Analytical Techniques and Instrumentation PDF

Analytical techniques and instrumentation Analytic techniques It is a method of determining the chemical composition of sample (specimen) qualitatively and quantitatively. The foundation of measurement of modern clinical chemistry. It pertains to instrument, system or device used for the measuremen...

Analytical techniques and instrumentation Analytic techniques It is a method of determining the chemical composition of sample (specimen) qualitatively and quantitatively. The foundation of measurement of modern clinical chemistry. It pertains to instrument, system or device used for the measurement of an analyte in a solution. Analytical examination Includes: 1.Separation 2.Identification 3.Quantification which employs different analytical technique. ANALYTICAL EXAMINATION Uses Analytical To separate Analyte in techniques biological To identify and specimen To quantify instrumentat ion Analytic techniques III. Nephelometry and turbidimetry I. Photometry IV. Electro chemistry a. Photoelectric colorimetry a. Electrophoresis b. Spectrophotometry b. Ion selective electrode (ISE) c. Atomic absorption V. Chromatography spectrophotometry (AAS) a. GC-gas chromatography d. Flame emission spectrophotometry b. HPLC- high pressure liquid (FES) chromatogaphy e. Infrared spectrophotometry f. Mass spectrometry (MS) II. Luminescence spectrometry a. Fluorometry b. Chemiluminescence Spectrometry The study of interaction between light and matter. The study of electromagnetic radiation (light) emitted or absorbed by a chemical in a solution. All molecules absorbed light. Electromagnetic radiation (EM) Electromagnetic radiation Electromagnetic radiation (light) is a combination of electronic and magnetic vibration that travels in a wave- like manner (oscillation). Wavelength and frequency Wavelength – the distance between 2 peaks of wave, measured in nanometer (nm) Frequency- the number of waves produce in a specific time (1 second). Wavelength and frequency Wavelength is inversely proportional to frequency and energy. The shorter the wavelength the higher the frequency and energy and vice versa. Electromagnetic spectrum region Infrared light Visible light (narrow. UV light Above 780 nm. Long Below 380 nm. region) wavelengths Can see by the eye, Short wavelengths Low frequency waves, Harmful to body made up of colors. not harmful. Non- From 380nm to 780nm. due to extreme ionizing high energy which Examples radio waves, causes ionization. microwaves. Example gamma rays and X rays. Visible light spectrum (region) Note: visible light is the most commonly used electromagnetic radiation in measuring different analyte in the sample. Beer-Lambert’s Law Beer’s law States that the intensity of transmitted light is inversely proportional to concentration of the solution while absorbed light is directly proportional concentration of the solution. Beer’s law mathematical expression Equation: A= abc A- The absorbance of the solution ( no unit) a- Molar absorptivity- a measure of how well the molecule absorbs the particular wavelength of radiation b- path length -the distance travel by light through the solution ( in cm) c- The concentration of the solution (mol/L) Absorbance is directly proportional to, molar absorptivity, path length and the concentration of solution. Beer’s law Transmitted light or Transmittance (T) can be calculated as follows: : T =I/Io I= the intensity of light (transmitted light) after it passed through the sample. Io= the intensity of light (incident light) striking the sample. Beer’s law How to calculate percentage transmittance (%T) Simply: %T=T X 100 Beer’s law Absorbance (Abs) is a called optical density (OD). Can be calculated as follow: A= -log (T), or 2 -log (%T), no unit for absorbance. T is calculated from transmittance (T =I/Io) %T is inversely proportional to absorbance. Which means: The higher the transmittance the lower the absorbance and vice versa. Transmittance vs Absorbance Transmittance Absorbance 100 % Determine the absorbance of the 75 % following % transmittance 50 %. 25 % 10 % A= -log (T) or 2 -log (%T) Transmittance is inversely proportional to the concentration of solution Absorbance is directly proportional to the concentration of solution Limitations of Beer’s law a. simultaneous absorption at multiple wavelengths b. absorption of light by other species c. transmission of light by other mechanisms like fluorescent solution d. when a very wide range of concentration is measured, deviation from Beer’s law occurs REMEDIES 1. use of blank solutions a. water blank – corrects interference by instrument itself b. reagent blank – corrects interference by colored reagents c. serum blank – corrects interference by test sample 2. use of Allen correction measure absorbance at peak wavelength and at two wavelengths equidistant from the peak Lambert’s law The intensity of transmitted light decreases as the thickness or path length (diameter of test tube) increases through which the light travels. Beer- Lambert’s Law States that the amount of absorbance by a color solution is directly proportional to the concentration of the solution and the length of a light path through the solution.  