is the extraterrestrial intensity weakened by absorption, Rayleigh scattering and Mie scattering along the light path: absorption by all trace gases j **extinction** by Mie scattering **extinction** by Rayleigh scattering unattenuated intensity integral over light path scattering efficiency exponential from **Lambert** **Beer**’s **law** Introduction to Measurement Techniques in Environmental Physics, A. Richter, Summer Term 2006 14 DOAS equation II if the absorption cross-sections/

is unity. - Molar absorptivity or epsilon ( ) -If the unit of concentration is 1M, (a) is known as molar absortivity or epsilon ( ) or molar **extinction** coefficient. (Unit of is L mol -1 cm -1 ) - If unity of concentration is 1%, (a) is known as A (1%, 1cm). A (1/ stable with time. 4- The reaction of its formation, must be rapid and quantitative. 5- The colored product, should obey **Beer**-**lambert**’s **law**, i.e on plotting A versus C at fixed b, we obtain straight line passing through the origin. 37 38 A- /

The attenuation of light, from the light beam (source) to the photodetector (signal), is typically modeled by the **Beer**- **Lambert** **law**. This **law** states that in a homogeneous medium, light intensity decays exponentially as a function of path length (l) and light /precise wavelength. The absorption spectrum shown in Figure 4 represents the optical behavior in terms of coefficient of absorption/**extinction** of a particular molecule with respect to light wavelength. The main constituent of tissue, namely water (H2O),/

the far UV. Phe is trivial. Uses of Absorbance Spectra: Quantitating Protein by Amino Acid absorption **Beer**-**Lambert** (**Beer**’s) **Law** Absorbance, A= lc = Log (1/T) Molar **extinction** coefficient has units of M -1 cm -1 and is a constant of proportionality that /.5 units is considered inaccurate). A useful expression relating the parameters of , concentration (c) and A are derived from the **Beer**- **Lambert** **law** (assuming 1 cm path length): A/ 280nm = c For example, if a sample of bovine insulin was observed to/

Absorbance, AOptical Density, D Transmittance, TTransmission, T Path Length, bl, d Absorptivity, aExtinction Coefficient, k Molar Absorptivity, Molar **Extinction** Coefficient Transmittance Absorbance, like the previous table shows, can be defined as the base-ten logarithm of the reciprocal of the transmittance/obtained with the equation: A = log P solvent /P solution x log P o /P **Beer**’s **Law** Bouguer, and later **Lambert**, observed that the fraction of the energy, or the intensity, of radiation absorbed in a thin/

a spectrometer for producing light of any selected color (wavelength), and a photometer for measuring the intensity of light. **BEER** **LAMBERT** **LAW** As the cell thickness increases, the intensity of I (transmitted intensity of light ) decreases. T- Transmittance T /–if **extinction** coeff ( ( )) and path length are known Use **Beer**’s **Law** to find C –might try at a number of different wavelengths as a self-check –develop calibration Check linearity vs concentration Deviations from the **beer** **Lamberts** **law** Radiation /

/f1=exp(DE/kT)=f(T) N – number of molecules N = c NAS dx Absorption of light the **Lambert** **Beer** **law** DE = hv0 I I0 2 Nf2 molecules DE = hv0 1 Nf1 molecules I0 dx I the **Lambert** **Beer** **law** e the molar **extinction** coefficient (molar absorptivity) Absorption: measurement The **Beer** **Lambert** **Law** Absorption (Optical Density) = log Io / I = c l l is the path length of the sample (1/

at the lowest vibrational energy level of the ground state Suppose a molecule is illuminated with light at a resonance frequency Light is absorbed; for dilute sample, **Beer**-**Lambert** **law** applies where e is molar absorption (**extinction**) coefficient (M-1 cm-1); its magnitude reflects probability of absorption and its wavelength dependence corresponds to absorption spectrum Excitation - following light absorption, a chromophore is excited/

