Optical Absorption of Hemoglobin

by Scott Prahl, Oregon Medical Laser Center

A model of hemoglobin at low resolution. A model of heme. Equivalents One of the most confusing things about looking at hemoglobin (Hb) spectra is that the values are typically tabulated in equivalents. The term equivalent is here used to indicate the amount of hemoglobin which contains 1 gm atom of Fe and combines with 1 gm molecule of O2 or CO. One equivalent of hemoglobin is assumed to be 64,500/4 or 16,125 gm. A concentration of 10-6 equivalent is 16.125mg of hemoglobin per cc. Thus there are four times as many equivalents as there are hemoglobin molecules. Despite the fact that others use equivalents, I will present the oxy and deoxy-hemoglobin spectra in terms of molar extinction coefficient. To convert from the molar extinction coefficient e to absorbance A, multiply by the molar concentration and the pathlength. For example, if x is the number of grams per liter and a 1 cm cuvette is being used, then the absorbance is given by
A = (e) [(1/cm)/(moles/liter)] (x) [g/liter] (1) [cm] --------------------------------------------------64,500 [g/mole]

using 64,500 as the gram molecular weight of hemoglobin. Binding changes the spectrum If the hemoglobin molecule is bound to oxygen then one has oxy-hemoglobin or Hb02. If the hemoglobin molecule is bound to carbon monoxide then one has carboxy-hemoglobin or HbCO. If the hemoglobin molecule is bound to nothing then one has deoxy-hemoglobin or Hb. If the hemoglobin molecule has broken down then one has met-hemoglobin. These all have different spectra. Spectra

A best estimate of the spectrum of Hb and HbO2 from a variety of sources by Scott Prahl. A tabulation of the data is available.

Comparison of Moaveni's data (points) with my compiled values (curve).

Comparison of Takatani's data (points) with my compiled values (curve).

Blood Hemoglobin has a normal concentration of 150g/liter of blood permits whole blood to carry 65 times more oxygen than does plasma at a PO2 of 100 mmHG. Hematocrit determines the fraction of the blood that is red blood cells. The red blood cells are primarily composed of hemoglobin (95% of the dry mass). When arterial blood is 90% saturated, some of the hemoglobin molecules have four oxygens bound, some have three, and a few have tow or one. The statistical average of all oxygen bound to hemoglobin molecules relative to the total amount that can be bound is its oxygen staturation. One gram of O2 of functional hemoglobin combines with 1.34ml O2, the O2 capacity of normal blood is (150g Hb/liter)(1.34ml O2 g Hb) = 200ml O2/liter. But I just want a typical spectrum for blood in units I can understand... Quit whining. Assume 150 g Hb/liter. Then to convert the molar extinction coefficient e to an absorption coefficient, multiply by the molar concentration and 2.303, a(lambda) = (2.303) e(lambda) (150 g/liter)/(64,500 g Hb/mole) = 0.0054e(lambda) where a is in (cm-1) and e(lambda) is the molar extinction coefficient for the wavelength of interest. SAP 15 Dec 1999
THE ABSORPTION SPECTRA OF HEMOGLOBIN AND ITS DERIVATIVES IN THE VISIBLE AND NEAR INFRA-RED REGIONS
BY B. L. HORECKER (From the Division of Industrial Hygiene, National Institute of Health, Bethesda, Maryland)
(Received for publication, December 3, 1942)

Since the pioneering investigations of Vierordt and of Hiifner and his group, numerous reports on the absorption spectra of hemoglobin and its derivatives have appeared in the literature. A detailed summary of these reports is to be found in Heilmeyers monograph (1). For the most part these investigations have been confined to the visible portion of the spectrum, although in a few cases the observations were extended into the ultraviolet,. The near infra-red, however, has been singularly neglected, despite the fact that as early as 1914 Hartridge and Hill (2) published some qualitative results indicating the presence of an interesting oxyhemoglobin band in this region. In this laboratory, it became necessary to develop a rapid.and accurate spectroscopic method for the estimation of carbon monoxide in blood which could be adapted to a simple portable instrument. The visible portion of the spectrum proved unsuitable for this purpose, since the character of the oxyhemoglobin and carbonylhemoglobin bands in this region require the use of narrower spectral regions than can conveniently be isolated in such an instrument. The infra-red spectra were investigated in the hope that more suitable absorption bands might be found. The spectrophotometric data for the various hemoglobin derivatives which may be found in the literature are characterized by discrepancies with respect to the absolute values of the absorption coefficients, depending

