Absorption Photometry

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					      Chapter 6 Absorption Photometry
     The analysis method, which is based on
    selective absorption of the matter to the light,
    includes Color method, ultraviolet obvious
    spectrophotometer method, and infrared
    spectrometry and so on. This chapter discusses
    absorption photometry in the visible range
    (spectrophotometer method or luminosity
    analytic method).

   1 、What is Characteristic of Absorption
   2 、How to design Absorption Photometry?
   3 、How many sources of the error to
    Absorption Photometry?
     The contents of this chapter

   6.1 Outline
   6.2 The design of Absorption Photometry
   6.3 The error of Absorption Photometry
   6.4 Other Absorption Photometry and The
    application of Absorption Photometry
               6.1 Outline

   1 The basic principle, characteristic of the
    Absorption Photometry
   2 The basic law of the Light absorption
   3 Colorimeter and Absorption Photometry
    and their instruments
   1 The characteristic of the Absorption Photometry

   (一) The sensitivity is high. The lower limit concentration of the
    measured matter (lowest concentration) when using the absorption
    photometry is generally up to 1~10-3 % micro component. It can
    measured 10-4 % to solid specimen. If the measured components
    gathering after measured, it can improve 10~100 times.
   (二) The accuracy is high. The relative error is 2~5 %, but it is quite
    satisfying to the micro ingredient. Because, in this case, the titration
    analysis and the weight method are also inaccurate, even it is
    unable to carry on the determination.
   (三) Easy to operate, the determination speed is quickly.
   (四) Wildly application , nearly all inorganic ions and the organic
    compound could use Absorption Photometry conduct directly or
    indirectly measurement.
   2 The basic property of the light

     Light is one kind of electromagnetic wave and has
    the adulatory property and the corpuscular property.
     Light in dissemination displays the adulatory
    property. For example, light’ diffraction, refraction,
    interference, polarization and so on. The parameter
    that to describe the wave property includes: λ υ c.
     Light has the corpuscular property at the same time,
    for example, Photoelectric effect, Light ’absorption,
    light’ launch and so on. Energy:E .
   wave-particle duality of the
                                  light’ refraction

                                light’ diffraction
                                  light’ polarization

                                   light’ interference

property            E               Photoelectric
                         E: Photon ’energy (J, joule)
             hc           :Photon ’frequency (Hz, hertz)
E  h                    :Photon ’wave length (cm)
                         c: Speed of light(2.99791010 cm.s-1)
                          h: Plank Constant 6.625610-34 J.s

★Manifesting: The above equation manifests that the light has the
adulatory property and the corpuscular property as well as the
relations between them.
Indicating: the photon energy of the light is different when the
★light has different wave length or the frequency. If the wave
length is shorter, then the frequency is higher, the photon energy
is bigger.
Spectrum           Wavelengt     Transition Types              Emitter                         Analysis methods
                   h range
X-ray              10-1~l0 nm    electrons of K and L layer x-ray tube                         x-ray spectrometry
Far ultraviolet    10~200 nm     middle level electron hydrogen ,deuterium , xenon lamp        Vacuum ultraviolet spectrophotometry
Near ultraviolet   200~400 nm    valence electron hydrogen , deuterium , xenon lamp            UV spectrophotometry
Visible light      400~750 nm                                                                  colorimetric method
Near infrared      0.75~2.5 μm   valence electron tungsten lamp                                near-infrared Spectrophotometry
Middle infrared    2.5~5.0 μm    The molecule shakes the hot silicon-carbide stick             Middle infrared spectrophotometry
Far infrared       5.0~1000 μm   molecule shaking       the hot silicon-carbide stick          far-infrared spectrophotometry
Micro wave         0.1~100 cm    molecule rotating and shaking the hot silicon-carbide stick   microwave spectroscopy,
Radio wave         1~1000 m      molecule turning the electromagnetic wave                     NMR spectroscopy
     10-2 nm 10 nm     102 nm 104 nm          0.1 cm 10cm    103 cm     105 cm