Beer’s law Lambert’s Beer- law Lambert’s Law Transmitted light is Transmitted light is The absorbance is inversely proportional inversely proportional directly proportional to to the concentration of to the path length (the the concentration of solution. diameter of the tube). the solution and the diameter of the test Absorbed light is The wider the diameter tube or path length. directly proportional to of the test tube the the concentration of lower the transmitted solution light and vice versa. Sample problem The intensity of light incident on a sample is 50 w/m2 and the intensity of light transmitted is 25 w/m2 at 500 nm. Find the transmittance and absorbance of the sample. : T =I/Io % T= Tx100 A= -log (T) or 2 -log (%T) Standard curve/calibration curve Use to find the concentration of unknown solution. Tube Concentrat absorbanc Procedure ion (mg/dl) e 1. Make a series of standard solution. 2. Measure the absorbance of each standard solution. 1 60 0.15 3. Graph the concentration vs 2 70 0.26 absorbance (standard curve). 4. Measure the absorbance of the 3 80 0.55 unknown and graph to the standard curve. 4 90 0.9 Note: the concentration of unknown must be within the concentration of 5 100 1.25 standard curve. 6 110 1.30 Standard curve/calibration curve Tube Concentrati absorbance on (mg/dl) 1 60 0.15 2 70 0.26 3 80 0.55 4 90 0.90 5 100 1.25 6 110 1.30 Unknown ? 0.80 Finding the concentration of unknown solution Au Concentration of unknown = -------- x concentration of standard As PHOTOELECTRIC COLORIMETRY PRINCIPLE: 1. White light passes through an appropriate filter which absorbs light of all wavelength except the wavelength denoted by the particular filter. 2. Light passes through the colored solution (unknown or standard) and this colored solution absorbs some of the light depending on the intensity of the color. 3. The transmitted light impinges on a photoelectric cell which converts the light energy to electrical energy and this is recorded on a galvanometer as either percent transmittance or absorbance (optical density). Note: photoelectric colorimetry uses only visible light and measures only colored solution. Spectrophotometry Spectrophotometer A combination of spectrometer and photometer that measures the amount of light absorbed through light transmittance to determine concentration in the solution. It can measures concentration of solution in visible or UV lights. much like photoelectric colorimetry except the use of monochromator like diffraction grating or quartz prism to disperse white light into a systemic array of its component colors or wavelengths while colorimeter uses filters of specific wavelength. Note: spectrophotometer used visible, UV and infrared light for measurement of analyte. It can analyzed both colored and non-colored solution. Spectrophotometer PRINCIPLE: Light passes through a monochromator and is spread into a spectrum of colors. Slit blocks off all but a narrow band of the light. Sample absorbs some of this light and transmits the rest. The transmitted light strikes a detector which sends a signal to an amplifier. This small signal is amplified and presented as transmittance, absorbance or concentration units by a read-out devices like meters, digital displays, printed read-outs and recorders. Basic components of spectrophotometer 1. Light source 2. Monochromator (with entrance and exit slits) 3. Sample cells (cuvettes) 4. Photodetector 5. Readout device Basic components of spectrophotometer Light source UV light (below 300 Visible light (300-700 Infrared light (above nm) nm) 700 nm) 1. Hydrogen lamp 1. Tungsten-iodine lamp 1. Merst glower 2. Deuterium lamp 2. Mercury/sodium vapor 2. Globar (silicone 3. Mercury arc lamp lamp carbide) 4. Xenon lamp 3. Hollow cathode 5. Hollow cathode Note: Tungsten light bulb- commonly used light source in visible and near infrared region. Deuterium lamp- used in UV region. Basic components of spectrophotometer Tungsten light bulb- commonly used light source in visible and near infrared region. Deuterium lamp- used in UV region. Basic components of spectrophotometer Monochromator- isolates the specific wavelength or band of light from the light source. - It has entrance and exit slits which regulates the degree of light the passes through. Types 1. Glass filter- less expensive, but not precise. Made up of semi transparent silver film used in photoelectric colorimetry. 2. Prism- it disperse the white light into several spectrum. It is rotated to isolate desired wavelength. 