Absorbance

Absorbance and applications

1. Absorbance, spectrophotometry, Nanodrop Presented by Yedhu Mohan

2. 2 BASIC CONCEPTS

3. What is light? We see light as color and brightness ,It’s actually electromagnetic radiation electromagnetic radiation is an oscillating waves of electric and magnetic fields propagating through space-time, carrying electromagnetic radiant energy. It includes radio waves , microwaves , infrared , (visible) light , ultraviolet , X-rays , and gamma rays .

4. How does light form It all starts with ATOMS. A nucleus surrounded by electrons that orbit. If you add energy to an atom (heat it up), the electrons will jump to bigger orbits. When atom cools, electrons jump back to original orbits. As they jump back, they emit light, a form of energy The bigger the jump, the higher the energy.The energy determines color; a blue photon has more energy than a red

5. Electromagnetic spectrum The electromagnetic spectrum is the range of frequencies (the spectrum) of electromagnetic radiation and their respective wavelengths and photon energies .

6. What is a wavelength? A wavelength is the distance between the points where a wave repeats itself.

7. Wavelength

8.             The waves can pass through the object The waves can be absorbed by the object. The waves can be reflected off the object. The waves can be scattered off the object. The waves can be refracted through the object.

9. Theory of absorbance Light absorption occurs when atoms or molecules take up the energy of a photon of light, thereby reducing the transmission of light as it is passed through a sample . In order for a photon to be absorbed by the electron, it has to have exactly the right amount of energy, which means a specific frequency (i.e., wavelength). Photons that don't have the right frequency will not interact with that atom

10. Transmittance The amount of monochromatic light absorbed by a sample is determined by comparing the intensities of the incident light (Io) and transmitted light (I). The ratio of the intensity of the transmitted light (I) to the intensity if the incident light (Io) is called transmittance (T) . T = I / Io In practice, one usually multiplies T by 100 to obtain the percent transmittance (%T), which ranges from 0 to 100%. %T = T * 100 If the T of a sample is 0.40, the %T of is 40%. This means that 40% of the photons in the incident light emerge from the sample as transmitted light and reach the photodetector. If 40% of the photons are transmitted, 60% of the photons were absorbed by the sample .

11. Absorbance Absorbance is the amount of light absorbed by a sample. It is calculated from T or %T using the following equations: A = - log10 T or A = log10 (1/T) A = 2 - log10 %T

12. Beer–Lambert law The concentration (c) of a substance in a sample is one of three factors that affect the amount of light absorbed by a sample.  The other two are path length (l), that is the distance the light travels through the sample, and the extinction coefficient of the absorbing substance (ε).  The extinction coefficient is simply a measure of how strongly a substance absorbs light of a given wavelength.  The relationship between transmittance or absorbance and these three factors is expressed by  Beer's Law Beer's Law states that the intensity of transmitted light decreases exponentially as any one of these factors increases.   That is,

13. T = I/Io = 10 -εlc Putting this in terms of absorbance, absorbance is directly proportional to each of these factors. That is, A = log 10  (1/T) = εlc

14. Beer’s Law is followed only if the following conditions are met: Incident radiation on the substance of interest is monochromatic Solvent absorption is insignificant compared to the solute absorbance Solute concentration is within given limits Optical interferant is not present Chemical reaction does not occur between the molecule of interest and another solute or solvent molecule

15. 18 Spectrophotometry Photometry is the measurement of the amount of luminous light (Luminous Intensity) falling on a surface from a source. Spectrophotometry is the measurement of the intensity of light at selected wavelengths. The method depends on the light absorbing property of either the substance or a derivative of the substance being analysed

16. 4 When light passes through a solution, a certain fraction is being absorbed. This fraction is detected, measured and used to relate the light absorbed or transmitted to the concentration of the substance. This enables both qualitative and quantitative analyses of substances. The spectrophotometric technique is used to measure light intensity as a function of wavelength. It does this by :

17. INSTRUMENTATION: The Spectrophotometer 20

18. Basic components of a spectrophotometer a light source . a means to isolate light of desired wavelength . Cuvets . Photodetector. readout device. recorder and a computer.

19. Light Sources: 16 This provides a sufficient amount of light which is suitable for making a measurement. The light source typically yields a high output of polychromatic light over a wide range of the spectrum . Types of light sources used in spectrophotometers Incandescent lamps lasers .