                                      Visible light
The principle of the absorption spectrum

  The absorption spectrum divides into :

  1、Atomic absorption spectrum
  2、Molecular absorption spectrum
   ■Atomic absorption spectrum:It is produced by the outer-
    shell electron of the atom selectively absorbs certain lengths
    wave ’ electromagnetic wave. And it is line spectrum, Atomic
    absorption spectrophotometer method is established based on
    this property.
   ■Molecular absorption spectrum: it is band spectrum which is
    produced by the electron level jumping. The difference of the
    electronic energy interstate energy is 1~20 (eV). The molecular
    spectrum produced by the valence electron jumping is called
    the electronic spectrum
   ■Infra-red absorption spectrum: it is produced by the
    molecular vibration energy level (Energy difference
    approximately 0.05~l eV) and the rotational level (energy
    difference is smaller than 0.05 eV) jumping, which is called
    band spectrum. Infrared Spectrophotometer is used to the
    research of molecular structure
     The complexity of Molecular absorption
   ★Because of the complexity of the molecular structure ,the
    molecular spectrum is quite complex. There are several
    vibration energy levels in identical electron level. But there
    are also several rotational levels in the identical vibration
    energy level.
   ★When electron level is changing, the molecular vibration
    and the rotational level are following changing at the same
    time. Therefore the molecular spectrum is much more
    complex than the atomic spectrum, and it becomes band
   Noun terminology

   ★monochromatic light:the light with identical wave length .
   ★Multiplex light: the light with different wave length
   Ultraviolet light: wave length 200~400 nm
   ★Visible light: the light we can feel with our eyes, wave length
    400~750 nm. It is mixed by red, the orange, yellow, green, blue, blue,
    purple and so on according to the certain proportion.
   ★Wave band: Each kind of color light has different wave length
   ★Complementary color light: two kind of color lights, which mixed
    with each other according to the certain proportion, is called
    complementary color light mutually.
   ★Absorption curve: Surveys something confrontation different
    wave length monochromatic light the absorption degree, wave
    length() as Horizontal coordinates , Extinction degree of light
    as vertical coordinates, then draw a curve of extinction degree
    change following the change of wave length, so the curve is
    absorption curve maximum absorption wavelength: the max
    wavelength when light absorption is λmax.
     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 complementary color
    light mutually .and this phenomena is called
    complementary of light.

                                                         green     Yellow
                                        green                               yellow

                                Green                                         orange

             The absorption of Material to the light
                 The relation of the color of material and light

             The spectrum signals hint   Multiplex   The form view phenomenon signals hint

Completely absorption

Completely through

  Absorb yellow light
   Table 6-2 The relation between material color and absorption

    The color of the               The absorbed light
       material                color           Wave rangeλ/nm
      Yellow green            purple              400~450
         yellow                 blue              450~480
         orange             Green blue            480~490
           red              Blue green            490~500
       Purple red              green              500~560
         purple            Yellow green           560~580
          blue                Yellow              580~600
       Green blue             orange              600~650
       Blue green               red               650~750
  The color of the material is produced because the matter has the
selective absorption function to the different wave length light .
•Looking at the absorption light and the color relations
from the absorption curve

   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. λmax= 525 nm. When
    concentration are different, the
    curve shape of the light
    absorption is same, λmax
    invariable, absorbance are
   The characteristic of Colorimetry and Absorption

★High Sensitivity: Commonly used to determine the micro
  component in the test specimen whose quality score is 1%~10-5,
  even can determine the trace amount component. whose quality
  score is lower to 10-6~10-8.
★The accuracy is high: the relative error of Colorimetry is 5%~10%;
  and Absorption Photometry is 2%~5%.
★Applies broadly: almost all inorganic ions and many organic
  compounds may be determined directly or indirectly by the
  Colorimetry or the Absorption Photometry.
★The instrument is simple, easy to operate.
    2.The fundamental law of the light absorption

     When a bunch of parallel monochromatic lights through any
    even, non-scattering solid, liquid or gas medium, partly is
    absorbed, partly is penetrate medium, partly is reflected by
    household utensils superficial. Like chart 6-3 shows, supposes
    the intensity of the insert light is I'0, the intensity of the
    absorption light is Ia, the intensity of the penetration light is It,
    the intensity of the reflection light is Ir.

