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B.V.
Absorption photometry
B. V. a.1. Electromagnetic radiations
The most important types of electromagnetic radiation are illustrated in Figure 30. Radiant energy is characterized by its wavelength. For absorption
photometry the wavelength range between 200 nm and 2000 nm is especially
important. Radiation between 400 and 760 nm is visible to the human eye as
colored light. Adjacent to visible light at the long-wavelength end is the infrared. It is invisible, but its intensity can indeed be measured with the appropriate photocells. Of shorter wavelength than visible light are the ultraviolet
rays which are also invisible to the human eye but can be measured with
suitable photocells. The great majority of all materials have the ability to absorb selectively radiant energy of definite wavelengths with the concomitant
generation of heat. This observation is the basis for a large numberof analytical
methods. The human eye is relatively insensitive in distinguishing different
colors and intensities of light. Therefore, absorption is usually measured with
the aid of photocells. This field of analytical technique is known as absorption
photometry.
I1'""0
.<
10'
10'
1 km
10'
10'
1m
10'"
10-'
10....
1 mm
11, m
1P
10-'
1 nm
1 mp
1 pm
1/11'
10~'
10-"
I
I
I
Infra red
Ultraviolet
I
_
Visible light
"ot,,, roy,
~ro"
10-"
Figure 30. Electromagnetic spectrum.
107
Hertzian rays
•
Cosrriic rays
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B.V.a.
Physical principles
The term "colorimetry" should be avoided because the colorless ultraviolet
and infrared regions are just as useful in photometric procedures as visible
light.
B.V.a.2. Visible light
Electromagnetic oscillations with a wavelength between 400 and 760 nm are
visible to the human eye as light. White or "colorless" light is a mixture of
many different colored light rays, i. e. light rays with different wavelengths.
It is described as being polychromatic. If such white light is passed through a
prism or a diffraction grating lig ht rays of different wavelengths are refracted
or diffracted through different angles. Projection of the emitted rays on to a
screen produces the well-known spectrum with violet at the short wavelength
end and red at the long wavelength end. The wavelengths of the individual
color regions are given in Table 20.
When a solution appears colored to the human eye it is because the solution
absorbs all the incident light with the exception of the wavelengths perceived
by the eye. Thus, a blue solution appears blue because the long wavelength
red rays are absorbed. A red solution, on the contrary, absorbs the short wavelength blue light and transmits only the red light rays. This relationship is
schematically represented in Figure 31.
B.V. a. 3. The law of Bouguer and Lambert
It is easierto understand the important physical relationships of light when it is
considered not as waves but as rays. Let us provisionally assume that
the light be of a single wavelength only, i. e. monochromatic light.
I.R.l--
II
II
II
Visible
•
-
----1 U•V•
-
Characteristic
color
white
yellow
orange
red
purple
700
red
600
500
orange yellow green
green
400
nm
blue spectrum color
Figure 31. Characteristic colors and spectrum colors.
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blue
Table 20. Wavelength range of colors in visible light
Color
Wavelength range
Violet
Blue
Green
Yellow
Orange
Red
400-450 nm
450-500 nm
500-570 nm
570-590 nm
590-620 nm
620-760 nm
When monochromatic light, with a definite initial intensity (10) passes through
an optically homogeneous medium or layer, the intensity ofthe emergent light
(11) is less than that of the incident light (10): i. e. some of the light is absorbed.
The quotient 1,/1 0 is known as the transmittance, abbreviation T. If 10 is equal
to 100% the intensity of the emergent light may be measured as a percentage
%T = T x 100
In 1729 Bouguer investigated the relationship between a decrease of light
intensity and the thickness of the absorbing medium (d). He observed that
optically homogenous layers of equal thickness, lying one behind the other,
all absorb a constant proportion of the incident light. The principle of this law
of Bouguer and Lambert is shown schematically in Figure 32. Incident light
(10) traverses three identical layers of thickness 1. The first layer absorbs 25%
of 10 and therefore transmits 75%. Layer 2 again absorbs 25% of the incident
light, i. e. 25% of 75% and accordingly transmits 56.2%. Layer 3 absorbs 25%
of the remaining 56.2% and thus only 42.2% of the original light intensity
emerges from layer 3. The corresponding percentage transmissions are 75%
after layer 1,56.2% after layer 2, and 42.2% after layer 3.
