Biochemical Techniques BY Augustine I. Airaodion PDF

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LECTURE NOTE ON BIOCHEMICAL TECHNIQUES (BCH 402) BY MR AIRAODION A. I.

Course Outline    

Spectrophotometry Chromatography Centrifugation Isotopic Techniques SPECTROPHOTOMETRY

Electromagnetic radiation (EM radiation or EMR) refers to the waves of the electromagnetic field, propagating (radiating) through space carrying electromagnetic radiant energy. Electromagnetic radiation has been put to many uses in our daily routine. The radio and television broadcasting, medical x-ray etc. are some common examples. The use of electromagnetic radiation in analytical chemistry and biochemistry gained much importance during the last 50 years for characterization of materials. Electromagnetic radiation in the region of 200 to 700nm is generally termed as light. The eye can perceive radiation between 340 to 650 nm and can distinguish it as various ROYGBIV. ROYGBIV is an acronym for the sequence of colour commonly described as making up a rainbow: Red, Orange, Yellow, Green, Blue, Indigo, and Violet. A rainbow spans a continuous spectrum of colours; the distinct bands are an artifact of human colour vision. In ROYGBIV, the colours are arranged in the order of decreasing wavelengths, with red being 650 nm and violet being about 400 nm. Spectrophotometry is the quantitative measurement of the reflection or transmission properties of a material as a function of wavelength. It is more specific than the general term electromagnetic spectroscopy in that spectrophotometry deals with visible light, near-ultraviolet, and near-infrared, but does not cover time-resolved spectroscopic techniques. Spectrophotometry uses photometers, known as spectrophotometers that can measure a light beam's intensity as a function of its colour (wavelength). Important features of spectrophotometers are spectral bandwidth (the range of colours it can transmit through the test sample), the percentage of sample-transmission, the logarithmic range of sample-absorption, and sometimes a percentage of reflectance measurement. A spectrophotometer is commonly used for the measurement of transmittance or reflectance of solutions, transparent or opaque solids, such as polished glass, or gases. Although many biochemicals are coloured, as in, they absorb visible light and therefore can be measured by colorimetric procedures, even colourless biochemicals can often be converted to coloured compounds suitable for chromogenic colour-forming reactions to yield compounds suitable for colorimetric analysis. However they can also be designed to measure the diffusivity on any of the listed light ranges that usually cover around 200 nm – 2500 nm using different controls and calibrations. Within these ranges of light, calibrations are needed on the machine using standards that vary in type depending on the wavelength of the photometric determination. PRINCIPLE OF SPECTROPHOTOMETER The spectrophotometer is a much more refined version of a colorimeter. In a colorimeter, filters are used which allow a broad range of wavelengths to pass through, whereas in the spectrophotometer, a prism or grating is used to split the incident beam into different wavelengths. By suitable mechanisms, waves of specific wavelengths can be manipulated to fall on the test solution. The range of the wavelengths of the incident light can be as low as 1 to 2nm. The spectrophotometer is useful for measuring the absorption spectrum of a compound, that is, the absorption of light by a solution at each wavelength. This is the basic Principle of spectrophotometry in biochemistry. Spectrophotometer works with the principle of Beer-Lambert‘s Law. Beer-Lambert‘s law (or Beer's law) is the linear relationship between absorbance and concentration of an absorbing species. It states that the absorbance of a solution is directly proportional to the concentration of the solution. The general Beer-Lambert‘s law is usually written as: A abc 1

A = a( ) * b * c where A is the measured absorbance, a( ) is a wavelength-dependent absorptivity coefficient, b is the path length, and c is the analyte concentration. When working in concentration units of molarity, the Beer-Lambert‘s law is written as: A=

