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Clinical Chemistry I

ANALYTIC TECHNIQUES AND INSTRUMENTATION Mandy A. Delfin, RMT

Analytic techniques and instrumentation provide the foundation for all measurements made in a modern clinical chemistry laboratory. The majority of techniques fall into one of four basic disciplines within the field of analytic chemistry: 1. Spectrometry spectrophotometry atomic absorption mass spectrometry [MS]

2. Luminescence fluorescence chemiluminescence nephelometry


3. Electroanalytic methods electrophoresis potentiometry amperometry


4. Chromatography gas liquid thin-layer


With the improvements in optics, electronics, and computerization, instrumentation has become miniaturized. This miniaturization has enabled the development of point-ofcare testing (POCT) devices that produce results as accurate as those provided by large laboratory based instrumentation.

COLORIMETRY PHOTOMETRY AND SPECTROPHOTOMETRY COLORIMETRY:

Colorimetry is a branch of quantitative analysis in which the quantity of colored constituent is determined by measuring the relative amount of absorption of light passing through a solution of the constituent. It has made possible the development of methods for analysis of even small amount of blood containing very minute concentrations of substance. The two primary considerations in colorimetric analysis are quality of color and

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Clinical Chemistry I
ANALYTIC TECHNIQUES AND INSTRUMENTATION Mandy A. Delfin, RMT

its intensity, and there are two types of colorimeters.

1. Visual Colorimetry:

As the name implies, visual colorimeters rely on human eye to match the intensities of color. This involves the comparison of an unknown solution with a series of colored standard solutions.

2. Photoelectric Colorimetry:

This involves the measurement of light intensity independently of wavelength. An electrical signal sensed by a detector measures the relative absorbance. Many determinations made in clinical laboratory are based upon measurements of radiant energy emitted, transmitted, absorbed, or reflected under controlled conditions. The term photometric measurement was defined originally as making a measurement of light intensity independently of wavelength, and most instruments used at present have some means of isolating narrow wavelength range of the spectrum for measurements. Those that use filters for this purpose are referred to as filter photometers, whereas those that use prisms or gratings are called spectrophotometers.

BASIC CONCEPTS

Electromagnetic radiation includes radiant energy from short wavelength gamma rays to long wavelength radio waves. The term light will be used to describe radiant energy with wavelengths visible to, and bordering to those visible to the human eye. The wavelength of light is defined as the distance between two peaks as light travels in a wavelike manner. This distance is expressed in nanometers (nm) for wavelengths commonly used in photometry. Formation of wavelengths Infra red (over 680 nm) (over 700 nm) Visible (350-680 nm) (380-700 nm) Below 350 (ultraviolet) (Below 380 nm)

NOTRE DAME OF MARBEL UNIVERSITY

Clinical Chemistry I
ANALYTIC TECHNIQUES AND INSTRUMENTATION Mandy A. Delfin, RMT

WAVELENGTH REGION COLOR COMPLEMENTARY NAME ABSORBED COLOR 180-200 Short Not visible U.V 220-380 U.V. Not visible 380-430 Visible Violet Yellow-green 430-475 Visible Blue Yellow 475-495 Visible GreenOrange blue 495-505 Visible Blue-green Red 505-555 Visible Green Purple 555-575 Visible Yellow Violet green 575-600 Visible Yellow Blue 600-620 Visible Orange Green blue 620-700 Visible red Blue green

Complementary Color

If a solution absorbs all the light between certain wavelengths, it will appear as different to the eye. Example if a solution absorbs all light between 575-600 nm which yellow, it will appear blue to the eye. Therefore blue is the complementary color of yellow.

SPECTROPHOTOMETRY This is one of the most important of all analytical methods because majority of the determination done in clinical chemistry laboratory are based on spectrophotmetric measurements. Basic Components of a spectrophotometer:

Principle: Light from the lamp is passed through a monochromator to provide a selection of the desired region of the spectrum to be used for measurement.

Slits are used to isolate a narrow beam of light source (the entrance slit provides light to come from a well defined point) and improve it chromatic purity. Light next passes through the cuvet containing the solution where a portion of the radiant energy is absorbed depending on its nature and concentration.

NOTRE DAME OF MARBEL UNIVERSITY

Clinical Chemistry I
ANALYTIC TECHNIQUES AND INSTRUMENTATION Mandy A. Delfin, RMT

Any light not absorbed is transmitted to the phototube which converts light energy to electrical signal, the output of which is registered on the meter

1. LIGHT SOURCE: It provides a continuous spectrum of white light which can be resolved to different wavelengths of interest.
Tungsten light bulb.