upon the hemoglobin preparations examined, the analytical methods used, and the dispersing power of the spectrophotometers employed by the various investigators. Since the precision of any spectrophotometric method of analysis depends upon the accuracy with which the corresponding absorption coefficients are known, a redetermination of these coefficients was undertaken. The objectives of this investigation were to determine as accurately as possible the absorption coefficients of various hemoglobin derivatives in whole hemolyzed human blood and to compare these with constants obtained with pure hemoglobin, in order that the contribution of hemoglobin to the absorption of whole blood might be evaluated. A similar study was made of the spectra of these substances in the near infra173

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174 hBSORYTION SPECTRA OF HEMOGLOBIN red region from 7000 to 10,000 8. The new absorption bands found in this region promise to be extremely useful for analytical work. According to the Lambert-Beer Law, the specific absorption coefficients of an absorbing material may be defined by the equation,
Log,, : = cucl where 10 = intensit,y of incident light I= (1 transmitted light I = length of light path through solution c = conceutration of absorbing material LY = specific absorption coefficient

This relationship is valid only when monochromatic light is used and provided that the material investigated contains no absorbing impurities. With instruments available at the present time, sufficiently narrow wavelength intervals can be isolated to permit the determination of accurate absorption coefficients. Much greater difficulty has been encountered in preparing pure hemoglobin and in establishing adequate criteria for its purity. The earlier workers (3-7) employed crystalline hemoglobin prepared by alcohol precipitation or other means, and estimated the concentration from the dry weight of the crystals. It is now generally recognized, however, that crystallinity in the case of proteins is no assurance of homogeneity (8); other criteria for purity must be applied. With the development of precise gasometric methods by Van Slyke and his coworkers, it became possible to determine accurately the concentration of hemoglobin in solution and in whole blood. Using this method of analysis, Newcomer (9) and, more recently, Kennedy (10) determined the absorption of oxyhemoglobin and carbonylhemoglobin in hemolysed human and dog blood. In each case, however, only the concentration of active hemoglobin (HbOz) was measured; the contribution of other blood components, including other hemoglobin derivatives, to the total light absorption was not evaluated. The same is true, to a lesser extent, of the work of Drabkin and Austin (II), although these workers demonstrated that absorption constants obtained from washed, hemolyzed erythrocytes are highly reproducible. The effective slit width obtained with the instruments used by Kennedy and by Drabkin and Austin was about 30 b. in the green and 50 8. in the yellow. Measurable differences between the absorption curves of these authors and the present writer may be attributable t, the fact that in the present paper narrower slits are used, 7 to 12.5 A., in the visible spectrum. The narrower slit yields the more precisely defined absorption constants. The first important measurements in the infra-red were made by Merkelbath (12) in 1935. He found oxyhemo lobin to have a broad absorption band with a maximum at about 9100 d ., while carbonylhemoglobin had
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B. L. HORECKER 175

practically no absorption in the infra-red. A small portion of the infrared spect>rum has also been described by Sidwell, Munch, Barron, and Hogness (13). Although they report a band for reduced hemoglobin at 7550 A., their observations did not extend beyond 7700 8.; thus they failed to observe the oxyhemoglobin band. Carbonylhemoglobin was not examined.
EXPERIMENTA4L

Preparation of Purified Hemoglobin-Iurified hemoglobin was prepared from calf blood by the method of Altschul, Sidwell, and Hogness (14), involving treatment with aluminum hydroxide gel. This method was selected because hemoglobin solutions so prepared showed, at low O2 tensions, a higher affinity for oxygen than did any other preparations, including hemoglobin crystallized by the method of Heidelberger. The percentage saturation of hemoglobin with oxygen at low 02 tension was used by these investigators as a criterion of purity, in accordance with their finding that impurities lowered the percentage saturation. From 500 cc. of whole calf blood about 300 cc. of a clear red solution are obtained, having about one-half the hemoglobin content of the original blood. Determination of Purity-The concentration of active hemoglobin was determined on 2.0 cc. samples by the carbon monoxide capacity method of Van Slyke and Hiller (15). Some preparations were also analyzed for methemoglobin by determination of the carbon monoxide capacity after reduction with sodium hydrosulfite. Since the values for total hemoglobin obtained in this way always agreed closely with values calculated from the dry weight of the preparations, t.his determination was found to be unnecessary. Dry weight determinations were made by evaporating aliquot, s of the solution to constant weight at 100-105. From the dry weight the total hemoglobin concentration was calculated, with 66,800 as the molecular weight,. This value, calculated by Svedberg and Fahraeus (16) from sedimentation measurements, agrees well with 67,000 calculated by Adair (17) from osmotic pressure data and 66,000 calculated by Morrison and Hisey (18) from the iron content and gas capacity. The following is a t,ypical analysis of a hemoglobin solution purified by the above method. The assumption is made that hemoglobin has four iron-containing groups and that the equivalent weight is one-fourth the molecular weight.
equivalents HbOz Dry weight, = 83.9 mg. per cc. = 5.02 X 10-S cc. equivalents Hb02 concentration from CO capacity = 4.99 X low6 --____
CC.

