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Prof. of Clinical Chemistry, Mansoura University.

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1 Prof. of Clinical Chemistry, Mansoura University

2 Most methods in clinical chemistry are based on quantitative measurement of a coloured compound produced when a sample containing the substance to be measured is mixed with appropriate reagents and subjected to certain reaction conditions. The radiation most often employed in photometric analysis has the following wave lengths.

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4 glass prismdiffraction grating White light can be dispersed into its constituent wave lengths by being refracted through a glass prism or a diffraction grating Anatural dispersion of light occurs when a rainbow is formed with the light from the sun being dispersed into its various colours by the rain drops from acloud. redblue green If a solution absorbs light completely it appears completely black but if a solution absorbs only part of the light energy passing through it, it will appear coloured so a solution of haemoglobin appears to be red because it absorbs blue green light and transmitts the complementary colour of red.

5 the molecular and atomic structure of the substance. The ability of a substance to absorb selectively certain wave lengths of light while transmiting others is determined by the molecular and atomic structure of the substance. The wave – length of choice is generally the one at which the greatest absorbance occurs.

6 Absorptivity constant: This depends upon: The wave length of the radiation. The wave length of the radiation. The nature of the absorbing material. The nature of the absorbing material. It is reasonabe that a more concentrated solution or longer light path should absorb more light since in either case there are more light absorbing molecules placed in the path of light. i-e-a yellow blue light A cromophore exhibits the complementary colour to that which it absorbs i-e-a yellow component appears yellow because it absorbs blue light. Thus it must be estimated in the blue region of the spectrum.

7 Photometric measurements measure Light intensity without Consideration to wavelength. To isolate a narrow range of the incident wave length use: Filters ( photometer ) prisim or gratings ( spectrophotometer ) Electromagnetic radiation is photons of energy packets travelling in waves Electromagnetic radiation includes radian energy from short wavelength ( x rays, 6 rays )to long wavelength (radio) waves, Visible ligh falls in between

8 Light: radiant energy with wavelength visible to the human eye and with wavelength bordering on those visible to the human eye ( nm ). Energy (E) is inversely proportional to the wavelength. UV rays with short λ has energy more than the infrared ( 750 nm E ). A wave length of light is defined as the distance ( ) peaks as the light is envisioned to travel in a wave like manner.

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10 The distanace ( ) peaks in the UV and visible is measeured in Angstroms (Ao), nanometers (nm) or millimicrons (mu): There are Ao, 10 9 nm, or 10 9 mu in 1 meter (SI unit is nm = 10 A = 1 mu) Radient energy that passes through an object will be parlially Reflected Absorbed And transmitted Beers law concentration Light absorbed transmitted Light The concentration of a substance is directly proportional to the amount of Light absorbed or inversely proportional to the Logarithm of the transmitted Light.

11 (A) % of original incident light transmitted by equal layers of light- absorbing solution; (B) % Tver­sus concentration on linear graph paper (C) % Tversus concentration on semilog graph paper; (D) A versus concentration on linear graph paper.

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15 Absorbance (A) = Extinction (E) Extinction coefficient (EC ): is the extinction measured with a light path 1 cm long. specific ( EC ) : is that measured with a light 1 cm long and concentration of 1% Molecular ( EC ) : is the extinction measured with a light path 1 cm long and a concentration of a gram molecule per litre ( molar Absorpitivity )

16 Components of spectrophotometer

17 (1) Light source Range of spectrum Stability of radiant energy Temperature A ) Visible region : Tungesten lamp and quartz – halogen ( nm). Suitable for moderatly dilute soln = colour changes significantly with change in concentration Operate for hr 15% visible + mostly near infrared Quartz: withstand higher temp. Aheat absorbed filter,between sample and the lamp to absorb the infrared is used.

18 B) U.V : Low pressure mercury – vapour lamp: Used at certain wavelength emits a sharp. Line spectrum with both uv and visible lines - medium and high pressure mercury lamp emits from uv to mid – visible region. Hydrogen and deuterium lamps ( nm): Provide continous spectra Deuterium : More stable Longer half life than hydrogen lamp

19 C) Laser source To obtain an intense, narrow – wavelength ligh source light EmissionLASER The technique of light Amplification by Stimulated Emission of Radiation (LASER ) is tried to be used. Certain material has the capability of absorbing energy → excited state when change to low energy level (decay) emitte light (highly quantified light) Different materials to give different wavelengths (e.g argon nm.)

20 (2) Monochromators Isolalation of part of the spectrum (individual wavelength of light ) depend on: Monochromator. Monochromator. Width of entrance and exit slits. Width of entrance and exit slits. (a) Filters : The spectral purity of a filter or other monochromat or is described in term of its spectral bandwidth: B.W is measured in nm at a point equal to one half the peak transmitance of the spectral transmittance curve The use of high intensity light favors the use of narrow bandpass interference filters.

