Analytic Techniques PDF

CHAPTER 4 CHAPTER OUTLINE Spectrophotometry Analytic Techniques Beer’s Law Spectrophotometric Instruments Components of a Spectrophotometer Spectrophotometer Quality Assura...

CHAPTER 4 CHAPTER OUTLINE Spectrophotometry Analytic Techniques Beer’s Law Spectrophotometric Instruments Components of a Spectrophotometer Spectrophotometer Quality Assurance Atomic Absorption Spectrophotometry Karen K. Apolloni Fluorometry Fluorometry Instrumentation Fluorescence Polarization Advantages and Disadvantages of Fluorometry Chemiluminescence Turbidimetry Nephelometry Laser Applications Electrochemistry Galvanic and Electrolytic Cells Half-Cells Ion-Selective Electrodes pH Electrodes Gas-Sensing Electrodes Enzyme Electrodes Coulometric Titration Anodic Stripping Voltammetry Electrophoresis Procedure Support Materials Treatment and Application of Sample Detection and Quantitation Electroendosmosis Isoelectric Focusing Immunofixation Electrophoresis (IFE) Capillary Electrophoresis Two-Dimensional Electrophoresis Osmometry Freezing Point Osmometer Newer Optical Techniques Chromatography Modes of Separation Chromatographic Procedures High-Performance Liquid Chromatography Gas Chromatography Mass Spectrometry Sample Introduction and Ionization Mass Spectrometer Analyzer Detector Applications of MS in the Clinical Laboratory Small Molecule Analysis Mass Spectrometry in Proteomics and Pathogen Identification Mass Spectrometry at the Point of Care References KEY TERMS Atomic absorption spectrophotometry Chemiluminescence Chromatography Electrochemistry Electrophoresis CHAPTER OBJECTIVES Fluorescence Upon completion of this chapter, the clinical laboratorian should be able to: Fluorometry Explain the general principles of each analytic method. Gas chromatography Discuss the limitations of each analytic technique. High-performance liquid chromatography Compare and contrast the various analytic techniques. Ion-selective electrodes State existing clinical applications for each analytic technique. Describe the operation and component parts of the following instruments: spectrophotometer, atomic absorption Mass spectrometry spectrometer, fluorometer, ion-selective electrode, pH electrode, osmometer, gas chromatograph, and mass Osmometry spectrometer. Spectrophotometry Outline spectrophotometer quality assurance procedures. A variety of analytic techniques are incorporated into instrumentation and are in widespread use in the modern clinical chemistry laboratory. The majority of analytic techniques fall into one of four basic disciplines within the field of analytic chemistry: spectrometry (including spectrophotometry, atomic absorption spectrometry, and mass spectrometry [MS]); luminescence (including fluorescence and chemiluminescence); electroanalytic methods (including electrophoresis, potentiometry, and amperometry); and chromatography (including gas, liquid, and thin layer). Spectrophotometry Instruments that measure electromagnetic radiation have several concepts and components in common. Shared instrumental components are discussed in some detail in a later section. Photometric instruments measure light intensity without consideration of wavelength. Most instruments today use filters (photometers), prisms, or gratings (spectrometers) to select (isolate) a narrow range of the incident wavelength. Radiant energy that passes through an object will be partially reflected, absorbed, and transmitted. Electromagnetic radiation is described as photons of energy traveling in waves. The relationship between wavelength and energy E is described by Planck’s formula: (Eq. 4.1) where h is a constant (6.62 × 10–27 erg sec), known as Planck constant, and v is frequency. Because the frequency of a wave is inversely proportional to the wavelength, it follows that the energy of electromagnetic radiation is inversely proportional to wavelength. Figure 4.1A shows this relationship. Electromagnetic radiation includes a spectrum of energy from short- wavelength, highly energetic gamma rays and x-rays on the left in Figure 4.1B to long- wavelength radiofrequencies on the right. Visible light falls in between, with the color violet at 400 nm and red at 700 nm wavelengths being the approximate limits of the visible spectrum. Figure 4.1 Electromagnetic radiation—relationship of energy and wavelength. © Wolters Kluwer. Description CASE STUDY 4.1, PART 1 Remember Miles and Mía from Chapter 1? The laboratory is placing a spectrophotometer back in service after being in storage for 6 months. The instrument manuals are no longer available for this model. Miles and Mía, who manage quality control for the laboratory, are tasked with getting it ready for use. 1. What procedures should Miles and Mía develop to validate that the instrument is working properly for clinical use?  The instruments discussed in this section measure either absorption or emission of radiant energy to determine the concentration of atoms or molecules. The two phenomena, absorption and emission, are closely related. For a ray of electromagnetic radiation to be absorbed, it must have the same frequency as a rotational or vibrational frequency in the atom or molecule that it strikes. Levels of energy that are absorbed move in discrete steps, and any particular type of molecule or atom will absorb only certain energies and not others. When energy is absorbed, valence electrons move to an orbital with a higher energy level. Following energy absorption, the excited electron will fall back to the ground state by emitting a discrete amount of energy in the form of a characteristic wavelength of radiant energy. Absorption or emission of energy by atoms results in a line spectrum. Because of the relative complexity of molecules, they absorb or emit a band of energy over a large region. Light emitted by incandescent solids (tungsten or deuterium) is in a continuum. The three types of spectra are shown in Figure 4.2.1–3 © dotshock/Shutterstock. Figure 4.2 Characteristic absorption or emission spectra. Data from Coiner D. Basic Concepts in Laboratory Instrumentation. Bethesda, MD: ASMT Education and Research Fund; 1975- 1979. Beer’s Law The relationship between absorption of light by a solution and the concentration of that solution © Ariel Skelley/DigitalVision/Getty Images. has been described by Beer and others. 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. Percent transmittance (%T) and absorbance (A) are related photometric terms that are explained in this section. © Wolters Kluwer. Figure 4.3A shows a beam of monochromatic light entering a solution. Some of the light is Description absorbed. The remainder passes through, strikes a light detector, and is converted to an electric signal. Percent transmittance is the ratio of the radiant energy transmitted (T) divided Equal thicknesses of an absorbing material will absorb a constant fraction of the energy by the radiant energy incident on the sample (I). If all light is absorbed or blocked, %T is equal incident upon the layers. For example, in a tube containing layers of solution (Figure 4.4A), the to zero. A level of 100% T is obtained if no light is absorbed. In practice, the solvent without the first layer transmits 70% of the light incident upon it. The second layer will, in turn, transmit 70% constituent of interest is placed in the light path, as in Figure 4.3B. Most of the light is of the light incident upon it. Thus, 70% of 70% (49%) is transmitted by the second layer. The transmitted, but a small amount is absorbed by the solvent and cuvette or is reflected away third layer transmits 70% of 49%, or 34% of the original light. Continuing on, successive layers from the detector. The electrical readout of the instrument is set arbitrarily at 100% T, while the transmit 24% and 17%, respectively. The %T values, when plotted on linear graph paper, yield light is passing through a “blank” or reference. The sample containing absorbing molecules to the curve shown in Figure 4.4B. Considering each equal layer as