Total Hb concentration from CO capacity = 5.03 X 10-O equivalents cc.

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176 ABSORPTION SPECTRA OF HEMOGLOBIN The sample thus contains only 0.6 per cent of methemoglobin and no other impurities. The term equivalent is here used to indicate the amount of hemoglobin which contains 1 gm. atom of Fe and combines with 1 gm. molecule of 02 or CO. 1 equivalent of hemoglobin is assumed to be 66,800/4, or 16,700 gm. A concentration of 1 X 10e6 equivalent is 16.7 mg. of hemoglobin per cc. Stability of Pure Hemoglobin Preparations-The hemoglobin solutions obtained could be stored in the refrigerator for over a month with no apparent decrease in their carbon monoxide-combining power. The absorption spectrum in the visible region of the spectrum also remained unchanged. After a week or two, however, the preparations began to show evidence of an absorption band at 8200 8. which was absent in the fresh preparations, and which increased in intensity with the age of the preparation. The position of this band would indicate that the preparations were becoming contaminated with methemoglobin, despite the fact that the Van Slyke analysis showed no decrease in the active hemoglobin content. In practice, preparations were discarded at the first appearance of this band. Preparation of Solutions-The absorption spectra of oxyhemoglobin (Hb0.J and carbonylhemoglobin (HbCO) were determined with pure calf hemoglobin. For the visible spectrum the stock solution was diluted 1: 50. For the infra-red spectrum the stock solution was diluted 1: 2. All measurements were made in a cell of length 0.500 cm. The dilutions were

made in borate buffer of pH 9.2 to a final buffer concentration of 0.1 M. Carbonylhemoglobin was prepared by equilibrating the diluted solutions in a rotating tonometer through which pure carbon monoxide was passed for 20 to 30 minutes. The absorption cell was then filled with carbon monoxide gas and the solutions transferred directly from the tonometer to the cell without exposure to air. From hemolyzed human blood, solutions were prepared for the determination of the spectra of reduced hemoglobin (Hb), alkaline and acid methemoglobin (MHb), and metcyanhemoglobin (MHbCN), as well as HbOs and HbCO. The hemoglobin concentration was determined on the whole unhemolyzed blood samples by the O2 capacity method of Sendroy ((15) p. 338). For the determination of the infra-red spectra of HbOa, HbCO, and Hb, the blood was diluted 1:5 with saponin and borate buffer, pH 9.2, to a final saponin concentration of 0.3 per cent and a final buffer concentration of 0.02 M. A portion of this solution was saturated with CO as described above. A second portion was washed with pure Na in a rotating tonometer until the violet color of reduced hemoglobin was produced. The solution
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B. L. HORECKER 177 FIG. 1. Absorption spectra of HbOs and HbCO in the visible region. Curves I

and II represent absorption constants obtained from pure calf hemoglobin for HbOn and HbCO, respectively. 0 and o represent constants for HbOt and HbCO, respectively in hemolyzed human blood. FIG. 2. Absorption spectra of HbO, and HbCO in the infra-red region. Curves I and Ia represent HbOs in hemolyzed human blood and pure calf hemoglobin, respectively. Curves II and IIa represent HbCO in hemolyzed human blood and pure calf hemoglobin, respectively.

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178 ABSORPTION SPEOTRA OF HE~MOGLOBIN

FIG. 3.

