chem chapt 5: analytic techniques

Spec QA

wavelength, accurary, stray light and linearity

atomic absorption spect: analyzed sample

- must contain the reduced metal in the atomic vaporized state -this is done by using the heat of a flame to break the chemical bonds and form free, unexcited atoms -flame (instead of cuvette): common burner is the premix long-path burner -sample in solution: is aspirated as a spray into a chamber, where it is mixed with air and fuel -mixture passes through baffles, where large drops fall and are drained off. Only fine droplets reach the flame -Light from the hollow-cathode lamp passes through the sample of ground state atoms in the flame. The amount of light absorbed is proportional to the concentration -When a ground state atom absorbs light energy, an excited atom is produced. The excited atom then returns to the ground state, emitting light of the same energy as it absorbed -The flame sample thus contains a dynamic population of ground state and excited atoms, both absorbing and emitting radiant energy. The emitted energy from the flame will go in all directions, and it will be a steady emission -the hollowcathode 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 -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 -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

isoelectric focusing

-Charged proteins migrate through a support medium that has a continuous pH gradient -Individual proteins move in the electric field until they reach a pH equal to their isoelectric point, at which point they have no charge and cease to move

coulometric chloridometers and anodic stripping voltammetry

-Chloride ISEs have largely replaced coulometric titrations for the determination of chloride in body fluids -Anodic stripping voltammetry was widely used for the analysis of lead and is best measured by electrothermal (graphite furnace) atomic absorption spectroscopy or, preferably, inductively coupled plasma-mass spectrometry (ICP-MS)

liquid juctions

-Electrical connection between the indicator and reference electrodes is achieved by allowing a slow flow of electrolyte from the tip of the reference electrode -is always set up at the boundary between two dissimilar solutions because of positive and negative ions diffusing across the boundary at unequal rates -resultant junction potential may increase or decrease the potential of the reference electrode -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+, NO3 −, 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, assuming there is no liquid junction potential -isopotential is that potential at which a temperature change has no effect on the response of the electrical cell. Manufacturers generally achieve this by making the midscale (pH 7.0) correspond to 0 V at all temperatures -use an internal buffer whose pH changes due to temperature compensate for the changes in the internal and external reference electrodes.

membrane electrode

-H+-sensitive gel layer of the glass pH electrode -change in the glass formulation makes the membrane more sensitive to sodium ions (Na+) than to H+, creating a sodium ISE -Other solidstate 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 change -ex:; F−-selective electrodes of LaF3, Cl−-sensitive electrodes with AgCl crystals, and AgBr electrodes for the detection of Br−.

monochromators

-Isolate individual wavelength of light -bandpass: defines the range of wavelengths transmitted and is calculated as width at half the maximum transmittanc

Beer law

-Relationship between absorption of light by a solution and the concentration of the solution -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 -%T: Transmittance -A: Absorbance

blank

-The solvent without constituent of interest (analyte) is placed in the light path -Most of the light is transmitted -Small amount is absorbed by solvent and cuvette (or reflected away from detector) -The electrical readout is set at 100%T -Sample containing absorbing molecules to be measured is placed with the solvent in the light path → Analyte -Difference in the amount of light transmitted by the blank and the sample mixture is due to the analyte being measured.

surface plasmon resonance

-This technique enables the study of the binding of ligands to surface receptors such as membrane proteins in real time -A light source passes through a prism and onto a back side of a sensor chip containing a metal such as gold and reflects off the back of the chip onto a detector -At the proper angle of light, the light is absorbed by the electrons in the metal causing a resonance -decreases the amount of light reaching the detector. SPR can be used to measure interactions between immobilized antibodies and freely circulating analytes -used for the analysis of low molecular weight compounds, proteins and biomarkers, hormones, nucleic acids, circulating antibodies, and infectious microorganisms from biological matrices -analytical sensitivity of SPR is similar to that of conventional immunoassays. The costs for developing and using SPR may be less expensive since there are no labels involved. Moreover, sensors can be placed into an array for multiplexing purposes