many monomolecular layers, be measured is then placed in the light path. The difference in amount of light transmitted by the we can translate layers of material to concentration. If semi log graph paper is used to plot the blank and that transmitted by the sample is due only to the presence of the compound being same figures, a straight line is obtained (Figure 4.4C), indicating that, as concentration measured. The %T measured by commercial spectrophotometers is the ratio of the sample increases, %T decreases in a logarithmic manner. transmitted beam divided by the blank transmitted beam, multiplied by 100. Figure 4.4 (A) Percent of original incident light transmitted by equal layers of light-absorbing solution. (B) Percent T versus concentration on linear graph paper. (C) Percent T versus concentration on semi log graph paper. (D) A versus concentration on linear graph paper. © Wolters Kluwer. Description Absorbance A is the amount of light absorbed. It cannot be measured directly by a Figure 4.3 Percent transmittance (%T) defined. spectrophotometer but rather is mathematically derived from %T as follows:  (Eq. 4.2) where I0 is the incident light and I is the transmitted light. Absorbance is defined as follows: (Eq. 4.3) According to Beer’s law, absorbance is directly proportional to concentration (Figure 4.4D): Figure 4.5 Single-beam spectrophotometer. © Wolters Kluwer. (Eq. 4.4) Description where ε = molar absorptivity, the fraction of a specific wavelength of light absorbed by a given type of molecule; b is the length of light path through the solution; and c is the concentration of Components of a Spectrophotometer absorbing molecules. Absorptivity depends on the molecular structure and the way in which the absorbing Light Source molecules react with different energies. For any particular molecular type, absorptivity changes The most common source of light for work in the visible and near-infrared regions is the as wavelength of radiation changes. The amount of light absorbed at a particular wavelength incandescent tungsten or tungsten-iodide lamp. Only about 15% of radiant energy emitted falls depends on the molecular and ion types present and may vary with concentration, pH, or in the visible region, with most emitted as near-infrared.1–3 Often, a heat-absorbing filter is temperature. inserted between the lamp and the sample to absorb the infrared radiation. Because the path length and molar absorptivity are constant for a given wavelength, The lamps usually used for ultraviolet (UV) work are the deuterium discharge lamp and the absorbance is directly proportional to concentration. mercury arc lamp. Deuterium provides continuous emission down to 165 nm. Low-pressure mercury lamps emit a sharp line spectrum, with both UV and visible lines. Medium- and high- A~ c pressure mercury lamps emit a continuum from UV to the mid-visible region. The most Unknown concentrations are determined from a calibration curve that plots absorbance at a important factors for a light source are range, spectral distribution within the range, the source specific wavelength versus concentration for standards of known concentration. For calibration of radiant production, stability of the radiant energy, and temperature. curves that are linear and have a zero y-intercept, unknown concentrations can be determined from a single calibrator. Not all calibration curves result in straight lines. Deviations from linearity Monochromators are typically observed at high absorbances. The stray light within an instrument will ultimately Isolation of individual wavelengths of light is an important and necessary function of a limit the maximum absorbance that a spectrophotometer can achieve, typically 2.0 absorbance monochromator. The degree of wavelength isolation is a function of the type of device used and units. the width of entrance and exit slits. The band-pass of a monochromator defines the range of wavelengths transmitted and is calculated as width at half the maximum transmittance (Figure Spectrophotometric Instruments 4.6). A spectrophotometer is used to measure the light transmitted by a solution to determine the concentration of the light-absorbing substance in the solution. Figure 4.5 illustrates the basic components of a single-beam spectrophotometer, which are described in subsequent sections.  side of the filter and are reflected at the second surface. Wavelengths that are twice the space between the two glass surfaces will reflect back and forth, reinforcing others of the same wavelengths and finally passing through. Other wavelengths will cancel out because of phase differences (destructive interference). Because interference filters also transmit multiples of the desired wavelengths, they require accessory filters to eliminate these harmonic wavelengths. Interference filters can be constructed to pass a very narrow range of wavelengths with good efficiency. The simple glass prism is another type of monochromator. A narrow beam of light focused on a prism is refracted as it enters the denser glass. Short wavelengths are refracted more than long wavelengths, resulting in dispersion of white light into a continuous spectrum. The prism can be rotated, allowing only the desired wavelength to pass through an exit slit. Diffraction gratings are commonly used as monochromators. A diffraction grating consists of many parallel grooves (15,000 or 30,000 per inch) etched onto a polished surface. Diffraction, the separation of light into component wavelengths, is based on the principle that wavelengths bend as they pass a sharp corner. The degree of bending depends on the wavelength. As the wavelengths move past the corners, wave fronts are formed. Those that are in phase reinforce one another, whereas those not in phase cancel out and disappear. This results in complete spectra. Gratings with very fine line rulings produce a widely dispersed spectrum. They produce linear spectra, called orders, in both directions from the entrance slit. Because the multiple spectra have a tendency to cause stray light problems, accessory filters are used. Sample Cell The next component of the basic spectrophotometer is the sample cell or cuvette, which typically has a flat surface. The light path must be kept constant to have absorbance proportional to concentration. This is easily checked by preparing a colored solution to read midscale when using the wavelength of maximum absorption. Each cuvette to be tested is filled, readings are taken, and results are compared against an acceptable tolerance (e.g., ±0.25% T). Cuvettes are sold in matched sets. Square cuvettes have plane-parallel optical surfaces and a constant light path. Cuvettes with scratched optical surfaces scatter light and should be discarded. Inexpensive glass cuvettes can be used for applications in the visible range, but they absorb light in the UV region. Quartz cuvettes enable transmission of light and are used when substances absorb in this region (e.g., NADH at 340 nm). Photodetectors Figure 4.6 Spectral transmittance of two monochromators with band pass at half height of 5 and 20 nm. The purpose of the detector is to convert the transmitted radiant energy into an equivalent © Wolters Kluwer. amount of electrical energy. The least expensive of the devices is known as a barrier-layer cell, Description or photocell. The photocell is composed of a film of light-sensitive material, frequently selenium, on a plate of iron. A thin, transparent layer of silver overlays the light-sensitive material. When Numerous devices are used for obtaining monochromatic light. The least expensive are exposed to light, electrons in the light-sensitive material are excited and released to flow to the colored glass filters. These filters usually pass a relatively wide band of radiant energy and highly conductive silver. In comparison with the silver, a moderate resistance opposes the have a low transmittance of the selected wavelength. Although not precise, they are simple and electron flow toward the