E=h or E=hc/ h=Planks Constant = 6.6260 6 x 10 34 J. s Where in the spectrum are these transitions? **Beer**-**Lambert** **Law** AKA - **Beer**’s **Law** The Quantitative Picture Transmittance: T = P/P 0 b(path through sample) P 0 (power in) P (power out) Absorbance: A / nanometers (nm) 0.0 400 800 1.0 200 UV Visible max with certain **extinction** Make solution of concentration low enough that A≤ 1 (Helps to Ensure Linear **Beer**’s **law** behavior) UV bands are much broader than the photonic transition event. This is because vibration/

h or E=hc/ h=Planks Constant = 6.6260 6 x 10 34 J. s Where in the spectrum are these transitions? **Beer**-**Lambert** **Law** AKA - **Beer**’s **Law** The Quantitative Picture Transmittance: T = P/P 0 b(path through sample) P 0 (power in) P (power out) Absorbance/generally in nanometers (nm) 0.0 400 800 1.0 200 UV Visible max with certain **extinction** Make solution of concentration low enough that A≤ 1 (Helps to Ensure Linear **Beer**’s **law** behavior) UV/Vis and Molecular Structure The UV Absorption process * transitions: /

and Emission spectra Electron excitation Principles of Spectrophotometry **Beers** **law** Measurement of absorbance Action spectra Difference spectra/**Beers**-**Lambert** **Law** o Absorbance is related to the concentration of the absorbing substance as well as the thickness of the layer o Mathematical relationship between concentration and absorbance is called **Beers** **Law** o Mathematical relationship between concentration and thickness of the layer is called **Lamberts** **Law** A = εcl where A = absorbance ε = **extinction**/

+ ] is equal to the original thiocyanate concentration [SCN - ] A standard curve was prepared to determine the molar **extinction** coefficient of [FeSCN 2+ ] A standard curve was prepared to determine the molar **extinction** coefficient of [FeSCN 2+ ] **Beer**’s **law**, or the **Beer**-**Lambert** equation shown below. **Beer**’s **law**, or the **Beer**-**Lambert** equation shown below. A = abc or A = bc A = abc or A = bc sample size was around/

via a lookup table For absolute measurement calibration with blood sample required Does show relative changes - still clinically useful 1 **Beer**-**Lambert** **Law** Vout normalised Empirical Calibration 0 % 100 - SpO2 Section Summary PPG produces a measure of blood perfusion changes in a / and path length L held constant the absorbitivity or **extinction** coefficient varies with Saturation percent of Hb as Sp02=Hb/THb 0 100% SpO2 Also we can say Pulse Oximetry Theory - II … **Beer**’s **Law** I0 I Br is the non-Hb absorption of/

s yields: (7) where Lυ(0) is the radiance entering the medium at x=0. ►This is known as **Beer**-Bouguer-**Lambert** **law**. The ratio Lυ(s)/Lυ(0) is called the spectral transmittance of the medium. Transmittance and opical depth ►The optical / be approximated by the Planck function. ►In the scaling approximation the absorption optical depth, , is replaced by an effective **extinction** optical depth, , defined as The parametrization of scattering in a fast radiative transfer model Here is the scattering optical depth/

com) Slide 20 /classes/BMS524/524lect003.ppt© 1993-2014 J. Paul Robinson - Purdue University Cytometry Laboratories Parameters **Extinction** Coefficient – refers to a single wavelength (usually the absorption maximum) Quantum Yield –Q f is a measure/ ln (I o /I) = C d (**beer** –**Lambert** **law**) =absorption coefficient C = concentration Converting to decimal logs and standardizing quantities we get Log (I 0 /I) = cd = A Now is the decadic molar **extinction** coefficient A = absorbance or optical density (OD) a /

com) Slide 18 t:/classes/BMS524/524lect003.ppt© 1993-2008 J. Paul Robinson - Purdue University Cytometry Laboratories Parameters **Extinction** Coefficient – refers to a single wavelength (usually the absorption maximum) Quantum Yield –Q f is a /or ln (I o /I) = C d (**beer** –**Lambert** **law**) =absorption coefficient C = concentration Converting to decimal logs and standardizing quantities we get Log (I 0 /I) = cd = A Now is the decadic molar **extinction** coefficient A = absorbance or optical density (OD) a /