20. Incandescent Lamps: Hydrogen Gas Lamp and Mercury Lamp, Xenon (wavelengths from 200 to 800 nm): high-pressure mecury and xenon arc lamps are commonly used in UV absorption measurements as well as visible light. Globar (silicon carbide rod): Infra-Red Radiation at wavelengths: 1200 - 40000 nm NiChrome wire (750 nm to 20000 nm); ZrO2 (400 nm to 20000 nm): for IR Region 17 Tungsten Filament Lamp: The most common source of visible and near infrared radiation ( at wavelength 320 to 2500 nm) Deuterium lamp: Continuous spectrum in the ultraviolet region is produced by electrical excitation of deuterium at low pressure. (160nm- 375nm)

21. Laser Sources: These devices transform light of various frequencies into an extremely intense, focused, and nearly non- divergent beam of monochromatic light Used when high intensity line source is required Unique properties of laser sources include: Spatial coherence: a property that allows beam diameters in the range of several microns Production of monochromatic light 18

22. Spectral Isolation: 25 A system for isolating radiant energy of a desired wavelength and excluding that of other wavelength is called a Monochromator . Monochromator consists of these parts : Entrance slit Collimating lens or mirror Dispersion element: A special plate with hundreds of parallel grooved lines. The grooved lines act to separate the white light into the visible light spectrum Devices used for spectral isolation include: Filters, Prisms, and Diffraction gratings.

23. 26 Cuvets: This is a small vessel used to hold a liquid sample to be analyzed in the light path of a spectrophotometer. May be round, square or rectangular and are constructed from glass, silica (optical grade quartz) or plastic. It should be without impunities that may affect spectrophotometric readings Quartz or fused crystalline silica cuvettes for UV spectroscopy. Glass cuvettes for Visible Spectrophotometer. NaCl and KBr Crystals for IR wavelengths.

24. 27 Photodetectors: These are devices that convert light into an electric signal that is proportional to the number of photons striking its photosensitive surface. The photocell and phototube are the simplest photodetectors, producing current proportional to the intensity of the light striking them The Photomultiplier tube (PMT) is a commonly used photodetector for measuring light intensity in the UV and Visible region of the spectrum. They are extremely rapid, very sensitive and slow to fatigue.

25. 28 The PMT consists of : A photoemissive cathode (a cathode which emits electrons when struck by photons) Several dynodes (which emit several electrons for each electron striking them) An anode – Produces an electric signal proportional to the radiation intensity Examples : Phototube (UV); Photomultiplier tube (UV-Vis); Thermocouple (IR); Thermister (IR)

26. Display or Readout Devices: Electrical energy from the detector is displayed on a meter or readout system such as an analog meter (obsolete), a light beam reflected on a scale, or a digital display, or LCD Digital readout devices operate on the principle of selective illumination of portions of a blank of light emitting diodes (LEDs), controlled by the voltage signal generated. Compared to meters, digital read out devices have faster response and are easier to read 29

27. APPLICATIONS: Measurement of Concentration: Prepare samples Make series of standard solutions of known concentrations Set spectrophotometer to the λ of maximum light absorption Measure the absorption of the unknown, and from the standard plot, read the related concentration 30

28. 2. Detection of impurities: UV absorption spectroscopy is one of the best methods for determination of impurities in organic molecules Additional peaks can be observed due to impurities in the sample and it can be compared with that of standard raw material 31

29. 32 3. Elucidation of the structure of Organic Compounds : From the location of peaks and combination of peaks UV spectroscopy elucidate structure of organic molecules: the presence or absence of unsaturation, the presence of hetero atoms 4. Chemical Kinetics: – Kinetics of reaction can also be studied using UV spectroscopy. The UV radiation is passed through the reaction cell and the absorbance changes can be observed

30. NANODROP

31. Nanodrop is a HUGELY useful machine for doing spectroscopy on extremely small volumes. This instrument scans over a range of wavelengths, does some baseline correction, and adjustment for Beer’s Law, and then measures, quantitatively, levels of absorption at a specific wavelength.

32. Instrument Specifications Minimum Sample Size : 0.5 μ L Pathlength : 1 mm (auto-ranging to 0.05 mm) Light Source : Xenon flash lamp Detector Type : linear silicon CCD array Wavelength Range : 190-840 nm Wavelength Accuracy : +1 nm Detection limit : 2 ng/ μ L dsDNA Maximum Concentration : 15,000 ng/ μ L (dsDNA) Measurement Time : < 5 seconds

33. Process 1 - 2 μL sample is pipetted onto a measurement pedestal. A smaller, 0.5 μL volume sample, may be used for concentrated nucleic acid and protein A280 samples . A fiber optic cable (the receiving fiber ) is embedded within this pedestal . A second fiber optic cable (the source fiber ) is then brought into contact with the liquid sample causing the liquid to bridge the gap between the ends of the two fibers . A pulsed xenon flash lamp provides the light source and a spectrometer utilizing a linear CCD array analyzes the light passing through the sample

34. Blanking and Absorbance Calculations When a NanoDrop spectrophotometer is blanked, a spectrum is taken of the reference solution ( blank)and stored in memory as an array of light intensities by wavelength. When a measurement of a sample is taken, the intensity of light that was transmitted through the sample is recorded. The sample intensities along with the blank intensities are used to calculate the sample absorbance according to the following equation:

35. The Beer-Lambert equation is used to correlate the calculated absorbance with concentration : A = ε * b * c A = the absorbance represented in absorbance units (A) ε = the wavelength-dependent molar absorptivity coefficient (or extinction coefficient) with units of liter / mol -cm b = the pathlength in cm c = the analyte concentration in moles/ liter or molarity (M )