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22 (i) Transmission absorption filter : colored- glass filters coloured gelatin sandwiched between two glass plates inexpensivesimple not precise (ii) Interference filters: used to obtain spectral purity and to eleminate the harmonic wave lengths it can be constructed to pass a very narrow range of wavelength with good efficiency

23 (b) Prism: glass prism for visibl. quartez prism for uv. short wave refracted more than the long. (c) Diffraction gratings : most common many parrallel grooves on polished surface ( e.g alloy of alum. copper on flat galass plate ) diffract the light = seprate it into component wavelengths (wavelengths are bent as they pass a sharp corner ) because the multiple spectra cause stray Light, accessory filters are used provide much narrower wavelength than the filters. the spectrum or plate is moved so that only the specific wave –length band desired pass only through the slit.

24 Fiber optics = Light pipes are a bundles of thin transparent fibers of glass, quartz or plastic enclosed within material of a lower refractive index, transmit light throughout their lengths by internal reflection. Adv.: better directional control and single beam multiplexity. Adv.: better directional control and single beam multiplexity. Disadv: stray light and solarization (loss of energy and decreased optical sensitivity. Disadv: stray light and solarization (loss of energy and decreased optical sensitivity.

25 (3) Cuvets (cell) : Glass or plastic ( 320 – 1000 nm ) Silica ( quartz ) ( below 320 nm ) May be square or round The square is better because of : a) flat surface to light ( no reflection or refraction) b) easier to line up the same side The cuvet must be: Optically clear. No scratching on the surface to avoid scattering of light Clean in soln of: conc HCL: water: ethanol (1 : 3 : 4 ), or distilled water. Avoid hot acids or alkalies. Optical bath 1cm – macrocuvet, microcuvet, flow through.

26 (4) Photodetectors : To convert the transmitted radiant energy into an equivalent amount of electrical energy

27 a) Photocell or barrier – Layercell : The least expensive. Composed of: film of light sensitive material on plate of iron (selenium ) thin transparent layer of silver over the light sensitive material Light excitation of the electrons on the light sensitive material which release and flow to the highly conductive silver where electromotive force can be measured used in filter photometers with a wide band pass producing high level of illumination so that there is no need to amplify the signal - temp sensitive and non linear

28 b) phototube : Outside voltage is required for operation Cathod composed of rubiduim or lithium which emit electrons when exposed to light c) Photomultiplier ( PM ) tube: Detect and ampilify radiant energy Light stricks coated cathode,whichs absorbs light and emit electrons Attrating to a series of anodes (dynodes ) which composed of material give off many secondary electrons(multiple cascade of electrons) current signal measured in ampers. 200 times more sensitive than the phototube Extremly sensitive to very low light levels and short duration light flashes.

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30 d) photodiode : produces current proportonal to the incident radient power. the cell put before the gratting used where light is adequate photodiode array (PDA ) detectors are available in integrated circiuts containing 256 to 2048 photodiodes in a linear arrange ment Each photodiode responds to a specific wavelength and so complete uv / visible spectrum can be obtained in one second has excellent linearity, speed,small size

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32 5- Galvanometer : 1. Direct reading 2. Ampilified reading 3. Digital 4. Microprocessor Future instruments 1-Thermo – Lens effect Laser → heating → refractive index change of the soln This thermolens effect can be extremely sensitive as an absorption detector 2- Piezelectric detector : Absorption of energy gas volume changes or slight temp changes sensed by membrane current or potential generating device.

33 Types of spectrophotometers 1. Single beam sp. 2. Double beam sp:

34 All the compenents are duplicated except the light source and the meter. In the single beam instrument any alteration in wavelength of the beam will necessitate readjustment of the output device to zero absorption for the blank solution before an absorption on the sample is possible. This requirement is no longer present for the double beam instrument which permits automatic change of wave – length and continuous display of absorbance. Compansate for changes in intensity of light source And also for changes in absorbance of the reagent blank as the

35 - Double beam in time spectrophotomet : if the mirror is used after exit

36 Source of errors in photometic measurements ( performance characteristics ) Photometric measurements involve finding extinction at a particular wave length and calculation of concentraton from this measurement, many procedures involve direct comparision with standards In some cases the extenction of unknwn compared directly with that pridicted for relevant pure substance when the molecular extinction coefficiert is known as enzyme determinations linked to NADH or to p nitr ophenol in the standardization of thyroxine or bilirubin solutions and for the determination of proteins.

37 Factors which need consideration are: Accuray of the wavelength Accuray of measurements of extinction The effect of stray light The linearity of calibration curve 1-Wave length accuracy: molar absorptivity constant Knowledge of exact wavelength becomes critical when using published molar absorptivities for identification of substances in toxicological studies and in the use of differential absorption techniques e.g Enzyme assay using NAD – N ADH reaction are based on a molar absorptivity constant for NADH of 6.22x10 at 340 nm.

