Spectrophotometric Instruments (Detector) and Application of UV – VIS spectroscopy and Immunoassays
4- Detector :
• The purpose of the detector is convert the transmitted radiant energy into an
equivalent amount of electrical energy .
• Two types of detectors are frequently used :-
1- Photomultiplier tube ( PMT ) :
• 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 .
• Theses dynodes are of a material that gives off many secondary electrons when hit
by single electrons .
• Initial electron emission at the cathods triggers a multiple cascade of electrons
within the PMT itself .
• Because of this amplification , PMT are used in instruments designed to be
extremely sensitive to very low light levels .
• the accumulation of electrons striking the anode produces a current signal that can
be fed into a meter or recorder .
2- Photodiode arrays ( PDA ) :
• are new detectors being used in modern
• photodiodes are composed of silicon crystals
that are sensitive to light in the wavelength range
170-1100 nm .
• Upon photon absorption by the doiode , a current is generated in the photodiode
that is proportional to the number of photons .
• Although photodiodes are not as sensitive as PMT because of the lack of internal
amplification , their excellent linearity , speed , and small size make them useful in
applications where light levels are adequate .
• PDA detectors are available in integrated circuits containing 256 to 2,048
photodiodes in a linear arrangement .
• Each photodiode responds to a specific wavelength, and as a result, a complete
UV/visible spectrum can be obtained in less than 1 second .
◘ Application of UV – VIS spectroscopy :
• Although many different types of operations can be carries out on a
spectrophotometer, all applications fall in one of two categories.
1- Measurement of absorbance at a fixed wavelength :-
• Are most often used to obtain quantitative information , such as the concentration
of a solute in solution or the absorption coefficient of a chromophore .
• For fixed-wavelength measurements with a single-beam instrument, a cuvette
containing solvent only is placed in the sample beam and the instrument is adjusted
to read “Zero” absorbance .
• A matched cuvette containing sample plus solvent is then placed in the sample
chamber and the absorbance is read directly from the display .
• The adjustment to zero absorbance with only solvent in the sample chamber allows
the operator to obtain a direct reading of absorbance for the sample .
• Fixed-wavelength measurements using a double-beam spectrophotometer are made
by first zeroing the instrument with no cuvette in either the sample or reference
• Alternatively , the spectrophotometer can be balanced by placing matched cuvettes
containing water or solvent in both sample chambers .
• Then, a cuvette containing pure solvent is placed in the reference position and a
matched cuvette containing solvent plus sample is set in the sample position .
• The absorbance reading given by the instrument is that of the samples; that is, the
absorbance due to solvent is subtracted by the instrument.
2- Absorbance measurement as a function of wavelength :-
• It provides qualitative information that
assists in solving the identity and structure
of a pure substance by detecting
characteristic grouping of atoms in a
◘ General considerations :
• Immunoassays are available for analysis of over 100 different analytes .
• Most immunoassay methods used specimens without any pretreatment and used
very small sample volumes ( 10 µl-50 µl ) .
• Antibodies ( Abs ) are incorporated into many clinical laboratory tests .
• They are useful because of their unique properties in recognizing and distinguishing
among closely related antigens ( Ags ) .
• Ab molecules is an immunoglobulin protein binds to a site in the Ag .
• An Ag is relatively large and complex and usually has multiple sites that can bind
to Abs with different specificities; each site on the Ag referred to as an antigenic
determinant or epitope .
• Some tests aid in diagnosis of infections through the detection of Abs to infectious
agents in patient specimens; examples are HIV , influenza , hepatitis , and rubella
• Other laboratory tests use the Ag-Ab reaction to measure or detect a substance not a
part of the immune system, such as using Abs to measure drug or hormone levels .
◘ Monoclonal and polyclonal antibodies :
• Abs used in immunological testes can be monoclonal or polyclonal .
• Monoclonal Abs are of one class and one specificity ( react with only one epitope )
and are derived from one ( mono ) clone, or cell line .
• Monoclonal Abs are produced in laboratories and used as reagents in many
immunodiagnostic kits .
• polyclonal Abs are mixtures of antibodies produced by more than one ( poly ) cell
• For instance , one bacterial infection will stimulate
many plasma cells ( poly clones) to respond, each
producing and secreting antibodies to a different
bacterial epitope .
• this results in a mixture of Abs in plasma that taken
together can react with multiple Ags .
◘ Test sensitivity and specificity
• Sensitivity refers to the lower limit of
detection, or the lowest concentration
capable of being detected by a test method .
• Failure to detect small amounts of a
substance in a test will result in a false-
• Specificity refers to the ability to detect only the substance for which the test is
• Reaction with other substances ( cross-reactivity ) decreases the specificity of the
test and can cause false positive results .
◘ Qualitative, Semi-qualitative and quantitative tests :-
• Many immunological procedures are reported only as negative or positive
; these are called qualitative tests .
• Other procedures are semi-quantitative or quantitative .
• Semi-quantitative procedures usually estimate concentrations of
antibodies or sometimes antigen .
• The antibodies concentration can be
estimated by making serial dilutions
and determining the maximum
dilution still capable of causing a
visible reaction in the test procedure .
• Semi-quantitative estimates of
antibody concentration can be
expressed as a titer , the reciprocal of
the highest dilution showing a
• Quantitative immunological tests are less frequently performed .
• Examples of some quantitative tests are drug assays .
◘ Principles of Ag-Ab tests :-
♠ Examples of tests that incorporate the Ag-Ab reaction include :
1- Agglutination :
• It is the visible clumping or aggregation of cells or particles as a result of reaction
with a specific antibody .
• IgM is the antibody class that reacts best in agglutination reactions because of its
large size and multivalent binding capacity .
• Antigen-coated cells or particles , such as red blood cells or latex beads, become
linked together and form visible clumps when reacted with sufficient antibody .
• In most agglutination test designs, the presence of agglutination indicates a positive
• Blood typing, bacterial identification , and the classic latex test for rheumatoid
arthritis are tests based on the agglutination reaction .
• Agglutination tests can be qualitative or semi-quantitative .
• Semi-quantitative tests can performed on slides, in tubes, or in special microtiter
plates having wells with rounded bottoms .
• Serial dilutions of serum are made and
tested to determine the maximum dilution
capable of causing agglutination , which is
reported as a titer .
• When agglutination tests are performed in
microtiter plates , a negative test indicated
by the presence of non-agglutinated
particles concentrated in a small dot in the
bottom of the well .
• A diffuse pattern of cells spread over the
bottom of a round-bottomed well indicates a
positive test .