In Figure 33a the relationship between the percentage transmission and the
number of optical layers is plotted on ordinary millirn9ter paper. Obviously
I
"-1,
I,
Light ray
2
I
Light intensity (T) 100%
3
..-I
I
I
..-1,
I,
I
75%
75%01
75%
75%01
56.2%
Transmission in %
100%
75%
56.2%
42.2%
log (%T)
2.0
1.875
1.750
1.625
I
0.125
Differences
2.0-log (%T)
i
~
E
0
Figure 32. Law of Bouguer and Lambert.
109
II
I
II
I
0.125
0.125
0.125
I
0.250
0.375
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2
Optical layers
there is not a linear relationship between layer thickness and transmission.
However, if the values are plotted on semi-logarithmic paper as in Figure 33b,
the observed points lie on a straight line. In other words, there is a linear
relationshi p between the layer thickness and the logarithm of the percentage
transmission, as shown in Figure 33c. Therefore, if we plotthe logarithm ofthe
percentage transmission against the layer thickness on ordinary millimeter
paper, a straight line results as is shown in Figure 33c.lf, instead ofthe number
of optical layers, the layer thickness b is employed, a direct proportionality
between layer thickness and the logarithm of the transmission is observed in
accordance with the Bouguer-Lambert law:
-log T
=
bxk
This may be expressed: The transmission of light decreases logarithmically
with linear increase in the layer thickness.
B.V. a. 4. Transmittance and absorbance
In photometric measurements we are not directly interested in how much light
is transmitted by a solution but in how much it absorbs. Obviously there must
be a relationship between transmittance and absorption. The measure of
absorption is the absorbance. abbreviated A (also called the optical density,
0.0., or extinction). This is equal tothe negative logarithm of the transmission:
A
= -
log T
=
-log I, as % of 10
100
and likewise
A
=
2-log %T.
If we recalculate the experimental data given in Figure 32 and in Figure 33
employing the absorbance A, a direct relationship between the number of
optical layers and the extinction can be demonstrated. The greater the layer
thickness (b) the higher is the extinction. Accordingly the Bouguer-Lambert
law can also be written as follows:
A = bk
The human eye finds it easier to read the equal subdivisions of a percentage transmittance scale and consequently the readings are somewhat more exact, especially
at higher absorbance values. It is sometimes advisable to read the results as percentage transmittance readings and to convert these mathematically to absorbance.
However, if the photometer has a fairly large scale, the increased work entailed by
this procedure is scarcely justified by the increase in exactness of the readings.
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It is important to emphasize that all of the curves given in Figure 33 describe
the same experiment. It is obviously clearer to express the result as absorbance than as transmittance. We recommend therefore that all results should
be read and calculated as absorbance. Transmittance readings can readily
be converted into absorbance and back again with a slide rule and the equation A = 2-log %T,or more simply by means of a table (Appendix IV, page 506).
% transmission (%T)
100
a
~
\
80
1
1.
'\.
.'"
"- '\l
'"
20
~
,.
2
3
4
5
6
'\.
\~
20
10
o
~.
40
l~
40
log (%T)
c
\.
2.0
"-
60
\
b
I\.
80
60
o
% transmission (%T)
100
)~
o
2
3
4
5
6
Absorbance (A) = 2-log (%T)
d
1.0
~\
1.8
0.8
"'~
".'\
1.6
1.4
0.6
I\.
7
0.4
\.
)'
,,~
1.2
"
o
0.2
o
1.0
2
3
4
5
6
/
tV
)~
/
o
tV
)r
2
3
4
5
6
Figure 33. Relationship between layer thickness or concentration of a solution (abscissa) and transmittance or absorbance of the solution (ordinate).
B.V. a. 5. Beer's law
If the number of dissolved molecules in the absorbance layer, i.e. the concentration (C) is substituted for the layer thickness, the equation becomes
=
Cxk.
In other words, absorbance is proportional to concentration. This physical
relation, usually known as the Beer-Lambert law, is the fundamental basis of
photometry with modern apparatus (b= constant).
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A
B.V. b.1. Absorbing molecules
Attempts to determine substances after a color-producing reaction are based
upon an old analytical principle which has many useful applications:
a) The substance to be measured may exhibit characteristic absorption in visible light
(hemoglobin, carotenes), in the ultraviolet (protein, nucleic acid) or in the infrared
(steroids).
b) The substanceto be measured may form a colored compound with an added reagent
(cholesterol, ammonia).
c) The substance investigated may release a colored compound from an added reagent
(chloride).
d) The substance may decolorise an added colored compound.