*b*c

where is the wavelength-dependent molar absorptivity coefficient with units of M-1 cm-1. Data are frequently reported in percent transmission (I/I0 * 100) or in absorbance [A = log (I/I0)]. The latter is particularly convenient. Sometimes the extinction coefficient is given in other units; for example, A = E1% * b * c where the concentration C is in gram per 100 ml of solution. This is useful when the molecular weight of the solute is unknown or uncertain. Experimental measurements are usually made in terms of transmittance (T), which is defined as: T = I / Io where I is the light intensity after it passes through the sample and Io is the initial light intensity. The relation between A and T is: A = -log T = - log (I / Io). Modern absorption instruments can usually display the data as either transmittance, or absorbance. An unknown concentration of an analyte can be determined by measuring the amount of light that a sample absorbs and applying Beer's law. If the absorptivity coefficient is not known, the unknown concentration can be determined using a working curve of absorbance versus concentration derived from standards. Limitations of the Beer-Lambert’s Law The linearity of the Beer-Lambert‘s law is limited by chemical and instrumental factors. Causes of non linearity include: i.

Deviations in absorptivity coefficients at high concentrations (>0.01M) due to electrostatic interactions between molecules in close proximity

ii.

Scattering of light due to particulates in the sample

iii.

Fluoresecence or phosphorescence of the sample

iv.

Changes in refractive index at high analyte concentration

v.

Shifts in chemical equilibria as a function of concentration

vi.

Non-monochromatic radiation, deviations can be minimized by using a relatively flat part of the absorption spectrum such as the maximum of an absorption band

vii.

Stray light SPECTROPHOTOMETER INSTRUMENTATION

The essential components of a spectrophotometer instrumentation include: 1. A Stable and cheap radiant energy source

2

2. A monochromator, to break the polychromatic radiation into component wavelength or bands of wavelengths. 3. Transport vessels (cuvettes), to hold the sample 4. A Photosensitive detector and an associated readout system

1. Radiant Energy Sources Materials which can be excited to high energy states by a high voltage electric discharge or by electrical heating serve as excellent radiant energy sources. a. Sources of Ultraviolet Radiation: Most commonly used sources of UV radiation are the hydrogen lamp and the deuterium lamp. Xenon lamp may also be used for UV radiation, but the radiation produced is not as stable as the hydrogen lamp. b. Sources of Visible Radiation: ―Tungsten filament‖ lamp is the most commonly used source for visible radiation. It is inexpensive and emails continuous radiation in the range between 350 and 2500nm. ―Carbon arc‖ which provides more intense visible radiation is used in a small number of commercially available instruments. c. Sources of Infra–Red Radiation: ―Nernst Glower‖ and ―Global‖ are the most satisfactory sources of IR radiation. Global is more stable than the nearest flower. 2. Wavelength Selectors Wavelength selectors are of two types, viz: filters and monochromators 1. Filters: ―Gelatin‖ filters are made of a layer of gelatin, coloured with organic dyes and sealed between glass plates. 2. Monochromators: A monochromator resolves polychromatic radiation into its individual wavelengths and isolates these wavelengths into very narrow bands. The essential components of a monochromator are: 

Entrance slip – admits polychromatic light from the source



Collimating device – Collimates the polychromatic light onto the dispersion device.



Wavelength resolving device like a PRISM or a GRATING



A focusing lens or a mirror 3



An exit slip – allows the monochromatic beam to escape.

The kinds of the resolving element (prisms and gratings) are of primary importance PRISMS: A prism disperses polychromatic light from the source into its constituent wavelengths by virtue of its ability to reflect different wavelengths to a different extent. The degree of dispersion by the prism depends on upon: i. ii.

The optical angle of the Prism (usually 600) The material of which it is made

Two types of Prisms are usually employed in commercial instruments, viz: 60 0 cornu quartz prism and 300 Littrow Prism.

GRATINGS: Gratings are often used in the monochromators of spectrophotometers operating ultraviolet, visible and infrared regions. 3. Sample Containers Sample containers are also one of the parts of Spectrophotometer instrumentation. Samples to be studied in the ultraviolet or visible region are usually glasses or solutions and are put in cells known as ―CUVETTES‖. Cuvettes meant for the visible region are made up of either ordinary glass or sometimes Quartz. Most of the spectrophotometric studies are made in solutions, the solvents assume prime importance. The most important factor in choosing the solvent is that the solvent should not absorb (optically transparent) in the same region as the solute. 4. Detection Devices Most detectors depend on the photoelectric effect. The current is then proportional to the light intensity and therefore a measure of it. Important requirements for a detector include: i.

