NIKHIL.K.

POTDUKHE
Outline of UV spectrophotometer

Outline of Recombinant DNA technology

Application of UV spectroscopy in recombinant DNA

technology

References
Lambert law:
“When a beam of light is allowed to pass through a
transparent medium, the rate of decrease of intensity
with the thickness of medium is directly proportional
to the intensity of the light”.

Beer law:
“The intensity of beam of monochromatic light
decreases exponentially with the increase in
concentration of the absorbing substance
arithmetically”.
A = abC or A = ξbC

A: absorbance
b : Sample Path length
C : Sample Concentration
a : Absorbance Constant
ξ : Molecular Absorbance
Constant
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
 Fluorescence or Phosphorescence of the sample
 Changes in refractive index at high analysis concentration
 Shifts in chemical equilibria as a function of concentration

 The recorder assembly

The spectrometer itself – this houses the

lamps, mirrors, prisms and detector. The
spectrometer splits the beam of radiation
into two and passes one through a sample
and one through a reference solution (that is
always made up of the solvent in which you
have dissolved the sample). The detector
measures the difference between the sample
and reference readings and communicates
this to the recorder.
The samples are dissolved in a solvent which is transparent to UV
light and put into sample cells called cuvettes. The cells themselves
also have to be transparent to UV light and are accurately made in all
dimensions. They are normally designed to allow the radiation to
pass through the sample over a distance of 1cm
Cont…..

Spectrometric instruments have a common set of general

features. Here we look at specific features for the
UV/Visible experiment.

Sources: D2 lamp, W filament (halogen lamp), and

Xe arc lamp.
Wavelength Selectors: Filters and Monochromators.
Sample Containers: Fused silica, quartz, and glass.
Detectors: Phototube, PMT, photodiode, photodiode
array,CCD array
Strike a low voltage DC arc
in a lamp filled with D2.
Gives continuum emission
from 160 to 400 nm.
 Reflecting focusing assembly

Lamp

Condenser Lens

A heated W filament, gives off blackbody

radiation. Add a small amount of a halogen
gas. Sublimated W reacts with halogen to
form tungsten halide; does not deposit on
quartz cover (no blackening) but does
redeposit on filament (extends life).
 Tube filled with Xe (or
sometimes a mixture of Hg
and Xe), invented in 1940,
 commercialized in 1961 by
Osram. Pass a low voltage
DC current to excite Xe.
 The broad spectral output
closely resembles natural
daylight, and is often used in 150 Watt Xe lamp
projection systems (e.g. 15
kW IMAX systems)
• A filter is device that allows the required wavelength to pass
but absorb of other wavelength wholly or partially .
• Filters are of two type -
Absorption filter – It work by selective absorption of unwanted
light. Absorption filter is solid sheet of glass colored by
pigment which is dispersed in glass.
Interference filter - It work by reflection of unwanted light. A
semitransparent metal film is deposited on a plate of glass .
Then it is coated with dielectric material . When ray of light
incident on it part of light reflects back whereas remaining
light is transmitted
 A monochromator is an optical device that transmits a mechanically
selectable narrow band of wavelengths of light or other radiation
chosen from a wider range of wavelengths available at the input.
 Accepts polychromatic input light from a lamp and outputs
monochromatic light.
 Components : Entrance slit, Dispersion device, Exit slit
 Entrance slit provides narrow source light to avoid overlapping of
monochromatic light.
 Exit slit select narrow band of dispersed spectrum for observation of
detector.
•Diffraction grating is an optical component with a regular
pattern, which splits (diffracts) light into several beams traveling
in different directions.

•Grating consist of large number of parallel lines ruled on highly

polished surface such as alumina.

•The directions of these beams depend on the spacing of the

grating and the wavelength of the light so that the grating acts as a
dispersive element.
•Prism is a transparent
optical element with flat,
polished surfaces that refract
light.

