Chemistry Instrumentation SOLO 2

spectrophotometry

measurement of intensity of light at selected wavelengths

frequency

• how often a repeating event takes place

• the number of waves occuring per unit of time

wavelength

the distance between two crests that is measured in nanometers

Frequency and wavelength relationship

the frequency of the wave is inversely proportional to the wavelength

protons

• discrete energy packets in light

• energy is inversely proportional to the wavelength

Ultraviolet radiation

200 nm

infrared radiation

750 nm

visible light

falls between 380 nm (violet) and 700-750 nm (red)

% Transmittance equation

• I/I₀ × 100

• T/I x 100

%Transmittance

the ratio of radiant energy transmitted divided by the radiant energy incident on the sample

If no light is absorbed…

%T=100%

If all light is absorbed…

%T=0%

Absorbance

amount of light absorbed as incident light passes through a sample

Absorbance equation from transmittance

-logT

Absorbance equation from %T

2-log%T

Beer’s law

the concentration of a substance is directly proportional to the amount of light absorbed or inversely proportional to the logarithm of the transmitted light

Beer’s law equation

A= εbc

• A= absorbance

• ε= proportionality constant defined as absorptivity by an analyte at a specific wavelength; constant for a particular analyte at a particular wavelength

• b= distance that light travels through the solution in centimeters

• c= concentration of the absorbing compound, usually in grams/liter

Absorbance~c

absorbance is directly proportional to concentration

spectrophotometer

an instrument used to measure the light transmitted by a solution to determine the concentration of the light-absorbing substance in the solutionlight

Visible range light source

incandescent tungsten or tungsten iodide lamp

UV range light source

deuterium discharge lamp and mercury arc lampm

Monochromator

optical device that provides a means to isolate a single wavelength or band of wavelengths of light while excluding other wavelengths

Monochromator examples

• colored glass filters

• simple glass prism

• diffraction gratings

Sample cuvette considerations

• quality

• light path

• shape

• surfaces

• types

Photodetectors

convert light into an electrical signal that is proportional to the number of protons striking its photosensitive surface

Photodetector examples

• barrier-layer cells (photocells)

• phototube

• photomultiplier tube

• photodiode

single beam spectrophotometer

the absorbance reading from the smaple must be blanked using an appropriate reference solution that does not contaain the sompound of interest

Double-beam spectrophotometers

permit automatic correction of sample and reference absorbance

What checks validate spectrophotometer function?

• wavelength accuracy

• stray light

• linearity

wavelength accuracy

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

Stray light

• refers to any wavelength outside the band transmitted by the monochromator

• stray light causes include reflection of light from scratched on the optical surfaces or from dust particles anywhere in the light path

• the major effect is absorbance error in the high absorbance range

Linearity

demonstrated when a change in concentration results in a straight-line calibration curve

Methods should be designed to read an absorbance or transmittance interval of what?

• A= 0.1-0.7

• T= 20-80%

What would you do if the transmittance is <20% or absorbance is >0.7?

dilute

What would you do if the transmittance is >80%?

report as below the AMR

Stray light in a spectrophotometer places limits on the instrument’s ability to accurately measure:

absorbance at the upper end of the range of linearity

Two examples of luminescent labels in chemiluminescence are:

luminol and acridinium ester

The prescence of Human anti-mouse antibodies in patients leads to?

false positive signal in immunoassay

To provide specificity for the antigen of interest, most immunoassays use which type of immunoglobulin as reagent?

IgG

What may be done if a signal is below the analytical measurement range?

The result maybe reported as less than the lower end of the dynamic range or less than the lowest calibrator used in the lab

What type of certificate is issued to a lab that enables an entity to conduct moderate/high complexity testing?

Certificate of registration

Atomic Absorption Spectrophotometry

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

• sensitive and precise; used to measure the concentration of trace metals

• hollow cathode lamp used

Fluorometry

• fluorescence occurs when a molecule absorbs light at one wavelength and reemits light at a longer wavelength

• fluorometers measure concentrations of solutions that contain fluorescing molecules

• measurements related to molar absorptivity of the compound

stokes shift

the difference between the excitation wavelength and the emitted wavelength

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 roate (attached to large molecule)

• small molecule smits depolarized light because it will rotate out of the plane of polarization

• widely used for therapeutic and abused drugs

• The lower the concentration of the sample analyte, the higher the concentration of macromolecular antibody-analyte-fluorophore formed and the lower the depolarization of radiant light

Fluorometry advantages

specificity and sensitivity

fluorometry disadvantages

very sensitive to environmental changes

What compound is only found in a fluorometer used to make measurements of emitted fluorescent light as opposed to in a spectrophotometer used to make measurements of absorbed/transmitted light?

an excitation source

Chemiluminescence

• chemical energy generated produces excited intermediates that decay to a ground state with the emission of photons

• light produced during the chemical reaction is measured

• emission of light when an electron returns to a lower energy state from a higher energy state

• the excitation event is caused by a chemical reaction

• no excitation radiation is required and no monochromators are needed

Barrier-layer cells

• contains a photosensitve material that gives off electrons when energy strikes it

• requires no external voltage source but relies on internal electron transfer to produce a current in an external circuit

phototube

• contains cathodes that emit electrons when exposed to light

• an outside voltage is required for operation

Photomultiplier tube

• detects and amplifies radiant energy

• uses amplification techniques to make this type of photodetector 200 times more sensitive than a phototube

photodiode

• not as sensitive as PM tubes but have excellent linearity, speed and small size

• has the ability to read the whole visible spectrum in less than 1 second

What type of reactions are chemiluminescence?