    I0     I a  It  I r
      In the Absorption Photometry analytic method, the
    test solution and the blank solution are put in the
    same material and thickness absorption pond
    separately , then let monochromatic light whose
    intensity is I `0 through these two absorption pond
    separately, again surveys its penetration light
    intensity. Then the intensity of the reflect light is
    invariable, also its influence may counterbalance
    mutually. The relation between the intensity of the
    insert luminous and the intensity of the penetration
    luminous is a equation below

                 I 0  I a  It
     The ratio of penetration light intensity It and insert
    light intensity Io is called the transitivity or the
    transmittance, indicated with T. The bigger the
    transitivity of the solution is, indicating it is smaller to
    the light absorption; On the contrary, the transitivity is
    smaller, indicating it is bigger to the light absorption.

                 T 
           insert light I0               penetrate light Ir

                               The T value is 0.0% ~ 100.0%.
The definition of
Transmittance          T        Absorb completely T=0.0%
                          I0   Penetrate completely T=100.0%
                       Lambert-Beer’s law
      Lambert J H and Beer 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
    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:

              Io                                Io      1
       A  lg     Kbc                   A  lg     lg
              I                                 I       T
      Lambert-Beer’s law indicates that, after a bunch of
    monochromatic lights through 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 matter 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. This
    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
           Absorbance and transmittance
                                          A            Kbc
 lg T  Kcb  A                 T  10          10
T: transmittance A: Absorbance
                                    T = 0.0 %
                                     T = 100.0 %
                                      A = 0.0

                                     T = 36.8 %


                                      A = 0.434

              molar absorptivity and Sandell
    ★ Molar absorptivity
       When concentration c is indicated with mol·L-1, fluid
     layer thickness b is indicated with cm, then K is indicated
     with the mark ε. ε is called molar absorptivity, the unit is
     L·mol-l·m-1, it expresses the solution Absorbance when the
     material quantity concentration is the l mol·L-1, fluid layer
     thickness is l cm.

                         A  bc
      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 μg·cm-2,

      S        bcM  10 6

       bcM  10 3
        0.001             M
              M  10 

                          
The relation of S 、ε and the molar quality of
              the matter M is

         S 
3.Visual colorimetry and Absorption Photometry
              and their instruments
   ★Colorimetry
    It is a method to determine the content of the matter
    by using eyes to observe the color depth of the
   ■Merit: The instrument is simple, the operation is
    simple, Visual colorimetry is suitable for the analysis
    of a large quantity of test specimen. The sensitivity is
    high, may be carried on under the compound of light -
    white light, so It is also available this law carried on the
    determination when certain color reactions do not
    conform to Lambert - the Bill law.
   ■Main shortcoming: the accuracy is not high enough,
    the standard series cannot last for a long time and
    needs to prepare temporarily.
   colorimeter                                 Standard

     Use nature light

     Compare the supplementary
     colored light of absorption light

     The accuracy is low(Semi-
     quantitative)                       Unknown
     the multi-components that
     Cannot distinguish