iron, forming a hypothetical barrier to flow in that direction. inexpensive. Consequently, this cell generates its own electromotive force, which can be measured. The Interference filters produce monochromatic light based on the principle of constructive current produced is proportional to the incident radiation. Photocells require no external voltage interference of waves. Two pieces of glass, each mirrored on one side, are separated by a source but rely on internal electron transfer to produce a current in an external circuit. Because transparent spacer that is precisely one-half the desired wavelength. Light waves enter one of their low internal resistance, the output of electrical energy is not easily amplified. Consequently, this type of detector is used mainly in filter photometers with a wide bandpass, The third major type of light detector is the photomultiplier (PM) tube, which detects and producing a fairly high level of illumination so that there is no need to amplify the signal. The amplifies radiant energy. As shown in Figure 4.8, incident light strikes the coated cathode, photocell is inexpensive and durable; however, it is temperature sensitive and nonlinear at very emitting electrons. The electrons are attracted to a series of anodes, known as dynodes, each low and very high levels of illumination. having a successively higher positive voltage. These dynodes are made of a material that gives A phototube (Figure 4.7) is similar to a photocell in that it has photosensitive material that off many secondary electrons when hit by single electrons. Initial electron emission at the gives off electrons when light energy strikes it. It differs in that an outside voltage is required for cathode triggers a multiple cascade of electrons within the PM tube itself. Because of this operation. Phototubes contain a negatively charged cathode and a positively charged anode amplification, the PM tube is 200 times more sensitive than the phototube. PM tubes are used enclosed in a glass case. The cathode is composed of a material (e.g., rubidium or lithium) that in instruments designed to be extremely sensitive to very low light levels and light flashes of acts as a resistor in the dark but emits electrons when exposed to light. The emitted electrons very short duration. The accumulation of electrons striking the anode produces a current signal, jump over to the positively charged anode, where they are collected and return through an measured in amperes, that is proportional to the initial intensity of the light. The analog signal is external, measurable circuit. The cathode usually has a large surface area. Varying the cathode converted first to a voltage and then to a digital signal through the use of an analog-to-digital material changes the wavelength at which the phototube gives its highest response. The converter. Digital signals are processed electronically to produce absorbance readings. photocurrent is linear with the intensity of the light striking the cathode as long as the voltage between the cathode and the anode remains constant. A vacuum within the tubes avoids scattering of the photoelectrons by collision with gas molecules. Figure 4.8 Dynode chain in a photomultiplier. © Wolters Kluwer. Figure 4.7 Phototube drawing and schematic. In a photodiode, absorption of radiant energy by a reverse-biased PN junction diode (PN: © Wolters Kluwer. positive–negative) produces a photocurrent that is proportional to the incident radiant power. Although photodiodes are not as sensitive as PM tubes because of the lack of internal amplification, their excellent linearity, speed, and small size make them useful in applications where light levels are adequate.4 Photodiode array (PDA) detectors are available in integrated circuits containing 256 to 2048 photodiodes in a linear arrangement. A linear array is shown in Figure 4.9. Each photodiode responds to a specific wavelength, and as a result, a complete UV/visible spectrum can be obtained in less than 1 second. Resolution is 1 to 2 nm and depends on the number of discrete elements. In spectrophotometers using PDA detectors, the grating is positioned after the sample cuvette and disperses the transmitted radiation onto the PDA detector (Figure 4.9).  Figure 4.9 Photodiode array spectrophotometer illustrating the placement of the sample cuvette before the monochromator. Stray light refers to any wavelengths outside the band transmitted by the monochromator. © Wolters Kluwer. Figure 4.11 shows the performance of a spectrophotometer to measure high absorbance in the presence of stray light. The most common causes of stray light are reflection of light from scratches on optical surfaces or from dust particles anywhere in the light path and higher order For single-beam spectrophotometers, the absorbance reading from the sample must be spectra produced by diffraction gratings. The major effect is absorbance error, especially in the blanked using an appropriate reference solution that does not contain the compound of interest. high absorbance range. Stray light is detected by using cutoff filters, which eliminate all Double-beam spectrophotometers permit automatic correction of sample and reference radiation at wavelengths beyond the one of interest. To check for stray light in the near-UV absorbance, as shown in Figure 4.10. Because the intensities of light sources vary as a region, for example, a filter that does not transmit in the region of 200 to 400 nm is inserted. If function of wavelength, double-beam spectrophotometers are necessary when the absorption spectrum for a sample is to be obtained. Computerized, continuous zeroing, single-beam the instrument reading is greater than 0% T, stray light is present. Certain liquids, such as NiSO4, NaNO2, and acetone, absorb strongly at short wavelengths and can be used to detect spectrophotometers have replaced most double-beam spectrophotometers. stray light in the UV range. Figure 4.10 Double-beam spectrophotometer. © Wolters Kluwer. Description Spectrophotometer Quality Assurance Performing at least the following checks should validate instrument function: wavelength accuracy, stray light, and linearity. Wavelength accuracy means that the wavelength indicated on the control dial is the actual wavelength of light passed by the monochromator. It is most Figure 4.11 Spectrophotometer’s ability to measure high absorbance with stray light. (A) No stray light, with no deviation from the commonly checked using standard absorbing solutions or filters with absorbance maxima of actual absorbance. (B) Some stray light within the instrument showing deviations from the actual at high absorbance. (C) A known wavelength. Didymium or holmium oxide in glass is stable and frequently used as filters. higher degree of stray light showing further deviation from the actual absorbance. The filter is placed in the light path, and the wavelength control is set at the wavelength at which © Wolters Kluwer. maximal absorbance is expected. The wavelength control is then rotated in either direction to Description locate the actual wavelength that has maximal absorbance. If these two wavelengths do not match, the optics must be adjusted to calibrate the monochromator correctly. Linearity is demonstrated when a change in concentration results in a straight-line calibration Some instruments with narrow bandpass use a mercury vapor lamp to verify wavelength curve, as discussed under Beer’s law. Colored solutions may be carefully diluted and used to accuracy. The mercury lamp is substituted for the usual light source, and the spectrum is check linearity, using the wavelength of maximal absorbance for that color. Sealed sets of scanned to locate mercury emission lines. The wavelength indicated on the control is compared different colors and concentrations are available commercially. They should be labeled with with known mercury emission peaks to determine the accuracy of the wavelength indicator expected absorbance for a given bandpass instrument. Less