SPECTROPHOTOMETRY OBJECTIVES 1. Describe the mechanism by which by dfgh4bnmu




1.    Describe the mechanism by which atoms and molecules absorb light. What
      atomic/molecular characteristic determines the wavelength of light that is absorbed?

2.    Calculate absorbance from %T. Use Beer's law to relate the absorbance or %T of a
      solution to the concentration of the absorbing compound. List the assumptions inherent in
      Beer's law.

3.    List the components of a spectrophotometer. List examples of each component. Describe
      how each component works.
                lamp (visible and UV)
                monochromator (fixed wavelength and variable wavelength)
                detector (PMT and photodiode)

4.    Describe bichromatic analysis including the principle, arrangement of components and
      advantages. Extrapolate the principle to multiple-wavelength spectrophotometry. List
      examples of common interferences that can be corrected using bichromatic analysis.

5.    Describe check systems for verifying the performance of spectrophotometer components.
      List the components that require checking, and describe the errors that would be caused by
      malfunctions. Describe the reference components used for checking.

6.    Define band-width (band-pass) and stray light, and describe their effects on absorbance

7.    Define fluorescence. Describe the atomic mechanisms that result in fluorescence.
      Describe the relationship between amount of fluorescence emitted and concentration of the

8.    Define nephelometry. Describe the relationship between amount of light scattered and the
      concentration of particles. List the limitations of using nephelometry.

9.    Compare the components of a fluorometer and a nephelometer to the components of a
      spectrophotometer. Contrast their arrangement and functions.

10.    Define reflectance spectrophotometry. Correlate it to transmission spectrophotometry.

I. Wavelength absorbed by an atom or molecule corresponds to energy levels available to
the valence electrons.

                                                                  K shell (max 2 e-)

                                                                      L shell (max 8 e-)

                                                                     M shell (max 18 e-)
                                                                     1st comfort level @ 8 e-

      The energy and wavelength of a photon are inversely related to each other.

      Colors perceived by the eye correspond to certain ranges of wavelengths.

II.Beer's law relates concentration of solute to the transmission of light through its solution.

                                                                      (opaque cuvet or shutter)
                                        Defined as 0% T

                                                                      (cuvet with solvent)

                                                                = I/Io X 100

       Each successive "layer" of solute absorbs a constant % of the light that is incident on it

       Thus, %T is a logarithmic function of solute concentration

               1. Define a new term: Absorbance = A = Abs
                        A = - log I = -[log I - log Io] = log Io - log I

                        when Io is set at 100 % then log Io = 2 and I is %T

                        ∴ A = 2 - log I = 2 - log %T

             2. Beer's Law: A = abc
                     where A = absorbance
                            c = concentration
                            b = length of light path thru solution (i.e., width of cuvet)
                            a = proportionality constant
                                   also called extinction coefficient or molar absorptivity

                            In short: A ∝ concentration

                            Assumptions: only 1 chromogen and only 1 wavelength of light

III. Spectrophotometer

      A. Light source = lamp

             Must emit the desired wavelength with enough intensity to reach the detector

             Types: tungsten (& tungsten halogen) → ~320 to >1000 nm = visible & infrared
                    xenon → ~200 to ~800 nm = UV and visible               require HV
                    deuterium & hydrogen → ~200 to ~450 nm = UV             power supply
                    mercury → several sharp spikes in UV range

B. Monochromator

      Chromogen ABSORBS λ that corresponds to energy level of its e-,
            and TRANSMITS the rest of the wavelengths

             Beer's law : concentration ∝ absorbance
             ∴ select λ that is absorbed in order to measure concentration

              λ absorbed is complementary to the λ that is transmitted
                                                                                        V       R
                     yellow and blue are complementary to each other I                              O
                     red (and violet) are complementary to green
                                                                             B                          Y

      1. Fixed wavelength types of monochromators:                                          G

             Colored glass or plastic
                   absorbs λ complementary to its color (appearance)

             Interference filter
                    transmits λ that is even multiple of distance between its sides
                                                     If distance between the surfaces
                                                     is an even multiple of λ, then
                                                     reflecting waves stay in phase →
                                                     constructive interference →
                                                     If distance between the surfaces
                                                     is NOT an even multiple of λ,
                                                     then reflecting waves get out of
                                                     phase → destructive
                                                     interference→ damping.

