cc lec chapter 3

UV light

used for excitation can cause photochemical changes.

Neutral density filters and dichromate solution

verify absorbance accuracy on linearity

Photomultiplier (PM)

• Detects and amplifies radiant energy. • Incident light strikes the coated cathode, emitting electrons • The electrons are attracted to a series of anodes, known as dynodes, each having a successively higher positive voltage. • Used in instrument designed to be extremely sensitive to very low light levels and light flashes of very short duration.

ISE Membrane

• Glass aluminum silicate (sodium), valinomycin gel (potassium) organic liquid membrane ion exchangers (calcium and lithium), gas and enzyme electrodes.

Ion Selective Electrode

• Is an electrochemical transducer capable of responding to one given ion. • It is very sensitive and selective for the ion it measures- it measures the activity of one ion much more than other ions present in the sample. • Its ionic selectivity depends on the membrane/barrier composition used. • ISE analyzers measure the electrolyte dissolved in the fluid phase of the sample in mmol/L of plasma water. • It is sensitive method but not specific- it does not discriminate between ions in causing voltage differences between the measuring electrode and the standard electrode. • ISE analyzers using undiluted samples are not subject to pseudohyponatremia caused hyperlipemic samples.

Potentiometry

• Is the measurement of electrical potential due to the activity of free ions- change voltage indicates activity of each analyte. • It is also the measurement of differences in voltage (potential) at a constant current. • It follows the Nermst equation. • Concentration of ions in a solution can be calculated from the measured potential difference between the two electrodes. • Reference electrode: calomel and silver-silver chloride • Use: pH and pCO2 tests

Coulometry

• Is the measurement of the amount of electricity (in coulombs) at a fixed potentia. • Is an electrochemical titration in which the titrant is electrochemically generated and the endpoint is detected by amperometry. • It follows the Faraday's Law

Barrier Layer Cell/Photocell/Photovoltaic Cell

• It is the simpliest detector, inexpensive; temperature sensitive • Composed of a film of light-sensitive material, frequently selenium , on a plate of iron. Over the light-sensitive material is a thin, transparent layer of silver. • 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 faily high level of illumination

. Diffraction gratings

• Most commonly used as monochromators. • Consist of many parallel grooves (15,000 or 30,000 per inch ) etched onto a polish surface. • Based on the principle that wavelengths bend as they pass a sharp corner. The degree of bending depends on the wavelength. • Gratings with very fine line rulings produced a widely dispersed spectrum

Interference

• Protein coating the ISE membrane would cause a response error-If interference is due to excess protein, an alternative mode of analysis such as ISE in undiluted specimen can be employed to yield correct electrolyte activity

Prism

• Wedge-shaped pieces of glass , quartz or sodium chloride. • 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.

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 tubes because of the lack of internal amplifications, their excellent linearity, speed and small size make them useful in applications where light levels are adequate

Phototube

• contains negatively charged cathode and a positively charged anode enclosed in a glass case. The cathode is composed of material (e.g rubidium or lithium) that act as a resistor in the dark but emits electron when exposed to light. • Outside voltage is required for operation

Continuum source

- emits radiation that changes in intensity . Widely used in the laboratory

>700 nm

- infrared region

Absorbance

- is the amount of light absorbed. It is proportional to the inverse log of transmittance

Frequency

- is the number of vibrations of wave motion per second.

Alumina silica glass

- most commonly used ( 350-2000nm)

Zeeman effect

- shift in wavelength

Nominal wavelength

- the wavelength which a wavelength selector is set to pass. It represents the wavelength in nanometers at peak transmittance.

< 400

- ultraviolet region

Quartz/Plastic

- used for measurement of solution requiring visible and ultraviolet spectra (NADH at 340 nm)

Deuterium lamp

- used to provide UV Radiation in analytic spectrophotometers. Provides continuous emission down to 165 nm

Incandescent tungsten or tungsten-iodide lamp

- visible and near-infrared region

400-700nm

- visible spectrum

Energy

-is transmitted via electromagnetic waves that are characterized by their frequency and wavelength

rotational or vibrational frequency in the atom or molecule that it strikes

For a ray of electromagnetic radiation to be absorbed , it must have the same frequency as a

orders

. Diffraction gratings Gratings with very fine line rulings produced a widely dispersed spectrum. They produce linear spectra, called ______, in both directions from the entrance slit.

Electrodeless discharge lamps

Atomic Absorption Spectrophotometry - new light source for AAS. A bulb is consist of argon and the element to be tested. A radiofrequency generator around the bulb supplies the energy to excite the element

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.