**Beer**-**Lambert** **law**: Principle: The absorption of light by a solution is described by the **Beer**-**Lambert** **law**: The **law** implies that there is linear relationship between absorbance and concentration of an absorbing species. A = εlc A= is the absorbance. ε = **extinction**/ the ” unknown solution” Concentration of the ” unknown solution” To determine the concentration of an unknown solution: **Beer**-**Lambert** **law**: From standard curve: -Measure the absorbance of the “unknown solution” in order to determine the concentration. /

of fluorescence relative to distance through the solution. This rate of decay can be used to determine the **extinction** coefficient ( ɛ ) of the molecule using the **Beer**-**Lambert** **law**, shown in Equ. 1. Log (I/I 0 ) = - ɛ cl Where… I =/ of detected light I 0 = Intensity of incident light c = Concentration l = Path length ɛ = **Extinction** coefficient Equ. 1. the **Beer**-**Lambert** **Law** Experimental setup The experiment required a light sensor to periodically take measurements while travelling parallel to the laser beam./

of transmitted light I is related to incident light Io is given by, **Beer** **Lambert**’s **law**: I=I 0 -KCb, where C is concentration of absorbing medium, thickness b and K is **extinction** coeffecient. KCb is absorbance A. Reflection oximetry: Based on scattering of light by/averaging and storage Central processor Fibre optic ear probe Optica l sync, pin holes The instrument is based on the **Beer**-**Lambert** **law**. The mathematical statement of this **law** for wavelength can be written as: Aj = E 1j C 1 D l + E 2j C 2 D /

length (b) Absorbance (A) = log 10 P 0 /P **Extinction** co-efficient ( ) P0P0 P A = *b*[compound] **Beer**-**lambert** **law** Constant Emitter Linear relationship between dye concentration and absorbance at low [dye] (**Beer**-**Lambert** **law**) 34,000*Fura-2 100,000*Fluo-4 (cm -1 / fluorescent indicator. 2. Detector sensitivity, instrumental efficiency in collecting photons. 3. Quantum efficiency 4. **Extinction** coefficient Summary Light Path in an Inverted Microscope Designed to detect Fura-2 signals Experimental Fura-2 /

= hc/ = h ( photon) E = energy h = Plancks constant c = speed of light = wavelength = frequency absorption maxima ( max ) chromophores exhibit unique absorption spectra d d IoIo I **Beer**-**Lambert** **Law** I = I o 10 - dc I = light intensity = **extinction** coefficient d = thickness c = concentration Absorption (A) = -log(I/I o ) = dc Spectrophotometer -log(I/I o ) = A photo multiplier tube (photo electric cell) d d/

**Beer** - **Lambert** **law** : where A is the measured absorbance, in Absorbance Units ( AU ), is the intensity of the incident light at a given wavelength, is the transmitted intensity, L the path length through the sample, and c the concentration of the absorbing species. For each species and wavelength, ε is a constant known as the molar absorptivity or **extinction**/ energy levels and electronic transitions in UV? What is **Beer** ’ s **Lambert** **law** of absorption? Explain the principle of absorption spectroscopy? /

pathlength (L) and the chromophore concentration (c). **Beer**-**Lambert** **law** (**Beer**’s **law**) The fraction of the incident light absorbed by a solution at a given wavelength is related to: a. thickness of the absorbing layer (path length). b. concentration of the absorbing species. Concentration amount of light absorbed A = aLC = log(100/%T) A = absorbance a = **extinction** coefficient L = light path of the solution c/

=s yields: (7) where Lυ(0) is the radiance entering the medium at x=0. This is known as **Beer**-Bouguer-**Lambert** **law**. The ratio Lυ(s)/Lυ(0) is called the spectral transmittance of the medium. Transmittance and opical depth The optical depth/ be approximated by the Planck function. In the scaling approximation the absorption optical depth, , is replaced by an effective **extinction** optical depth, , defined as The parametrization of scattering in a fast radiative transfer model Here is the scattering optical depth/

sample cell c = molar concentration of solute l = length of sample cell (cm) = molar absorptivity (molar **extinction** coefficient) 3 7 2 UV / visible Spectroscopy The **Beer**-**Lambert** **Law** is rigorously obeyed when a single species is present at relatively low concentrations. 3 8 2 UV / visible Spectroscopy The **Beer**-**Lambert** **Law** is not obeyed: High concentrations Solute and solvent form complexes Thermal equilibria exist between the ground/