Turbidity

-Turbidimetric measurements are made with a spect determine the concentration of particulate matter in a sample -The amount of light blocked by a suspension of particles depends not only on concentration but also on size -particles tend to aggregate and settle out of suspension, sample handling becomes critica -Measuring light scatter at an angle other than 180° that is employed in turbidimetry minimizes error from colored solutions and increases sensitivity

electrophoresis buffe

-Two buffer properties that affect the charge of ampholytes are pH and ionic strength -ions carry the applied electric current and allow the buffer to maintain constant pH during electrophoresis -ampholyte:molecule, such as protein, whose net charge can be either positive or negative -if the buffer is more acidic: than the isoelectric point (pI) of the ampholyte, it binds H+, becomes positively charged, and migrates toward the cathode -more basic: than the pI, the ampholyte loses H+, becomes negatively charged, and migrates toward the anode -particle with no net charge: will not migrate, remaining at the point of application -during this process: ions cluster around a migrating particle. The higher the ionic concentration, the higher the size of the ionic cloud and the lower the mobility of the particle -Greater ionic strength produces sharper protein-band separation but leads to increased heat production, can cause denaturation of heat-labile proteins -optimal buffer concentration should be determined for any electrophoretic system

agarose gel

-Used as a purified fraction of agar, it is neutral and, therefore, does not produce electroendosmosis -after electrophoresis and staining: it is detained (cleared), dried, and scanned with a densitometer -requires small amounts of sample (~2 mL); it does not bind protein and, therefore, migration is not affected

photodiode

-absorption of radiant energy by a reverse-biased pn-junction diode (pn, positive-negative) produces a photocurrent that is proportional to the incident radiant power -not as sensitive as PM, lack of internal amplification, their excellent linearity, speed, and small size make them useful in applications where light levels are adequate -photodiode array (PDA): detectors are available in integrated circuits containing 256 to 2,048 photodiodes in a linear arrangement - each potodiode respond to a specific wavelength: complete UV/visible spectrum can be obtained in less than 1 second -resolution: to 2 nm and depends on the number of discrete elements

absorbance

-amount of light absorbed -needs to be mathematically derived from T% -%T = I/Io x 100 Io= incidcent light I= transmitted light A=-log (I/Io)= log(100%)-log%T=2-log%T

flourescence concentration

-are related to molar absorptivity of the 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 -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 -The solution must absorb less than 5% of the exciting radiation for a linear response to occur

laser applications

-based on the interaction of radiant energy with suitably excited atoms or molecules -interaction leads to stimulated emission of radiation -wavelength, direction of propagation, phase, and plane of polarization of the emitted light are the same as those of the incident radiation -laser light: polarized and coherent and has narrow spectral width and small cross-sectional area with low divergence -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, making these applications about three to six orders more sensitive than conventional spectrometers -can also be used for the determination of structure and identification of samples, as well as for diagnosis -

reference electrode

-calomel: paste of predominantly mercurous chloride, is in direct contact with metallic mercury in an electrolyte solution of potassium chloride -electrolyte concentration and the temperature remain constant, a stable voltage is generated at the interface of the mercury and its salt -cable connected to the mercury leads to the voltmeter. The filling hole is needed for adding potassium chloride solution -liquid junction consists of a fiber or ceramic plug that allows a small flow of electrolyte filling solution -must generate a stable electrical potential -generally consist of a metal and its salt in contact with a solution containing the same anion -mercury/mercurous: is a frequently used reference electrode; the disadvantage is that it is slow to reach a new stable voltage following temperature change and it is unstable above 80°C -Ag/AgCl is another common reference electrode. It can be used at high temperatures, up to 275°C, and the AgCl-coated Ag wire makes a more compact electrode than that of mercury -chloride contamination must be avoided, a mercury sulfate and potassium sulfate reference electrode may be used

elecropheresis power supply

-can be constant current or constant voltage -heat is produced when current flows through a medium that has resistance, resulting in an increase in thermal agitation of the dissolved solute (ions) and leading to a decrease in resistance and an increase in current. -increase leads to increases in heat and evaporation of water from the buffer, increasing the ionic concentration of the buffer and subsequent further increases in the current -migration rate can be kept constant by using a power supply with constant current. This is true because, as electrophoresis progresses, a decrease in resistance as a result of heat produced also decreases the voltage