High sensitivity to allow the detection of low levels of radiant energy

ii.

Short response time

iii.

Long term stability

iv.

An electric signal which easily amplified for typical readout apparatus.

5. Amplification and Readout Radiation detectors generate electronic signals which are proportional to the transmitter light. These signals need to be translated into a form that is easy to interpret. This is accomplished by using amplifiers, Ammeters, Potentiometers and Potentiometric recorders.

4

APPLICATION OF SPECTROPHOTOMETER 1. Qualitative Analysis The visible and UV spectrophotometer may be used to identify classes of compounds in both the pure state and in biological preparations. This is done by plotting absorption spectrum curves. Absorption by a compound in different regions gives some hints to its structure. Absorption Range (nm) 220 to 280nm 220 to 250 nm

Structure or Type of compounds Aliphatic or alicyclic hydrocarbons or their derivatives The compounds contain two unsaturated linkages in conjugation or ―Benzene derivatives‖ 250 to 330 nm Presence of more than two conjugated double bonds usually gives rise to absorption. 450 to 500nm Beta-carotene, a precursor of Vitamin A has eleven double bonds in a conjugated system and appears yellow. 250 to 330 nm Vitamin K (249nm; 260nm and 325nm) (Due to the presence of ―NAPTHAQUINONE‖) 2. Quantitative Analysis Quantitative analysis method developing for determining an unknown concentration of a given species by absorption spectrometry. Most of the organic compounds of biological interest absorb in the UV-visible range of the spectrum. Thus, a number of important classes of biological compounds may be measured semiquantitatively using the UV-visible spectrophotometer. Nucleic acids at 254nm and protein at 280nm provide good examples of such use. The absorbance at 280nm by proteins depends on their ―Tyrosine‖ and ―Tryptophan‖ content. 3. Enzyme Assay This is the basic application of spectrophotometry. This assay is carried out most quickly and conveniently when the substrate or the product is coloured or absorbs light in the UV range. Let‘s consider few examples: a. Lactate Dehydrogenase (LDH) Lactate + NAD + ↔ Pyruvate + NADH + H+ 

The LDH is engaged in the transfer of electrons from lactate to NAD+.



The products of the reaction are pyruvate, NADH, and a proton



One of the products, NADH, absorbs radiation in the UV range at 340 nm while its oxidized counterpart, NAD+ does not.



The reaction in the forward direction can be followed by measuring the increment in the light absorption of the system at 540nm in a spectrophotometer.

b. Pyruvate Kinase Phosphoenolpyruvate + ADP ↔ Pyruvate + ATP Pyruvate + NADH + H+ ↔ Lactate + NAD + We have added a large excess of NADH to the system, the system now absorbs at 340nm. According to the above-given reactions, each molecule of pyruvate formed in the reaction, a molecule of NADH is oxidized to NAD+. Since NAD+ does not absorb at 340nm the absorbance goes on decreasing with increased pyruvate generation. Such measurements are known as ―Coupled assays‖. 5

4. Molecular Weight Determination Molecular weights of amine picrates, sugars and many aldehyde and ketone compounds have been determined by this method. Molecular weights of only small molecules may be determined by this method. a. Study of Cis-Trans Isomerism: Geometrical isomers differ in the spatial arrangement of groups about a plane, the absorption spectra of the isomers also differs. The trans-isomer is usually more elongated than its cis counterpart. Absorption spectrometry can be utilized to study Cis-Trans isomerism. b. Control of Purification: Impurities in a compound can be detected very easily by spectrophotometric studies. ―Carbon disulfide‖ impurity in carbon tetrachloride can be detected easily by measuring absorbance at 318nm where carbon sulfide absorbs. A lot many commercial solutions are routinely tested for purity spectroscopically. 5. Other Physiochemical Studies Spectrophotometry (UV-VIS) has been used to study the following physiochemical phenomena: i.

Heats of formation of molecular addition compound and complexes in solution

ii.

Determination of empirical formulae

iii.

Formation constants of complexes in solution

iv.

Hydration equilibrium of carbonyl compounds

v.

Association constants of weak acids and bases in organic solvents

vi.