work? **Beer**-**Lambert** **law**: Principle: The absorption of light by a solution is described by the **Beer**-**Lambert** **law** as: There is linear relationship between absorbance and concentration of an absorbing species. A = εlc A= is the absorbance. ε = **extinction**(absorption)/ curve: -Measure the absorbance of the “solution with unknown concentration” in order to determine the concentration. **Beer**-**Lambert** **law**: Using available information of any standard solution to determine the “ε”, then using these information to get the /

factors that determines the amount of light that passes through a solution. Identify two of these factors. –What is **Beer**’s **Law**? »determines the quantitatively analysis using spectroscopy. Spectroscopy studies em radiation emitted or absorbed by chemical species. **Beer**-**Lambert** **Law** Is molar absorptivity or molar **extinction** coefficient (L/mol*cm) Length of path, distance light travels through the solution Concentration of solution (M) Questions… Therefore/

range: Tungsten lamp (400 – 700 nm) UV range: Deutrium lamp (200 – 400 nm) Sample cells Detector Mirrors Grating Monochrometer Application **Lambert**-**Beer** **Law** A = εcl where: A = absorbance ε = molar **extinction** coefficient (L mmol -1 cm -1 ) c = molar concentration (mM) l = pathlength (cm) The **Lambert**-**Beer** **law** is used to accurately determine the concentration of a substance by measure absorbance at a specific wavelength Determining Concentration The/

Nellcor pulse oximeter PRINCIPLES ABSORBTION SPECTRO PHOTOMETRY **BEER** **LAMBERT** **LAW** **LAMBERT**’S **LAW** states that when a light falls on a homogenous substance,intensity of transmitted light decreases as the distance through the substance increase **BEER**’S **LAW** states that when a light is /to measure 4 species of haemoglobin It =I o e –Ecd [Ecd –absorbance] wavelength Oxy Hb 660nm 940nm **Extinction** coefficient MetHb Deoxy Hb COHB 660nm 940nm wavelength DESIGN OF PULSEOXIMETER 2 WAVELENGTHS- 660nm [red] & 940nm[infra red/

or functional saturation values. PRINCIPLES **Beer** **Lambert** **Law** All atom and molecules absorbed specific wavelength of light.this property is the basis for an optical technique known as spectrophotometry. **Beer** **Lambert** **Law** **Lambert**’s **Law**- states that when a light falls/infrared region(940nm)The opposite is true,and oxygenated Hb absorbed more light effectively than deoxygenatedHb. wavelength **Extinction** coefficient MetHb Deoxy Hb COHB Oxy Hb 660nm Operating Principles The pulse oximeter computes the ratio between/

**Beer**-**Lambert** **law** log = [J] l –I o = incident light –I = transmitted light –l = length of light path – = molar absorption coefficient –[J] = molar concentration of J I = I o 10 – [J]l IoIo I Molar **extinction** coefficient? Molar absorption coefficient ( ) was known as the “molar **extinction** coefficient” Use of the term “molar **extinction**/ Dictionary of Spectroscopy, 2nd ed. (Wiley, 1982), p.119-20. Spectrophotometry **Beer**’s **law**: [J] = Thus, absorbance is directly proportional to the molar concentration A =/

fluorescence/fluorescenceintro.html © 1990-2016 J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt Exctinction Using **Beer**’s **law** (**Beer**-**Lambert** **law**) for light travelling through a curvette thickness d cm containing n molecules/cm 3 ln (I o /I/ J.Paul Robinson, Purdue University BMS 631- Flow Cytometry lecture3.ppt Properties of Fluorescent Molecules n Large **extinction** coefficient at the region of excitation n High quantum yield n Optimal excitation wavelength n Photostability n Excited-/

, EDTA and phenol all absorb at 260 nm) How can concentration be determined by absorbance? DNA has a characteristic “molar **extinction** coefficient” The **Beer**-**Lambert** **law**: I = I o 10 - dc I = intensity of transmitted light I o = intensity of incident light = molar **extinction** coefficient d = optical path length c = concentration of absorbing material How much light gets through a solution depends on what’s/

radiation and connot be used below 360 nm –So silica cells are employed below thos wavelength Spectrum of Radiation **Beer** – **Lambert** **Law** As the cell thickness increases, the transmitted intensity of light of I decreases. transmitted light beam with intensity incident/ I I0I0 I 1 T I A CL = ECL by definition and it is called the **Beer** - **Lambert** **Law**. A = ECL A = ECL E =Molar **Extinction** Coefficient ---- **Extinction** Coefficient of a solution containing 1g molecule of solute per 1 liter of solution UNITS A = ECL /