two-dimensional electrophoresis

-combines two different electrophoresis dimensions to separate proteins from complex matrices such as serum or tissue -first dimension, proteins are resolved according to their isoelectric points (pIs), using immobilized pH gradients -second dimension, proteins are separated according to their relative size (molecular weight), using sodium dodecyl sulfate-polyacrylamide gel electrophoresis -Gels can be run under denaturing or nondenaturing conditions (e.g., for the maintenance of enzyme activity) and visualized by a variety of techniques, including the use of colorimetric dyes (e.g., Coomassie blue or silver stain) and radiographic, fluorometric, or chemiluminescence of appropriately labeled polypeptides -These latter techniques are considerably more sensitive than the colorimetric dyes

electrochemical cell

-consists of two half-cells and a salt bridge, which can be a piece of filter paper saturated with electrolytes -the electrodes can be immersed in a single, large beaker containing a salt solution -solution serves as the salt bridge

spect sample cell

-cuvette or cell: has a flat surface -light path: must be kept constant to have absorbance proportional to concentration -can be checked by: preparing a colored solution to read midscale when using the wavelength of maximum absorption -cuvettes with scratch optical surfaces scatter light should be discarded -quartz cuvettes: enable transmission of light and are used when substances absorb in this region

linearity

-demonstrated when a change in concentration results in a straight line calibration curve -colored solutions can be diluted and use to check: using the wavelength of maximal absorbance for that color or neutral-density filters

chemiluminescence advantages

-detection limits, speed, ease of use and simple instrumentation

galvanic cell

-electrodes are connected, there is spontaneous flow of electrons from the electrode with the lower electron affinity -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 -In short, a galvanic cell can be built from an electrolytic cell

sample flourophore

-excited: emits polarized light along the same plane as the incident light if the fluorophore does not rotate in solution -a small molecule emits depolarized light because it will rotate out of the plane of polarization during its excitation lifetime -used for the detection of therapeutic and abused drugs. -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 macromolecular antibody-analyte-fluorophore formed and the lower the depolarization of the radiant light

flourometry basic instrumentation

-filter flourometers: measure the concentrations of solutions that contain fluorescing molecules -source emits short-wavelength high-energy excitation light -primary filter: placed between the radiation source and the sample, selects the wavelength that is best absorbed by the solution to be measured -flourescing sample: in the cuvette emits radiant energy in all directions -detector: and a secondary filter that passes the longer wavelengths of fluorescent light prevent incident light from striking the photodetector -electrical output of the photodetector is proportional to the intensity of fluorescent energy. In spectrofluorometers, the filters are replaced by a grating monochromator

flourometry disadvantage

-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 change solvent may change the structure -UV light used for excitation can cause photochemical changes -

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 -ex: determination of pO2, glucose, and peroxidase. -chemical reactions of the pO2 electrode (Clark electrode), an electrochemical cell with a platinum cathode and an Ag/AgCl anode, -The electrical potential at the cathode is set to −0.65 V and will not conduct current without oxygen in the sample -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

flourometry lamps

-gas discharge lamps (mercury and xenon arc):most frequently used sources of excitation radiant energy -incandescent tungsten lamps: seldom used because they release little energy in the UV region -mercury vapor lamps: commoly used in filter flourometers -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 broad band of longer wavelengths. -xenon lamp: high-pressure, has a good continuum, which is necessary for determining the excitation spectra