In any of these methods the following conditions must be fulfilled:
1. The concentration of the colored compound must be exclusively dependent on the
concentration of the measured substance (specificity).
2. The color must be stable.
3. Color development must be nearly independent of the environmental conditions
(temperature, light).
4. The colored compound must follow the Beer-Lambert law.
Before any steps can be taken to measure a colored compound the spectral
properties of the compound must be fully investigated.
B.V. b. 2. Absorption spectrophotometry
Instead of inspecting the entire spectrum by eye we may use an absorption
spectrophotometer which enables the entire wavelength range to be surveyed.
The absorbance is first measured with monochromatic light at a wavelength
of 400 nm, then with monochromatic light of wavelength of410 nm and so on
until the entire spectrum is measured. The concentration of the solution is
maintained constant throughout. Thus, a picture of the relative absorption of
the compound at different wavelengths is obtained. Examples of such absorption spectrums are given in Figure 45 (page 137) and Figure 70 (page 330).
B.V. b.3. The absorptivity
The absorption spectrum is a stable and reproduci ble property of a great many
compounds. It is useful not only for the identification of a given compound but
also enables its concentration to be determined by measuring the absorbance
with monochromatic light. The following are the conditions necessary for
using molar absorptivity in measuring the concentration:
1. Absolute calibrated pipets must be used for preparing the necessary dilutions.
2. A photometer, which gives absolute measurements and which is stabilized against
electrical transients in the power supply, must be used.
3. Monochromatic light is essential.
4. Cuvets with plane parallel sides, usually of 1 cm light path must be used.
5. Measurement must be made at constant temperature.
If even a single one of these conditions is not fulfilled, calculation of the concentration using the molar absorptivity is no longer reliable. It is clear that this
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B.V.b.
Optical properties
of molecules
principle applies not only for the measurement of components with characteristic colors, but also for the determination of all colored reaction products with
stable spectral properties.
As a rule readings are taken at the wavelength of the absorption maximum.
This is, however, not an unconditional requirement. It is more important that
the wavelength should always be exactly reproducible. The most exact results
are thus obtained with photometers which use isolated monochromatic lines
(e.g. the mercury lamp+a filter).
A value often employed is the percentage absorptivity, a, which is defined as
a
A
c% x
b
in which
A
c
b
is the measured absorbance,
is the concentration of the solution in g/100 ml, and
is the thickness of the absorbing layer (in most cuvets b = 1 cm).
After the percentage absorptivity has first been empirically determined concentrations can subsequently be calculated from single measurements at the
defined wavelength. Accordingly
A
ab
c%
If the molecular weight of a substance is known the percentage absorptivity
is replaced by the molar absorptivity. This is defined as
e
A
cM x b
where
A
cM
b
is the measured absorbance,
is the concentration in moles/1000 ml, and
is the thickness of the absorbing layer in cm.
Also in this case the molar concentration can easily be evaluated from the
known coefficients by measurement of the absorbance:
A
exb
Occasionally it will be more practical to use the millimolar or micromolar absorptivity instead of the molar coefficient. In German-speaking areas the
absorptivity is occasionally referred to the units of moles!ml (instead of moles!
liter). Care must be taken not to confuse these two values which differ by a
factor of 1000.
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cM
Unfortunately the scientific literature [1-5] employs the concept of the absorptivity in a surprising variety of ways. We have therefore collected the most important definitions and abbreviations in the following table:
Definitions and abbreviations of photometric concepts
Definition
Nomenclature
Abbreviation
-log T
Absorbance, extinction [2,3,4,5], optical density
A,OD
Alb
Absorptivity [2], absorbance index [2],
extinction coefficient
K
A
c% x b
Absorptivity [3,4,5], absorbancy index, specific
extinction coefficient
%
)
a ( A 11c~xA
cM x b
Molar absorptivity [3, 4, 5], molar absorbancy
index [5], molar extinction coefficient
The absorptivity is therefore of special value when the standard is difficult to
obtain orto keep, e.g. in the estimation of protein with the biuret reagent, or
in the determination of bilirubin as azobilirubin. Butthe absorptivity is used in
many other determinations. Occasionally it is necessary to calcUlate the percentage absorptivity from the molar absorptivity or vice versa:
e =
a x MW
10
or a =
eX 10
MW
Literature
1. Lothian, G. F.: Analyst 88: 678, 1963. - 2. Cooper, B.S., A. E.Gillam, G. F. Lothian and R.A. Morton:
Analyst 67: 164, 1942. - 3. Brode, W. R.: J.opt.Soc. Amer. 39: 1022, 1949. - 4. Hughes, H. K.: Anal.