•The prism disperse the light

radiation in to individual
colors or wavelength.
•Prisms are typically made out of glass, but can be made from
any material that is transparent to the wavelengths for which
they are designed.

•The resolution depends upon the size and refractive index.

• Prisms have higher dispersion in the UV region.
Successful spectroscopy requires that all materials in the beam path other than
the analyte should be as transparent to the radiation as possible. Also, the
geometries of all components in the system should be such as to maximize the
signal and minimize the scattered light.
 Polystyrene
 340-800 nm
 Methacrylate
 280-800 nm
 Glass
 350-1000 nm
 Suprasil Quartz
 160-2500 nm

•Keep the cuvette clean.

•Don’t clean with paper products (Kim-wipe); use optical paper.
•Store dry.
•Don’t get finger prints on them.
•Store carefully and gently.
Detectors :

• Phototube

• Photomultiplier tube

• Photodiode

• Channeltron
Phototube:
 Detector is composed of :
1. Photo cathode- It is coated with elements of
high atomic volume like Potassium or silver
oxide.
2. Collector anode
 Photo cathode liberates electron towards anode
when light incident on it
 Photomultiplier tube:
 It is most sensitive of all detector .
 In this detector multiplication of photoelectron by secondary
emission of electron achieved by using photo diode and series of
anode (dynodes)
 Up to 10 dynodes are used , maintained at 75 – 100 V higher than
preceding one
 It can detect very week signal.
light
dynodes
anode
electrons
photocathode

voltage divider network

high voltage
Photodiode:
A photodiode is formed by sandwiching an undoped layer of Si
between a heavily doped p-layer and a heavily doped n-layer.
Photons whose wavelength is between 400 nm and 1100 nm can be
absorbed in the intrinsic layer, producing an electron-hole pair. The
bias potential sweeps these carriers to the opposite regions,
producing a current in the external circuit.

Photodiodes are more sensitive than phototubes , but far less sensitive
than PMT’s, since they only generate ~1 electron-hole pair per photon.
On the other hand they are about the size of a transistor and require no
high voltage support.
A continuous dynode chain is built into a
single unit. Excellent and widely used electron
multiplier. If the front end is a photo emissive
surface then you have a compact “PMT”.
Channeltrons require high vacuum to operate.
Mechanism:
Cont….

A beam of light from a visible and/or UV light source

is separated into its component wavelengths by a prism
or diffraction grating. Each monochromatic (single
wavelength) beam in turn is split into two equal
intensity beams by a half-mirrored device. One beam,
the sample beam , passes through a small transparent
container (cuvette) containing a solution of the
compound being studied in a transparent solvent. The
other beam, the reference (colored blue), passes
through an identical cuvette containing only the
solvent. The intensities of these light beams are then
measured by electronic detectors and compared. The
ultraviolet (UV) region scanned is normally from 200
to 400 nm, and the visible portion is from 400 to 800
nm.
Single beam spectrophotometer:

This consist of tungsten lamp as source of light . This light

radiation focused on slit by using concave mirror. This light
passes through simple absorption filter where the only required
wavelength of light passed through it and passed through it and
falls on the sample cell where the solution to be analyzed is
present . The sample or standard solution absorbs apart of the
radiation and rest is transmitted . The intensity of transmitted
light is determined by photovoltaic cell.
Double beam spectrophotometer:

It is similar to that of single beam instrument . Here the

light beam after passing through filter is split into sample
beam and reference beam by using beam splitter . These
beam pass through sample and reference solution and fall
onto detector separately The final read out is in
absorbance or transmittance , obtained after electronic
manipulation of 2 detectors.
DNA is the keeper of the all the information needed to
recreate an organism.
 All DNA is made up of a base consisting of sugar,
phosphate and one nitrogen base.
 There are four nitrogen bases, adenine (A), thymine (T),
guanine (G), and cytosine (C). The nitrogen bases are found
in pairs, with A & T and G & C paired together.
The sequence of the nitrogen bases can be arranged in an
infinite ways, and their structure is known as the famous
"double helix".
 The sugar used in DNA is deoxyribose.
The four nitrogen bases are the same for all
organisms.
The sequence and number of bases is what creates
diversity. DNA does not actually make the organism,
it only makes proteins.
The DNA is transcribed into mRNA and mRNA is
translated into protein, and the protein then forms the
organism.
Recombinant DNA is the general name for taking a
piece of one DNA and combining it with another
strand of DNA. Thus, the name recombinant!
Recombinant DNA is also sometimes referred to as
"chimera."
Determining the Concentration of Double-Stranded
DNA

Determining the Concentration of Single-Stranded

DNA Molecules

Molecular Weight of DNA

Oligonucleotide Quantitation

To determine the purity of Nucleic acid

Quantitation is typically performed by taking absorbance
measurements at 260 nm, 280 nm, and 320 nm.
Absorbance at 260 nm is used to specifically detect the
nucleic acid component of a solution.
 Absorbance at 280 nm is used to detect the presence of
protein (since tryptophan residues absorb at this
wavelength).
 Absorbance at 320 nm is used to detect any insoluble
light-scattering components.
 A spectrophotometer capable of providing a scan from
200 to 320 nm will yield maximum relevant information
Each of the four nucleotide bases has a slightly
different absorption spectrum, and the spectrum of
DNA is the average of them.
To determine the concentration of single-stranded
DNA (ssDNA) as a µg/ml amount, the following
conversion factor is used:

1 OD of ssDNA - 33µg /ml

 The average molecular weight of a DNA base is approximately
330 Daltons (or 330 grams/mole). The average molecular weight
of a DNA base pair is twice this, approximately 660 Daltons (or
660 grams/mole). These values can be used to calculate how
much DNA is present in any biological source. For small single-
stranded DNA molecules, such as synthetic Oligonucleotide, the
molecular weight of the individual nucleotides can be added to
determine the strand's total molecular weight (MW) according to
the following formula:

MW = (n A x 335.2) + (n c x 311.2) + (n c x 351.2) +

(n T x 326.2) + P
 where n x is the number of nucleotides of A, C, G, or T in the
Oligonucleotide and P is equal to-101.0 for dephosphorylated
(lacking an end phosphate group) or 40.0 for phosphorylated
Oligonucleotide.
An amount of an Oligonucleotide express in terms of
optical density (OD) units. An OD unit is the amount of
Oligonucleotide dissolved in 1.0 ml giving an A260 of
1.00 in a cuvette with a 1-cm light path length. It is
calculated by the equation:

OD units = (A260 ) x (Oligonucleotide volume) x (dilution

factor)
 Certain of the subunits of nucleic acids (purines) have an absorbance
maximum slightly below 260 nm while others (pyrimidines) have a
maximum slightly above 260 nm.
An A260/A280 ratio of 2.0 is characteristic of pure RNA

An A260/A280 ratio of 1.8 is characteristic of pure DNA

An A260/A280 of 0.6 is characteristic of pure protein

A ratio of less than 1.7 means there is probably a contaminant in

the solution, usually either protein or phenol.

DNA Purity (A260/A280) = (A260 reading – A320 reading)÷

(A280 reading – A320
 William Kemp(1991), Organic Spectroscopy ,3rd edition, MACMILAN,
Canada.

 Kori S.S. , Halkai M.A. (2000), Pharmaceutical Biotechnology, First

Edition, Vallabh Prakashan, Efficient Offset Printers, Delhi.

 Robert M. Silverstein, Francis x. Webster, David J. Kiemle (2005),

Spectrometric Identification of Organic Compound, 7th Edition, John
Wiley and Sons, USA.

 www.matcmadison.edu
 www.UVspectrum_2.html
 www.chem.purdue.edu/sciexpress
Questions?