Del Rosario, Ma. Veronica M.

BSMT - 3

CLINICAL CHEMISTRY

Discuss the principles and concepts of the following instrumentation methods:

1. Photometry
2. Spectrometry
a. Atomic absorption spectrometry
b. Flame emission spectrometry
c. Reflectance
d. Spectrophotometry
3. Nephelometry
4. Turbidimetry
5. Fluorometry
6. Electrophoresis
7. Chromatography
8. Ultracentrifugation
9. Chemiluminescence
10. Electrochemistry
11. Immunochemistry
12. Dry chemistry

 PHOTOMETRY

PRINCIPLE:
 The basis of photometric working is that the species of alkali metals and alkaline earth
metals are separated due to the thermal energy provided by the flame source, and some
of the atoms are expelled to a higher energy level where they are not stable.
 Direct absorption techniques can be used to evaluate the light absorbance due to
electron excitation.
 As a result of the following loss of energy, departed atoms will go to a low energy ground
state, emitting certain radiations that can be seen in the visible spectrum.
 The direct absorption technique can be used to measure the absorbance owing to
electron excitation, while emission techniques can be used to assess the intensity of the
emitting radiation.
 For specific elements, the wavelength of emitted light is specific.

Uses of Photometry
 Photometry is utilized in a wide range of sectors, including chemicals, soils, agriculture,
pharmaceuticals, glass, and ceramics, as well as in plant materials and water and
biological and microbiological laboratories.
 It is employed in the routine determination of potassium, sodium, magnesium, and
calcium in biological fluids such as serum, plasma, and urine.
 In many laboratories, analyzing industrial and natural water to determine the constituents
that cause hard water is common operation.
CONCEPTS:
 The basic unit of photometry is the lumen, which is related to its radiometric analog, the
Watt, by: Im = 683 x W x Vλ
 Where Vλ is the relative luminosity, a coefficient scaled to visual response. Unity occurs
at the eye’s peak response wavelength, 555 nanometers.
 Two useful laws in photometry recur: the inverse square law and the cosine law. The
first defines the relationship between illumination from a constant-intensity light sources
and its distance from a surface. It states that the intensity per unit-area on the surface,
varies in inverse proportion to the square of the distance between the source and
surface or: ΔIm/M2α1/Δd2

 SPECTROMETRY

A. ATOMIC ABSORPTION SPECTROMETRY

PRINCIPLE:
 The theory behind the technology is that free atoms (gas) produced in an atomizer can
absorb radiation at a given frequency.
 Atomic-absorption spectroscopy measures how much ultraviolet or visible light is
absorbed by ground state atoms in the gaseous state, allowing them to shift to higher
electronic energy levels. The amount of absorption is used to determine the analyte
concentration.
 After calibrating the device with known concentration standards, concentration values
are usually calculated from a working curve.
 For detecting metals and metalloids in environmental samples, atomic absorption is a
typical approach.

CONCEPTS:
 The extent to which radiation of a particular frequency is absorbed by an atomic vapour
is related to the length of the path transversed and to the concentration of absorbing
atoms in the vapour
 This is analogues to the Beer – Lamberts law relating to samples in samples in solution.
Thus , for a collimated monochromatic beam of radiation of incident I0 passing through
an atomic vapour of thickness I
Iv = I0 e –kvI
 Where
Iv is the intensity of the transmitted radiation
kv is absorption co efficient
 The value of kv is determined by the concentration of atoms which can absorb at
frequency v is given by the expression :
ʃ kv dv = xe2 Nv f
 Where
m & e are the mass and charge of the electron
Nv is the number of atoms per cm3 capable of absorbing radiation of frequency v ( ie.,
ground state atoms )
f is the oscillator strength ( defined as the number of electrons per atom capable of being
excited by the incident radiation )
 Hence , for transitions from the ground state , integrated absorption is proportional to
Nv ,which approximates to the concentration (c) of the element in the sample
 B. FLAME EMISSION SPECTROMETRY

PRINCIPLE:
 Flame emission spectroscopy (FES) is a chemical analysis method that determines the
quantity of an element in a sample by measuring the intensity of light emitted from a
flame, arc, or spark at a certain wavelength.
 The principle of flame photometric functioning is that the thermal energy delivered by the
flame source dissociates the species of alkali metals (Group 1) and alkaline earth metals
(Group II) metals.
 Some of the atoms are stimulated to a higher energy level, where they are unstable, as
a result of this thermal excitation.
 Direct absorption techniques can be used to evaluate the light absorbance due to
electron excitation.
 As a result of the following loss of energy, excited atoms will migrate to a low energy
ground state, emitting certain radiations that can be seen in the visible spectrum.
 The wavelength of emitted light is distinct for specific elements, and the strength of
radiation released by these excited atoms returning to the ground state provides the
basis for analytical determination in FES.
 The wavelength of spectral lines determines the identity of components in flame
emission spectroscopy.
 The amount of atoms present determines the intensity of the radiated light.
 The wavelength of the emitted radiation is a property of the elements and is used to
identify them (Qualitative Analysis). The amount of radiation emitted is proportional to
the concentration of the element being studied (Quantitative Analysis).
The wavelength of the radiation emitted is given by the following equation –
 λ=hc/E2−E1λ=hc/E2−E1
 where hh = Planck's constant, cc = Velocity of light, E2−E1E2−E1 = energy levels of
excited and ground state respectively.