3. Diffraction gratings- the most commonly used because it isolates a linear and widely distributed wavelength. It can operates in visible and UV light. Basic components of spectrophotometer Prism- made up of glass, quartz or sodium chloride (wedge shape). A narrow light focus on a prism is refracted as it enters the more dense glass. Rotated to a specific wavelength. Diffraction gratings- made up of parallel groves into aluminized surface of a flat piece of crown glass. Wavelenght is bent when it strikes the diffraction gratings.  Types of monochromator Entrance slit- concentrate the white light towards the monochromator. Minimizes the entrance of stray light and scattered light into monochromator. Stray light –is the most common cause of loss of linearity at high analyte concentration. Exit slit- concentrates the specific wavelength towards the sample cell. Allows a narrow of light beam to reach the cuvette or sample cell. HOLMIUM OXIDE – use to check the accuracy of wavelength setting in spectrophotometer. Band of visible light (350-700 nm) Wavele Color 476-495- green blue 496-505- blue green ngth 556-575- yellow green (nm) 350- 430 Violet 431-475 Blue 506-555 Green 576- 600 Yellow 601- 650 Orange 651- 700 Red  Band of visible light (350-700 nm) Lambda max (λmax)- The wavelength at which a substance has its maximum absorbance of light. Basic components of spectrophotometer Sample cells- also known as cuvettes, made of glass round or square shape. - It holds the colored solution or test sample. - Must be free from scratches to prevent the bouncing of lights that passes through. Types 1. Glass cuvettes- for visible light spectrophotometer. E.g. borosilicate, alumina silica. 2. Quartz cuvettes- for UV light spectrophotometer. Basic components of spectrophotometer Light/Photo detector- converts light energy transmitted into electrical energy. The reading is directly proportional to the intensity of light. e.g. photocell, photo tube, photomultiplier tube, photo iodide Photomultiplier tube (PMT)- most commonly used detector due to its sensitivity and rapid response. Has cathode, a light sensitive metal, that absorbs light and emits electrons in proportion to the radiant energy that strikes the surface of the light-sensitive material. Basic components of spectrophotometer Readout system- displays the output of photodetector. It could be meter or digital display e.g. galvanometer, ammeter, LED display Types of spectrophotometer 1.Single beam spectrophotometer 2.Double beam spectrophotometer Types of spectrophotometer Single beam spectrophotometer - Uses single beam of light - Wavelength is at 325 nm to 1000 nm. - The light travels in a linear direction and the test solution and blank are read in the same. - One measurement at a time at one specific wavelength. Types of spectrophotometer Double beam spectrophotometer - Operates at 185 nm to 1000 nm wavelength. - It has two beam of light split by monochromator. One beam is used for reference and the other for sample reading. - Advantage is it eliminates the error which occurs due to fluctuations in the light output and the sensitivity of the detector. APPLICATION OF SPECTROPHOTOMETER 1. Analyses the concentration of a solution qualitatively and quantitatively. It can measure: - Colored solution using visible light spectrophotometer - Colorless solution using UV light spectrophotometer. 2. Analyses the rate of reaction in a solution. - Example: enzyme analysis. 3. Determines the molecular weight of some molecules such as proteins and sugar. 4. Detection of impurities of a solution. Types of spectrophotometer Chemistry analyzer whether semi automated or fully-automated uses the principle of spectrophotometry. It calculates the absorbance, concentration of unknown as well as it can generate QC chart. Inside of a spectrophotometer  Atomic absorption spectrophotometry (AAS) AAS Principle: The measurement of the concentration of the sample is determined by measuring the light absorb by an atom instead by a molecules. It uses hollow-cathode lamp as source of light. The atoms in the sample are vaporized (atomized) using a flame, the sample absorbs this light in proportion to its concentration and the intensity of the transmitted light for the particular element can be measured by photo detectors Modern AAS is flameless. AAS very sensitive technique which gives rapid results and avoids time-consuming chemical extractions Clinical application: Analyses trace metals in blood and urine such 1. magnesium 2. copper 3. lead. 4. Calcium  Flame emission spectrophotometry (FES) FES Principle The sample is atomized by flame, emitted light produced by excited atom is detected and measured to determine the concentration of solution. this emitted is proportional to the concentration of the analyte. Metal ion Color of flame Lithium Red Sodium Yellow Potassium Lilac Calcium Brick-red/red orange Magnesium Blue FES Clinical application: measures electrolytes such as Na, K, or Li. Development of Ion selective electrode (ISE) made FES obsolete.  