Atomic Absorption Spectrophotometry Light source:

deuterium lamp

Atomic Absorption Spectrophotometry The most common approach uses a ____________ as a secondary source and measures the difference between the two absorbance signals

background correction is considerably more necessary and critical for electrothermal techniques than for flame-based atomic absorption methods.

Atomic Absorption Spectrophotometry major problem

Molecular spectra

Band Spectrum Known as

Continuous and closely spaced with one another

Band Spectrum Nature

Appears as continuous form of bands viewed from a spectroscope

Band Spectrum View

environmental changes.

Biggest disadvantage is that fluorescence is

defective ISE membrane, buildup of countervoltages from liquid junction potentials at the salt bridge and buidup of proteins at the electrodes.

Causes of Malfunction of ISE:

NiSO4, NaNO2, and acetone,

Certain liquids, such as _______________ absorb strongly at short wavelengths and can be used to detect stray light in the UV range.

include subpicomolar detection limits, speed (with flash-type reactions, light is only measured for 10 seconds), ease of use (most assays are one-step procedures), and simple instrumentation.

Chemiluminiscence Advantages

Photomultiplier tube (luminometer)

Chemiluminiscence Photodetector:

Immunoassays

Chemiluminiscence Use:

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

Chemiluminiscence main disadvantage

light source entrance slit monochromator exit slit sample cell photodetector

Components of Spectrophotometer

structure

Contaminating chemicals or a change of solvents may change the

electrolytic cell

Electrochemistry Current may be forced to flow through the dead cell only by applying an external electromotive force E. This is called

Potentiometry Coulometry Amperometry Voltammetry

Electrochemistry types

quenching- pH and temperature changes, chemical contaminants, UV light changes.

FLUOROMETRY It is affected by

Resonance lines at 365 to 366 nm are commonly used

FLUOROMETRY Resonance lines

less than 5%

FLUOROMETRY The solution must absorb_________ of the exciting radiation for a linear response to occur

Mercury vapor lamps

FLUOROMETRY are commonly used in filter fluorometers.

Gas discharge lamps (mercury and xenon arc )

FLUOROMETRY are the most frequently used sources of excitation radiant energy.

1000x

FLUOROMETRY Is about__________more sensitive that spectrophotometer-emitted radiation is measured directly

Lithium/Cesium-corrects variation in flame and atomizer characteristics

Flame Emission Photometry Internal Standard:

Flame (also serves as the cuvette)

Flame Emission Photometry Light Source:

Indirect Internal Standard Method

Flame Emission Photometry Method:

Excitation of electrons from lower to higher energy state.

Flame Emission Photometry Principle:

1. Molar absorptivity of the compound. 2. Intensity of the incident radiation. 3. Quantum efficiency of the energy emitted per quantum absorbed. 4. Length of light path

Fluorescent concentrations measurement are related to

Electrochemistry

In galvanic cell, as the electrodes are connected , there is spontaneous flow of electrons from the electrode with 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 is, the cell "is dead" and cannot produce electrical energy to the external meter.

SPECTROPHOTOMETRY

It involves measurement of the light transmitted by a solution to determine the concentration of the light absorbing substances in the solution

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

Flame Emission Photometry

It measures the light emitted by a single atom burned in a flame. • It is used for the measurement of excited ions (sodium and potassium) • Flickering light indicates changes in the fuel reading of the instrument

BEER LAW

It states that the concentration of the unknown substance is directly proportional to the absorbed light (absorbance or optical density) and inversely proportional to the amount of transmitted light (% Transmittance)

Barrier Layer Cell/Photocell/Photovoltaic Cell Phototube Photomultiplier (PM) Photodiode

Kind of Detectors

Filters Prism Diffraction gratings Holographic gratings

Kinds of Monochromators

Alumina silica glass Quartz/Plastic Borosilicate Glass Soft glass

Kinds of cuvet:

Continuum source Line Source

Light/Radiant Source 2 Types:

1. Mercury Arc (UV and visible) 2. Deuterium lamp (165nm) UV 3. Hydrogen lamp- UV 4. Xenon lamp- UV 5. Merst glower -IR 6. Globar (silicone carbide)- IR

Light/Radiant Source Alternatives for Tungsten Bulb

mercury and sodium vapor lamps in spectrophotometers (UV and visible region), and the hollow cathode lamp (AAS)

Line Source Example

Atomic spectra

Line Spectrum Known as

Distinct and Unique

Line Spectrum Nature

Appears as parallel lines when viewed from slit

Line Spectrum View

AAS, molecular and fluorescent spectroscopy

Line sources that emit a few discrete lines find wide use in

1. Double-beam in space 2. Double-beam in time

SPECTROPHOTOMETRY Two Types:

Band Spectrum

Spectrum consist of groups or bands closely spaced lines in emission or absorption, characteristics of molecular gases and chemical compounds.