CONCEPTS:
 Liquid sample contaning metal salt solution is introduced into a flame,
 Solvent is first vaporized, leaving particles of solid salt which is then vaporised into
gaseous state
 Gaseous molecule dissociate to give neutral atoms which can be excited (made
unstable) by thermal energy of flame
 The unstable excited atoms emit photons while returning to lower energy state
 The measurement of emitted photons forms the basis of flame photometry.
 Under constant and controlled conditions, the light intensity of the characteristic
wavelength produced by each of the atoms is directly proportional to the number of
atoms that are emitting energy, which in turn is directly proportional to the concentration
of the substance of interest in the sample.
 Various metals emit a characteristic colour of light when heated.
 C. REFLECTANCE

PRINCIPLE:
 A reflectance photometer measures the reflectance of a surface as a function of
wavelength. The surface is illuminated with white light, and the reflected light is
measured after passing through a monochromator. This type of measurement has
mainly practical applications, for instance in the paint industry to characterize the colour
of a surface objectively.
 In reflectance photometry, diffused light illuminates a reaction mixture in a carrier, and
the reflected light is measured. Alternatively, the carrier is illuminated, and the reaction
mixture generates a diffuse reflected light that is measured.

CONCEPTS:
 Beam of light is directed at a flat reaction surface & the reflected light is quantified.
 Reaction mixture in a carrier is illuminated with diffuse light, & the intensity of the
reflected light from the chromogen is compared with the intensity of the light reflected
from a reference surface.
 The reflected light intensity is non linear in relation to conc of analyte.
DR = log ( Ro/Rtest)
 Kubelka-Munk or Clapper-Williams transformation equation used to convert the data into
linear format.

D. SPECTROPHOTOMETRY

PRINCIPLE:
 Spectrophotometry is a scientific approach that uses two laws of light absorption to
determine the amount of light absorbed by a substance.
 The Beer-Lambert Law (also known as Beer's Law) asserts that the absorbance of a
sample is proportional to its concentration.
 The Beer–Lambert law can be used to analyze a combination using Spectrophotometry
without the requirement for substantial sample pre-processing.

Lambert’s Law
The intensity of incident light has no bearing on the proportion of light absorbed by a medium.
No matter how strong the light source is, a sample that absorbs 75% of the light (25 percent
Transmittance) will always absorb 75% of the light.
Lambert’s law is expressed as I / I o = T

Where, I = Intensity of transmitted light

I o = Intensity of Incident light
T = Transmittance

This enables spectrophotometers using diverse light sources to generate equivalent

absorption results regardless of the light source's power.
Beer’s Law
The concentration of the absorbing medium and the thickness of the medium are directly related
to light absorption. The PATH LENGTH is the thickness of the medium in Spectrophotometry.

Because a conventional spectrophotometer utilizes a cuvette that is 1 cm wide, or is always

presumed to be 1 cm wide, the route length is measured in centimeters. Beer's law allows us to
measure samples with varying journey lengths and immediately compare the findings.

The Beer-Lambert Law (also known as Beer's Law) asserts that the absorbance of a sample is
proportional to its concentration.
A = log 10 I / Io = ϵ l c

Where
 A is the measure of absorbance (amount of light absorbed by a sample.)
 ϵ is the molar extinction coefficient or molar absorptivity or absorption coefficient (It is
defined as a measure of a chemical's ability to absorb light at a specified wavelength.)
 l is the path length, and
 c is the concentration.

The absorbance is directly proportional to the concentration (c) of the solution of the sample
used in the experiment.
A∝c
The absorbance is directly proportional to the length of the light path (l), which is equal to the
width of the cuvette.
A∝l
Combining the two relationships,
A∝cl
This proportionality can be converted into an equation by including a constant.
A=ϵlc

CONCEPTS:
 A specific fraction of light is absorbed when it passes through a solution; this fraction is
detected, quantified, and utilized to connect the amount of light absorbed or transmitted
to the concentration of the material. This allows for both qualitative and quantitative
substance analysis.
 Light intensity is measured as a function of wavelength using the spectrophotometric
approach. It accomplishes this by:
 Diffracting the light beam into a spectrum of wavelengths
 Direct it to an object
 Receiving the light reflected or returned from the object
 Detecting the intensities with a charge-coupled device
 Displaying the results as a graph on the detector and then the display device
  NEPHELOMETRY

PRINCIPLE:
 The scattering or absorption of light by solid or colloidal particles suspended in solution
is the basis for nephelometry.
 When light passes through the suspension, absorption, reflection, and reaction dissipate
some of the incident radiant energy, while the remainder is transferred.
 Light is directly transmitted through the sample solution (suspended particles) in
nephelometry. At 90 degrees Celsius, the amount of dispersed radiation is measured.
 The basis of nephelometry analysis is the measurement of scattered light intensity as a
function of dispersed phase concentration.
 It is critical to remember that incident and scattered light in nephlometry have the same
wavelength, whereas scattered light in a fluorimeter (in fluorimetry) has a larger
wavelength than incident light.