**extinction**’ or optical Density (OD.)) rather than Transmittance for Quantitative analysis. Remember-0% T=Infinite A 0.1 %=3.0 A 1.0=2.0 A 10%=1.0 A 100%=0.0 A. 40 ¼ ¼ ¼ ( 1- ¼) 75%100% IoIo I (10mm ) 56.25% (75 - ¼) I 42.19% (56.25% - ¼) I 41 42 43 The **Beer**-**Lambert** **Law**/ of the solution the light is passing through. Both concentration and solution length are allowed for in the **Beer**-**Lambert** **Law**. Applications of UV – visible Spectrometry. 1]Quantitative Analysis-How much analyte is in the sample. 2]Qualitative/

, EDTA and phenol all absorb at 260 nm) How can concentration be determined by absorbance? DNA has a characteristic “molar **extinction** coefficient” The **Beer**-**Lambert** **law**: I = I o 10 - dc I = intensity of transmitted light I o = intensity of incident light = molar **extinction** coefficient d = optical path length c = concentration of absorbing material How much light gets through a solution depends on what’s/

throughout the night. Use of average **extinction** coefficients should be avoided. In a stable homogeneous atmosphere, the log of the transmitted flux decreases linearly with atmospheric path length (airmass) **Beer**’s **Law** (**Beer**-**Lambert** **Law**) Real Observations Most nights do / http://www.webshots.com/g/33/621-sh/19001.html Wind Flow Commonly the data indicates that different **extinction** coefficients are required for different parts of the night Pre-meridian observations Post-meridian observations One should expect /

… From the **beer**’s **law**, the absorbance against the conc. A straight line passing through origin is obtained (linear graph) However, deviation might occurs. Deviation is due to the following factors:- A foreign substance having colour particle may affect the absorption & **extinction** coefficient. Deviation also occur if colored solute ionized or/ atomic emission is far more convenient to qualitative emission. Quantitative analysis Quantitative analysis with spectroscopic methods is based on the **Beer**-**Lambert** **Law**.

1 < A < 1.0 Figure © David P. Goldenberg, University of Utah, 2003 The **Beer**-**Lambert** **Law**: A = C · l · A = absorbance C = concentration l = cuvette pathlength = **extinction** coefficient – specific for a particular wavelength – specific for a particular compound The electromagnetic spectrum http://www/optical density (OD) – often, the wavelength of light is denoted, e.g. OD 600 = absorbance at 600 nm = A 600 The **Beer**-**Lambert** **Law**: A = C · l · For double stranded DNA, – has been found to be 20 L/(g cm) – C /

**Beer**’s **law**(also called **Beer**-**Lambert**’s **law**) : If I 0 is incident light intensity and I t is transmitted light intensity, dependence of I t /I 0 on the concentration c and the path length l Molar **extinction** coefficient /the ‘level’ of molecule molecule - Study term symbol and application - **Beer**-**Lambert**’s **Law** : connection between theoretical allowed and forbidden transition to theoretical allowed and forbidden transition to experimental spectroscopy experimental spectroscopy - Real application : genome /

increase as the concentration of the molecule increases. The amount of light that blocked by the fixed amount of solution is called absorbance The **Beer**-**Lambert** **Law** The **Beer**-**Lambert** **law** sortof has the wrong name… Pierre Bouguer (1698-1758) Johan **Lambert** (1728-1777) **Extinction** coefficient Concentration Path length UV- Vis Spectrophotometer Spectrophotometer: use of electromagnetic radiation to measure the absorbance that is directly proportion with concentration UV/

J. Paul Robinson, Purdue University Lecture0004.ppt © 1990-2012 J. Paul Robinson, Purdue University Lecture0004.ppt **Extinction** Using **Beer**’s **law** (**Beer**-**Lambert** **law**) for light travelling through a curvette thickness d cm containing n molecules/cm3 ln (Io/I) = / PM © 1990-2012 J. Paul Robinson, Purdue University Lecture0004.ppt Properties of Fluorescent Molecules Large **extinction** coefficient at the region of excitation High quantum yield Optimal excitation wavelength Photostability Excited-state lifetime Minimal/