Acceptable for making measurements of moderately dilute solutions in which change in color intensity varies significantly with small changes in concentration. Quartz Halogen Lamp: with bromine or iodine vapour in the filament Does not supply sufficient radiant energy however for measurmenets below 320 nm.
Mercury Arc Lamp

Emits discontinuous of line spectrum and useful for calibration purposes but not practical for absorbance measurements.
Deuterium Lamp and Hydrogen Vapor Lamp

Provides source of continuous spectra in the UV region and are commonly used in UV absorption measurements. Deuterium provides continuous emission down to 165 nm
Laser sources LIGHT AMPLIFICATION BY STIMULATED EMISSION OF RADIATION.

Transform light of various frequencies into an extremely intense focused and nearly non-divergent beam of monochromatic light.

2. ENTRANCE SLIT. Minimizes stray light emitted by the lamp (energy source) and prevents scattered light from entering the monochromator. 3. MONOCHROMATOR.

A system for isolating radiant energy of a desired wavelength and excluding that of other wavelengths.
A.

Prisms. Triangular (wedged) piece of transparent material capable of resolving spectral wavelength.

NOTRE DAME OF MARBEL UNIVERSITY

Clinical Chemistry I
ANALYTIC TECHNIQUES AND INSTRUMENTATION Mandy A. Delfin, RMT

Separates white light into a continuous spectrum by refraction with shorter wavelengths as they passed through the prism
B.

Diffraction Gratings. Grooved piece of transparent material wherein each groove functions as individual prisms. Diffraction gratings are prepared by depositing a thin layer of aluminium-copper alloy on the surface of a flat glass plate, it is arranged at precise angle to allow selection of the wavelength of interest.

C.

Interference filters. Are composed of semi-transparent silver films on both sides of dielectric such as magnesium fluoride.

4. EXIT SLIT. Controls the amount of emergent light that passes into the analytical cell or cuvet. 5. CUVETTE. This is also known as the analytical cell or absorption cell which holds the solution whose concentration has to be assayed or simply contains the analyte (substance of interest).

Cuvettes differ from ordinary receptacles since their walls are precisely arranged and without optical flaws. It can be a soft glass (for acidic solutions) borosilicate glass (for alkaline solution), quartz or plastics.

6. DETECTOR OR PHOTOCELL. Measures the intensity of the emergent light from the solution. A. Barrier Layer Cells. Operates on the principle that when light falls on certain metals, electron falls in proportion to the intensity of the emergent light. B. Photomultiplier Tubes. Electron tubes that is capable of significantly amplifying a current. 7. METER OR READ OUT DEVICE. Numerically presents absorbance (A) or percent Transmittance (%T). BEER'S LAW

The relationship between absorption of light by a solution and the concentration of that solution. Beer's law states that the concentration of a substance is directly proportional to the amount of light absorbed or inversely proportional to the logarithm of the transmitted light.

NOTRE DAME OF MARBEL UNIVERSITY

Clinical Chemistry I
ANALYTIC TECHNIQUES AND INSTRUMENTATION Mandy A. Delfin, RMT

OPTICAL DENSITY. Synonymous with absorbance which refers to the amount of light blocked by a solution. TRANSMITTANCE. Ratio of the Incident light to the emergent light.

A = log Is/Io = log T Since, A = log (1/T) A = log 100 log %T Then, A = 2 log %T = a b c

Mathematical Expression is: A=abc Where:

A is absorbance

a = absorptivity b = depth of the solution c = concentration of the colored substance

As =as x bs x cs

and

Au = au x bu x cu

Since as=au and bs=bu, rearrangement of the preceding formula gives this working equation:

NOTRE DAME OF MARBEL UNIVERSITY

Clinical Chemistry I
ANALYTIC TECHNIQUES AND INSTRUMENTATION Mandy A. Delfin, RMT

Three Types of Spectrophotometric Methods 1. Endpoint Colorimetric Spectrophotometry: A colored product or chromogen forms that is measured by its ability to absorb visible light. 2. Endpoint Enzymatic Spectrophotometry: The final product, often a coenzyme that absorbs light strongly at lower wavelengths in the visible or near-UV spectrum. 3. Kinetic Spectrophotometry: coupled sequential enzymatic reactions that produce NAD or NADP are commonly used for the measurement of other chemicals in the reaction since the change in absorbance at 340 nm is simple and relatively free from interference. Either the consumption of NADH or the production of ionized NAD may be measured as a reflection of the concentration of other chemicals in the reaction.
NOTRE DAME OF MARBEL UNIVERSITY

Clinical Chemistry I
ANALYTIC TECHNIQUES AND INSTRUMENTATION Mandy A. Delfin, RMT

Factors to consider in using the spectrophotometer.