38 A narrow bandwidth is needed for light sensitivity and beers law is more likely to be obeyed over a wider rang in monochromatic light. Avariety of methods is available to check wavelength & extinction accuracy: using spectral lamp sources : certain lamp give eimission at certain wave length using glass filter with earth element give band at certain wavelength as holmium oxide glass which is used for the narrow spectral bandwidth instruments holmium oxide glass may be scanned over the range of 280 to 650 nm

39 This material shows very sharp absorbance peaks at well defined wavelengths A solution of holmium oxide in dilute perchloric acid may also be used. W.L. accuracy

40 For broader bandpass instruments a didymium filter may be used this filter shows a minimum percent transmittance at 530 nm against an air blank. W.L. accuracy

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43 Stray light

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46 Turbidimety & Nephelometry Some analytical methods result in the formation of an insoluble product in finely divided form, so that the particles remain in suspension. if abeam of ligh is passed through such a suspension some of the light is scattered the tyndall effect, this result in reduction of intensity of the original beam. the variation in the intensity of the scattered light in various directions depends on the size and shape of the scattering particles, the wavelength of the light and the refractive indices of the solvent and particles.

47 It involves the measurement of the reduction of the intensity of the incident beam and so similar to the study of the absorption of light in colorimeters or specterephotometers. Turbidimetry Are made with the usual types of photemeter. The extinction & therefore the sensitivity increases with decrease in wavelength. However the selection of the suitable wavelength is affected by the position of the absorption peaks of other substances which may be present. If small stable and reproducible particle size can be obtained with out sedement, the extinction is proportional to the concentration of the insoluble material in some cases over awid range.

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49 Nephlometry It studies the intensity of the scattered light at right angls to beam incident to the cuvet similar to the measurement of the emitted light using a fluorimeter

50 Advantages over specterophotometry: Sensitivity Wide nange of concentration measurable Greater precisien Specific Ag- Ab complexes and a laser saurce have been combind to provide high spicificty and high precision Example for nephlomotric procedurs: lipoperoteins proteins by immunologicol methools amylase (amyloclastic method).

51 Precautions for turbidimetry and nephilometry: Particle size of the standard must be the same as the substance measured Avoid setting of particle by adust time for reading and use gelatin or arabic gum to provide a viscous meduim retard settling

52 Atomic absorption spectrophotometry light source. hollow –cathod lamp (inert gas,anode and cylindrical cathod) when voltage is applied the gas is ionized ions attracted to the cathode collide with the metal excited metal ions emite light (this metal the same as that will be measured (alloye may be used). burner ( O2, H2, air ):

53 The sample is aspirated atomized and excited → to the ground state and emitte light The emitted light from the light source excite the sample atoms in the ground state which excited, then emited light which passed to monochromator to isolate the specific wavelength measured by PM tube.

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55 To differentiate between light beem emitted by the hollow-cathod lamp and that emitted by excited atoms in the flam use: A mechanical rotating chopper between light and the flam or; by pulsing the electric supply to the lamp.

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57 Used to measure light emitted by excited atoms e.g., Na+, K+. The flame is used to break up the chemical bands to produce atoms. Then the atoms absorb energy and become excited. Return to the ground state and emote light energy → monochromator → detector.

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60 Fluorescence is a physical energy process that occurs when certain compounds absorb electromagnetic radiation, become excited and then return to their original energy level. Since the energy given off is less than or equal to that absorbed, the waver length of the light being given off will longer (will be emitted). The excited state persists for less than 10 nanoseconds (decay time 10-8 sec.).

61 Components of the fluoromiter: (1) Energy source: Mercury are lamps or xenon lamp Will produce enough energy. Absorption transion to high energy level within the molecular. (2) Inlet and exit slits: Perpendicular Prevent the incident high energy from reaching the detector.

62 (3) Monochromators: The first to isolate wavelengths before excitation of the substance in the cell. The second will selectively remove unwanted wavelengths before they fall upon the detector.

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64 Types: Filter fluorimeter Spectrofluorimeter Precautions (Interference): PH and temp. control. Avoid contamination to avoid extrafluorescence or diminished fluorescence by other substances (quenching). The use of high purity solvents (fluorescence grade). Avoid turbidity and air bubbles, to avoid light scattering. Nitric acid is a good cleaning agent for glassware.

65 Checking performance: A set of fluorescence glass standards. Known concentrations of quinine sulphate. Uses: Fluoroimmunoassay and immunofluorimetric assay.

66 Chemiluminescence or bioluminescence

67 The excitation results from chemical reaction. This chemical reaction is oxidation process require O 2 or H 2 O 2. e.g.; Luminol reaction, acridinium esters.

68 In the luminol reaction → dicarboxylate is excited → emits photoms when return to it's ground state. No monochromators are required because chemiluminescence arise from one species.

69 Adv.: Subpicomolar detection lemit. Disadv.: impurities can cause background signal that degrade sensitivity and specificity.

70 Enhanced chemiluminescence technique: Increase efficiency by enhancer system in the reaction with an enzyme. The time course of the high intensity is much longer (60 min.) vs 30 sec. in the conventional.

71 Mass spectrometry

72 It is a technique that can provide information concerning the elemental composition and structure of organic compounds: The arrangement of the functional groups. The molecular weigh (upto a.m.u). Submicrogm quantities in biological matrix (compounds, drugs, metabolites).

73 Principle: Ionization of the compound. Filtration through electrostatic or magnetic field. Identification according to mass (m) and to charge (z) ratio (m/z). Sufficient excess energy can be imported to the molecular ion to generate many fragment ions that can be separated, measured and recorded from the smallest fragment to the intact ion to produce a mass spectrum.

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