E=hv

-h is planck constant: 6.62 x 10^-27 -v: frequency

gas sensing electrodes

-imilar to pH glass electrodes but are designed to detect specific gases (e.g., CO2 and NH3) in solutions and are usually separated from the solution by a thin, gas-permeable hydrophobic membrane -The membrane in contact with the solution is permeable only to CO2, which diffuses into a thin film of sodium bicarbonate solution -The change in pH of the HCO3 − is detected by a pH electrode -pCO2 electrode is widely used in clinical laboratories as a component of instruments for measuring serum electrolytes and blood gases -NH3 gas electrode, the bicarbonate solution is replaced by ammonium chloride solution, and the membrane is permeable only to NH3 gas. As in the pCO2 electrode, NH3 changes the pH of NH4Cl -The amount of OH− produced varies linearly with the log of the partial pressure of NH3 in the sample

half cells

-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 -potential generated by the hydrogen-gas electrode is used to rate the electrode potential of metals in 1 mol/L solution -hydrogen electrode is used to determine the accuracy of reference and indicator electrodes, the stability of standard solutions, and the potentials of liquid junctions

3 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

polycrylamide gel

-involves separation of protein on the basis of charge and molecular size -Layers of gel with different pore sizes are used -is prepared before electrophoresis in a tube-shaped electrophoresis cell -small-pore separation gel is at the bottom, followed by a large-pore spacer gel and, finally, another large-pore gel containing the sample -At the start of electrophoresis, the protein molecules move freely through the spacer gel to its boundary with the separation gel, which slows their movement -allows for concentration of 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 by cellulose acetate or agarose

cellulose acetate electrophoresis

-is acetylated to form cellulose acetate by treating it with acetic anhydride -a dry, brittle film composed of about 80% air space, is produced commercially -the film is soaked in buffer, the air spaces fill with electrolyte and the film becomes pliable -After electrophoresis and staining, cellulose acetate can be made transparent for densitometer quantitation -dried transparent film can be stored for long periods -Cellulose acetate prepared to reduce electroendosmosis is available commercially. Cellulose acetate is also used in isoelectric focusing

absorbance in beer law

-is directly proportional to concentration A = e x b x c . ɛ → molar absorptivity → the fraction of a specific wavelength of light absorbed by a given type of molecule b → length of light path through the solution c → concentration of absorbing molecules

osmometry

-is used to measure the concentration of solute particles in a solution osmolality = o x n x C -o: osmotic coefficient -n: number of dissociable particles (ions) per molecule in the solution -C: concentration in moles per kg of solvent -osmotic coefficient is an experimentally derived factor to correct for the fact that some of the molecules, even in a highly dissociated compound, exist as molecules rather than as ions -four physical properties of a solution that change with variations in the number of dissolved particles in the solvent are osmotic pressure, vapor pressure, boiling point, and freezing point -measure osmolality indirectly by measuring one of these colligative properties, which change proportionally with osmotic pressure -measure either freezing point depression or vapor pressure depression; results are expressed in milliosmolal per kilogram (mOsm/kg) units

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 -when energy is absorbed, valence electrons move to an orbital with a higher energy level -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

photocell

-least expensive -composed: film of light-sensitive material, frequently selenium, on a plate of iron -Over the light sensitive material is a thin, transparent layer of silver -when exposed to light: electrons in the light-sensitive material are excited and released to flow to the highly conductive silver -resistance oposes the electron flow toward the iron, forming a hypothetical barries to flow in that direction - require no external voltage source but rely on internal electron transfer to produce a current in an external circuit -used mainly in filter photometers with a wide bandpass, producing a fairly high level of illumination so that there is no need to amplify the signal it is temperature sensitive and nonlinear at very low and very high levels of illumination

flourometry

-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 -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 -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 -difference between the maximum wavelengths, excitation, and emitted fluorescence is called Stokes shift -Both excitation (absorption) and fluorescence (emission) energies are characteristic for a given molecular type -ex: shows the absorption and fluorescence spectra of 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 energy) fluorescent spectrum

Nephelometry

-light scattered by the small particles is measured at an angle to the beam incident on the cuvette -Light scattering depends on wavelength and particle size -macromolecules: size close to or larger than the wavelength of incident light, sensitivity is increased by measuring the forward light scatter -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 -instruments use light amplification by stimulated emission of radiation (laser) as a source of monochromatic light; however, any monochromator may be used -

absorption or emission of energy by atoms results in

-line spectrum: because of relative complexity of molecules they absorb or emit a bank of energy over a large region

calcium ISE

-liquid membrane electrode -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 -Ag/AgCl internal reference in a filling solution of CaCl2 is in contact with the carrier by means of the membrane