AAS and FES both use flame to atomized the atom (ground state to excited state) AAS determine the FES determine the concentration of sample by concentration of sample by measuring the light absorbed measuring the light emitted by ground state atom. by excited atom. INFRARED SPECTROPHOTOMETRY - designed to measure the vibrational spectrum of a sample by passing infrared radiation through it and recording the frequencies that have been absorbed and the extent to which they have been absorbed. - since the amount of absorbed energy is a function of the number of molecules, infrared spectrophotometry provides both qualitative and quantitative information - use to analyze the composition of calculi or stones Luminescence spectrometry Fluorometry Principle Occurs when a molecule absorbed light at a specific wavelength and reemits light at a longer wavelength (fluorescence). This fluorescence is detected and measured to determine the concentration of solution. It is more sensitive than spectrophotometry due direct measurement of emitted light. But is sensitive to environment changes. Fluorescence the intensity of the fluorescent light emitted by a sample under constant input of light intensity is directly proportional to the concentration. Fluorometer basic component Light source 1. Mercury arc lamp- above 350 nm 2. Xenon arc lamp- 470 nm to 600 nm 3. Tungsten lamp- for visible region only 4. Turnable dye lasers- 360 to 650 nm Fluorometer basic component Monochromators 1. Excitation monochromators- isolates light absorbed by molecules. 2. Emission monochromators- isolates light reemitted by molecules. Filters Primary filter- absorbs visible light and transmits UV light Secondary filter- Absorbs UV light and transmits visible light. Fluorometer basic component Cuvettes Made of glass or silica that is round or rectangular test tube. The path length or diameter is 1 cm. The surface should free from scratches and dust to prevent interferences. Fluorometer basic component Detectors 1. Photo cell 2. Photo tube 3. Photomultiplier- widely used because of accuracy. FLUOROMETRY Advantages : It is sensitive and specific due to selection of optimal wavelength. Disadvantage: sensitive to environment. Performance may affect by changes in pH and temperature. Fluorometry application 1. Detection of for the determination of porphyrins, hormones, amino acids, vitamins and catecholamine. 2. Detects molecules separated from Chromatography and Electrophoresis.. 3. Used in microbiology for detection of infectious microorganism. 4. Used in molecular biology for detection of specific nucleic acid sequences. 5. Used in Serology for detection of target antibodies. Chemiluminescence Principle A chemical reaction (oxidation reaction) emits light which then detect and measure. The luminescence produced is directly proportional to the concentration of solution. It does not require excitation of sample. No monochromator is needed. Enzyme is employed to maximize the efficiency. Chemiluminescence Advantage 1. Speed of reading 2. Easy to use 3. Simple instrumentation Application: mostly in immunoassays (Ag-Ab reaction). Nephelometry and Turbidimetry Nephelometry and Turbidimetry Nephelometry and Turbidimetry used in determining the concentration of insoluble particulate suspended in a sample as a result of Antigen-Antibody interaction. These technique both measure concentration and size of the particulate. In Chemistry it is used in determining proteins in biological sample. In Serology it is used in determining the types of antibody. Nephelometry Light scattered (reflected) is measure at 90 º angle with respect to beam of light. The intensity of light scattered is directly proportional to the concentration of solution. Used for the measurement of Ag-Ab complexes. Turbidimetry Light transmitted (passing through the solution) is measured at 180 º angle with respect to beam of light. The transmitted light is inversely proportional to the concentration of solution. The more light is blocked the more concentrated the sample. Turbidimetry Used for 1. Measurement of protein in urine and CSF 2. Detection of bacterial growth in broth culture 3. Broth antimicrobial test 4. Detect clot formation Electrochemistry Ion selective electrode (ISE) Measures the activity of ions in a solution through detection of electrical potential. The concentration of an analyte in a sample is measured through its activity. Most automated machine that analyses electrolytes such Na, K, Cl and gases in blood sample employ ISE technique. ISE components 1. Ion selective electrode- selects the ion of interest and excluding the other 2. Internal reference electrode- found inside the ISE made up of silver wire coated with AgCl suspended in KCl this solution contains the same ion to be analysed. 