Chem. 24: 1349, 1952. - 5. U.S. National Bureau of Standards, Circular No.484, Washington D.C.
1949.
B.V.c.
Apparatus
for photometry
B.V. c.1. Principle of the photometer
The photometer is used to measure the absorbance of unknown solutions at
a predetermined wavelength in absolute and reproducible terms [1]. A diagram of such an apparatus is shown in Figure 34. Every instrument employs
a light source. If an incandescent lamp, which produces white light, is used
then potentially every wavelength in the visible region can be isolated. When
gas discharge tubes are used as the light source, then monochromatic light
of only the specific wavelengths generated by the lamp can be obtained. The
lines generated by a mercury lamp are tabulated in Table 21. The optics of the
instrument serve to concentrate the light rays. For the resolution of the light
into the desired wavelengths several different systems can be employed. First
the wavelength range of white light can be narrowed by the use of filters. The
band-width of a simple filter photometer is, however, too wide to be spoken
of as monochromatic. The ideal small band-width of 1 nm can be obtained,
however, by combination of a special filter with a mercury lamp as light source.
This type of apparatus is known as a spectral line photometer. There remains
the possibility of splitting the incident light beam into approximately mono114
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where MW is the molecular weight of the compound sought.
I
Light
source
III
Optical
system
II
Diaphragm
IV
Light
decomposition
V
VI
Analytical Photocell
sample
VII
Measuring
apparatus
(galvanometer)
I I I
II'~~~I
Figure 34. Schematic diagram of a photometer.
chromatic rays with a prism or diffraction grating and then to center the isolated ray by the appropriate optics. This apparatus is called the spectrophotometer.
When the light ray is rendered as monochromatic as possible it is passed
through the solution to be analyzed. Plane parallel cuvets of 1 cm optical thickness are much to be preferred to round test tubes. The intensity of light passing through the cuvet is then measured by the photocell which produces a
signal capable of being read on a galvanometer scale.
1. Kortum, G.: Kolorimetrie, Photometrie und Spektrometrie. Springer, Berlin-Giittingen-Heidelberg. 4. Auf!. 1962.
Table21. The lines of mercury and cadmium lamps (nm) which can be isolated
by a filter.
Hg
Cd
313
326
334
366
404/407
435/436
468
480
492
509
546
577/579
623
644
691
772
1014
B.V. c. 2. Types of photometer
At the present time some 50 different types of photometer are commercially
available. The prices of these instruments range from 70 to 11,000 U.S. dollars.
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Literature
The quality ofthe apparatus is largely proportional tothe price. The photometer
is today the central and most important piece of equipment in the clinical
chemistry laboratory, and consequently only highly stable apparatus should
be purchased. It should provide approximately monochromatic light, i.e. a
band-width of less than 5 nm. Only two basic types of apparatus have practical
significance:
1. Spectral-line photometers. As light source these use a mercury or cadmium lamp
which emit a discontinuous spectrum. The wavelengths of the lines obtainable are
listed in Table 21. With the aid of appropriate filters the Ii nes can be isolated to produce
predominantly monochromatic light having a bandwidth of about 1 nm. These instruments are especially useful for routine work as well as forthe measurement of absolute
absorbances.
2. Spectrophotometers. In this equipment any wavelength is selected and isolated
with a prism or diffraction grating. The band-width differs from one apparatus to another but in the highest quality instruments it reaches approximately the band-width
of the spectral-line photometer. Commercially available instruments can be roughly
divided into two groups: (a) with outstanding performance and a price above 2,500
U.S. dollars and (b) with fairly good performance and price approximately 1,000 U.S.
dollars. There is still another group of less expensive instruments whose performance,
however, does not reach sufficiently high standards.
The following points are of particular importance in choosing a photometer:
1. Its wavelength range should extend down to 340 nm for enzyme determinations.
2. The apparatus should employ cuvets and if possible it should also be provided with
semi-micro cuvets with a light path of 1.0 cm and volume of 0.5 ml. If possible flow
cuvets should also be available.