Protein-dye interactions

vii.

Chlorophyll-Protein complexes.

viii.

Vitamin-A aldehyde – Protein complex

ix.

Determination of reaction rates

x.

Dissociation constants of acids and bases

xi.

Association of cyanine dyes

CHROMATOGRAPHY Chromatography is a physical method of separation that distributes components to separate between two phases (stationary and mobile phase) moving in a definite direction. It is a laboratory technique for the separation of a mixture. The mixture is dissolved in the mobile phase, which carries it through the stationary phase. The various constituents of the mixture travel at different speeds, causing them to separate. The separation is based on differential partitioning between the mobile and stationary phases. Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus affect the separation. The mobile can either be a liquid or a gas while the stationary phase is either a solid or liquid. Chromatography may be preparative or analytical. The purpose of preparative chromatography is to separate the components of a mixture for later use, and is thus a form of purification. Analytical chromatography is done normally with smaller amounts of material and is for establishing the presence or measuring the relative proportions of analytes in a mixture. The two are not mutually exclusive. Terms Used in Chromatography 1.

Analyte: is the substance to be separated during chromatography. It is also normally what is needed from the mixture. 6

2.

Chromatograph: is equipment that enables a sophisticated separation, e.g. gas chromatographic or liquid chromatographic separation.

3.

Chromatogram: is the visual output of the chromatograph. In the case of an optimal separation, different peaks or patterns on the chromatogram correspond to different components of the separated mixture.

4.

Eluate: is the mobile phase leaving the column. This is also called effluent.

5.

Eluent: is the solvent that carries the analyte.

6.

Eluite: is the analyte, the eluted solute.

7.

Eluotropic Series: is a list of solvents ranked according to their eluting power.

8.

Immobilized Phase: is a stationary phase that is immobilized on the support particles, or on the inner wall of the column tubing.

9.

Stationary Phase: is the substance fixed in place for the chromatography procedure. Examples include the silica layer in thin layer chromatography.

10.

Mobile Phase: is the phase that moves in a definite direction. It may be a liquid (LC and Capillary Electro-chromatography (CEC)), a gas (GC), or a supercritical fluid (supercritical-fluid chromatography, SFC). The mobile phase consists of the sample being separated/analyzed and the solvent that moves the sample through the column. In the case of HPLC the mobile phase consists of a non-polar solvent(s) such as n-hexane in normal phase or a polar solvent such as methanol in reverse phase chromatography and the sample being separated. The mobile phase moves through the chromatography column (the stationary phase) where the sample interacts with the stationary phase and is separated.

11.

Preparative Chromatography: is used to purify sufficient quantities of a substance for further use, rather than analysis.

12.

Analytical Chromatography: is used to determine the existence and possibly also the concentration of analyte(s) in a sample.

13.

Bonded Phase: is a stationary phase that is covalently bonded to the support particles or to the inside wall of the column tubing.

14.

Retention Time: is the characteristic time it takes for a particular analyte to pass through the system (from the column inlet to the detector) under set conditions.

15.

Sample: is the matter analyzed in chromatography. It may consist of a single component or it may be a mixture of components. When the sample is treated in the course of an analysis, the phase or the phases containing the analytes of interest is/are referred to as the sample whereas everything out of interest separated from the sample before or in the course of the analysis is referred to as waste.

16.

Solute: refers to the sample components in partition chromatography.

17.

Solvent: refers to any substance capable of solubilizing another substance, and especially the liquid mobile phase in liquid chromatography.

18.

Detector: refers to the instrument used for qualitative and quantitative detection of analytes after separation.

19.

Forward Phase Chromatography: is the chromatographic technique in which the matrix support or stationary phase is polar (e.g. paper, silica etc.).

20.

Reverse Phase Chromatography: is the chromatographic technique in which the matrix support or stationary phase is non-polar (e.g. C-18) 7

Planar Chromatography Planar chromatography is a separation technique in which the stationary phase is present as or on a plane. The plane can be a paper, serving as such or impregnated by a substance as the stationary bed (paper chromatography) or a layer of solid particles spread on a support such as a glass plate (thin layer chromatography). Different compounds in th...