than expected absorbance is an control. indication of stray light or of a bandpass that is wider than specified. Sets of neutral-density filters to check linearity over a range of wavelengths are also commercially available. There are various designs; however, the most common burner is the premix long-path burner. The sample, in solution, is aspirated as a spray into a chamber, where it is mixed with air and Atomic Absorption Spectrophotometry fuel. This mixture passes through baffles, where large drops fall and are drained off. Only fine droplets reach the flame. The burner is a long, narrow slit, to permit a longer path length for The atomic absorption spectrophotometer is used to measure concentration by detecting the absorption of incident radiation. Light from the hollow-cathode lamp passes through the sample absorption of electromagnetic radiation by atoms rather than by molecules. The basic of ground state atoms in the flame. The amount of light absorbed is proportional to the components are shown in Figure 4.12. The usual light source, known as a hollow-cathode concentration. When a ground state atom absorbs light energy, an excited atom is produced. lamp, consists of an evacuated gas-tight chamber containing an anode, a cylindrical cathode, The excited atom then returns to the ground state, emitting light of the same energy as it and an inert gas, such as helium or argon. When voltage is applied, the filler gas is ionized. Ions absorbed. The flame sample thus contains a dynamic population of ground state and excited attracted to the cathode collide with the metal, knock atoms off, and cause the metal atoms to atoms, both absorbing and emitting radiant energy. The emitted energy from the flame will go in be excited. When they return to the ground state, light energy is emitted that is characteristic of all directions, and it will be a steady emission. Because the purpose of the instrument is to the metal in the cathode. Generally, a separate lamp is required for each metal (e.g., a copper measure the amount of light absorbed, the light detector must be able to distinguish between hollow cathode lamp is used to measure this metal). the light beam emitted by the hollow-cathode lamp and that emitted by excited atoms in the flame. To do this, the hollow cathode light beam is modulated by inserting a mechanical rotating chopper between the light and the flame or by pulsing the electric supply to the lamp. Because the light beam being absorbed enters the sample in pulses, the transmitted light will also be in pulses. There will be less light in the transmitted pulses because part of it will be absorbed. There are, therefore, two light signals from the flame—an alternating signal from the hollow- cathode lamp and a direct signal from the flame emission. The measuring circuit is tuned to the modulated frequency. Interference from the constant flame emission is electronically eliminated by accepting only the pulsed signal from the hollow cathode. The monochromator is used to isolate the desired emission line from other lamp emission lines. In addition, it serves to protect the photodetector from excessive light emanating from flame emissions. A PM tube is the usual light detector. Flameless atomic absorption requires an instrument modification that uses an electric furnace to break chemical bonds (electrothermal atomization). A tiny graphite cylinder holds the sample, either liquid or solid. An electric current passes through the cylinder walls, evaporates the solvent, ashes (heats at a high temperature to leave an ash residue for analysis) the sample, and, finally, heats the unit to incandescence to atomize the sample. This instrument, like the spectrophotometer, is used to determine the amount of light absorbed. Again, Beer’s law is used for calculating concentration. A major problem is that background correction is much more necessary and critical for electrothermal techniques than for flame-based atomic absorption methods. Currently, the most common approach uses a deuterium lamp as a secondary source Figure 4.12 Single-beam atomic absorption spectrophotometer—basic components. and measures the difference between the two absorbance signals. However, there has also been extensive development of background correction techniques based on the Zeeman effect.1 © Wolters Kluwer. The presence of an intense static magnetic field will cause the wavelength of the emitted Description radiation to split into several components. This shift in wavelength is the Zeeman effect. Atomic absorption spectrophotometry is sensitive and precise. It is routinely used to measure Electrodeless discharge lamps are a relatively new light source for atomic absorption concentration of trace metals that are not easily excited. It is accurate, precise, and specific. spectrophotometers. A bulb is filled with argon and the element to be tested. A radiofrequency One disadvantage, however, is the inability of the flame to dissociate samples into free atoms. generator around the bulb supplies the energy to excite the element, causing a characteristic For example, phosphate may interfere with calcium analysis by formation of calcium phosphate. emission spectrum of the element. This may be overcome by adding cations that compete with calcium for phosphate. Routinely, The analyzed sample must contain the reduced metal in the atomic vaporized state. lanthanum or strontium is added to samples to form stable complexes with phosphate. Another Commonly, this is done by using the heat of a flame to break the chemical bonds and form free, possible problem is the ionization of atoms following dissociation by the flame, which can be unexcited atoms. The flame serves as the sample cell in this instrument, instead of a cuvette. decreased by reducing the flame temperature. Matrix interference, due to the enhancement of light absorption by atoms in organic solvents or formation of solid droplets as the solvent evaporates in the flame, can be another source of error. This interference may be overcome by pretreatment of the sample by extraction. Recently, inductively coupled plasma (ICP) has been used to increase sensitivity for atomic emission. The torch, an argon plasma maintained by the interaction of a radiofrequency field and an ionized argon gas, is reported to have used temperatures between 5500 and 8000 K. Complete atomization of elements is thought to occur at these temperatures. Use of ICP as a source is recommended for determinations involving refractory elements such as uranium, zirconium, and boron. ICP with MS detection is the most sensitive and specific assay technique for all elements on the periodic chart. Atomic absorption spectrophotometry is used less frequently because of this newer technology. Fluorometry As seen with the spectrophotometer, light entering a solution may pass mainly through or may be absorbed partly or entirely, depending on the concentration and the wavelength entering that particular solution. Whenever absorption occurs, there is a transfer of energy to the medium. Each molecular type possesses a series of electronic energy levels and can pass from a lower energy level to a higher energy level only by absorbing an integral unit (quantum) of light that is equal in energy to the difference between the two energy states. There are additional energy levels owing to rotation or vibration of molecular parts. The excited state lasts about 10–5 seconds before the electron loses energy and returns to the ground state. Energy is lost by collision, heat loss, transfer to other molecules, and emission of radiant energy. Because the molecules are excited by absorption of radiant energy and lose energy by multiple interactions, the radiant energy emitted is less than the absorbed energy. The difference between the Figure 4.13 Absorption and fluorescence spectra of quinine in 0.1 N sulfuric acid. maximum wavelengths, excitation, and emitted fluorescence is called Stokes shift. Both excitation (absorption) and fluorescence (emission) energies are characteristic for a given Data from Coiner D. Basic Concepts in Laboratory Instrumentation. Bethesda, MD: ASMT Education and Research Fund; 1975– molecular type; for example, Figure 4.13 shows the absorption and fluorescence spectra of 1979. quinine in sulfuric acid. The dashed line on the left shows the short-wavelength excitation energy that is maximally absorbed, whereas the solid line on the right is the longer wavelength (less Fluorometry Instrumentation energy) fluorescent spectrum. Filter fluorometers measure the concentrations of solutions that contain fluorescing molecules. A basic instrument is shown in Figure 4.14. The source emits short-wavelength high-energy excitation light. A mechanical attenuator controls light intensity. The primary filter, placed between the radiation source and the sample, selects the wavelength that is best absorbed by the solution to be measured. The fluorescing sample in the cuvette emits radiant energy in all directions. The detector (placed at right angles to the sample cell) and a secondary filter that passes the longer wavelengths of fluorescent light prevent incident light from striking the photodetector. The electrical output of the photodetector is proportional to the intensity of fluorescent energy. In spectrofluorometers, the filters are replaced by a grating monochromator.  compound, intensity of the incident radiation, quantum efficiency of the energy emitted per quantum absorbed, and length of the light path. In dilute solutions with instrument parameters held constant, fluorescence is directly proportional to concentration. Generally, a linear response will be obtained until the concentration of the fluorescent species is so high that the sample begins to absorb significant amounts of excitation light. A curve demonstrating nonlinearity as concentration increases is shown in Figure 4.15. The solution must absorb less than 5% of the exciting radiation for a linear response to occur.5 As with all quantitative measurements, a standard curve must be prepared to demonstrate that the concentration used falls in a linear range. Figure 4.14 Basic filter fluorometer. Data from Coiner D. Basic Concepts in Laboratory Instrumentation. Bethesda, MD: ASMT Education and Research Fund; 1975– 1979. Gas discharge lamps (mercury and xenon arc) are the most frequently used sources of excitation radiant energy. Incandescent tungsten lamps are seldom used because they release little energy in the UV region. Mercury vapor lamps are commonly used in filter fluorometers. Mercury emits a characteristic line spectrum. Resonance lines at 365 to 366 nm are commonly used. Energy at wavelengths other than the resonance lines is provided by coating the inner surface of the lamp with a material that absorbs the 254-nm mercury radiation and emits a broad band of longer wavelengths. Most spectrofluorometers use a high-pressure xenon lamp. These lamps produce a nearly continuous spectrum of wavelengths. Monochromator fluorometers use gratings for isolation of incident radiation. Light detectors are almost exclusively PM tubes because of their higher sensitivity to low light intensities. Double-beam instruments are used to compensate for instability due to electric power fluctuation. Fluorescence concentration measurements are related to molar absorptivity of the  In fluorescence polarization, radiant energy is polarized in a single plane. When the sample fluorophore is excited, it emits polarized light along the same plane as the incident light if the fluorophore does not rotate in solution (i.e., if it is attached to a large molecule). In contrast, a small molecule emits depolarized light because it will rotate out of the plane of polarization during its excitation lifetime. This technique is widely used for the detection of therapeutic and abused drugs. In the procedure, the sample analyte is allowed to compete with a fluorophore- labeled analyte for a limited antibody to the analyte. The lower the concentration of the sample analyte, the higher the concentration of macromolecular antibody–analyte–fluorophore formed and the lower the depolarization of the radiant light. Advantages and Disadvantages of Fluorometry Fluorometry has two advantages over conventional spectrophotometry: specificity and sensitivity. Fluorometry increases specificity by selecting the optimal wavelength for both absorption and fluorescence, rather than just the absorption wavelength seen with spectrophotometry. Fluorometry is approximately 1000 times more sensitive than most spectrophotometric methods.5 One reason is because the emitted radiation is measured directly; it can be increased simply by increasing the intensity of the exciting radiant energy. In addition, fluorescence measures the amount of light intensity present over a zero background. In absorbance, however, the quantity of the absorbed light is measured indirectly as the difference between the transmitted beams. At low concentrations, the small difference between 100% T and the transmitted beam is difficult to measure accurately and precisely, limiting the sensitivity. The biggest disadvantage is that fluorescence is very sensitive to environmental changes. Changes in pH affect availability of electrons, and temperature changes the probability of loss of energy by collision rather than fluorescence. Contaminating chemicals or a change of solvents may change the structure. UV light used for excitation can cause photochemical changes. Any decrease in fluorescence resulting from any of these possibilities is known as quenching. Because so many factors may change the intensity or spectra of fluorescence, extreme care is required in analytic technique and instrument maintenance. Chemiluminescence In chemiluminescence reactions, part of the chemical energy generated produces excited intermediates that decay to a ground state with the emission of photons.6 The emitted radiation is measured with a PM tube, and the signal is related to analyte concentration. Chemiluminescence is different from fluorescence in that no excitation radiation is required and no monochromators are needed because the chemiluminescence arises from one species. Most importantly, chemiluminescence reactions are oxidation reactions of luminol, acridinium esters, and dioxetanes characterized by a rapid increase in intensity of emitted light followed by a gradual decay. Usually, the signal is taken as the integral of the entire peak. Enhanced Figure 4.15 Dependence of fluorescence on the concentration of fluorophore. chemiluminescence techniques increase the chemiluminescence efficiency by including an Data from Guilbault GG. Practical Fluorescence, Theory, Methods and Techniques. New York, NY: Marcel Dekker; 1973. enhancer system in the reaction of a chemiluminescent agent with an enzyme. The time course for the light intensity is much longer (60 minutes) than that for conventional chemiluminescent Fluorescence Polarization reactions, which last for about 30 seconds (Figure 4.16).  