Absorption spectra of Hb, MHb, and MHbCN in hemolyzed human blood in the visible region. Curve I represents MHbCN; Curve II, MHb at pH 9.18 to 9.20; Curve III, MHb at pH 6.29 to 6.51; Curve IV, Hb. WAVLEC NGTxH [i] FIG. 4. Absorption spectra of Hb, MHb, and MHbCN in hemolyzed human blood in the infrared region. Curve I represents Hb; Curve II, MHb at pH 8.88 to 8.92; Curve III, MHb at pH 6.30 to 6.72; Curve IV, MHbCN; Curve V, HbOz.
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179 was then transferred directly from the tonometer into an absorption cell filled with pure Nz and containing a trace of dry NazSz04 to insure complete reduction, For the visible spectra the blood diluted 1:5 was further diluted with 24 volumes of 0.01 M borate buffer to a final dilution of 1: 125. From this solution, HbCO and Hb were prepared as above. For the preparation of acid and alkaline methemoglobin, blood was hemolyzed with saponin and treated with a 6-fold excess of K,Fe(CN),. The mixture was then buffered with either phosphate or borate buffer and diluted with water to 5 times the volume of the original blood sample. The saponin concentration was then 0.3 per cent and the buffer concentration was 0.05 M. For the visible spectra 1 cc. of each of these solutions was further diluted to 25 cc. with 0.02 M buffer. The pH of each solution was measured with the glass electrode and is indicated in Table I. From the borate-buffered solutions MHbCN was prepared by the addition of a small amount of solid KCN. Visible and Infra-Red Absorption Xpectra-The absorption measurements were made on an automatic recording spectrophotometer constructed in this laboratory by F. S. Brackett and J. B. H. Kuper. This instrument is essentially similar in construction to the one descriied by them in 1940 (19), but extending into the infra-red to 10,000 A. The effective slit widths at various wave-lengths are as follows:
B. L. HORIKRER
Wave-length, d.. Slit width, b., ,~;~~+!&~+ l+i:

The values of incident and transmitted light intensity were read from curves obtained on photographic paper. The concentration of hemoglobin was determined as described above and the values of the specific absorption coefficients calculated from the relation

log10 + a=cl

c is given in equivalents per cc. and 1 in cm. The units of CY are then sq. cm. per equivalent. The absorption spectra are plotted in Figs. 1 to 4. In each instance, the constants are the average values obtained from at least two hemoglobin preparations or blood samples. In Table I are summarized the values of the absorption constants at the maxima and minima, showing the spread of the determinations.
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TABLE I

Absorption Constants for Hemoglobin Derivatives

Pure calf hemoglobin HbOz Hemolyzed human blood
I II I II , J I L I 6 I 1 I L

Substance
HbCO HbO, HbCO Hb MHbCN MHb MHb

-No. of mmples

3 3 4 3 4 4 4 4 2 3 3 3 3*

2t
3$ 3#

A. i
5100 5400 I 5600 5765 6800 i 9200 5000 5375 1 5550 5680 9200 i 5100 5400 I 5600 5765 7000 i 9200 5000 5375

I 5550 5680 9200 (4800 1 5550 7310 7600 I 8400 9000 5040 \ 5400 8000 5100 5400 5600 5770 6850 7200 8175 9400 )4700 5000 6000 6200 7000 9800

Specific absorption constants, (Ix 1o-a(S~)

Average
5.19 15.0 8.88 15.9 0.064 0.274 5.50 15.0 11.9 15.0 0.004 5.07 15.0 8.87 16.0 0.093 0.296 5.44 14.8 11.7 14.8 0.010 3.50 13.6 0.310 0.395 0.179 0.198 7.03 11.0 0.033 7.18 9.68 7.68 8.51 0.297 0.336 0.525 0.259 7.72 9.47 3.01 3.68 0.151 0.794
.-

High

5.28 15.1 9.06 16.0 0.069 0.279 5.54 15.2 12.0 15.2 0.005 5.12 15.2 9.03 16.3 0.097 0.299 5.34 15.0 11.9 15.1 0.011 3.52 13.8 0.336 0.417 0.190 0.206 7.11 11.4 0.036 7.32 9.78 7.76 8.61 0.306 0.341 0.526 0.263 7.93 9.65 3.09 3.75 0.158 0.804

