Fourth Level Biochemistry Lecture Notes PDF

# Don't study Bio.... Love It ## For the Fourth Level Biochemistry Students ### Introduction to Spectroscopic Methods #### I. INTRODUCTION Spectrophotometry and colorimetry are analytical methods of measuring the amount of light absorbed by a substance in a solution. - Absorbance is characteris...

# Don't study Bio.... Love It ## For the Fourth Level Biochemistry Students ### Introduction to Spectroscopic Methods #### I. INTRODUCTION Spectrophotometry and colorimetry are analytical methods of measuring the amount of light absorbed by a substance in a solution. - Absorbance is characteristic for each substance, i.e. each substance absorbs light at a specific wavelength and transmits light at another specific wavelength, wave number(X). Since absorbance is related to the amount of a substance in the solution, it can be used for quantitative determination of the amount (concentration) of a substance in a solution. #### II. ELECTROMAGNETIC RADIATION & SPECTRA Electromagnetic radiation is composed of "photons" that move in a wave that oscillates along the pathway of motion. - WAVELENGTH (λ): is the distance between two adjacent peaks. - FREQUENCY (ν): is the number of waves passing a certain point per unit time. #### Mathematically, C = λν Where c is the speed of light (3 x 10⁸ m/s). - Photons of different wavelengths have different energies that are given by the equation; $E = hv = hc/λ$ Where h is "PLANCK'S CONSTANT" = 6.62 x 10⁻³⁴ J.s. ##### Regions of electromagnetic radiation | Region | Ranges from | | -------- | ----------- | | Ultraviolet (UV) region | 200-400 nm | | Visible region | 400-700 nm | | Infrared (IR) region | 700 nm-400 µm | | Radiowave region | 1-5 m | #### How does the spectrum arise?! - In a molecule, the energy of bonding electrons can be divided into three types: Electronic, Vibrational, and Rotational. - A molecule whose electronic, vibrational, and rotational energies are all at the lowest values is said to be in the "ground state". - The absorption of light can affect the energy of the bonding electrons, i.e. absorption of electromagnetic radiation involves a change in one or more of the three bonding energies. - Spectra arise when a molecule is irradiated by photons that correspond to the difference in energy between the ground state and some higher or excited state of the molecules. - Spectra in the UV or visible region arise due to transitions between electronic levels. - Spectra in the near IR region arise due to vibrational transitions. - Spectra in the far IR region arise due to rotational transitions #### Chromophore - A chromophore or "chromophoric group" is a specific grouping of atoms having unsaturation and absorbing light, e.g C=C, C≡C, carbonyl, carboxyl, amido, nitrile, nitro, nitroso, imidazole, indole, purine, pyrimidine, etc. - Absorption of UV and visible light occur only when a chromophore or more is present in a substance, due to conjugation of unsaturated bonds. #### Quantitative aspects of light absorption - Transmittance (T): the amount of light passing through a substance. Mathematically, T=T% = I/I₀ x 100 Where I is the "intensity of the transmitted light" I₀ is the "intensity of the incident light" - Absorbance (A): is the minus log of transmittance (T). Mathematically, A=-log T = -log I/I₀ - Absorbance depends on: - Length of the light path (b) through which the light passes. Mathematically, Aab = -log T = -log I/I₀ = A = k₁b "LAMBERT LAW" - Concentration (c) of absorbing substance. Mathematically, Aac = -log T = -log I/I₀ = A = k₂c "BEER'S LAW" - Combining the two laws (3) and (4) as the "LAMBERT-BEER LAW" gives equation 5: -log T = -log I/I₀ = A = abc - Where a is known as "extinction coefficient" incorporating k₁ and k₂. #### "Lambert law” states that "the negative logarithm of the transmittance, i.e. the absorbance, is directly proportional to the amount of light absorbed and to the length of the light path". Aab #### “Beer's law” states that "the negative logarithm of transmittance is directly proportional to the concentration of the absorbing substance". A/C #### Extinction coefficient (a) - The extinction coefficient depends on: - Wavelength of the light passing through the substance. - Chemical nature of the substance. - Concentration of the substance (c). - Length of the light path (b). - The extinction coefficient may be expressed as: - Molar extinction coefficient (am), and is then expressed in moles/liter. Hence, the units for (am) are liters per mole per centimeter (L mol⁻¹ cm 1) - Millimolar extinction coefficient (amm), and is then expressed in millimoles/liter. - Micromolar extinction coefficient (aµm), and c is then expressed in micromoles/liter. - The extinction coefficient can be obtained from: - Literature. - Measuring the absorbance of different concentrations of the substance, then a plot of the absorbance (A) versus concentration (c) should give a linear curve whose slope is the extinction coefficient (a) if b=1.00. #### N.B If the extinction coefficient is known and a fixed path length is established, the concentration of the substance can be determined by measuring the absorbance of the substance and using "Lambert-Beer law". A=abc → c=A/ab #### V. Limitations of Lambert-Beer law - As shown in Fig. (3-4), when the amount of light absorbed is greater than 50% (> 0.30 absorbance), errors become magnified. - Therefore, measurement of concentrations is best achieved between 0.05 - 0.30 absorbance or between 90 - 50% transmittance.