NIKHIL.K.POTDUKHE Outline of UV spectrophotometer Outline of - - PowerPoint PPT Presentation

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 projection systems (e.g. 15 kW IMAX systems)

150 Watt Xe lamp

• A filter is device that allows the required wavelength to pass

A filter is device that allows the required wavelength to pass but absorb of other wavelength wholly or partially . but absorb of other wavelength wholly or partially .

• Filters are of two type -

Filters are of two type - Absorption filter Absorption filter – It work by selective absorption of unwanted – It work by selective absorption of unwanted

• light. Absorption filter is solid sheet of glass colored by
• light. Absorption filter is solid sheet of glass colored by

pigment which is dispersed in glass. pigment which is dispersed in glass. Interference filter Interference filter - It work by reflection of unwanted

• 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.

• 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.
• Prism is a transparent
• ptical element with flat,

polished surfaces that refract light.

• The prism disperse the light

radiation in to individual colors or wavelength.

 Polystyrene  340-800 nm  Methacrylate  280-800 nm  Glass  350-1000 nm  Suprasil Quartz  160-2500 nm

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.

• 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

• xide.

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.

dynodes

electrons

anode high voltage

voltage divider network

light photocathode

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

• r 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

• ther 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

• nto 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

• rganisms.

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

• rganism.

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

• f 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

• ptical 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 reading)

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?