Spec wavelength accuracy

-means that the wavelength indicated on the control dial is the actual wavelength of light passed by the monochromator -checked using standard absorbing solutions or filters with known wavelength of maximum absorbance -didymiun or holmium oxide in glass: is used as filter 1. filter is placed in the light path, and the wavelength control is set at the wavelength at which maximal absorbance is expected 2.wavelength control is then rotated in either direction to locate the actual wavelength that has maximal absorbance 3. these two wavelengths do not match, the optics must be adjusted to calibrate the monochromator correctly.

photometric instruments (SPECT)

-measure light intensity without consideration of wavelength -use filters (photometers), prisms, or gratings (spectrometers) to select (isolate) a narrow range of the incident wavelength

flame photometry

-measures light emitted by excited atoms, was widely used to determine concentration of Na+, K+, or Li+

electrophoresis

-migration of charged solutes or particles in an electrical field - whereas zone electrophoresis is the migration of charged macromolecules in a porous support medium such as paper, cellulose acetate, or agarose gel film -electrophoretogram: result of zone electrophoresis and consists of sharply separated zones of a macromolecule -macromolecules of interest: proteins in serum, urine, cerebrospinal fluid (CSF), and other biologic body fluids and erythrocytes and tissue -consists of 5 components: the driving force (electrical power), the support medium, the buffer, the sample, and the detecting system -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

pH combination electrode

-most commonly used: has both the indicator and reference electrodes combined in one small probe -consists: Ag/AgCl internal reference electrode sealed in a narrow glass cylinder with a pH-sensitive glass tip -reference electrode: is an Ag/AgCl wire wrapped around the indicator electrode -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

electroendosmosis

-movement of buffer ions and solvent relative to the fixed support -Support media, such as paper, cellulose acetate, and agar gel, take on a negative charge from adsorption of hydroxyl ions -When current is applied to the electrophoresis system, the hydroxyl ions remain fixed while the free positive ions move toward the cathode -ions are highly hydrated, resulting in net cathodic movement of solvent -Molecules that are nearly neutral are swept toward the cathode with the solvent. -Support media such as agarose and acrylamide gel are essentially neutral, eliminating electroendosmosis -position of proteins in any electrophoresis separation depends not only on the nature of the protein but also on all other technical variables

absoprtion or emission

-of radiant energy to determine the concetration of atoms or molecules -closely related

indicator electrode

-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 membrane is only sensitive to hydrogen ions (H+). -Glass membranes: selectively sensitive to H+ consist of specific quantities of lithium, cesium, lanthanum, barium, or aluminum oxides in silicate -pH electrode placed into the test solution: movement of H+ near the tip of the electrode produces a potential difference between the internal solution and the test solution, which is measured as pH and read by a voltmeter -combination pH electrode also contains a built-in reference electrode, either Ag/AgCl or calomel (Hg/Hg2Cl2) immersed in a solution of saturated KCl -formulated glass: continually disolves from the surface -Cationic exchange occurs only in the gel layer—there is no penetration of H+ through the glass -glass is constantly dissolving, the process is slow, and the glass tip generally lasts for several years. pH electrodes are highly selective for H+; however, other cations in high concentration interfere, the most common of which is sodium

chemiluminescence

-part of the chemical energy generated produces excited intermediates that decay to a ground state with the emission of photons -emitted radiation is measured with a PM tube, and the signal is related to analyte concentration -different from fluorescence in that no excitation radiation is required and no monochromators are needed because the chemiluminescence arises from one species -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 -Enhanced chemiluminescence techniques increase the chemiluminescence efficiency by including an enhancer system in the reaction of a chemiluminescent agent with an enzyme -time course for the light intensity is much longer (60 minutes) than that for conventional chemiluminescent reactions, which last for about 30 seconds

phototube

-photosensitive material that gives off electrons when light energy strikes it. -contain a negatively charged cathode and a positively charged anode enclosed in a glass case. The cathode is composed of a material (e.g., rubidium or lithium) that acts as a resistor in the dark but emits electrons when exposed to light -emitted electrons jump over to the positively charged anode, where they are collected and return through an external, measurable circuit -The 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 -vaccum within the tubes avoids scattering of the photoelectrons by collision with gas molecules.