36. Nucleic Acid Calculations For nucleic acid quantification, the Beer-Lambert equation is modified to use a factor with units of ng-cm/microliter. The modified equation used for nucleic acid calculations is the following: c = (A * ε )/ b c = the nucleic acid concentration in ng/microliter A = the absorbance in AU ε = the wavelength-dependent extinction coefficient in ng- cm/microliter b = the pathlength in cm The generally accepted extinction coefficients for nucleic acids are: • Double-stranded DNA: 50 ng-cm/ μ L • Single-stranded DNA: 33 ng-cm/ μ L • RNA: 40 ng-cm/ μ L

37. Absorbance Absorbance at 260 nm Nucleic acids absorb UV light at 260 nm due to the aromatic base moieties within their structure . Purines (thymine, cytosine and uracil) and pyrimidines (adenine and guanine) both have peak absorbances at 260 nm, thus making it the standard for quantitating nucleic acid samples. Absorbance at 280 nm The 280 nm absorbance is measured because this is typically where proteins and phenolic compounds have a strong absorbance. Aromatic amino acid side chains ( tryptophan, phenylalanine , tyrosine and histidine) within proteins are responsible for this absorbance. Similarly , the aromaticity of phenol groups of organic compounds absorbs strongly near 280 nm . Absorbance at 230 nm Many organic compounds have strong absorbances at around 225 nm. In addition to phenol , TRIzol , and chaotropic salts, the peptide bonds in proteins absorb light between 200 and 230 nm

38. A260/280 ratio The A260/280 ratio is generally used to determine protein contamination of a nucleic acid sample. The aromatic proteins have a strong UV absorbance at 280 nm . For pure RNA and DNA, A260/280 ratios should be somewhere around 2.1 and 1.8, respectively. A lower ratio indicates the sample is protein contaminated. The presence of protein contamination may have an effect on downstream applications that use the nucleic acid samples

39. OD 260:280 RATIOS

40. A260/230 ratio The A260/230 ratio indicates the presence of organic contaminants, such as (but not limitedto ): phenol, TRIzol , chaotropic salts and other aromatic compounds . Samples with 260/230ratios below 1.8 are considered to have a significant amount of these contaminants that will interfere with downstream applications. In a pure sample, the A260/230 should be close to 2.0

41. Measurement Ranges Micro Array- The NanoDrop measures the absorbance of the fluorescent dye, allowing detection at dye concentrations as low as 0.2 picomole per microliter. The software automatically utilizes the optimal pathlength to measure the absorbance of each sample.

42. UV-Vis The UV-Vis application allows the NanoDrop 2000/2000c to function as a conventional spectrophotometer. Sample absorbance is displayed on the screen from 190 nm to 840 nm . Up to 40 wavelengths can be designated for absorbance monitoring and inclusion in the report.

43. Protein A280 The Protein A280 application is applicable to purified proteins that contain Trp , Tyr residues or Cys-Cys disulphide bonds and exhibit absorbance at 280 nm

44. Proteins & Labels The Proteins & Labels application can be used to determine protein concentration (A280 nm) as well as fluorescent dye concentration (protein array conjugates). It can also be used to measure the purity of metalloproteins (such as hemoglobin ) using wavelength ratios.

45. Protein BCA The BCA ( Bicinchoninic Acid) assay is a colorimetric method for determining the total protein concentration in unpurified protein samples . It is often used for dilute protein solutions and/or proteins in the presence of components that have significant UV (280 nm) absorbance . Unlike the Protein A280 application, the Protein BCA application requires a standard curve be generated before sample protein concentrations can be measured

46. Protein Lowry The Lowry assay is an alternative method for determining protein concentration based on the widely used and cited Lowry procedure for protein quantitation. Like the other colorimetric assays, the Lowry Assay requires a standard curve be generated before sample proteins can be measured.

47. Protein Bradford The Bradford Assay is a commonly used method for determining protein concentration. It is often used for more dilute protein solutions where lower detection sensitivity is needed and/or in the presence of components that also have significant UV (280 nm) absorbance. Like the other colorimetric assays, the Bradford assay requires a standard curve be generated before sample proteins can be measured.

48. 52 THANK YOU

49. 53 REFERENCES: http://www.u.arizona.edu/~ gwatts/azcc/InterpretingSpec.pdf http:// employees.oneonta.edu/kotzjc/LAB/Spec_intro.pdf http:// www.mlz-garching.de/files/nanodrop_2000_user_manual.pdf Tietz Textbook of Clinical Chemistry The principles of use of a spectrophotometer and its application in the measurement of dental shades[2003] Fundamentals of UV-visible spectroscopy, Tony Owen, 1996 Spectrophotometry FUNDAMENTALS (Chapters 17, 19, 20), Dr. G. Van Biesen , Win2011