.1

1 1 I I
1 I 1

1 1 I 3 LOW

5.11 14.8 8.76 L5.8 0.061 0.268 5.46 14.9 11.8 14.9 0.004 5.00 14.8 8.72 15.8 0.089 0.292

5.56 14.6 L1.5 14.6 0.010 3.49 13.5 0.302 0.376 0.169 0.190 6.95 10.8 0.039 7.06 9.50 7.56 8.37 0.288 0.331 0.524 0.255 7.45 9.23 2.88 3.58 0.139 0.788 180

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B. L. HORECKER

TABLE I-Concluded

181
* pH = 9.18,9.20, 9.12. t pH = 8.88, 8.92. $ pH = 6.29, 6.32, 6.51. 8 pH = 6.72, 6.30, 6.44. DISCUSSION

It is shown in Fig. 1 that there is no perceptible difference in the visible region between the absorption spectra obtained from hemolyzed human blood and pure calf hemoglobin. It may therefore be concluded that other blood constituents make a negligible contribution to the light absorption of hemolyzed blood in this region. The results also bear out the previous findings of Drabkin and Austin (11) and others with regard to the spectroscopic identity of the hemoglobins of various mammalian species. In the infra-red region, where the absorption of hemoglobin is much less intense, whole blood absorbs appreciably more than does pure hemoglobin (see Fig. 2). This difference may be attributed to the absorption of light by other blood constituents and to scattering of light by suspended material such as lipids and cell fragments. At the high dilutions used for the visible measurements the contribution of these materials is negligible. It is noteworthy that no special precautions were taken in the collection of the blood samples. The individuals were not required to fast before the venipunctures were made, the only limitation being that no samples were taken for several hours after lunch. The broad absorption band of oxyhemoglobin in the infra-red, together with the almost complete lack of absorption by carbonylhemoglobin, makes this region ideally suited for the determination of the CO content of blood. In any spectral interval beyond about 7500 8., the presence of HbCO will produce a marked decrease in the total absorption. The total hemoglobin concentration may be determined, independent of the presence or absence of CO, in the neighborhood of the isobestic point at 4965 A. The concentration of HbOz may be calculated from the infra-red absorption and the concentration of CO computed by subtracting HbOz from total hemoglobin. In order to eliminate the effect of reduced hemoglobin, which will usually be present in the samples diluted 1: 5 if blood is collected by venipuncture, the spectral region in the infra-red may be so selected that the oxyhemoglobin and reduced hemoglobin have the same absorption. An examination

of Fig. 4 will show that the isobestic point for these substances lies at about 8000 A. The two measurements described above are sufficient if only two hemoglobin derivatives, HbOz and HbCO, are present. For this purpose Hb and HbOz may be considered identical, since they will absorb alike in the infra-red and Hb will be converted to HbOz at the high dilutions required
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182 ABSORPTION SPECTRA OF HYMOGLOBIN for the determination at 4965 A. If, however, a third component, such as MHb, is present, a third measurement is necessary for the evaluation of the HbCO, HbOz, and MHb concentrations. The required data may be obtained from the absorption at a third spectral interval, such as that around 6400 A., or by repeating the absorption measurement at 8600 A. after the sample is saturated with CO. In this laboratory we have developed a portable photoelectric instrument for the determination of carbon monoxide in blood which is based on the principles outlined above. With this device, the carbon monoxide content of human blood can be rapidly and conveniently determined with an error of less than 1 per cent HbCO. The details of construction and operation of this instrument will be described in a subsequent publication.
SUMMARY

The visible and infra-red absorption spectra of oxyhemoglobin, carbonylhemoglobin, reduced hemoglobin, methemoglobin, and metcyanhemoglobin have been determined. Several new bands in the infra-red are described. The absorption spectra of oxyhemoglobin and carbonylhemoglobin in hemolyzed human blood are identical with those obtained from pure calf hemoglobin in the visible region of the spectrum. In the infra-red the whole hemolyzed blood has a somewhat higher absorption. A method of evaluating the absolute purity of purified hemoglobin preparations is described. A simple spectrophotometric method for determining the CO and methemoglobin contents of blood is indicated. The author is indebted to Dr. F. S. Brackett for his constant interest and valuable suggestions, to Mr. T. W. Allen for technical assistance in the spectrophotometric measurements, to Mr. E. R. Mitchell for the Van Slyke determinations on human blood, and to his associates in the Division of Industrial Hygiene who were kind enough to furnish the blood samples.
BIBLIOGRAPHY