      Note: several λ's will be even multiples; called 1st, 2nd, 3rd order wavelengths;
            remove them with cut-off filters before the monochromatic light hits the

      2. Variable wavelength types of monochromators spread the λ's of incident light
      into a continuous spectrum of different colors;
      then the desired color (λ) is selected by passing it to the cuvet through an exit slit.

             a. prism
                    obsolete because short λ's are spread out nicely (can pick out the λ
                    you need), but long λ's are crammed together (can't select just one

               b. diffraction grating "bends" light around sharp edged grooves inscribed
               in a reflective surface

                      Short wavelengths are diffracted through only a small angle; long
                      wavelengths through a large angle;

                      λ's that overlap either reinforce each other (constructive
                      interference) or cancel each other (destructive interference);
                      ∴ the incident light is spread out into one continuous spectrum
                      (like one rainbow from diffraction off of many water droplets)

                                         diffraction grating

C. Cuvet - holds sample in the light path; ∴ defines "b" in Beer’s Law A = abc

       must transmit essentially all of the incident light
              ordinary glass transmits > ~320 nm
              special acrylic plastic transmits > ~320 nm
              Fused silica or quartz glass transmits > 200 nm

       must use "matched set" of cuvets, i.e., all have identical, minimal absorbances
       (diameters, reflections, refractions, imperfections, etc.) and are clean (no left-over
       reagents or soap, no fingerprints from handling)

D. Detector - transduces light energy into electrical energy

       Light hits surface of selenium or cadmium oxide → dislodges e-'s → current or ∆

       Photocell fatigue = all the available e- have been removed, ∴ surface is "blind"

              Two common types:

              photomultiplier tube (PMT)                    photodiode
              multiplies and measures current               measures drop in voltage
              requires high voltage power supply
              has dark current

IV. Bichromatic analyzer splits the light beam in two and selects a different λ for each

       A. Method:

              One λ is the absorption peak of the analyte (desired chromogen),

                      ∴ Abs at this λ measures the concentration of the analyte
                      PLUS cross-over absorbance due to other interfering chromogens in the

              Second λ is NOT absorbed by the analyte but IS absorbed by the interfering
              chromogen, ∴ this absorbance indicates how much interfering chromogen is
                     The ratio of absorbances at the two wavelengths is must be known for the
                     interfering chromagen

              Then subtract the interfering absorbance to determine the concentration of the

       B. The function of the 2nd λ is to act as the "serum blank"

              "Blank" = solution in a cuvet that does not contain specific absorbing substance,
              e.g., you did not perform the color reaction.

                      ex: if reagent itself is colored, then total Abs =
                               Abs due to color reaction with the analyte plus Abs due to reagent

                      ∴ set the cuvet containing reagent (in the same proportion) at 100 %T
                      = “reagent blank”

                    ex: if sample itself is colored, then total Abs =
                    Abs due to color reaction with the analyte plus Abs due to background

                    ∴ set cuvet containing sample (in the same proportion) but NO COLOR-
                      REACTION at 100 %T = “serum blank”

                            Examples: hemolysis (red hemoglobin), jaundice (yellow
                            bilirubin), lipemia (turbidity due to triglycerides)

      Example of calculations and assumptions:
            analytical cuvet contains:                    serum blank cuvet contains:
                    Abs due to cuvet glass                       Abs due to cuvet glass
                    Abs due to colored reagent                   Abs due to colored reagent
                    Abs due to sample color                      Abs due to sample color
                    Abs due to reaction-chromogen

                    Total Abs in analytical cuvet                 Total Abs in blank cuvet

                    Set blank cuvet = 100 %T = 0 Abs, ∴ all Abs in blank cuvet has been
                    subtracted leaving only Abs due to the desired analytical reaction

V. Performance checks

      Principle: each component can malfunction and cause errors in the absorbance reading.

      A. Lamp check: Does the lamp emit sufficient light energy?

             Select a wavelength where the lamp only emits a little bit;
             can you set the meter reading at 100 %T?

