Enclonar, Kimberly / MLS 3A

Instruments of Measurement
Instrumentation & Analytical Two most common:
Techniques • Visible Spectrophotometer
August 20, 2020 o Amount of light absorbed by the dissolved
Ace Ronald Sarabia, RMT, MHPEd substance
• Atomic-Absorption
Analytical Run
1. Blank (Blanking) Two Methods to Detect Molecules:
o Measure interferences • Indicator
o For calibration o Quickest method
2. Standard o Indicator solutions change colors when a
o Assay will have a basis. molecule of interest is present
3. Control o Allows scientist to detect the colorless
4. Sample molecules in a solution
o Qualitative
Foundation for all measurements done in Clinical Chemistry • Spectrometer
• 4 basic disciplines o Shines a beam of light on a sample. The
o Spectrometry molecules in the sample interact with the light
o Luminescence waves in 3 ways:
o Electroanalytic Methods ▪ Absorb the energy
o Chromatography ▪ Reflect the energy
▪ Transmit the energy between and
Probe/aspirator through the atoms and molecules of the
sample.
Spectrophotometry o Measures the amount of light transmitted
• Spectrum - different levels, measurements, through the sample (Transmittance)
wavelengths. o By using an equation (Beer's Law), it converts
• Measurement of the different light wavelength. the transmittance data to an absorbance value.
o Quantitative
Properties of Light
• Electromagnetic radiation moves in waves Elements not to forget in stating the principle:
• Shorter wavelength, higher electron volt energy 1. Analyte
• Visible light - between ultraviolet and infrared rays 2. How it is measured
• 400nm(violet) to 700nm(red)
• Energy is indirectly proportional to wavelength Spectrophotometer "Spectro"
• An instrument that measures the amount of light that
passes through (is transmitted through) a sample.
• Spectrophotometry measures analyte concentration
by determining absorbance and transmittance of
light.

Colorimetry
• The solutions of many compounds have characteristic
colors.
• The intensity of such color is proportional to the
concentration of the compound (Beer's law)

Absorbance of light
• White light
o All colors
o Polychromatic light

Spectroscopy and Spectrophotometry

• Spectroscopes measure electromagnetic emission
• Spectrophotometers measure electromagnetic
absorption
 Enclonar, Kimberly / MLS 3A
Parts of a Spectrophotometer Quantitative Spectrophotometry
1. Light source
o Incandescent Tungsten / Tungsten-iodide lamp
- most common
o Deuterium
o Hollow cathode lamp
o Zenon arc
2. Monochromator/Prism
o Isolates white light into a specific wavelength
o Prism - oldest type of monochromator
o Diffraction gratings - commonly used
o Interference filter
o Colored glass filter
3. Slit/diaphragm
o Entrance slit - so that incident light will not Linearity
• If there is too much or too little analyte,
enter
o Exit slit - chooses specific wavelength spectrophotometer cannot read the absorbance
accurately
4. Cuvette/Sample cell
o Shape
Definitions & Symbols
▪ Round cuvette
▪ Square cuvette • Intensity (I)
o Material • Transmittance (T)
o Also referred to as %T or T x 100
▪ Quartz cuvettes - ideal cuvette especially
o T = I/Io
when measuring UV
• Glass and plastic absorb UV
Graphical Relationship
radiation
▪ Glass cuvettes • % transmission and % absorption are not linearly
related to concentration
▪ Plastic cuvettes
• For a graph to be useful, a straight line is needed
5. Photodetector
o Convert the transmitted radiant energy into an • Absorbance = log(1/T) = -log(T)
equivalent amount of electrical energy
o Types: Standardization graph
▪ Photocell • Standards (solution of known concentration) of the
compound of interest are made, treated, and their
• Simplest type
absorbances (ABS) and concentration values are used
• Converts light energy to electrical
to create a standardization graph.
energy
▪ Phototube • Standard curve
▪ Photomultiplier (PM) tube
▪ Photodiode
6. Read out device

Function
• The spectrophotometer can measure the amount of
absorbance or;
• Lack of absorbance of different colored light for a
given molecule

Applications of a Spectrophotometer
• Determines the presence and concentration of
samples
• Determines the purity of a sample
• Look at the change of samples over time.

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.
• The concentration of a substance is directly
proportional to the amount of light absorbed and the
negative logarithm of the transmitted light.
• The intensity of a ray of monochromatic light
decreases exponentially as the concentration of the
absorbing medium increases.
• More dissolved substance = more absorption and less
transmittance.
 Enclonar, Kimberly / MLS 3A
Other Instruments Electrophoresis
• Principle: Separation of molecules based on their
Mass Spectrometry electric charge densities in an electric field.
• Separation of ions is based on their mass-to-charge • Migration of particles in an electric field.
ratio.
• pH of 8.6, proteins become anionic and travel towards
anode.
Atomic Absorption Spectrophotometer
• Zone
• Principle: Ability of atoms, when excited and return to o Migration of charged macromolecules in
ground state, absorb light. porous support medium.
• Measures the light absorbed by atoms dissociated by • Agarose-gel
heat. o Requires small amount of sample and does not
• Atomizer/nebulizer converts molecules to atoms. bind to protein.
• Element is not excited by merely dissociated from its
chemical bond and place in an unionized, unexcited, Chromatography
ground state.
• Separation of mixtures without electricity.
• Monochromator is found after the analyte. • For measuring amount of antigen-antibody
• Flame serves as the cuvette. complexes.
• More sensitive than flame emission photometry. • TLC
• Light source is hollow-cathode lamp. o Most commonly used as a semiquantitative
screening test.
Flame Emission Photometry o Components of this instrument are mobile
• Principle: Atoms are burnt and upon excitement, they phase, stationary phase, column and eluate.
emit a particular wavelength of light which is
• HPLC
measured. o There is motor.
• Obsolete in CC. • GC
• Uses nebulizer/atomizer. o Useful for compounds that are volatile
• There is no light source or lamp.
• Light source may be a flame. GCMS
• Monochromator is after analyte. • Gold-standard for drug testing.
• Measures the light emitted by a single atom burned in
a flame, excitation of electrons from lower to higher Ion-selective Electrodes (Potentiometry)
energy state.
• An electrochemical transducer capable of responding
to one given ion.
Fluorometry
• Uses membrane for ion-selectivity.
• Principle: analytes that are fluorescing substances will • Measures electric potential formed across a
borrow high energy and emit low energy levels, and is membrane.
measured.
• Analyte is a fluorescing substance. Coulometry
• 90 degree angle. • Follows Faraday's law.
• Has two monochromators: primary and secondary • Measures amount of energy at a fixed potential.
filters.
o Excitation filter
Amperometry
o Emission filter
• Follows the likovic equation.
• Determines the amount of light emitted by a molecule • Measures the current flow produced by an oxidation
after electromagnetic radiation. reaction.
• Measures light intensity over a zero background.
• Affected by quenching gas.

Chemiluminescence
• Addition of chemical agent.
• Luminol - most common
• No excitation radiation is required.
• No monochromators and light source.
• Determines amount of light emitted through a
chemical reaction or an electrochemical reaction.

Non Absorptive Spectrometry

Turbidimetry
• Determines amount of light blocked; measurement
depends on specimen concentration and particle size.
• For measuring abundant large particles and bacterial
suspensions.

Nephelometry
• Determines the amount of light scattered.
• 95 or 45 degrees angle.
• Light scattering depends on wavelength and particle
size.