      The method is simple and
     the sensitivity is high.
   Spectrophotometer
      Surveys a series of standard solutions with the aid of the
    spectrophotometer, draw the standard curve (calibrated curve),
    we can obtains the concentration or the content of measured
    material from the standard curve, according to the testing of
    the fluid extinction.
   Standard curve- calibrated curve
   Working curve
   The Characteristics of Spectrophotometer
   (1) The insert light is the monochromatic light of high purity.
    The situation of deviating Lambert - the Bill law is reduced
    greatly, the straightway scope of the standard curve is bigger,
    the analysis result have high accuracy.
   (2)May select some wave length monochromatic light willfully,
    therefore using the extinction additive property, determines two
    kinds of or two kind of above components in the solution
   (3)Many colorless matters, so long as they have the absorption
    peak in ultraviolet or the infrared light region, also may be
                  Comparing the two methods

   ★The principle is different: Absorption Photometry and
    colorimeter have difference in methods. The Absorption
    Photometry is comparing the absorb situation of color
    solution to some wavelength light. But the colorimeter is
    comparing the intensity of the penetration light, For
    example, measured KMnO4's content, Absorption
    Photometry is measuring the absorb situation about
    KMnO4 solution to yellow green light, but colorimeter is
    comparing the penetrate intensity of the purple red light of
    the KMnO4 solution.
   ★Spectrophotometer
   The spectrophotometer may divide into the single light
    beam and the
   double light beam two kinds. There have numeral
    demonstrated single light
   beam, visualize spectrophotometer (for example 722)
    (recording spectrophotometer)

   ★The basic components
   Light source, monochromator, colorithrough,
    detector and manifest equipment
                   Working process

light source           monochromator          colorithrough

    transformation          Manifest record
   ★Light source :Using 6 ~ 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.
   ★monochromator:It is a device that
    decomposition the continuous
    spectrum which is sent out from the
    light source into mono-color. There
    have prism and grating two kinds。
 ★ The coloritrough also called absorbing
  pool. Be used for contain test solution.
  they are fabricated by glass and quartz
 Be marked according to thickness: 0.5 cm,
  l cm,2 cm,3 cm,5 cm,10 cm
   ★Detector: Accepting the 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. photomultiplier is widely used
    in modern spectrophotometry. In the early age it
    also use photocell.
   ★Manifest record system: It's 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 , tiny safety form , digital
    display record instrument.
    The type of the spectrophotometry
 Visible spectrophotometer , ultraviolet-
  visible spectrophotometer and infrared
 (一) Single light beam spectrophotometer

       721 , 722 , 751 type.
 (二) Double light beam spectrophotometer
         Single wavelength double light beam

                      Light beam                 ratio
                      split machine

Light     monochrom

                         Absorb       detector
    6.2 The design of spectrophotometer method

 1 Color reaction
 2 The choice of color condition

 3 The choice of measuring wavelength and
  the range of absorbance
 4 The choice of reference solution

 5 Design standard curve
                  1.Color reaction
   ★In spectrophotometric method ,the reaction
    which transform the measured component of the
    specimen into color compound is called color
   ★The color reaction can divided into two kinds,
    namely complexation reaction and oxidation-
    reduction reaction, but complexation reaction is
    the main coloration reaction.
   ★The reagent which composed with the
    measured composition into color material is
    called color-developing agent.
        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 agent have color ,so the difference of the
    maximal absorb wavelength between color compound and
    chromogenic agent must be big, general demand above 60
   (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 control.
                  Color developing reagent
 (1) Inorganic color developing reagent
 A lot of inorganic reagent can get color reaction with
  metal ion, for example Cu2+ can form a deep blue
  complex-ion Cu (NH4) 42+ with ammonia water, SCN-and
  Fe3+ form red complex compound Fe (SCN) 2+ or
 Fe (SCN)63-. But the sensitivity and selectivity of most
  inorganic color-developing agent are not high , among
  them the property is good and still have practical value's
  at present are sulfur cyanate , ammonium molybdate ,
  ammonia water and perhydrol etc.
 (2) Organic color developing reagent
 A lot of organic reagent, can generate the colored metal
  chelate with metal ion under given conditions, (have the
  architectural complex compound of cyclic annular).
       Chromophore and auxochuome.
      In organic compound molecular, any group that
    contains conjugation double bond for example: N = N , N
    = O , NO2,quinonyl, = C = O (carbonyl), = C = S (sulfur
    carbonyl) and so on , general have color ,because in
    these groups the launch of the electron only need a litter
    energy ,also can absorb light whose wavelength above
    200nm,so we call these groups chromophore.
      Some groups which are not have shared electron for
    example: amido-group ,—NH2,RHN—,R2N—(has a
    pair of shared electron), OH-(have two pairs of shared
    electron), as well as halog —F,—Cl,—Br,—I,and
    so on, they interact with the unfilled band of the
    auxochuome, cause the shift of the eternal electric, then
    lessen the activation energy made the maximal
    absorption of reagent to light "red shift“ (shift to length
    wave direction), made the color of the reagent deeper,
    these groups are called auxochuome.
    The choice of the color reaction condition
   1.The used dosage of the color-developing agent
   color is that let the measured compounds transformed
    into color compounds, indicate that:
           M +             R       =     MR
    (the measured compound) (color developing agent) (colored compounds)

