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Introduction

The Beer-Lambert law (or Beer's law) is the linear relationship between absorbance and concentration of an absorbing species. The general Beer-Lambert law is usually written as: A = a(lambda) * b * c where A is the measured absorbance, a(lambda) is a wavelength-dependent absorptivity coefficient, b is the path length, and c is the analyte concentration. When working in concentration units of molarity, the Beer-Lambert law is written as: A = epsilon * b * c where epsilon is the wavelength-dependent molar absorptivity coefficient with units of M-1 cm-1.

Instrumentation

Experimental measurements are usually made in terms of transmittance (T), which is defined as: T = I / Io where I is the light intensity after it passes through the sample and Io is the initial light intensity. The relation between A and T is: A = -log T = - log (I / Io). Absorption of light by a sample

Modern absorption instruments can usually display the data as either transmittance, %transmittance, or absorbance. An unknown concentration of an analyte can be determined by measuring the amount of light that a sample absorbs and applying Beer's law. If the absorptivity coefficient is not known, the unknown concentration can be determined using a working curve of absorbance versus concentration derived from standards.

The Beer-Lambert law can be derived from an approximation for the absorption coefficient for a molecule by approximating the molecule by an opaque disk whose cross-sectional area, sigma, represents the effective area seen by a photon of frequency w. If the frequency of the light is far

from resonance, the area is approximately 0, and if w is close to resonance the area is a maximum. Taking an infinitesimal slab, dz, of sample:

Io is the intensity entering the sample at z=0, Iz is the intensity entering the infinitesimal slab at z, dI is the intensity absorbed in the slab, and I is the intensity of light leaving the sample. Then, the total opaque area on the slab due to the absorbers is sigma * N * A * dz. Then, the fraction of photons absorbed will be sigma * N * A * dz / A so, dI / Iz = - sigma * N * dz Integrating this equation from z = 0 to z = b gives: ln(I) - ln(Io) = - sigma * N * b or - ln(I / Io) = sigma * N * b. Since N (molecules/cm3) * (1 mole / 6.023x1023 molecules) * 1000 cm3 / liter = c (moles/liter) and 2.303 * log(x) = ln(x) then - log(I / Io) = sigma * (6.023x1020 / 2.303) * c * b or - log(I / Io) = A = epsilon * b * c where epsilon = sigma * (6.023x1020 / 2.303) = sigma * 2.61x1020 Typical cross-sections and molar absorptivities are:

epsilon (M-1 cm-1) absorption - atoms molecules infrared Raman scattering sigma (cm2) 10-12 10-16 10-19 10-29 3x108 3x104 3x10 3x10-9

The linearity of the Beer-Lambert law is limited by chemical and instrumental factors. Causes of nonlinearity include:

deviations in absorptivity coefficients at high concentrations (>0.01M) due to electrostatic interactions between molecules in close proximity scattering of light due to particulates in the sample fluoresecence or phosphorescence of the sample changes in refractive index at high analyte concentration shifts in chemical equilibria as a function of concentration non-monochromatic radiation, deviations can be minimized by using a relatively flat part of the absorption spectrum such as the maximum of an absorption band stray light

Introduction

Many compounds absorb ultraviolet (UV) or visible (Vis.) light. The diagram below shows a beam of monochromatic radiation of radiant power P0, directed at a sample solution. Absorption takes place and the beam of radiation leaving the sample has radiant power P. The amount of radiation absorbed may be measured in a number of ways: Transmittance, T = P / P0 % Transmittance, %T = 100 T Absorbance, A = log10 P0 / P A = log10 1 / T A = log10 100 / %T A = 2 - log10 %T The last equation, A = 2 - log10 %T , is worth remembering because it allows you to easily calculate absorbance from percentage transmittance data. The relationship between absorbance and transmittance is illustrated in the following diagram:

So, if all the light passes through a solution without any absorption, then absorbance is zero, and percent transmittance is 100%. If all the light is absorbed, then percent transmittance is zero, and absorption is infinite.