1. Heilmeyer, L., Medizinische Spektrophotometrie, Jena (1933). 2. Hartridge, H., and Hill, A. V., J. Physiol., 48, p. li (1914). 3. Butterfield, E. E., 2. physiol. Chem., 62,173 (1909). 4. Hari, P., Biochem. Z., 82,229 (1917). 5. Charnass, D., in Abderhalden, E., Handbuch der biologischen Arbeitsmethoden, Berlin and Vienna, Abt. IV, Teil4,1109 (1926). 6. Haurowitz, F., 2. physiol. Chem., 136, 147 (1924). 7. Welker, W. H., and Williamson, C. S., J. Biol. Chem., 41,75 (1920). 8. McMeekin, T. L., J. Am. Chem. Sot., 61,2834 (1939). 9. Newcomer, H. S., J. BioZ. Chem., 37,465 (1919).
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B. L. HORECKER 183

10. Kennedy, R. P., Am. J. Physiol., 79,346 (1927). 11. Drabkin, D. L., and Austin, J. H., J. Biol. Chem., 98,719 (1932) ; 112,51 (1935-36). 12. Merkelbach, O., Schweiz. med. Woch., 66,1142 (1936). 13. Sidwell, A. E., Jr., Munch, R. H., Barron, E. S. G., and Hogness, T. R., J. Biol. Chem., 123,335 (1938). 14. Altschul, A. M., Sidwell, A. E., Jr., and Hogness, T. R., J. Biol. Chem., 127, 123 (1939). 15. Peters, J. P., and Van Slyke, D. I)., Quantitative clinical chemistry, Methods, Baltimore, 341 (1932). 16. Svedberg, T., and Fahraeus, R.; J. Am. Chem. Sot., 49,430 (1926).

17. Adair, G. S., Proc. Roy. Sot. London, Series A, 108,627 (1925). 18. Morrison, D. B., and Hisey, A., J. Biol. Chem., 109,233 (1935). 19. Kuper, J. B. H., and Brackett, F. S., Physic. Rev., 67,1069 (1940).
Downloaded from www.jbc.org by guest, on September 25, 2011In

optics, the BeerLambert law, also known as Beer's law or the LambertBeer law or the BeerLambertBouguer law (named after August Beer, Johann Heinrich Lambert, and Pierre Bouguer) relates the absorption of light to the properties of the material through which the light is traveling.

Contents
[hide]

1 Equations 2 Derivation 3 Prerequisites 4 Chemical analysis 5 BeerLambert law in the atmosphere 6 History 7 See also 8 References 9 External links

[edit] Equations

Diagram of BeerLambert absorption of a beam of light as it travels through a cuvette of width .

The law states that there is a logarithmic dependence between the transmission (or transmissivity), T, of light through a substance and the product of the absorption coefficient of the substance, , and the distance the light travels through the material (i.e., the path length), . The absorption coefficient can, in turn, be written as a product of either a molar absorptivity (extinction coefficient) of the absorber, , and the concentration c of absorbing species in the material, or an absorption cross section, , and the (number) density N' of absorbers.

For liquids, these relations are usually written as:

whereas for gases, and in particular among physicists and for spectroscopy and spectrophotometry, they are normally written

where I0 and I are the intensity (or power) of the incident light and the transmitted light, respectively; is cross section of light absorption by a single particle and N is the density (number per unit volume) of absorbing particles. The base 10 and base e conventions must not be confused because they give different values for the absorption coefficient: . The transmission (or transmissivity) is expressed in terms of an absorbance which, for liquids, is defined as . However, it is easy to convert one to the other, using

whereas, for gases, it is usually defined as

This implies that the absorbance becomes linear with the concentration (or number density of absorbers) according to

and

for the two cases, respectively. Thus, if the path length and the molar absorptivity (or the absorption cross section) are known and the absorbance is measured, the concentration of the substance (or the number density of absorbers) can be deduced. Although several of the expressions above often are used as BeerLambert law, the name should strictly speaking only be associated with the latter two. The reason is that historically,

the Lambert law states that absorption is proportional to the light path length, whereas the Beer law states that absorption is proportional to the concentration of absorbing species in the material.[1] If the concentration is expressed as a mole fraction i.e., a dimensionless fraction, the molar absorptivity () takes the same dimension as the absorption coefficient, i.e., reciprocal length (e.g., m1). However, if the concentration is expressed in moles per unit volume, the molar absorptivity () is used in Lmol1cm1, or sometimes in converted SI units of m2mol1. The absorption coefficient ' is one of many ways to describe the absorption of electromagnetic waves. For the others, and their interrelationships, see the article: Mathematical descriptions of opacity. For example, ' can be expressed in terms of the imaginary part of the refractive index, , and the wavelength of the light (in free space), 0, according to