      Reaction is reversible to a certain extent. For cutting
    reversibility of the reaction, according to common ion
    effect, it is necessary to adding excessive color-
    developing agents ,but it is also not excessive too
    much ,otherwise it will be cause by-reaction which is
    harmful to measuring.
                     2.The acidity of the solution

     The impact of solution acidity to color reaction is very big , this
    is because the solution acidity is having direct impact to the
    existing form of the metal ion and the color developing reagent,
    also the stability and the composition of the colored complex
    compound of the color complexes. Therefore, control proper
    acidity of solution is one of the important condition for ensuring
    the fine result of the spectrophotometry analysis.
   (1)The acidity have impact on the existing form of the measured
   (2)The impact of the acidity to the concentration and color of the
   (3)The impact of the acidity to the composition and the color of
    the complex
                         3.Time and temperature
     The 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, reagent's decompose or be volatized. The proper
    coloration time and 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 heat 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 the color depth, therefore the temperature of
    the standard sample and the reference sample should be the same one.
    4.Organic solvent and surface active agent
      The impact of solvent to color reaction mainly impressed by the
    following aspects:
   (1)The solvent impact the dissociation of the complex
   (2)Reasons of the solvent modify the color of the complex are the
    difference polarity and dielectric constant of each solvent
    molecular ,therefore impact the stability of the complex, changing
    the inside state of the complex molecular or form different lyonium
   3)The solvent impact the speed of the color reaction
      the addition of the surface active agent can improve the sensitivity
    of the color reaction and the stability of the color compound, the
    principle of the function are on the one hand is the increases
    dissolves of the mice, and on the other hand is can form the
    multivariate complex compound which contains the surface active
    5.The disturbance of the coexist ions and its
     When coexistent ion existence there has the following
    several kinds of influence to the luminosity determination:
    (1) produce colored complex compound with reagent.
    (2) disturbs ion itself have the color.
    (3)connect with regent to produce colorless complex
    compound that consume massive reagents cause the
    measured ion did not complex completely.
    (4)Be connected with the measured ions that can produce
    another compound whose dissociation is smaller than the
    measured ions.
     Methods to eliminate the disturbance

   Mainly has the following three kinds:
    (1) controls the acidity
    (2) joins the masking agent
    (3) uses the extract luminosity method
    (4) measures the absorbance of the two colored
    complexion under different wavelength, carries on
    determination at the same time .
   (5) seeks the new color reaction
   (6) separates the disturbance ions
   In addition, we can also through the choice of suitable
    survey condition to eliminate the interference of the
    disturbance ion.
      The measuring error and the choice of measuring
          condition of the absorption photometry