3. The scale of the photometer must be adequate for a wide range of absorbance.
4. Provision of an automatic recording attachment offers great advantage.
The different types of cuvet are compared in Table 22. It is advisable to work
only with plane parallel cuvets. If round measuring test tubes must be used
these must first be calibrated because the optical properties vary according
to the position of rotation of the tube. Valuable comparative investigations of
the quality of commonly available photometers have been carried out by Pohl
[1-5].
1. Pohl, H.: Chemiker ltg. 79: 401, 471, 551, 629, 1955. - 2. Pohl, H.: Chemiker ltg. 80: 819, 855, 1956. 3. Pohl, H.: Chemiker ltg. 81: 785, 1957. - 4. Pohl, H.: Chemiker ltg. 83: 513, 1959. - 5. Pohl, H.:
Chemiker ltg. 85: 12, 1961.
Table 22. Usable types of cuvet (light path d = 1 cm)
Type
Normal
Material
Plateglass Quartz
Optical glass
(OS)
Transmission limit
Width of light beam
Fluid volume required for
1 cm sample path length
Filling height
350 nm
10 mm
200 nm
10 mm
300 nm
10 mm
4mm
2.5 ml
25 mm
2.5 ml
25 mm
2.5 ml
25 mm
0.5 ml
0.1 ml
12.5 mm 5mm
116
Normal
Normal
Semi micro Micro
2mm
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Literature
B.V. c.3. The significance of monochromatic light
The great significance of monochromatic light in making exact measurements
is apparent from consideration of Figures 35a and band 36. When a solution
with the absorption spectrum indicated is measured monochromatically at
492 nm, monochromatically at 546 nm and with a polychromatic light of half
band width 20 nm and a maximum of 520 nm, the resulting standard curves
are given in Figure 35 b. When monochromatic light is used, a straight line is
obtained which is independent of whether the maximal absorbance of the
solution (J. max.) is used or whether a wavelength on either shoulder is used.
There is no absolute requirement to use the absorption maximum when making exact measurements. On the other hand, measuring with polychromatic
light produces a standard curve which deviates from a straight line at increasing concentrations and therefore deviates from Beer's law. Measurement is
therefore made more difficult since many standards must be determined and
hence absolute measurements are impossible.
a
E
b
E
3.0
2.0
1.0
400
500
600 nm
c
B.V.d.
Evaluation
of the results
B.V. d.1. The standard curve
For every new method the validity of Beer's law must be thoroughly tested.
This is most quickly done by the preparation of a standard or calibration curve.
A series of dilutions of the analytical solution in arithmetic (i.e. not in a geometric) progression is prepared and the absorbance is measured using the
reagent blank. The results are plotted on millimeter paper and a straight line
is drawn through the points. The equation of the curve is deduced from the
regression equation and the standard deviation and the correlation coefficient
are calculated. From the equation a straight line is calculated and constructed.
The graphical and the mathematical results are compared.
The first question to be decided is whether the calibration line passes through
the point of origin. If this is not the case it is advisable to check the blank value.
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Figure 35. Calibration curves for the use of measurement wavelengths of different
band widths: 1. Monochromatic light at 492 nm; 2. Polychromatic light from a filter
with a maximum at 520 nm and a half band width of 20 nm; 3. Monochromatic light
of 546 nm.
It may perhaps be possible to make the calibration curve pass through the point
of origin by alteration of the order of addition of the reagents. Those methods
in which it is not possible to obtain a coincidence of the absorbance zero with
the concentration zero are difficult to employ for routine analysis as they introduce unavoidable complications in the method of calculation.
Secondly, the question ofthe validity of the laws of Bouguer-Lambert and Beer
must be considered, i.e. is there a direct proportionality between the absorbance and the concentration? If this is in fact the case and if the correlation
coefficient exceeds 0.98, the method is suitable for clinical chemistry.
Ifthe cali bration curve does not show a linear relationship between absorbance
and concentration, one speaks of a deviation from Beer's law. Such deviations
can be grouped in one or the other of the following categories:
1. Chemical basis:
The so-called true deviations are due to the effects of the solvent medium, e.g. the salt
and protein errors of indicators. More frequently, however, they may be caused by the
fact thatthe chromogenic color reaction is not strictly stoichiometric. Examples of this
type are ammonia determination with Nessler's reagent and the determination of
protein by the Folin reaction (page 243).