In molecular absorption spectrometry, the absorption cross section is expressed in terms of a linestrength, S, and an (area-normalized) lineshape function, . The frequency scale in molecular spectroscopy is often in cm1, wherefore the lineshape function is expressed in units of 1/cm1, which can look funny but is strictly correct. Since N is given as a number density in units of 1/cm3, the linestrength is often given in units of cm2cm1/molecule. A typical linestrength in one of the vibrational overtone bands of smaller molecules, e.g., around 1.5 m in CO or CO2, is around 1023 cm2cm1, although it can be larger for species with strong transitions, e.g., C2H2. The linestrengths of various transitions can be found in large databases, e.g., HITRAN. The lineshape function often takes a value around a few 1/cm1, up to around 10/cm1 under low pressure conditions, when the transition is Doppler broadened, and below this under atmospheric pressure conditions, when the transition is collision broadened. It has also become commonplace to express the linestrength in units of cm2/atm since then the concentration is given in terms of a pressure in units of atm. A typical linestrength is then often in the order of 103 cm2/atm. Under these conditions, the detectability of a given technique is often quoted in terms of ppmm. The fact that there are two commensurate definitions of absorbance (in base 10 or e) implies that the absorbance and the absorption coefficient for the cases with gases, A' and ', are ln 10 (approximately 2.3) times as large as the corresponding values for liquids, i.e., A and , respectively. Therefore, care must be taken when interpreting data that the correct form of the law is used. The law tends to break down at very high concentrations, especially if the material is highly scattering. If the light is especially intense, nonlinear optical processes can also cause variances.

[edit] Derivation
The derivation is quite simple in concept. There are many details, so think of this first paragraph as a conceptual overview. Divide the absorbing sample into thin slices that are perpendicular to the beam of light. The light that emerges from a slice is slightly less intense

than the light that entered because some of the photons have run into molecules in the sample and did not make it to the other side. For most cases where measurements of absorption are needed, a vast majority of the light entering the slice leaves without being absorbed. Because the physical description of the problem is in terms of differencesintensity before and after light passes through the slicewe can easily write an ordinary differential equation model for absorption. The difference in intensity due to the slice of absorbing material dI is reduced; leaving the slice, it is a fraction of the light entering the slice I. The thickness of the slice is dz, which scales the amount of absorption (thin slice does not absorb much light but a thick slice absorbs a lot). In symbols, dI = Idz, or dI / dz = I. This conceptual overview uses to describe how much light is absorbed. All we can say about the value of this constant is that it will be different for each material. Also, its values should be constrained between 1 and 0. The following paragraphs cover the meaning of this constant and the whole derivation in much greater detail. Assume that particles may be described as having an absorption cross section (i.e., area), , perpendicular to the path of light through a solution, such that a photon of light is absorbed if it strikes the particle, and is transmitted if it does not. Define z as an axis parallel to the direction that photons of light are moving, and A and dz as the area and thickness (along the z axis) of a 3-dimensional slab of space through which light is passing. We assume that dz is sufficiently small that one particle in the slab cannot obscure another particle in the slab when viewed along the z direction. The concentration of particles in the slab is represented by N. It follows that the fraction of photons absorbed when passing through this slab is equal to the total opaque area of the particles in the slab, AN dz, divided by the area of the slab A, which yields N dz. Expressing the number of photons absorbed by the slab as dIz, and the total number of photons incident on the slab as Iz, the fraction of photons absorbed by the slab is given by

Note that because there are fewer photons which pass through the slab than are incident on it, dIz is actually negative (It is proportional in magnitude to the number of photons absorbed). The solution to this simple differential equation is obtained by integrating both sides to obtain Iz as a function of z

The difference of intensity for a slab of real thickness is I0 at z = 0, and Il at z = . Using the previous equation, the difference in intensity can be written as,

rearranging and exponentiating yields,

This implies that

and

The derivation assumes that every absorbing particle behaves independently with respect to the light and is not affected by other particles. Error is introduced when particles are lying along the same optical path such that some particles are in the shadow of others. This occurs in highly concentrated solutions. In practice, when large absorption values are measured, dilution is required to achieve accurate results. Measurements of absorption in the range of I1 / I0 = 0.1 to 1 are less affected by shadowing than other sources of random error. In this range, the ODE model developed above is a good approximation; measurements of absorption in this range are linearly related to concentration. At higher absorbances, concentrations will be underestimated due to this shadow effect unless one employs a more sophisticated model that describes the non-linear relationship between absorption and concentration.