      The source of the luminosity analysis method has two aspects, one
    aspect is error that led into by various chemistry factor ,another aspect
    is that instrument accuracy is insufficient, measurement is inaccurate.
   (一) Instrument measure error
      Instrument measurement error is refer to the instability intensity of
    the light source, the nonlinearity of the photoelectric effect, the
    nonlinearity of the potentiometer, miscellaneous astigmatism effect ,the
    bad quantity of the optical filtering or mono-color (the spectrum is too
    wide ),colorimetric vessel transmitting rate disaccords, transmitting rate
    and the scale of the absorbance is not accurate. To the given
    photometer, the accurate degree of the transmitting rate or absorbance
    is one of the main indictor of the precise of the instrument, it also an
    important factor to measure the accurate degree of the result.
  The mainly instrument measured error of the

    The reading numerical error of the transmission. The scale of the
 transmission T is homogeneous on the photometer numerical
 reading scale ,so the numerical error ΔT(absolute error) has no
 concerned with T , it is a constant to a given instrument ,it is general
 between 0.002-0.01.
   A  0.434 ln T  bc                 c            ln T
           0.434 dT
  dc          
            b T
            dc dT (T ln T )' dT (1  ln T )
           ( )'            2
                                         2
                                            Let ln T  1  0
            c     (T ln T )     (T ln T )
           SoT  e 1  0.368,
  Namely when absorbance A=0.434, the relative error of the measured
concentration is the smallest.
(二)The choice of the measuring condition
1.The choice of the measuring wavelength
   Because of the select absorbance of colored matter
to light, in order to let measurement result have higher
accurate and sensitivity ,we must select the maximal
sorption wavelength as the insert light ,if there has
disturbance we can select the sensitivity lower but it
can avoid disturbance as insert light, then we can gain
satisfied measurement result.
2. Controlling the range of the absorbance
   When absorbance between 0.20 to 0.80,measuring
accurate degree is higher
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
■(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 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
    The application of the absorption photometry
  The absorption photometry not only widely used to determine
the micro ingredient, 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.
一.Quantitative analysis
(1).Single component determination
1. General method: A-c standard curve method
2. Differential 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=κb(cx-cs)=κbΔc cx=cs+Δc
(二).Measuring many kind of components 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.

 A1  Ax,1  Ay ,1   x,1 bcx   y ,1 bc y
 A2  Ax,2  Ay ,2   x,2 bcx   y ,2 bc y
  二.Determine the composition of the compound and acid-
                   base dissociation constant

(一)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 + nR=MRn
  Usually we fix the concentration of the metallic ion M , increase
 the concentration 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, measuring its luminosity, then draw the curve, we
 can obtain the complexing ratio from the curve.
        2.Equal mole continuously changes

     This method is maintains M+R is a constant in the
    solution, preparing a series of solution by continuously
    changes cR/cM . Separately surveys the absorbance A of
    the series solution ,mapping by A to M/ (M+M) ,the R/M
    value which was corresponding by the curve of the curve
    was equal to the complex rate n.
     The method of equal mole continuously changes can
    practical in the determination of the composition of the
    compound whose complex rate is low , the stability is high.
     It also can determine the stability constant of the
(二)Measuring the acid-base dissociation constant

   If there has monohydric weak acid, dissociation according to the
following equations:
             HB =H+ + B-

   Preparing three kinds of solution whose analytical concentration
c = [HB] + [B-] is equal, but its pH is different. pH of 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:

      A  AHB  AB   HB [ HB ]   B [ B ]                  

                      [ H  ]c           Kac
       A   HB         
                                 B
                    [H ]  K a        [H  ]  K a
  The second kind of solution is the acid solution whose pH
is above two units lower than its pKa , then the 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 length.

   AHB   HB [ HB ]   HB c
   HB  AHB / c

  AB          B [B  ]   B c
 B         AB  / c
Reorganize the overall equations:

        AHB  A    
 Ka             [H ]
        A  AB 
                       A  AB 
  pK a  pH  lg
                       AHB  A