A light shield should be used to protect cuvette from unwanted light that may cause measurement errors. Rectangular cuvettes are preferably used in spectrophotometer because they reduce incident light. To check wavelength calibration of spectrophotometer, holonium oxide or didymium filters are used. Stray light may be detected by using filters or solutions with sharp cut off wavelength for transmission such as nickel sulfate.

DOUBLE BEAM SPECTROPHOTOMETER

Principle Absorbance is measured at two different wavelengths using a light from either single or two monochromators that would analyze samples and reference cuvette. Types of Double Beam Spectrophotometery a. Double beam in space: Passes light in two directions one beam is directed to sample and the other beam is directed to reference cuvette simultaneously.

b. Double beam in time:

Splits light using a rotator chopper. As the chopper rotates, it alternately presents a mirror and an opening that produces an interval of light towards the cuvette. One beam is directed to the sample and the other beam is for reference cuvette with an interval of time.

NOTRE DAME OF MARBEL UNIVERSITY

Clinical Chemistry I
ANALYTIC TECHNIQUES AND INSTRUMENTATION Mandy A. Delfin, RMT

EMISSION FLAME PHOTOMETER

Filter or Flame Photometry is basically used in the quantitative analysis of electrolytes in the body fluids such as sodium and potassium. Aside from these electrolytes, lithium determination is also carried out in relevance with therapeutic use of Lithium as Lithium carbonate in psychotic patients.

Principle: Atoms of different electropositive elements when excited by heat given off by a hot flame will emit energy in the form of a colored light that is characteristic of that element.

Involves the excitation of electron of different ions from a lower energy level to a higher excited state. When these electrons return to their ground state, it emits energy in the form of light, the color of which is characteristic of the ions of interest. Some of the colors produced by different electrolytes: Potassium: Violet Sodium: Yellow Calcium: Red Orange Magnesium: Blue

NOTRE DAME OF MARBEL UNIVERSITY

Clinical Chemistry I
ANALYTIC TECHNIQUES AND INSTRUMENTATION Mandy A. Delfin, RMT

Basic components of a Flame Photometer is almost similar with that of the spectrophotometer except that the light source is replaced by a burner assembly which consists of

THE BURNER ASSEMLY. Aspirator: Which draws the sample into the flame. The vacuum feed and gravitational feed are noted examples of aspirators. Atomizer. This sprays the sample into fine mists of droplets such that the excitation will be facilitated. Flame. Provides the energy for excitation. Blue flame the non-luminous flame is used since it gives off more heat. Acetylene, propane, or methane (natural gas) in combination with an oxidant, are gases used in the operation of a flame photometer. The temperature ranges from 2000 oC to 3000 oC

1.

2.

3.

Aside from the burner assembly, other basic parts of a flame photometer include: entrance slit, monochromator, exit slit, detector and meter. The monochromator in EFP is called optical filter or filter alone which is a colored glass or dyed gelatin sheets mounted between glass plates. EFP METHODOLOGIES: Direct EFP. o A standard solution of sodium or potassium is aspirated directly into the flame to provide a series of meter reading against a series of meter reading against which an unknown solution can be compared.

A.

Indirect. EFP. o Lithium is used as standard, and also acts as a radiation buffer in preventing mutual excitation. However when Lithium is measured Cesium can be used as a standard. In EFP, non-ionic surface-active agents or wetting agents such as Sterox and Acantionox are added into the standards and samples, which lower the viscosity and promote amore uniform flow of the sample into the burner.
B.

A dilution of the sample into 1:100 or 1:200 adjusts the concentration of measured ion intensity of emitted light.
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Clinical Chemistry I
ANALYTIC TECHNIQUES AND INSTRUMENTATION Mandy A. Delfin, RMT

ATOMIC ABSORPTION SPECTROPHOTOMETRY Atomic Absorption Spectrophotometry is a photometric technique particularly suited to assays of metals, which are not easily excited by heat coming from a filter photometer like calcium and magnesium. Principle: (Kirchoffs Law): In AAS there is no excitation of electrons but merely dissociation of their chemical bonds as free atoms in the neutral ground state. In such state, these atoms are capable of absorbing radiation equal to the energy that these atoms will emit if they have been excited. Components of AAS

1.

The hollow cathode lamp is the light source it is made of the metal to be analyzed and is different for each metal analysis. In some cases, when an alloy is used to make the cathode, a multielement lamp is produced. A nebulizer sprays the sample into the flame; the monochromator, slits and detectors have the functions describe for spectrophotometry. Burners:
a.

2.

3.

Total Consumption burner: gasses (hydrogen and air) mix within the sample within the flame. Disadvantage: relatively large droplets produced in the flame scatter light and thus cause signal noise. Advantage: Flame is more concentrated and it can be made hotter than that of other burners.

b.