Shapiro p 85 4 th Ed. Shapiro p 110 © 1990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt Slide 4 **Extinction** Using **Beer**’s **law** (**Beer**-**Lambert** **law**) for light travelling through a curvette thickness d cm containing n molecules/cm 3 ln (I o /I) = /Phycobiliproteins are stable and highly soluble proteins derived from cyanobacteria and eukaryotic algae with quantum yields up to 0.98 and molar **extinction** coefficients of up to 2.4 × 10 6 © 1990-2005 J.Paul Robinson, Purdue University Lecture0004.ppt Slide 22/

other materials Folie 14 “COLOUR” DEPENDENT ATTENUATION intensity after layer thickness x fraction of transmission fraction of absorption **Lambert**-**Beer** **law** IoIo I x dx Folie 15 additive base colours subtractive red, green, blue yellow, magenta, cyan TVcolour / intensity WHY DO BEES FIND THEIR DIRECTION HOME? 18DG CGSWHP 2016 Folie 19 POLARISATION one orientation survives light **extinction** by certain crossed filters CaCO 3 organic foils with macromolecules etc. 19 Folie 20 TRANSVERSAL WAVES separation of/

the analyte in the flame. The concentration value in the AAS is based on the **Lambert**-**Beer** **law**: with : E - **extinction**, T - light transmission expressed as a percentage (transmission), ID - intensity of the transmitted signal, I0 - intensity of the / in attenuation ( c) of the source radiation. Since most of the source radiation occurs at the peak of the absorption line, **Beer**’s **law** is obeyed. The mass spectrum of a standard rock sample obtained by laser ablation / ICP-MS. Atomizers 1) Flame atomization : the/

How to calculate the concentration According to beer_law The **Beer**-**Lambert** **law** (**Beer**’s **law**) mathematically establishes the relationship between concentration and absorbance in many photometric determinations. **Beer**’s **law** is expressed as A = abc The concentration of substance/ is not easy to get off. The solution is also quite acidic. The UV spectroscopy requires an **extinction** coefficient to be determined. Samples treated with the Bradford assay. The brown sample (lower absorbance) contains /

nm (OD280) when using a pathlength of 1 cm. **Lambert**-**Beer** LawLambert-**Beer** **Law**: OD or Abs = log 10 I o /I = -log 10 I/I o = c l 97.2%100% Suppose we know that a protein contains 3 Trp and 4 Tyr residues. What is the **extinction** coefficient at 280 nm of the protein? **Extinction** coeff. (1 M of substance) per Trp is 5/

of absorption depends on the conc. And path length given by **Beer**-**Lamberts** **law**. For Absorption in UV – visible intensity of absorption depends on the conc., and hence given by **Beer**’s **law** While in IR absorption Spe While in IR absorption Spe., it/ n e- on chlorine atom are comparatively difficult to excite, while n e- on iodine atoms are loosely bound Magnitude of molar **extinction** coefficient (εmax) for a particular absorption αnal prob. of particular electronic transition. εmax for CH3I= 400, εmax for CH3Cl=/

t Absorbance is what is generally recorded from a spectrophotometer. 17 **Beer**’s **Law** More dissolved substance = more absorption and less transmittance. **Beer**-**Lambert**’s **Law** is: A = l C Log I o = l C I t A= Absorbance (no units) I o = intensity of incident light I t = intensity of transmitted light = molar **extinction** coefficient, molar absorptivity c = concentration of the absorbing species (mol/L/

) is less than I o. ItIt IaIa I0I0 As the cell thickness increases, I t (transmitted intensity of light ) decreases. **LAWS** OF ABSORBTION OF LIGHT ItIt **Lambert**’s **law**: length-dependent I = I o e -kL or A=kL Where ‘k’ is a constant, e = base of natural / or A=kC Where ‘k’ is constant and ‘c’ = concentration solution. Combining both **Lambert**’s - **Beer**’s **law**, we have: I = I o e -kLC or A=kLC A =-lgT= k L C k: **extinction** coefficient L: length of the light path C: concentration Blank: This will help to exclude the/

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