) The cell is able to detect light because photons have enough energy to break covalent selenium bonds. the electron hole pairs that are produced provide current carriers that allow conduction across the selenium layer. The number of electron-hole pairs produced is directly proportional to the intensity of the radiation striking the selenium. therefore, there is a linear relationship between the detector photocurrent and the light intensity.;
 SLOWEST Cheap and STURDY
 Name the different light detection devices in light detection instruments. Their advantages and disadvantages and breifly describe how they function. PHOTOTUBE: A common detector that uses photoelectric effect to produce current (light induces the production of ejected electrons from a photoemissive surface.)
 More sensitive than a barrier cell. ; responds rapidly ; no fatigue with prolonged light exposure
 Name the different light detection devices in light detection instruments. Their advantages and disadvantages and breifly describe how they function. ; PHTOMULTIPLIER TUBE same as phototubes but has the ability tha amplify as much as 106 due to a step wise increase in potential through a series of Dynode units each stage causes amplified numbers.
 Fastest ;most sensitive more expensive ;
 Name the different light detection devices in light detection instruments. Their advantages and disadvantages and breifly describe how they function SILICON PHOTODIODE (PIN PHOTODIODE) Made of DOPED transistor construction. Hole electron pairs are formed from P-TYPE materal (readily gives up electrons) ;
 limited wavelength range Narrow bandwidth cheap sturdy
 What are the advantages of double beam over single beam spectrophotometer?
 ;A double beam will contineously monitor your blank and measures your sample at the same time.
 What are the wavelenghts in the visable range?
 Visable spectrum range 400-700. ; Measured by TUNGSTEN LAMP.
 What are the wavelenghts in the UV range?
 UV spectrum 220-360 ; Measured by Hydrogen or deuterium lamp
 What is a Turbidimeter? ; It is based on light scatter spectrophotometry.; How does light interact with a particle: When a beam of light strikes a particle in a solution the possible interactions are some light is reflected back, some light is scattered (bent), some is absorbed, some does not interact , hence is transmitted through the solution.
 Turbidimeter. measures the amount of light that is transmitted through a sample;. blocking filters are used .geometry relative to incident light is important, the angle is usually zero. laser lamps are used. ; ; ;
 What is a Nephelometer? ; Like turbidity It is based on light scatter spectrophotometry.; How does light interact with a particle: When a beam of light strikes a particle in a solution the possible interactions are some light is reflected back, some light is scattered (bent), some is absorbed, some does not interact , hence is transmitted through the solution. ;
 Nephelometry: Measured the amount of light SCATTERED, Usually the most sensitive of the two.
 What is Fluorimeter Emission spectrum is shifted to a longer wavelength and lower energy than the absorbtion spectrum. Stokes shift is the difference between the maxima wavelength of each spectrum (it is actually a reflection of the energy lost
 Fluorimeters measure Fluorescence a number of molecules possess chemical attributes that allow for some of the energy of absorbed light to be emmitted as photons of defined wavelength. this allows for the measurement of this remitted light by spectrophotometer methods.
 What are wavelength calibration filters? Variable glass filters with specific spectral maxima wavelengths DIDIMIUM CRYSTAL and HOLMIDE OXIDE CRYSTAL ; ;
 DIDIMIUM CRYSTAL Has absorbance maxima at 573, 586,685,741,803 nm good for calibrating at these wavelength. some peaks have broadish plateau (you lose detail) but is useful for general lab spectrophotometers. HOLMIDE OXIDE CRYSTAL Has absorbance maxima at 241,279,287,333.361,418,453,536,636nm The greater range, number of peaks and very narrow peak plateaus, makes this a choice for spectrophotometers at the high end / critical use as well as for checks in the UV range.