measured. The amount of light blocked by a suspension of particles at 180° depends not only on concentration but also on particle size. Because particles tend to aggregate and settle out of suspension, sample handling is critical for accurate measurement. Instrument operation is the same as for any spectrophotometer. Nephelometry Nephelometry is similar to turbidometry, except that light scattered by the small particles is measured at an angle to the beam incident on the cuvette, instead of at 180°. The amount of scattered light is proportional to the concentration of the analyte. Figure 4.17 demonstrates two possible optical arrangements for a nephelometer. Light scattering depends on wavelength and particle size. For macromolecules with a size close to or larger than the wavelength of incident light, sensitivity is increased by measuring the forward light scatter.7 Instruments are available with detectors placed at various forward angles, as well as at 90° to the incident light. Monochromatic light obtains uniform scatter and minimizes sample heating. Certain instruments use lasers as the source of monochromatic light; however, any monochromator may be used. The Siemens BN II analyzer is an example of one instrument that utilizes the principle of nephelometry. Figure 4.16 Representative intensity-versus-time curve for a transient chemiluminescence signal. © Wolters Kluwer. Figure 4.17 Nephelometer versus spectrophotometer—optical arrangements. Advantages of chemiluminescence assays include subpicomolar detection limits, speed (with © Wolters Kluwer. flash-type reactions, light is only measured for 10 seconds), ease of use (most assays are one- Description step procedures), and simple instrumentation.6 The main disadvantage is that impurities can cause a background signal that degrades the sensitivity and specificity. Measuring light scatter at an angle other than 180° minimizes error from colored solutions and increases sensitivity. Because both methods depend on particle size, some instruments Turbidimetry quantitate initial change in light scatter rather than total scatter. Reagents must be free of any Turbidimetric measurements are made with a spectrophotometer to determine the particles, and cuvettes must be free of scratches. concentration of particulate matter in a sample. The decrease in amount of light transmitted is Laser Applications Laser (light amplification by stimulated emission of radiation) is based on the interaction of radiant energy with suitably excited atoms or molecules. The wavelength, direction of Electrochemistry propagation, phase, and plane of polarization of the emitted light are the same as those of the Electrochemistry is the basis for many types of analyses used in the clinical laboratory, incident radiation. Laser light is polarized and coherent and has narrow spectral width and small including potentiometry, amperometry, coulometry, and polarography. The two basic types of cross-sectional area with low divergence. The radiant emission can be very powerful and either electrochemical cells involved in these analyses are galvanic and electrolytic cells. continuous or pulsating. Laser light can serve as the source of incident energy in a spectrometer or nephelometer. Some lasers produce bandwidths of a few kilohertz in both the visible and infrared regions, Galvanic and Electrolytic Cells making these applications about three to six orders more sensitive than conventional An electrochemical cell consists of two half-cells and a salt bridge, which can be a liquid or a spectrometers.8 piece of filter paper saturated with electrolytes, as shown in Figure 4.18. Each half-cell Laser spectrometry can also be used for the determination of structure and identification of contains one electrode, either an anode or a cathode. Instead of two beakers as shown, the samples, as well as for diagnosis. Quantitation of samples depends on the spectrometer used. electrodes can be immersed in a single, large beaker containing a salt solution. In such a setup, An example of the clinical application of laser is the hematology and flow cytometer analyzers the solution serves as the salt bridge. for the differential analysis of white blood cells.9 Figure 4.18 Electrochemical cell. © Wolters Kluwer. Description In a galvanic cell, as the electrodes are connected, there is spontaneous flow of electrons from the electrode with the lower electron affinity (oxidation). These electrons pass through the external meter to the cathode (reduction), where OH– ions are liberated. This reaction continues until one of the chemical components is depleted, at which point, the cell is “dead” and cannot produce electrical energy to the external meter. Current may be forced to flow through the dead cell only by applying an external electromotive force E. This is called an electrolytic cell. In short, a galvanic cell can be built from an electrolytic cell. When the external E is turned off, accumulated products at the pH Electrodes electrodes will spontaneously produce current in the opposite direction of the electrolytic cell. An ISE universally used in the clinical laboratory is the pH electrode. The basic components of a pH meter are shown in Figure 4.19. Half-Cells It is impossible to measure the electrochemical activity of one half-cell; two reactions must be coupled and one reaction compared with the other. To rate half-cell reactions, a specific electrode reaction is arbitrarily assigned 0.00 V. Every other reaction coupled with this arbitrary zero reaction is either positive or negative, depending on the relative affinity for electrons. The electrode defined as 0.00 V is the standard hydrogen electrode: H2 gas at 1 atmosphere (atm). The hydrogen gas in contact with H+ in solution develops a potential. The hydrogen electrode coupled with a zinc half-cell is cathodic, with the reaction 2H+ + 2e– → H2, because H2 has a greater affinity than does Zn for electrons. Cu, however, has a greater affinity than H2 for electrons, and thus the anodic reaction H2 → 2H+ + 2e– occurs when coupled to the Cu- electrode half-cell. The potential generated by the hydrogen-gas electrode is used to rate the electrode potential of metals in 1 mol/L solution. Reduction potentials for certain metals are shown in Table 4.1.10 A hydrogen electrode is used to determine the accuracy of reference and indicator electrodes, the stability of standard solutions, and the potentials of liquid junctions. TABLE 4.1 Standard Reduction Potentials Potential, V Zn2+ + 2e ↔ Z –0.7628 Cr2+ + 2e ↔ Cr –0.913 Ni2+ + 2e ↔ Ni –0.257 2H+ + 2e ↔ H2 0.000 Figure 4.19 Necessary components of a pH meter. Cu2+ + 2e ↔ Cu 0.3419 © Wolters Kluwer. Ag+ + e ↔ Ag 0.7996 Description Indicator Electrode Data presented are examples from Lide DR. CRC Handbook of Chemistry and Physics. 93rd ed. Boca Raton, FL: CRC Press; 2012–2013. The pH electrode consists of a silver wire coated with AgCl, immersed into an internal solution of 0.1 mmol/L HCl, and placed into a tube containing a special glass membrane tip. This Ion-Selective Electrodes membrane is only sensitive to hydrogen ions (H+). Glass membranes that are selectively sensitive to H+ consist of specific quantities of lithium, cesium, lanthanum, barium, or aluminum Potentiometric methods of analysis involve the direct measurement of electrical potential due to oxides in silicate. When the pH electrode is placed into the test solution, movement of H+ near the activity of free ions. Ion-selective electrodes (ISEs) are designed to be sensitive toward the tip of the electrode produces a potential difference between the internal solution and the individual ions. test solution, which is measured as pH and read by a voltmeter. The combination pH electrode also contains a built-in reference electrode, either Ag/AgCl or calomel (Hg/Hg2Cl2) immersed in assuming there is no liquid junction potential. The isopotential is that potential at which a a solution of saturated KCl. temperature change has no effect on the response of the electrical cell. Manufacturers The specially formulated glass continually dissolves from the surface. The present concept of generally achieve this by making the midscale (pH 7.0) correspond to 0 V at all temperatures. the selective mechanism that causes the formation of electromotive force at the glass surface They use an internal buffer whose pH changes due to temperature compensate for the changes is that an ion-exchange process is involved. Cationic exchange occurs only in the gel layer. in the internal and external reference electrodes. There is no penetration of H+ through the glass. Although the glass is constantly dissolving, the process is slow, and the glass tip generally lasts for several years. pH electrodes are highly Nernst Equation selective for H+; however, other cations in high concentration interfere, the most common of The electromotive force generated because of H+ at the glass tip is described by Nernst which is sodium. Electrode manufacturers should list the concentration