3. Milli-voltmeter- connects the ISE and the reference electrode. detects the electrical potential. To facilitate measurement the 2 electrodes is immersed in a solution. APPLICATION OF ISE Measures the following in a blood sample 1. Electrolytes such Na, K, Cl, Mg, Li 2. Blood pH and gases such as CO2 and O2 3. Haemoglobin ISE Advantage 1.Easy to use 2.Cheap 3.Real time measurement 4.Positive and negative ions can be measured Electrophoresis Analytical technique that is used for separation of molecules from mixture. It is accomplish by applying electric current on the molecules which separates them based on difference of net charges. It is not a diagnostic procedure and employed only for specimen preparation. It uses gel as medium to facilitate the separation. In clinical chemistry is mostly used for the separation of complex molecules like protein and lipid. ZONAL ELECTROPHORESIS use of solid support medium and is routinely done in the laboratory the material (serum) to be electrophoresed is applied as a small spot or narrow band and separation occurs into discreet spots or bands example of support media: paper, cellulose acetate, agarose gel, polyacrylamide gel dye such as bromphenol blue and Ponceau S is used to stain the various fractions so that they can be quantitated by densitometry important in the fractionation of serum and CSF proteins, lipoproteins, hemoglobin and isoenzymes IMMUNOELECTROPHORESIS(IEP) process by which serum proteins are first separated by electrophoresis and then allowed to react by diffusion against polyvalent or monospecific antiserum use in assessing protein abnormalities particularly dysgammaglobulinemia Electrophoresis application 1. In Chemistry it is used to separate different proteins from biological fluid such as CSF. 2. In molecular biology it used to separate DNA or RNA fragments. Chromatographic technique Chromatography Is a process of separating complex mixture (sample) on the basis on solubility (polarity) or ionic charges of molecules. Widely used in clinical laboratory for separation , identification and quantification of:Sugar, amino acids, proteins, lipids, drugs and its metabolites, hormones, enzymes, vitamins, DNA in biological fluid. Basic components of Chromatography chromatography Composed of layer of solid Stationary phase or liquid absorbed on a solid support or column. Mobile phase Composed of liquid or gas mixed with sample. Chromatography 1. High performance liquid chromatography (HPLC) 2. Gas chromatography (GC) High performance liquid chromatography (HPLC) Separation is based on difference in polarities. Uses pressure pump to force the solvent and the sample into a separation column. For fast separation. a. Stationary phase: polar b. Mobile phase: non-polar Reverse HPLC- non polar (stationary phase) polar (polar phase). Most commonly used technique in clinical laboratory for separation of drugs, hormones and drug metabolites due to less toxic and less flammable. Gas chromatography (gas-solid/gas- liquid) Components are separated as vapors Stationary phase- column coated Mobile phase- gas or volatile (sample mixed with gas and then vaporized). Mass spectrometry (MS) Measures mass at the molecular level. Separation is based on mass - charge ratio (m/z). Coupled with GC or HPLC for maximum sensitivity and specificity Chromatography Application in clinical laboratory 1. Drug testing 2. Neonatal screening 3. Hemoglobin analysis 4. TDM- therapeutic drug monitoring 5. Toxicology- identification of drugs in the blood. Chromatography Gas chromatography mass spectrophotometry (GC/MS)- gold standard for the detecting the drug metabolites in the blood. Confirmatory test for drugs of abuse. Liquid chromatography mass spectrophotometry (LC/MS)-more simple than GC/MS. Used in 1. Toxicology 2. Vitamin D determination 3. Testosterone determination ULTRACENTRIFUGATION - use to measure the rate of sedimentation of different classed of proteins with a gravitational field of about 250,000 grams. - rate of sedimentation is expressed in Svedberg unit (s) which is equal to 10-13 absolute units (cm per second per dyne per gram) - lipoproteins can be studied by using solvent with higher density so that lipoproteins float - groups found are HDL, LDL and VLDL - also used to clear lipemic serum Volumetric (titrimetric) Principle Unknown sample is made to react with known solution in the presence of indicator. volume of reagent, as a solution is added, is measured in a buret and the process of adding a reagent from a buret is called TITRATION. Example 1. Schales and schales method (chloride test) 2. EDTA titration method (Calcium test) References Bishop, Michael. 2018. Clinical Chemistry:Principles and Procedures, 8th ed. C and E Publishing Inc. Henry, John. 2002. Clinical Diagnosis and Management by Laboratory Methods, 22th ed. Merriam and Webster Inc. Tumamao, Aldrin. 2019. Clinical Chemistry Notes