[edit] Prerequisites
There are at least six conditions that need to be fulfilled in order for Beers law to be valid. These are:
1. 2. 3. 4. The absorbers must act independently of each other; The absorbing medium must be homogeneous in the interaction volume The absorbing medium must not scatter the radiation - no turbidity; The incident radiation must consist of parallel rays, each traversing the same length in the absorbing medium; 5. The incident radiation should preferably be monochromatic, or have at least a width that is narrower than that of the absorbing transition; and 6. The incident flux must not influence the atoms or molecules; it should only act as a noninvasive probe of the species under study. In particular, this implies that the light should not cause optical saturation or optical pumping, since such effects will deplete the lower level and possibly give rise to stimulated emission.

If any of these conditions are not fulfilled, there will be deviations from Beers law.

[edit] Chemical analysis

Beer's law can be applied to the analysis of a mixture by spectrophotometry, without the need for extensive pre-processing of the sample. An example is the determination of bilirubin in blood plasma samples. The spectrum of pure bilirubin is known, so the molar absorbance is known. Measurements are made at one wavelength that is nearly unique for bilirubin and at a second wavelength in order to correct for possible interferences.The concentration is given by c = Acorrected / .

For a more complicated example, consider a mixture in solution containing two components at concentrations c1 and c2. The absorbance at any wavelength, is, for unit path length, given by

Therefore, measurements at two wavelengths yields two equations in two unknowns and will suffice to determine the concentrations c1 and c2 as long as the molar absorbances of the two components, 1 and 1 are known at both wavelengths. This two system equation can be solved using Cramer's rule. In practice it is better to use linear least squares to determine the two concentrations from measurements made at more than two wavelengths. Mixtures containing more than two components can be analysed in the same way, using a minimum of n wavelengths for a mixture containing n components. The law is used widely in infra-red spectroscopy for analysis of polymer degradation and oxidation. The carbonyl group absorption at about 6 micrometres can be detected quite easily, and degree of oxidation of the polymer calculated.

[edit] BeerLambert law in the atmosphere

This law is also applied to describe the attenuation of solar or stellar radiation as it travels through the atmosphere. In this case, there is scattering of radiation as well as absorption. The BeerLambert law for the atmosphere is usually written

where each x is the optical depth whose subscript identifies the source of the absorption or scattering it describes:

a refers to aerosols (that absorb and scatter) g are uniformly mixed gases (mainly carbon dioxide (CO2) and molecular oxygen (O2) which only absorb) NO2 is nitrogen dioxide, mainly due to urban pollution (absorption only) w is water vapour absorption O3 is ozone (absorption only) r is Rayleigh scattering from molecular oxygen (O2) and nitrogen (N2) (responsible for the blue color of the sky).

m is the optical mass or airmass factor, a term approximately equal (for small and moderate values of ) to 1 / cos(), where is the observed object's zenith angle (the angle measured from the direction perpendicular to the Earth's surface at the observation site). This equation can be used to retrieve a, the aerosol optical thickness, which is necessary for the correction of satellite images and also important in accounting for the role of aerosols in climate. When the path taken by the light is through the atmosphere, the density of the absorbing gas is not constant, so the original equation must be modified as follows:

where z is the distance along the path through the atmosphere, all other symbols are as defined above.[2] This is taken into account in each x in the atmospheric equation above.

[edit] History
The law was discovered by Pierre Bouguer before 1729. It is often mis-attributed to Johann Heinrich Lambert, who cited Bouguer's Essai d'Optique sur la Gradation de la Lumiere (Claude Jombert, Paris, 1729) and even quoted from it in his Photometria in 1760. Much later, August Beer extended the exponential absorption law in 1852 to include the concentration of solutions in the absorption coefficient.