Now let us look at the Beer-Lambert law and explore it's significance. This is important because people who use the law often don't understand it - even though the equation representing the law is so straightforward:

A= bc

Where A is absorbance (no units, since A = log10 P0 / P ) is the molar absorbtivity with units of L mol-1 cm-1

c is the concentration of the compound in solution, expressed in mol L-1

b is the path length of the sample - that is, the path length of the cuvette in which the sample is contained. We will express this measurement in centimetres.

The reason why we prefer to express the law with this equation is because absorbance is directly proportional to the other parameters, as long as the law is obeyed. We are not going to deal with deviations from the law. Let's have a look at a few questions... Question : Why do we prefer to express the Beer-Lambert law using absorbance as a measure of the absorption rather than %T ? Answer : To begin, let's think about the equations...

A= bc %T = 100 P/P0 = e - bc

Now, suppose we have a solution of copper sulphate (which appears blue because it has an absorption maximum at 600 nm). We look at the way in which the intensity of the light (radiant power) changes as it passes through the solution in a 1 cm cuvette. We will look at the reduction every 0.2 cm as shown in the diagram below. The Law says that the fraction of the light

absorbed by each layer of solution is the same. For our illustration, we will suppose that this fraction is 0.5 for each 0.2 cm "layer" and calculate the following data: Path length / cm %T Absorbance 0 100 0 0.2 50 0.3 0.4 25 0.6 0.6 12.5 0.9 0.8 6.25 1.2 1.0 3.125 1.5

A = bc tells us that absorbance depends on the total quantity of the absorbing compound in the light path through the cuvette. If we plot absorbance against concentration, we get a straight line passing through the origin (0,0).

Note that the Law is not obeyed at high concentrations. This deviation from the Law is not dealt with here.

The linear relationship between concentration and absorbance is both simple and straightforward, which is why we prefer to express the Beer-Lambert law using absorbance as a measure of the absorption rather than %T. Question : What is the significance of the molar absorbtivity, ? Answer : To begin we will rearrange the equation A = bc :

= A / bc

In words, this relationship can be stated as " is a measure of the amount of light absorbed per unit concentration". Molar absorbtivity is a constant for a particular substance, so if the concentration of the solution is halved so is the absorbance, which is exactly what you would expect. Let us take a compound with a very high value of molar absorbtivity, say 100,000 L mol-1 cm-1, which is in a solution in a 1 cm pathlength cuvette and gives an absorbance of 1.

=1/1 c

Therefore, c = 1 / 100,000 = 1 10 -5 mol L-1 Now let us take a compound with a very low value of , say 20 L mol-1 cm-1 which is in solution in a 1 cm pathlength cuvette and gives an absorbance of 1.

=1/1 c

Therefore, c = 1 / 20 = 0.05 mol L-1 The answer is now obvious - a compound with a high molar absorbtivity is very effective at absorbing light (of the appropriate wavelength), and hence low concentrations of a compound with a high molar absorbtivity can be easily detected. Question : What is the molar absorbtivity of Cu2+ ions in an aqueous solution of CuSO4 ? It is either 20 or 100,000 L mol-1 cm-1 Answer : I am guessing that you think the higher value is correct, because copper sulphate solutions you have seen are usually a beautiful bright blue colour. However, the actual molar absorbtivity value is 20 L mol-1 cm-1 ! The bright blue colour is seen because the concentration of the solution is very high. -carotene is an organic compound found in vegatables and is responsible for the colour of carrots. It is found at exceedingly low concentrations. You may not be surprised to learn that the molar absorbtivity of -carotene is 100,000 L mol-1 cm-1 !

Equations

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

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 slice we 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,

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 non-invasive 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.

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.

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.