stray lights

-refers to any wavelengths outside the band transmitted by the monochromator. -cause by: reflection of light from scratches on optical surfaces or from dust particles anywhere in the light path and higher order spectra produced by diffraction gratings -major effect is absorbance error, especially in the high absorbance range -can be detected: cutoff filters, which eliminate all radiation at wavelengths beyond the one of interest -to check for stray light in the near-UV region: filter that does not transmit in the region of 200 to 400 nm is inserted - If 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 stray light in the UV range

flameless atomic absorption

-requires an instrument modification that uses an electric furnace to break chemical bonds -A tiny graphite cylinder holds the sample, either liquid or solid. An electric current passes through the cylinder walls, evaporates the solvent, ashes 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 -deuterium lamp as a secondary source and measures the difference between the two absorbance signals

freezing point osmometer

-sample in a small tube is lowered into a chamber with cold refrigerant circulating from a cooling unit -To measure temperature, a wire is used to gently stir the sample until it is cooled to several degrees below its freezing point -supercooled solution: cool water to as low as −40°C and still have liquid water, provided no crystals or particulate matter is present -Vigorous agitation when the sample is supercooled results in rapid freezing. Freezing can also be started by "seeding" a supercooled solution with crystals. -supercooled solution starts to freeze as a result of the rapid stirring, a slush is formed and the solution actually warms to its freezing point temperature. The slush, an equilibrium of liquid and ice crystals, will remain at the freezing point temperature until the sample freezes solid and drops below its freezing point -Impurities in a solvent will lower the temperature at which freezing or melting occurs by reducing the bonding forces between solvent molecules so that the molecules break away from each other and exist as a fluid at a lower temperature -decrease in the freezing point temperature is proportional to the number of dissolved particles present -thermistor is a material that has less resistance when the temperature increases. The readout uses a Wheatstone bridge circuit that detects temperature change as proportional to change in thermistor resistance. Freezing point depression is proportional to the number of solute particles. Standards of known concentration are used to calibrate the instruments in mOsm/kg

atomatic absorption spect characteristics

-sensitive and precise -measures: concentration of trace metals that are not easily excited -more sensitive than flame emission because the vast majority of atoms produced in the usual propane or air-acetylene flame remain in the ground state available for light absorption -disadvantage: inability of the flame to dissociate samples into free atoms ex: phosphate may interfere with calcium analysis by formation of calcium phosphate. This may be overcome by adding cations that compete with calcium for phosphate. Routinely, lanthanum or strontium is added to samples to form stable complexes with phosphate -ionization of atoms following dissociation by the flame, which can be 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

capillary electrophoresis

-separation is performed in narrow-bore fused silica capillaries (inner diameter 25 to 75 μm) -capillaries are only filled with buffer, although gel media can also be used -the capillary is filled with buffer and then the sample is loaded; applying an electric field performs the separation -fundamental CE concept is the electro-osmotic flow (EOF). -EOF is the bulk flow of liquid toward the cathode upon application of an electric field, and it is superimposed on electrophoretic migration -Cations migrate fastest because both EOF and electrophoretic attraction are toward the cathode; neutral molecules are all carried by the EOF but are not separated from each other; and anions move slowest because, although they are carried to the cathode by the EOF, they are attracted to the anode and repelled by the cathode -Widely used for monitoring separated analytes, UV-visible detection is performed directly on the capillary; however, sensitivity is poor because of the small dimensions of the capillary, resulting in a short path length -used for the separation, quantitation, and determination of molecular weights of proteins and peptides; for the analysis of polymerase chain reaction products; and for the analysis of inorganic ions, organic acids, pharmaceuticals, optical isomers, and drugs of abuse in serum and urine