of interfering cations that equation, which is shown in a simplified form: may cause error in pH determinations. Reference Electrode (Eq. 4.6) The reference electrode commonly used is the calomel electrode. Calomel, a paste of where ε is the electromotive force of the cell, F is the Faraday constant (96,500 C/mol), R is predominantly mercurous chloride, is in direct contact with metallic mercury in an electrolyte the molar gas constant, and T is temperature, in Kelvin. solution of potassium chloride. As long as the electrolyte concentration and the temperature As the temperature increases, H+ activity increases and the potential generated increases. remain constant, a stable voltage is generated at the interface of the mercury and its salt Most pH meters have a temperature compensation knob that amplifies the millivolt response (mercurous chloride). A cable connected to the mercury leads to the voltmeter. The filling hole when the meter is on pH function. pH units on the meter scale are usually printed for use at is needed for adding potassium chloride solution. A tiny opening at the bottom is required for room temperature. On the voltmeter, 59.16 is read as 1 pH unit change. The temperature completion of electric contact between the reference and indicator electrodes. The liquid compensation changes the millivolt response to compensate for changes due to temperature junction consists of a fiber or ceramic plug that allows a small flow of electrolyte filling solution. from 54.2 at 0°C to 66.10 at 60°C. However, most pH meters are manufactured for greatest Construction varies, but all reference electrodes must generate a stable electrical potential. accuracy in the 10°C to 60°C range. Reference electrodes generally consist of a metal and its salt in contact with a solution containing the same anion. Mercury/mercurous chloride, as in this example, is a frequently used Calibration reference electrode; the disadvantage is that it is slow to reach a new stable voltage following The steps necessary to standardize a pH meter are fairly straightforward. First, balance the temperature change and it is unstable above 80°C.1,2 Ag/AgCl is another common reference system with the electrodes in a buffer with a 7.0 pH (Zone A). The balance or intercept control electrode. It can be used at high temperatures, up to 275°C, and the AgCl-coated Ag wire shifts the entire slope, as shown in Figure 4.20. Next, replace the buffer with one of a different makes a more compact electrode than that of mercury. In measurements in which chloride pH (Zone B). If the meter does not register the correct pH, amplification of the response contamination must be avoided, a mercury sulfate and potassium sulfate reference electrode changes the slope to match that predicted by Nernst equation. If the instrument does not have may be used. a slope control, the temperature compensator performs the same function. Liquid Junctions Electrical connection between the indicator and reference electrodes is achieved by allowing a slow flow of electrolyte from the tip of the reference electrode. A junction potential is always set up at the boundary between two dissimilar solutions because of positive and negative ions diffusing across the boundary at unequal rates. The resultant junction potential may increase or decrease the potential of the reference electrode. Therefore, it is important that the junction potential be kept to a minimum reproducible value when the reference electrode is in solution. KCl is a commonly used filling solution because K+ and Cl– have nearly the same mobilities. When KCl is used as the filling solution for Ag/AgCl electrodes, the addition of AgCl is required to prevent dissolution of the AgCl salt. One way of producing a lower junction potential is to mix K+, Na+, NO–, and Cl– in appropriate ratios. Readout Meter Electromotive force produced by the reference and indicator electrodes is in the millivolt range. Zero potential for the cell indicates that each electrode half-cell is generating the same voltage,  Figure 4.20 pH meter calibration. Data from Willard HH, Merritt LL, Dean JA, et al. Instrumental Methods of Analysis. Belmont, CA: Wadsworth; 1981. Description pH Combination Electrode The most commonly used pH electrode has both the indicator and reference electrodes combined in one small probe, which is convenient when small samples are tested. It consists of an Ag/AgCl internal reference electrode sealed in a narrow glass cylinder with a pH-sensitive glass tip. The reference electrode is an Ag/AgCl wire wrapped around the indicator electrode. The outer glass envelope is filled with KCl and has a tiny pore near the tip of the liquid junction. The solution to be measured must completely cover the glass tip. Examples of other ISEs are shown in Figure 4.21. The reference electrode, electrometer, and calibration system described for pH measurements are applicable to all ISEs. Figure 4.21 Other examples of ion-selective electrodes. © Wolters Kluwer.  There are three major ISE types: inert metal electrodes in contact with a redox couple, metal electrodes that participate in a redox reaction, and membrane electrodes. The membrane can be solid material (e.g., glass), liquid (e.g., ion-exchange electrodes), or special membrane (e.g., compound electrodes), such as gas-sensing and enzyme electrodes. The standard hydrogen electrode is an example of an inert metal electrode. The Ag/AgCl electrode is an example of the second type. The electrode process AgCl + e– → Ag+ + Cl– produces an electrical potential proportional to chloride ion (Cl–) activity. When Cl– is held constant, the electrode is used as a reference electrode. The electrode in contact with varying Cl– concentrations is used as an indicator electrode to measure Cl– concentration. The H+-sensitive gel layer of the glass pH electrode is considered a membrane. A change in the glass formulation makes the membrane more sensitive to sodium ions (Na+) than to H+, creating a sodium ISE. Other solid-state membranes consist of either a single crystal or fine crystals immobilized in an inert matrix such as silicone rubber. Conduction depends on a vacancy defect mechanism, and the crystals are formulated to be selective for a particular size, shape, and charge. Examples include F–-selective electrodes of LaF3, Cl–-sensitive electrodes with AgCl crystals, and AgBr electrodes for the detection of Br–. The calcium ISE is a liquid membrane electrode. An ion-selective carrier, such as dioctylphenyl phosphonate dissolved in an inert water-insoluble solvent, diffuses through a porous membrane. Because the solvent is insoluble in water, the test sample cannot cross the membrane, but calcium ions (Ca2+) are exchanged. The Ag/AgCl internal reference in a filling solution of CaCl2 is in contact with the carrier by means of the membrane. Potassium-selective liquid membranes use the antibiotic valinomycin as the ion-selective carrier. Valinomycin membranes show great selectivity for K+. Liquid membrane electrodes are recharged every few months to replace the liquid ion exchanger membrane and the porous Figure 4.22 The pCO2 electrode. membrane. © Wolters Kluwer. Gas-Sensing Electrodes The change in pH of the is detected by a pH electrode. The pCO2 electrode is widely Gas electrodes are similar to pH glass electrodes but are designed to detect specific gases used in clinical laboratories as a component of instruments for measuring serum electrolytes (e.g., CO2 and NH3) in solutions and are usually separated from the solution by a thin, gas- and blood gases. permeable hydrophobic membrane. Figure 4.22 shows a schematic illustration of the pCO2 In the NH3 gas electrode, the bicarbonate solution is replaced by ammonium chloride solution, electrode. The membrane in contact with the solution is permeable only to CO2, which diffuses and the membrane is permeable only to NH3 gas. As in the pCO2 electrode, NH3 changes the into a thin film of sodium bicarbonate solution. The pH of the bicarbonate solution is