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Experimental Biochem (Molecular cloning/Recombinant DNA (Clone multiple…

- - - - Chromophores (parts of molecule that can absorb energy of UV-visible light region) [responsible for colour of compound - complementary colour of light not absorbed]: usually pi e- or heteroatom with unpaired valence e-
        sigma e-: form single bonds & usually strongly held (low energy level), thus difficult to excite. Usually requires EM waves with shorter wavelength (more energy) but cannot be observed since it exceeds UV wavelength.
        pi e-: used to form double or triple bonds & loosely held (high energy level), thus easier to excite. Usually occurs in EM waves with shorter wavelengths (eg visible light ard purple region or UV)
        lone pairs/n e-: not held (high energy level), hence easier to excite. Promotion to sigma anti-bonding orbital may occur at any region in UV range (dependent on presence of conjugation) and promotion to pi anti-bonding orbital is more easier & occurs at EM waves with longer wavelengths (eg visible light ard red region)
        Usually consists of saturated & unsaturated heterocycles, carbonyls, alkenes, alkynes, nitriles etc but for conjugated systems, delocalisation of pi e- allow for even greater ease of excitation, thus requiring EM waves with generally longer wavelengths.
        For proteins, they absorb light in UV range due to presence of aromatic side-chains in aa residues & for nucleic acids, they absorb light in UV range due to purine & pyrimidine bases [heterocycles]
        Molecules analysed at absorption maxima (wavelength for max absorption by compound) so as to determine minute changes in conc (more sensitive) eg b-carotene analysed at 455 to 483nm
        Molecules with little/no chromophores (Eg water, alcohol) are used as solvents for spectroscopy (limited absorption)
        **Complementary colours: 400 - 430nm [violet] (complementary colour - yellow). 430 - 490nm [blue] (complementary colour - yellow), 490 - 560nm [green] (complementary colour - red), 560 - 580nm [yellow] (complementary colour - violet), 580 - 600nm [orange] (complementary colour - blue), 620 - 800nm [red] (complementary colour - blue)
      - Measurement of light: Beer-Lambert's law - lg (IO/IT) = A = ebc, where IO is intensity of incident radiation & IT is intensity of transmitted radiation, A is absorbance value, b is path length/distance of cell, c is conc of sample [mol/L] & e is molar absorptivity/molar extinction coefficient [absorbance of 1M/L of solution at particular wavelength in 1cm cell]
        Every compound has a different molar absorptivity constant in different wavelengths of the EM spectra
        Absorbance value can be affected by the compound (instrinic chemical properties eg presence of how many chromophores, concentration of sample & presence/absence of auxochromes [fx grp with lone pairs that alters wavelength & intensity of absorption when attached to chromophore - electron-withdrawing or electron-donating groups]) or by the environment the compound is in (pH [affect structural integrity of molecule], temp [affect the ease of excitability of e-] & choice of solvent [affect the degree of ionisation/solubility])
        *Purity ratio used to compare purity of sample (fixed ratio if sample is pure) & absorbance value of different/same compounds compared using hypochromic [wavelength used remains unchanged but molar absorption coefficient decreases eg higher conc of impurity present/lower conc of product/presence of electron-withdrawing substituents on the aromatic rings that decrease electron density], hyperchromic [opp of hypochromic], hyposochromic [peak of molar absorption coefficient occurs at lower absorbance wavelength eg decrease in temp, hence more energy required] & bathochromic [opp of hyposochromic]
      - Presence of monochromator (entrance slit, dispersing source eg prism & exit slit [moveable - restrict wavelength of light transmitted to compound]) between source & sample to restrict wavelength of UV/VIS used. Detector: measure amount of light absorbed
      - Qualitative comparison of results
        UV absorbance: monitor bond breaking in DNA strands due to temp increase (single strands absorb more UV)
        Visible absorbance/colorimetric assay: differentiate fx groups present in compounds using transition metals eg Benedict's solution to test for reducing sugars & non-reducing sugars
        Quantitative comparison of results
        UV absorbance: determine unknown conc of sample by comparing absorbance values of known conc
        Visible absorbance/colorimetric assay: determine unknown conc of sample by comparing against values of know conc
        Absorbance value on spectrophotometer indicates ratio of incident ray to transmitted ray, thus must invert ratio and subtract by 100% to find percentage of light absorbed
    - - Presence of excitation monochromator (entrance slit, dispersing source eg prism & exit slit [moveable - restrict wavelength of light transmitted to compound]) between source & sample to restrict wavelength of UV/VIS used, followed by a fluorescence monochromator (more sensitive than that of UV/VIS as fluorescence is easier to detect) between sample & detector. Detector: measure amount of light emitted
      - Fluorophore (extended chromophore/auxochrome system with rigid structure eg atropisomer): may contain several fused aromatic groups (steroidal structure) or planar/cyclic compounds with multiple pi bonds & rigidity allows for the relaxation to V0 (radiationless deactivation) due to rotation/vibration of side chain.
        Molecules usually have 2 distinct spectra & are analysed at emission spectrum (similar peak to excitation spectrum but of a lower wavelength [Stoke Law/Shift])
      - Applications: Staining of nucleic acids (binds to DNA grooves or as intercalators) & dyes usually have a higher emission spectrum after binding [used to detect presence & size of DNA fragments in gel electrophoresis] or staining of antibodies/immunofluorescence (study mode of action/binding of antibodies on target antigens) & new dye colour indicates co-localisation (two different antibodies bind to/recognise similar antigen) so as to determine subcellular localisation of proteins/receptors during drug treatment