electrode process

-standard hydrogen: example of an inert metal electrode -Ag/AgCl: electrode is an example of the second type -electrode process: AgCl + e− → Ag+ + Cl− produces an electrical potential proportional to chloride ion (Cl−) activity -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

absorptivity depends on

-the molecular structure and the way in which the absorbing molecules react with different energies -changes as wavelength changes -the amount of light absorbed at a particular wavelength depends on the molecular and ion types present and on pH, temperature and concentration

calibration

-to stardardize a pH meter -balance the system with the electrodes in a buffer with a 7.0 pH. -The balance or intercept control shifts the entire slope -Next, replace the buffer with one of a different pH. If the meter does not register the correct pH, amplification of the response changes the slope to match that predicted by Nernst equation. If the instrument does not have a slope control, the temperature compensator performs the same function.

photomultiplier (PM)

-tube, which detects and amplifies radiant energy -incident light strikes the coated cathode, emitting electrons. -electrons are attracted to dynodes are of a material that gives off many secondary electrons when hit by single electrons -PM tube is 200 times more sensitive than the phototube. -accumulation of electrons striking the anode produces a current signal, measured in amperes, that is proportional to the initial intensity of the light -analog signal is converted first to a voltage and then to a digital signal through the use of an analog-todigital converter. Digital signals are processed electronically to produce absorbance readings.

monochromator flourometers

-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

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 membrane

coupled plasma (ICP)

-used to increase sensitivity for atomic emission -torch, an argon plasma maintained by the interaction of a radiofrequency field and an ionized argon gas, is reported to have used temperatures between 5,500 and 8,000 K -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 spect:: monochromator

-used to isolate the desired emission line from other lamp emission lines -it serves to protect the photodetector from excessive light emanating from flame emissions. A PM tube is the usual light detector

enzymes electrodes

-various ISEs may be covered by immobilized enzymes that can catalyze a specific chemical reaction -Selection of the ISE is determined by the reaction product of the immobilized enzyme -ex: 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

equal thickness of an absorbing material

-will absorb a constant fraction of the energy incident upon the layers -ex: *tube containing layer of solution first layer transmits 70% of the light incident upon it -2nd layer: 70% of 70% (49%) -The third layer transmits 70% of 49%, or 34% of the original light -next layers: 24% and 17%, respectively. -t%: yield a curve, meaning that we can translate layers of material to concentration

nerst equation

-ε is the electromotive force of the cell -F is the Faraday constant (96,500 C/mol) -R is the molar gas constant, and T is temperature, in Kelvin -temperature increases, H+ activity increases and the potential generated increases -pH function: temperature compensation knob that amplifies the millivolt response -voltmeter, 59.16 is read as 1 pH unit change -temperature compensation changes the millivolt response to compensate for changes due to temperature from 54.2 at 0°C to 66.10 at 60°C

% transmittance

1. bean of light entering the solution 2.some light is absorbed 3.remainder passes through and strikes a detector 4. detector converts to electrical signal 5: %T is the ratio of the radiant energy transmitted (T) divided by the radiant energy incident on the sample (I) *0%T → all light absorbed or blocked *100%T → no light absorbed

uknown concentrations are determined from

1. calibration curve that plots absorbance at a specific wavelength versus concentration for standards of known concentration. 2.calibration curves that are linear and have a zero y-intercept, unknown concentrations can be determined from a single calibrator 3.Not all calibration curves result in straight lines. Deviations from linearity are typically observed at high absorbances

used for monochromatic light

1. colored glass filters: filters usually pass a relatively wide band of radiant energy and have a low transmittance of the selected wavelength. Although not precise, they are simple and inexpensive 2. interference filters: produce light based on the principle of constructive interference of waves - Two pieces of glass, each mirrored on one side, are separated by a transparent spacer that is one-half the desired wavelength -Light waves enter one 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 until passing through -interference filters: transmit multiple desired wavelegths, required accesory filters to eliminate there harmonic wavelengths 3. glass prism: arrow 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. 4. diffraction gratings: consists of many parallel grooves (15,000 or 30,000 per inch) etched onto a polished surface -the separation of light into component wavelengths, is based on the principle that wavelengths bend as they pass a sharp corner -Those that are in phase reinforce one another, whereas those not in phase cancel out and disappear -result: complete spectrum

