This document discusses spectrophotometry and the Nanodrop instrument. Spectrophotometry involves measuring how much light is absorbed by a sample at specific wavelengths. The Nanodrop is a spectrophotometer that can measure extremely small sample volumes down to 0.5 microliters. It uses principles like Beer's law to calculate concentrations of nucleic acids, proteins, and other molecules from absorbance readings. Key applications of the Nanodrop include quantifying DNA, RNA, and proteins as well as measuring purity based on absorbance ratios.

1. Absorbance, spectrophotometry,
Nanodrop
Presented by
Yedhu Mohan

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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.

10. Wavelength

11. 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.

12. 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

13. 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.

14. 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

15. 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,

16. T = I/Io = 10-εlc
 Putting this in terms of absorbance, absorbance is directly
proportional to each of these factors. That is,
A = log10 (1/T) = εlc

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

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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

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• 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:

20. INSTRUMENTATION: The Spectrophotometer
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21. Basic components of a spectrophotometer
 a light source.
 a means to isolate light of desired wavelength.
 Cuvets.
 Photodetector.
 readout device.
 recorder and a computer.

22. Light Sources:
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• 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.

23. 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
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• 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)

24. LaserSources:
• 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
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25. Spectral Isolation:
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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.

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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.

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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.

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• 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)

29. 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
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30. APPLICATIONS:
1. 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
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31. 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
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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

33. NANODROP

34.  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.

35.  Instrument Specifications
 Minimum Sample Size : 0.5 μL
 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/μL dsDNA
 Maximum Concentration : 15,000 ng/μL (dsDNA)
 Measurement Time : < 5 seconds

36. 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

37. 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:

38. 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)

39. 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/μL
 • Single-stranded DNA: 33 ng-cm/μL
 • RNA: 40 ng-cm/μL

40. 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

42. 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

43. OD 260:280 RATIOS

44. 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

45. 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.

46. 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.

47. 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

48. 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.

49. 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

50. 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.

51. 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.

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THANK YOU

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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