Premix burner (Laminar Flow Burner): The gasses are mixed and the sample is atomized before it is burned.

NOTRE DAME OF MARBEL UNIVERSITY

Clinical Chemistry I
ANALYTIC TECHNIQUES AND INSTRUMENTATION Mandy A. Delfin, RMT

Advantages: Larger droplets go to waste while only the fine mist enters the flame; thus a less noisy signal is produced. The path length trough the flame of the burner is longer than that of the total consumption burner which produces a greater absorption and increases the sensitivity of the measurement. Interferences In Atomic Absorption Spectrophotometry: 1. Chemical Interference: Refers to the situation when the flame cannot dissociate the sample into free atoms. Example is calcium phosphate where this complex does not dissociate in flame. 2. Ionization Interference: Results when atoms become excited (instead of only dissociated) by the flame and then emit energy of the same wavelength that is being measured. This effect can be overcome by adding an excess of a more easily ionized substance that will absorb most of the flame energy, or by reducing the fame temperature. 3. Matrix Interference: Caused by organic solvents, which enhance light absorption and light absorption caused by the formation of solids from sample droplets as the solvent is evaporated into the flame.

NOTRE DAME OF MARBEL UNIVERSITY

Clinical Chemistry I
ANALYTIC TECHNIQUES AND INSTRUMENTATION Mandy A. Delfin, RMT

FLOUROMETRY

Principle:

It measures the amount of light emitted by a substance due to its excitation from a source rendering a higher or equal energy from its original state. The wavelength of emitted light is longer than the excited light.

Types of Luminescence:

Fluorescence:

An energy emission that occurs when certain compounds absorb electromagnetic radiation. The molecules become excited and return to an energy level that is usually higher than their original level.

Phosphorescence:

When the emitted light of the excited electrons is equal or lower than the absorbed energy.

Chemiluminescence and bioluminescence

Occurs from excitation of chemical or electrochemical compounds. It uses organic compounds that are oxidized with a presence of a catalyst. Organic compounds involve luminal, isoluminol, acridinium ester, luceferin.

Oxidants used are hydrogen peroxide, oxygen hypochlorite. Catalyst includes enzymes (alkaline phosphatase, horse radish peroxidase, microperoxidase) metal ions, and hemin.

Components of Flourometer

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Clinical Chemistry I
ANALYTIC TECHNIQUES AND INSTRUMENTATION Mandy A. Delfin, RMT

a. Light source (exciter lamp) uses high intensity light from mercury vapor lamp and xenon arc lamp. b. Excitation monochromator (primary) c. Cuvette d. Emission monochromatator (secondary) e. Photodector

Methods: 1. Direct: The compound itself has the fluorescent property 2. Conversion: The compound to be analyzed is converted to a fluorescent compound which can be achieved by application of certain dyes like rhodamine and auramine. Said to induce fluorescence. 3. Coupling. The formation of a fluorescent complex as those used in calcium assays. 4. Destruction. The compound to be analyzed has the ability to destroy or quench fluorescence. The reduction in fluorescence is proportional to the amount of unknown present. The single most important advantage of this method is its high sensitivity and some flourometric methods of determinations include porphyrins, magnesium, calcium and cathecolamines.

Factors affecting Fluorometry:


1.

Light Scattering: Occurs when the detector is positioned at right angle to the incident light Self quenching: As the absorbance in a simple with fluorophore concentration concentration increases, more of the excitation light is absorbed before it reaches the molecules near the center of the cuvette. Self Absorption: Occurs when excited light is absorbed before it gets out to the cuvette.

2.

3.

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Clinical Chemistry I
ANALYTIC TECHNIQUES AND INSTRUMENTATION Mandy A. Delfin, RMT

NEPHELOMETRY

Principle: o The light scattered by the small particles in the solution is measured at right angles to the beam incident in the cuvette. The amount of light that occurs is related to the number and size and particles in the light beam.

Application: o Measures particles large for absorptions spectrophotometry like the antigenantibody complexes formed in enzyme immunoassays.

TURBIDIMETRY

Turbdimetric measurements determine the amount of light blocked by the suspending particles (particulate matter) in the sample as light passes through the cuvette of a spectrophotometer. The determinants in turbidimetry are: Size and number of particles present Cross sectional area of each particle Depth of the cuvette Problems encountered in turbidimetric measurements Differences in particle size between the standard and test solutions Settling out of the particles in solution. The use stabilizing colloids like gum Arabic and gelatin provides a viscous medium which retards the rate of precipitation. Applications: Assay of low levels of protein in CSF and urine Detection of bacterial growth in bacterial culture Antibiotic sensitivity

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