      B. Monochromator check: Is the λ transmitted to the cuvet the same as the λ reading
      on the knob?

             Place a substance with known absorbance peaks and valleys in the cuvet;
             do the known maximum and minimum peaks and valleys correspond with the
             reading on the knob?

primary wavelength calibrators: didymium and holmium oxide filters

secondary calibrators: any solution with known peaks and valleys in its
absorption spectrum

       ex: NiSO4 or KMNO4 or CoCl2

Exit Slit

       1. Used in variable λ spectrophotometers to select the desired λ from the
       ∴ wide slit allows many surrounding λ's to reach the cuvet = wide
          narrow slit allows only 1 (or few) λ's to reach the cuvet = narrow

       2. Is the absorbance reading of a known calibrator as high as expected?

               Wide bandpass "averages" (integrates) the reading at the desired λ
               with all the readings at surrounding λ's → low overall reading

Exit slit is wider than expected (causing shallow peaks and valleys) if
        lamp is too dim
        slit gears malfunction
        slit becomes effectively wide by having dirt/dust collect on its edges

C. Cuvet Check: Is identical absorbance reading obtained when one solution is read in
different cuvets?

       Sources of error include:     warped glass
                                     cuvet diameter is not the same
                                     dirt inside (absorbs or reflects light)
                                     dirt outside (absorbs or reflects light) - including
                                              finger prints

D. Detector Check: Is the absorbance reading of a known calibrator equal to the
expected value?

       Primary calibrators are "neutral density" filters
              graded amounts of gray dye with known absorbance values

       Sources of error
              dirt or chemical film on the surface of the detector (absorbs or reflects
              wrong or variable voltage to the dynodes
              "photocell fatigue" = too bright light removed all the surface e-; wait a few


I. Atomic mechanism of fluorescence

       A fluorescent molecule absorbs a specific λ of light which raises an e- to a higher orbit
       (same as spectrophotometry)
       10-8 to 10--4 seconds later, the e- falls back to its ground state orbit and the energy is
       released as emitted light.

       During the 10-8 to 10-4 seconds, the electron vibrates and rotates in its chemical bond
       which uses up some energy.

       ∴ the λ of emitted light has less energy (longer λ) than the exciting light (shorter λ)

                                                                   λemission > λ excitation

II. Instrumentation:

       The exciting light is collimated into a narrow beam (same as spectrophotometer), but the
       emitted light leaves the cuvet in all directions
       ∴ the emission monochromator & detector are at right angle to the exciting light beam

               Need both primary (excitation) and secondary (emission) monochromators

III. Calculating concentration:
        Every excited molecule emits fluorescent light,∴ fluorescence ∝ concentration

                                                                    Fluorescence is ~ 1000 x more
                                                                    sensitive than spectrophotometry
                                                                    but exciting light must penetrate
                                                                    the whole cuvet to have
                                                                    Can detect the emitted light from
                                                                    a tiny amount of fluorescent
                                                                    molecule because 0 concentration
                                                                    → no fluorescence = blackness


I. Nephelometer (or turbidimeter) is used to measure turbid suspensions.

       When cuvet contains insoluble particles, incident light is scattered in all directions.

       Then less light reaches the detector of a spectrophotometer → pseudo-absorbance

               ∴ amount of turbidity appears to follow Beer's law; this is called Turbidimetry

       Used to measure insoluble product, e.g., aggregates of Ag-Ab complexes (precipitin

II. Instrumentation

       In a nephelometer, the scattered light is measured at an angle (like fluorometry) except
       photon was only scattered, not absorbed, ∴no change in λ ∴ no secondary

               Amount of light scattered ∝ concentration of insoluble particles;
               this is called nephelometry.

III. Calculating concentration

       In both turbidimetry and nephelometry, there is a very limited range of concentrations
       where light scatter is a function of particle concentration. The principle limiting factor is
       how far the incident light can penetrate the turbid suspension.

       Don't forget that suspensions tend to settle out of solution, ∴ must have a well-mixed

                                 REFLECTANCE PHOTOMETRY

I. Atomic Principle:

       When colored analyte sits on the surface of an opaque support, then it absorbs the same λ
       as if it was in solution and reflects the complementary λ's.

                                                                     Concentration ∝ inverse log of
                                                                     amount of light reflected
                                                                     ∴ follows an equation that resembles
                                                                     Beer's law.

       (This also works if the surface analyte is fluorescent, then it emits light the same as if it
       was in solution , and concentration ∝ amount of fluorescent light.)

II. Instrumentation

       Reflectance photometry is commonly used in instruments that measure antigens because
       the antigen-antibody complex formed in the analytical reaction can be adsorbed onto a
       filter while the unreacted materials flow through the filter to waste.

       Also commonly used in urine dipsticks where numerous solutes in a urine sample are
       tested in individual color reactions on separate pads of reagents tests


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