Beer-Lambert Law

Beer's law states that for a parallel beam of monochromatic radiation passing through homogeneous solutions of equal pathlength the absorbance is proportional to the concentration. i.e. For solutions 1 and 2 (Absorbance 1)/(Absorbance 2) = (Concentration 1)/(Concentration 2) Lambert's law states that for a parallel beam of monochromatic radiation passing through homogeneous solutions of equal concentration the absorbance is proportional to the pathlength. i.e. For pathlengths A and B (Absorbance A)/(Absorbance B) = (Pathlength A)/(Pathlength B)

The absorbance of a 2-cm layer of a 0.0006 %w/v solution is 0.48 What would be the absobance of a 1-cm layer of a 0.0009 %w/v solution of the same substance under the same conditions? 0.64 0.16 0.36 1.44 0.48

The Beer-Lambert law states that for a parallel beam of monochromatic radiation passing through a homogeneous solution the absorbance is proportional to the product of the concentration and pathlength. Absorbance = constant concentration pathlength The value of the constant depends on the substance, the solvent, the wavelength and the units used for concentration and pathlength. Two common methods of expressing the constant are A(1%,1cm) and molar absorptivity. A(1%, 1cm) is the absorbance of a 1-cm layer of a 1%w/v solution. A = A(1%, 1cm) C L Where C is the concentration as %w/v and L is the pathlength in cm.

2

A 2-cm layer of a 0.003 %w/v solution of Ethamivan exhibits a maximum at 280 nm with an absorbance 0.92. What is the A(1%, 1cm) of Ethamivan under these conditions?

304

3420

153

307

46700

A 4-cm layer of a 0.00003 mol litre-1 solution of Ethamivan has an absorbance of 0.411 at the maximum at 280 nm What is the molar absorpivity of Ethamivan under these conditions? 153 1120 2725 3425 13700

A(1%, 1cm) values are generally found in the pharmaceutical literature while molar absorptivities are found in the chemical literature. You may not always be able to find the information in the most convenient form.

A 1-cm layer of a 0.0008 %w/v solution of Thiabendazole in 0.1M hydrochloric acid has an absorbance of 0.47 at the maximum at 243 nm. Calculate the molar absorptivity (litre mol-1 cm-1) under these conditions. 1.18 103 1.18 104 1.43 104 588 517

When preparing solutions for absorbance measurements you will need to know what concentrations to prepare in order to obtain suitable absorbances. For routine analytical work absorbances greater than 1 should be avoided.

5

Sulphadiazine in ethanol has a molar absorptivity of 2.11104 mol-1 litre cm-1 at the maximum at 270 nm. From the list below, select the concentrations (mol litre-1 and %w/v) which would give an absobance of 1.0 in a 2-cm cell at 270 nm 4.7410-5 mol litre-1 1.1810-3 %w/v 3.2010-4 %w/v 2.3710-5 mol litre-1 6.4010-4 %w/v 6.4010-3 mol litre-1 5.9210-4 %w/v

Beer-Lambert Law

Beer-Lambert Law, more commonly known as Beer's Law, states that the optical absorbance of a chromophore in a transparent solvent varies linearly with both the sample cell pathlength and the chromophore concentration. Beer's Law is the simple solution to the more general description of Maxwell's far-field equations describing the interaction of light with matter. In practice, Beer's Law is accurate enough for a range of chromophores, solvents and concentrations, and is a widely used relationship in quantitative spectroscopy. Absorbance is measured in a spectrophotometer by passing a collimated beam of light at wavelength through a plane parallel slab of material that is normal to the beam. For liquids, the sample is held in an optically flat, transparent container called a cuvette. Absorbance (A) is calculated from the ratio of light energy passing through the sample (I0) to the energy that is incident on the sample (I): A = -log (I/I0) Beer's Law follows: A = bc = molar absorptivity or extinction coefficient of the chromophore at wavelength (the optical density of a 1-cm thick sample of a 1 M solution). is a property of the material and the solvent. b = sample pathlength in centimeters c = concentration of the compound in the sample, in molarity (mol L-1) In an absorbance experiment, light is attenuated not only by the chromophore, but also by reflections from the interface between air and the sample, the sample and the cuvette, and absorbance by the solvent. These factors can be quantified separately, but are often removed by defining I0 as the light passing

through a sample "blank" or "baseline" or reference sample (for example, a cuvette filled with solvent but zero concentration of the chromophore is used as the blank). Many factors can affect the validity of Beer's Law. It is usual to check for the linearity of Beer's Law for a chromophore by measuring the absorbance of a series of standards. This "calibration" can also remove errors in the experiment, the equipment, and the batch of reagents (such as cuvettes of unknown pathlength).

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