2. Physical basis:
False deviations from Beer's law are almost always due to the fact that the light used
for the measurement was not monochromatic.
A deviation from Beer's law does not by any means indicate that the method
cannot be used. Its use, however, is subject to one of the following limitations:
1. Frequently the relationship is linear up to a certain concentration and deviates only
at higher concentrations. Accordingly, the analytical sample should be diluted so that
the absorbance falls in the linear range.
0.6
Eppendorf
b = j em
A. = 405 nm
0.5
0.4
0.3
0.2
(Coleman(
b = 19 mm 0
A. = 405 nm
0.1
~----r----r----T---~----40
80
120
160 I.U.
Figure 36. Calibration curve for the estimation of alkaline phosphatase. Measurement
with monochromatic light (Eppendorf photometer) and with polychromatic light
(Coleman photometer).
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o
2. In many cases it is possible to adjust reagent concentrations so as to avoid deviation
from a straight line at higher concentrations.
3. Reactions which do not follow Beer's law never give absolute measurements. If the
two possibilities previously mentioned do not improve the procedure the sole remain·
ing possibility is to include at least five standards in every series of determinations
and to prepare a standard curve each time. The results are more reliable the closer the
concentration of the standard is to the concentration of the unknown.
B. V. d. 2. Calculation of results using standards
The result may be calculated either from the reading for a closely related
standard or directly from the extinction coefficient. The following illustrates
calculation with a standard.
The method is based on four determinations for each analysis:
T
TB
RB
S
the test analysis containing all reagents and the analytical sample,
the analysis blank containing only the analytical sample and serving as correc·
tion for intrinsic optical absorption,
the reagent blank containing the reagents without the analytical sample and
giving the correction for the intrinsic absorption of the reagents,
the standard containing the standard solution and all reagents.
In some methods one or more of these controls may be superfluous. For exam·
pie, in the determination of hemoglobin the analytical blank is not needed. All
readings should be taken with water in the reference cuvet. The RB value provides a control of the quality of the reagents. The TB value is important for
measuring the serum blank.
From the following readings:
A(To)
A(TBo)
A(RBo)
A(So)
the observed
the observed
the observed
the observed
absorbance of the test mixture,
absorbance of the test blank,
absorbance of the reagent blank,
absorbance of the standard solution,
the corrected absorbance ofthe standard and ofthe test sample are calculated:
A(Tc)
A(Sc)
= A(To)-A(TBo)-A(RBo),
=
A(So)-A(RBo).
The values are next corrected forthe difference of the volumes of T and S from
the nominal volumes of the method. If T and S are pipeUed with the same pipet
and the same volume no correction is necessary. If different pipets are employed the pure and volume·corrected value is calculated as follows:
A(T) = A(Tc)
x
A(S) = A(Sc)
x desired volume
desired volume
pipet volume
pipet volume
c(T) =
119
A(T)
A(S)
x
c(S)
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By this procedure the absorbance of the standard and the test sample are
corrected for the blank values and to the standard volume. The basic formula
for all further calculations, derived from the Beer-Lambert law, is as follows:
in which
c(A)
c(S)
is the concentration of the analytical sample, and
is the concentration of the standard.
A collection of photometric formulas is given on the front cover of this book.
B.V. d. 3. Calculation ofthe results using the molar absorptivity
When the following conditions apply the use of a standard is unnecessary:
1. The substance to be determined is itself colored or the colored product is formed in
a reproducible stoichiometric relationship to the substance to be determined.
2. Beer's law is valid over the whole range of concentrations.
3. Pipets with absolute calibrations are used.
4. Plane parallel cuvets with known light-paths are used.
In such a case it is sufficient to determine the analytical value only. When the
percentage absorptivity is known the following formula holds:
c(T)
=
A(T ) (dilution factor)
ax b
The result is given in g/100 ml.
In a similar manner the molar absorptivity can be employed:
c(T) = A(T ) (dilution factor)
eX b
From this equation the result is obtained in moles/liter.
120
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Université René Descartes Paris 5
193.51.85.197 - 10/28/2017 2:06:28 PM
In our laboratory we use molar absorptivities as much as possible. Nevertheless, we carry a standard through the analytical procedure which, however,
serves only as a primary control. The calculation is made from the molar absorptivity.
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