changed as pH of NH4Cl as follows: follows: (Eq. 4.8) (Eq. 4.7) The amount of OH– produced varies linearly with the log of the partial pressure of NH in the 3 sample. Other gas-sensing electrodes function on the basis of an amperometric principle—that is, measurement of the current flowing through an electrochemical cell at a constant applied electrical potential to the electrodes. Examples are the determination of pO2, glucose, and peroxidase. The chemical reactions of the pO2 electrode (Clark electrode), an electrochemical cell with a platinum cathode and an Ag/AgCl anode, are illustrated in Figure 4.18. The electrical potential at the cathode is set to –0.65 V and will not conduct current without oxygen in the sample. The membrane is permeable to oxygen, which diffuses through to the platinum cathode. Current passes through the cell and is proportional to the pO2 in the test sample. Electrophoresis Glucose determination is based on the reduction in pO2 during glucose oxidase reaction with glucose and oxygen. Unlike the pCO2 electrode, the peroxidase electrode has a polarized Electrophoresis is the migration of charged solutes or particles in an electrical field. platinum anode and its potential is set to +0.6 V. Current flows through the system when Iontophoresis refers to the migration of small ions, whereas zone electrophoresis is the peroxide is oxidized at the anode as follows: migration of charged macromolecules in a porous support medium such as paper, cellulose acetate, or agarose gel film. An electrophoretogram is the result of zone electrophoresis and consists of sharply separated zones of a macromolecule. In a clinical laboratory, the (Eq. 4.9) macromolecules of interest are proteins in serum, urine, cerebrospinal fluid (CSF), other biologic body fluids, erythrocytes, and tissue. Enzyme Electrodes Electrophoresis consists of five components: the driving force (electrical power), the support The various ISEs may be covered by immobilized enzymes that can catalyze a specific medium, the buffer, the sample, and the detecting system. A typical electrophoretic apparatus chemical reaction. Selection of the ISE is determined by the reaction product of the immobilized is illustrated in Figure 4.23. enzyme. Examples include urease, which is used for the detection of urea, and glucose oxidase, which is used for glucose detection. A urea electrode must have an ISE that is selective for NH4 + or NH3, whereas glucose oxidase is used in combination with a pH electrode. Coulometric Titration In coulometric titration a constant current is applied and the potential of a working electrode is monitored. When all of the analyte has changed state, the change in potential is registered. Coulometric titration is used clinically for sweat chloride determination. Anodic Stripping Voltammetry In anodic stripping voltammetry, the analyte is first concentrated onto the surface of an electrode at a constant potential and then goes back into solution as the voltage is changed. Anodic stripping voltammetry is used for the analysis of lead in point-of-care and laboratory settings, although lead testing in the laboratory is currently more commonly performed by electrothermal (graphite furnace) atomic absorption spectroscopy or, preferably, inductively coupled plasma-mass spectrometry (ICP-MS). Figure 4.23 Electrophoresis apparatus—basic components. © Wolters Kluwer. Charged particles migrate toward the opposite charged electrode. The velocity of migration is controlled by the net charge of the particle, the size and shape of the particle, the strength of the electric field, chemical and physical properties of the supporting medium, and the electrophoretic temperature. The rate of mobility11 of the molecule (μ) is given by  Cellulose Acetate (Eq. 4.10) Paper electrophoresis use has been replaced by cellulose acetate or agarose gel in clinical where Q is net charge of the particle, r is the ionic radius of the particle, and η is the viscosity laboratories. Cellulose is acetylated to form cellulose acetate by treating it with acetic of the buffer. anhydride. Cellulose acetate, a dry, brittle film composed of about 80% air space, is produced From the equation, the rate of migration is directly proportional to the net charge of the commercially. When the film is soaked in buffer, the air spaces fill with electrolyte and the film particle and inversely proportional to its size and the viscosity of the buffer. becomes pliable. After electrophoresis and staining, cellulose acetate can be made transparent for densitometer quantitation. The dried transparent film can be stored for long periods. Procedure Cellulose acetate prepared to reduce electroendosmosis is available commercially. Cellulose The sample is soaked in hydrated support for approximately 5 minutes. The support is put into acetate is also used in isoelectric focusing. the electrophoresis chamber, which was previously filled with the buffer. Sufficient buffer must Agarose Gel be added to the chamber to maintain contact with the support. Electrophoresis is carried out by applying a constant voltage or constant current for a specific time. The support is then removed Agarose gel is another widely used supporting medium. Used as a purified fraction of agar, it is and placed in a fixative or rapidly dried to prevent diffusion of the sample. This is followed by neutral and, therefore, does not produce electroendosmosis. After electrophoresis and staining, staining the zones with an appropriate dye. The uptake of dye by the sample is proportional to it is destained (cleared), dried, and scanned with a densitometer. The dried gel can be stored sample concentration. After excess dye is washed away, the supporting medium may need to indefinitely. Agarose gel electrophoresis requires small amounts of sample (~2 mL); it does not be placed in a clearing agent. Otherwise, it is completely dried. bind protein and, therefore, migration is not affected. Power Supply Polyacrylamide Gel Power supplies operating at either constant current or constant voltage are available Polyacrylamide gel electrophoresis involves separation of protein on the basis of charge and commercially. In electrophoresis, heat is produced when current flows through a medium that molecular size. Layers of gel with different pore sizes are used. The gel is prepared before has resistance, resulting in an increase in thermal agitation of the dissolved solute (ions) and electrophoresis in a tube-shaped electrophoresis cell. The small-pore separation gel is at the leading to a decrease in resistance and an increase in current. The increase leads to increases bottom, followed by a large-pore spacer gel and, finally, another large-pore gel containing the sample. Each layer of gel is allowed to form a gelatin before the next gel is poured over it. At in heat and evaporation of water from the buffer, increasing the ionic concentration of the buffer and subsequent further increases in the current. The migration rate can be kept constant by the start of electrophoresis, the protein molecules move freely through the spacer gel to its using a power supply with constant current. This is true because, as electrophoresis boundary with the separation gel, which slows their movement. This allows for concentration of progresses, a decrease in resistance as a result of heat produced also decreases the voltage. the sample before separation by the small-pore gel. Polyacrylamide gel electrophoresis separates serum proteins into 20 or more fractions rather than the usual 6 fractions separated Buffers by cellulose acetate or agarose. It is widely used to study individual proteins (e.g., Two buffer properties that affect the charge of ampholytes are pH and ionic strength. The ions isoenzymes). carry the applied electric current and allow the buffer to maintain constant pH during Starch Gel electrophoresis. An ampholyte is a molecule, such as a protein, for which the net charge can be either positive or negative. If the buffer is more acidic