• oxidation reactions of luminol, acridinium esters, and dioxetanes

• rapid increase in intensity of emitted light followed by a gradual decay

Turbidometry and nephelometry measure:

scattered light

Light scattering

a physical phenomenon that resuts from the interaction of light with particles in solution

Nephelometry

• detection and measurement of light energy scattered or reflected toward a detector that is not in the direct path of the transmitted light

• measure scattered light at right angles to the incident light

turbidimetry

• detection and measurement of a decrease in intensity of an incident beam of light as it passes through a solution of particles

• measurement made at 180 orientation to the incident light

Factors affecting light scatter:

• particle size

• wavelength of incident light

• distance of observation

• effect of polarization of incident light

• concentration of the particles

• molecular weight of particles

Limitations of light scattering methods

• antigen excess

• matric effects

Atomic Absorption Spectrophotometry limitations

• inability of the flame to dissociate samples into free atoms

• ionization of atoms following dissociation by the flame

• matrix interference

Stray light in a spectrophotometer places limits on the instrument’s ability to accurately measure:

upper range of linearity

Photometric analysis takes advantage of what characteristics of the analyte being measured?

• absorbance of light

• emission of light

• reflection of light

What does electrochemical analysis take advantage of?

electrical properties of the analyte

electrochemistry involves:

producing electrons or ions in a chemical reaction from nonionic analytes, such as oxygen

electrical potential

the work required to move an electrical charge and measured in volts

resistance

opposing force to flow of electrons in ohms

current

electrical charge

Ohm’s law

E=I x R

Potentiometry

measures an electrical potential difference(voltage) between two electrodes immersed in solution in an electrochemical cell

Voltammetry

process that measures the current at an electrode using a specific voltage generated at another electrode

Amperometry

process in which current is monitored in amperes at a fixed voltage between working and reference electrodes in an electrochemical cell

Conductometry

determines the quantity of an analyte present in a mixture by measuring the combined activity of all ions in a solution to conduct or carry an electrical charge

Coulometry

• measures aspects of current including rate of electron flow

• often used in titration of ions

• considered the gold standard to measure chloride in serum or plasma

electrode

• also referred as a half cell

• consists of a single metallic conductor that is in contact with an electrolyte solution

• one of the elctrolyte solutions is the sample containing the analyte to be measured

Potential difference

• the work required to move an electrical charge and measured in volts

• measured using a potentiometer

electromotive force

the maximum difference in potential between two electrodes obtained when the cell current is zero

direct reading potentiometer

a voltmeter that measures the potnetial in millivolts across an electrochemical cell

ionic activity

measurement of the concentration of free, unbound ions in solution

Types of electrochemical cells

• Glavanic cells

• electrolyte cells

Galvanic electrochemical cell

a device that consists of two electrodes that are connected by an electrolyte solution that conduct ions

Potentiometry is based on what elecrtochemical cells?

Galvanic cells

Electrolytic electrochemical cell

a device in which an external voltage is applied to a polarizable working electrode with the resulting cathodic or anodic current of the cell being monitored

Electrlytic cells are the basis for:

• voltammetry

• amperometry

• coulometry

• conductometry

redox couple

a conjugate pair of substances that consists of any substance that accepts electrons and any substance that donates electrons

Redox potential

the voltage difference due to electron transfer between a reductant and an oxidant in a balanced chemical reaction

Anode

the electrode where oxidation takes place

cathode

the electrode where reduction takes place

Hydrogen electrode

special redox electrode for pH measurement consisting of platinum or gold

ion selective electrodes

• most common method for analysis of many electrolytes

• only free, unbound ion is measured

• measure electrical potential difference across a membrane using potentiometry

indicator electrode (sensors)

• ISEs

• responds to changes in the activity of a particular ion

• designed to react to the activity of a particular species in solution

Reference electrode

• does not respond to changes in the activity of a particular ion

• has a stable, well known potential to allow the potential of the other half cell to be determined

ISE cell potential

the emasure of the potential difference between two half cells in an electrochemical cells

polymer membranes in ISEs

• predominant type of potentiometric electrode used in clinical laboratories

• ionophores may be used

Incorporation of valinomycin into a membrane allows for:

an ion selective elctrode that is highly selective for potassium

incorporation of methylmonensin allows for

an ion selective electrode that is highly selective for sodium

pH electrodes

commonly used glass reference electrode, usually for pH measurements on blood gas machines

PCO₂ electrodes

potentiometric cell inwhich carbon dioxide gas from the sample diffuses through a membrane and forms carbonic acid

Coulometric titration

• used in sweat chloride analysis

• silver-silver electrode

• start-stop time proportionate to Cl present in solution