Line Spectrum

Spectrum of radiation in which the quantity is studied such as frequency or energy takes discrete values

E = hv where h is constant (6.62 x 10-27 erg.sec), known as the Planck constant, and v is frequency. E= is the energy of a photon in Joules or eV

The relationship between wavelength and energy E is described by Planck's formula:

Turbidimeter

This device measures the reduction in light intensity caused by reflection, absorption, or scatter • Light then bounces off the molecules and travels in all directions. The amount of scatter is proportional to the size, shape, and concentration of molecules present in solution. • It is recorded in absorbance units, a measure of the ratio of incident light to that of transmitted light. The measurements are made using a spectrophotometer or an automated clinical chemistry analyzer.

"blanking" procedures or dual-wavelength methods

To correct for artifactual absorbance readings, ______________ may be used

Protein measurements(CSF and urine); to detect bacterial growth in broth cultures, antimicrobial test (broth method), and to detect clot formation

Turbidimetry Use:

Atomic Absorption Spectrophotometry

Used to measure concentration by detecting the absorption of electromagnetic radiation by atoms rather than molecules. • Sensitive and precise • Routinely used to measure 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

Changes in pH

affect availability of electrons, and temperature changes the probability of loss of energy by collision rather than fluorescence.

Double beam instruments

are used to compensate for instability due to electric power fluctuation

Reagent blank

corrects absorbance caused by the color of the reagents-the absorbance reagent is automatically subtracted from each of unknown reading

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 5,500 and 8,000 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

Turbidimetry

is a measure of the turbidity cloudiness of a solution. A detection device is placed in direct line with an incident light, collecting the light after it has passed through the solution.

Electromagnetic radiation

is described as photons of energy traveling in waves.

Wavelength

is the distance between two successive peaks and it is expressed in terms of nanometer

Wavelength accuracy

is the wavelength indicated on the control dial is the actual wavelength of light passed by the monochromator.

Didymium or holmium oxide filter

is used to check wavelength accuracy (wavelength calibration)

Blanking technique

means the blank contains serum but without the reagent to complete the assay

Sample blank

measures absorbance of the sample and reagent in the absence of endproduct, and corrects the measurement for optical interference, absorbing the wavelength of measurement.

Fluorescence

measures the amount of light intensity present over a zero background.

Nephelometry

measures the light that is scattered at a particular angle from the incident beam as it passes through a suspension. The amount of light scattered is an index of the solution's concentration. If a solution has excess antibody, adding increasing amounts of antigen results in an increase in antigen-antibody complexes and thus an increase in light scattering. The relationship between antigen concentration, as indicated by antigen-antibody complex formation, and the amount of light scattering will form a straight line if plotted on a graph. Light scatter may be directly extrapolated by a computer to give actual concentrations in milligrams per deciliter (mg/dL) or international units per milliliter (IU/mL), based on established values of standards.

Scattering

occurs when a beam of light passes through a solution and encounters molecules in its path.

Absorption

occurs when electrons absorb photons which causes them to gain energy and jump to a high energy levels.

Monochromator fluorometers

use gratings for isolation of incident radiation.

Xenon discharge lamp

produces a continuous source of radiation which covers both the UV and the visible range.

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 the sample, and, finally, heats the unit to incandescence to atomize the sample.

Filters

simple, least expensive, low transmittance of the selected wavelength, not precise. • Are made by placing semi-transparent silver films on both sides of a dielectric such as magnesium flouride • Interference filters produce monochromatic light based on the principle of constructive interference of waves. Two pieces of glass , each mirror on one side-light waves enter on one side of the filter and are reflected at the second surface. • It usually pass a wide band of radiant energy and have a low transmittance of the selected WL.

Nephelometers

typically measure light scatter at angles ranging from 10 degrees to about 90 degrees. If a laser beam is used, light deflected only a few degrees from the original path can be measured. Although the sensitivity of turbidity has increased, nephelometry is more sensitive, with a lower limit of detection of 1 to 10 mg/L for serum protein