light source of spec

1. incadescent tungsten or tungstem-iodide lamp -Only about 15% of radiant energy emitted falls in the visible region, with most emitted as near infrared -heat-absorbing filter is inserted between the lamp and the sample to absorb the infrared radiation -lamp for UV a)Deuterium provides continuous emission down to 165 nm b) mercury arc lamp: low pressure, emit a sharp line spectrum, with both UV and visible lines -medium and high pressure: emit a continuum from UV to the mid-visible region

flourometry advantage

1. increases specificity by selecting the optimal wavelength for both absorption and fluorescence, rather than just the absorption wavelength seen with spectrophotometry 2.1,000 times more sensitive: emitted radiation is measured directly; it can be increased simply by increasing the intensity of the exciting radiant energy -flourescence: measures the amount of light intensity present over a zero background -absorbance: quantity of the absorbed light is measured indirectly as the difference between the transmitted beams

mercury lamp to verify wavelength accuracy

1. mercury lamp is substituted for the usual light source, and the spectrum is scanned to locate mercury emission lines 2.The wavelength indicated on the control is compared with known mercury emission peaks to determine the accuracy of the wavelength indicator control

electrophoresis procedure

1. sample is soaked in hydrated support for approximately 5 minutes 2. The support is put into the electrophoresis chamber, which was previously filled with the buffer 3. Sufficient buffer must be added to the chamber to maintain contact with the support 4.is carried out by applying a constant voltage or constant current for a specific time. The support is then removed and placed in a fixative or rapidly dried to prevent diffusion of the sample 5.followed by staining the zones with an appropriate dye. The uptake of dye by the sample is proportional to sample concentration. 6. After excess dye is washed away, the supporting medium may need to be placed in a clearing agent

pH electrodes

ISE is the pH electrode

Ion-Selective electrodes

Potentiometric methods of analysis involve the direct measurement of electrical potential due to the activity of free ions

spectrophotometer

Used to measure the light transmitted by a solution to determine the concentration of the substance (analyte → light absorbing) in the solution

single-beam spectrophotometers,

absorbance reading from the sample must be blanked using an appropriate reference solution that does not contain the compound of interest

photodetectors

convert the transmitted radiant energy into an equivalent amount of electrical energy -Photocell -Phototube -Photomultiplier

atomic absorption spect components: light source

hollow-cathode lamp, consists of an evacuated gas-tight chamber containing an anode, a cylindrical cathode, and an inert gas, such as helium or argon. -When voltage is applied, the filler gas is ionized -Ions attracted to the cathode collide with the metal, knock atoms off, and cause the metal atoms to be excited -When they return to the ground state, light energy is emitted that is characteristic of the metal in the cathode -a separate lamp is required for each metal 2.electrode discharge lamps: new light source for atomic absorption spect -bulb is filled with argon and the element to be tested -radiofrequency generator around the bulb supplies the energy to excite the element, causing a characteristic emission spectrum of the element

glucose

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 platinum anode and its potential is set to +0.6 V. Current flows through the system when peroxide is oxidized at the anode

chemiluminescence disadvantage

is that impurities can cause a background signal that degrades the sensitivity and specificity

atomic absorption spect

is used to measure concentration by detecting the absorption of electromagnetic radiation by atoms rather than by molecules

double-beam spect

permit automatic correction of sample and reference absorbance -necessary when the absorption spectrum for a sample is to be obtained

electromagnetic radiation

photons of energy traveling in waves. E=hv -frequency of a wave is inversely proportional to the wavelength, it follows that the energy of electromagnetic radiation is inversely proportional to wavelength -includes: spectrum of energy from short-wavelength, highly energetic gamma rays and x-rays on the left -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

electrophoresis equation

u = Q/K- r - n Q: net charge of the particle K: constant r: ionic radius of the particle n: viscosity of the buffer -the rate of migration is directly proportional to the net charge of the particle and inversely proportional to its size and the viscosity of the buffer