Experimental Biochemistry

 You have studied pure chemistry in  General chemistry  Organic chemistry  Biochemistry  Now you will study the Applied part in your chemistry program

Course objectives  Analytical tools and instrumental techniques, by which, they can extract, purify, identify and determine the different types of biochemical compounds.  The theory and data interpretation of instruments that are used for elucidation of the chemical structure of biochemical compounds such as NMR, IR and mass spectrometer.  The instruments that are used in the quantitative analysis of different biochemical compounds and elements such as spectrophotometer, and atomic absorption spectrophotometer.  Quantitative analysis of several biochemical compounds from different sample types by using different instruments are also studied in the practical part.

Course contents Spectroscopy quantitative analyses UV-Vis Spectrophotometer Atomic Absorption Structure elucidation Mass spectrometer IR spectrophotometer NMR spectrophotometer

Chromatography Techniques Paper chromatography TLC HPLC GC Mechanism

Centrifugation Radioactive isotopes

ExamDateMarks Quiz 12/4/ MidtermStart on 9/4/ Quiz 214/5/ Performance attendance and Activities 10 Practical final exam 21/5/ Final examsStart on 4/6/ Total 150

Dr. M. Abd-Elhakeem Faculty of Biotechnology Experimental Biochemistry Chapter 1

9 WHAT IS A WAVE? a wave is a disturbance that travels through a medium from one location to another. a wave is the motion of a disturbance

Radiation: Is an energy or particulate matter, traveling in the space. Electromagnetic radiation: Is a stream of energy (photons), traveling through space in a wave-like pattern at the speed of light

TYPES OF RADIATION Radiation is classified into: Ionizing radiation Non-ionizing radiation

A radiation is said to be ionizing when it has enough energy to eject one or more electrons from the atoms or molecules in the irradiated medium. This is the case of α and β radiations, as well as of electromagnetic radiations such as gamma radiations, X- rays and some ultra-violet rays. Ionizing Radiation

SOME PRINCIPLES Wavelength: distance between crests or between troughs Frequency: the number of crests of the wave that pass by a point every second

Electromagnetic spectrum The electromagnetic spectrum is the entire range of electromagnetic waves arranged according to their frequencies or wavelengths.

nm

ELECTROMAGNETIC RADIATION

Visible light: 400 to 700 nm – This is the only form of electromagnetic wave visible to the human eye. Ultraviolet: 200 to 400 nm - these cause our skin to darken (tan) and can even damage our skin. The ozone layer protects us from most UV radiation from the sun. X-rays: these EM waves pass through much matter. It doesn’t pass through dense bone, so it is useful in determining whether a bone is broken or not.

Gamma rays: these waves are very penetrating and can severely damage cells. Infrared: these waves are responsible for the heat we feel. Heat seeking missiles detect infrared sources such as tanks or aircraft. Microwaves: these waves are used obviously to heat food. As these waves pass through food, it causes the particles to vibrate resulting in the heating of the food. Microwaves are also used in communication.

Radio waves: these waves include both TV and radio waves.

Interactions of Matter with E.M. radiation

Spectroscopy: Is the branch of chemistry that study the interaction between electromagnetic radiation and matter

RADIATION AND MATTER Emission Absorption Luminescence (fluorescence – phosphorescence) Scattering or Reflection Transmission

γ-rayschanges in nuclear structure X-rays Inner-shell electronic transition UV-radiation Outer-shell electron transitions Visible region atoms and molecules. MicrowaveRotation of nucleus

IR radiation: vibrations of atoms in molecules

Flame test

29

30 This excited configuration is unstable, and the electrons "fall" back to their normal positions of lower energy. As the electrons return to their normal levels, the energy that was absorbed is emitted in the form of electromagnetic energy. Some of this energy may be in the form of visible light. The colour of this light can be used as a means of identifying the elements involved. Such crude analyses are known as flame tests. Only metals, with their loosely held electrons, are excited in the flame of a laboratory burner.

31 Thus, flame tests are useful in the identification of atoms. Many metallic elements exhibit characteristic colours when they are vaporised in the burner flame. If the light emitted is carefully analysed for its characteristic frequencies, it can be shown that each element has a characteristic pattern or fingerprint.

UV- Vis Spectrophotometry:

Different wavelengths of light correspond to different colors All colors blended together is called white light The absence of all light is black Light of slightly shorter wavelengths is ultraviolet THEORY

WAVELENGTH OF VISIBLE LIGHT AND COLOR WAVELENGTHCOLOR PERCEIVED Violet Blue Greenish Blue Bluish Green Green Yellowish Green Yellow Orange Red

When light shines on a solution, it may pass through – be transmitted – or Some or all of the light energy may be absorbed according to the matter chemical composition

Absorption occurs when the specific amount of energy (light of distinct wavelength) is absorbed by an electron resulting in a transition to an excited state. The part of the molecule that cause absorption called “Chromophore”

THE ABSORPTION OF LIGHT AND COLOR OF SOLUTIONS WAVELENGTH OF LIGHT ABSORBED COLOR OF LIGHT ABSORBED COLOR OF SOLUTION Violet Y ellow Blue O range Green R ed Yellow V iolet Orange B lue Red G reen Matter absorb specific light (wavelength) and appear in a complementary color

COLOR PLATE 13 The color intensity increase as the concentration of the molecule increases. The amount of light that blocked by the fixed amount of solution is called absorbance

The Beer-Lambert Law The Beer-Lambert law sortof has the wrong name… Pierre Bouguer ( ) Johan Lambert ( ) Extinction coefficient Concentration Path length

UV- Vis Spectrophotometer

Spectrophotometer: use of electromagnetic radiation to measure the absorbance that is directly proportion with concentration

UV-Visible Spectroscopy: Instrumentation In absorption spectroscopy, we measure  as a function of wavelength The instrument we use to do this is called a UV/visible Spectrophotometer The Major Components Are: A light source A monochromator A detector A Sample Compartment

Light Sources Xenon, Mercury/Xenon Flash Arc-Lamps – light generated from Xe plasma Pure Xenon has very wide emission spectrum ~200 – 1200 nm

UV/Visible Spectroscopy: Light Sources Deuterium D 2 gas is discharged by contact with a high voltage tungsten cathode Continuous spectrum from ~150 nm - ~370 nm Usually used in conjunction with a tungsten/halogen source, which handles the visible spectrum

Monochromators The light sources we use produce continuous emission spectra. But we need single wavelengths, so we need monochromator

Sample Compartments/Holders These days, sample compartments are designed to accept accessories… The sample itself is held in a cuvette, usually plastic or quartz:

The detector Silicon diode: Basically a solar cell – light ionizes n-doped (phosphate) silicon, placing the electrons in the conduction band (i.e. having a voltage).

Usage of spectrophotometer Scanning: Spectrophotometer is used to scan a sample in wide range of wavelengths (for example from 200 to 800nm). This is carried out to obtain the wavelength that has the maximum absorbance of this sample (lambda (λ) max)

Quantitative analysis using Spectrophotometer This part will be discussed in practical part