CONCEPT:
 The light incident on the sample is scattered in all direction and the scattered light is at
thesame wavelength as the incident light
 In turbidimetry, the intensity of light transmitted through the medium, the unscattered
light, is measured at 180o from the incident light beam.
 In Nephelometry, the intensity of the scattered light is measured, usually, but not
necessarily, at right angles to the incident light beam.
 The two techniques differs only in the manner of measuring the scattered radiation
 Turbidity can be measured on most routine analysers by a spectrophotometer (absorbed
light)
 Reduced sensitivity and precision.
 Extent of light scattering increases as wavelength increases
 The intensity of scattered light is normally measured by Nephelometer. Fluorometers
are often used to perform Nephelometric measurements
 The choice of method used is dependent upon the amount of light scattered by
suspended particles present in solution.
 highly concentrated suspensions – turbidimetry
 Low concentration – nephelometry (more accurate results)

 TURBIDIMETRY

PRINCIPLE:
 Principle of Turbidimeter is established on the basis of scattering or absorption of light by
solid suspensions or colloids in the solution. When this light is processed through the
suspension, part of incident radiant energy is dissolute by absorption, reflection and
reaction while remaining is transmitted.
 SCATTERING OF LIGHT
 There are two ways in which light can be scattered:
o Elastic way of scattering
o Inelastic way of scattering
  TYNDALL’S EFFECT
 It is the effect of light scattering in multiple directions in colloidal dispersion, while
showing no light in a true solution.
 However, the scattering of light depends upon:
o Concentration of particles suspended in the medium.
o Size distribution of the particles.
o Refractive index of the particles
o Wavelength of light source employed.

CONCEPT:
 The light incident on the sample is scattered in all direction and the scattered light is at
thesame wavelength as the incident light
 In turbidimetry, the intensity of light transmitted through the medium, the unscattered
light, is measured at 180o from the incident light beam.
 In Nephelometry, the intensity of the scattered light is measured, usually, but not
necessarily, at right angles to the incident light beam.
 The two techniques differs only in the manner of measuring the scattered radiation
 Turbidity can be measured on most routine analysers by a spectrophotometer (absorbed
light)
 Reduced sensitivity and precision.
 Extent of light scattering increases as wavelength increases
 The intensity of scattered light is normally measured by Nephelometer. Fluorometers
are often used to perform Nephelometric measurements
 The choice of method used is dependent upon the amount of light scattered by
suspended particles present in solution.
 highly concentrated suspensions – turbidimetry
 Low concentration – nephelometry (more accurate results)

 FLUOROMETRY

PRINCIPLE:
 Electrons, electrons, and non-bonding (n) electrons are all present in a molecule. It's
possible that electrons are present in the bonding molecular orbital. The highest
occupied molecular orbital is referred to as this (HOMO). It is more stable and contains
less energy.
 When a molecule absorbs light energy, the bonding electrons may be advanced to an
antibonding molecular orbital (LUMO). Because it contains more energy, it is less stable.
 Excitation is the process of promoting electrons from HOMO to LUMO while absorbing
energy.
 Singlet state:-a state in which all the electrons in a molecule are paired 
 Doublet state:- a state in which un paired electrons is present  or 
 Triplet state:- a state in which unpaired electrons of same spin present 
 Singlet excited state:- a state in which electrons are unpaired but of opposite spin like 
(un paired and opposite spin)
CONCEPT:
 When a molecule absorbs light of the right wavelength, electrons are promoted from
singlet ground state to singlet excited state. Once the molecule is in this excited state, it
can relax through a variety of mechanisms. For example, by releasing radiation. The
procedure can be as follows:
1) Collisional deactivation
2) Fluorescence
3) Phosphorescence.

 Collisional deactivation: When all of the energy in a collision is dissipated and no

radiation is released.
 Fluorescence: the excited singlet state is extremely volatile. Emissions of light as
electrons relax from excited singlet to singlet ground state.
 Phosphorescence: When conditions are appropriate, such as low temperature and the
absence of oxygen, a transition from the excited singlet state to the triplet state occurs,
which is known as inner system crossing. Phosphorescence is the emission of radiation
when electrons move from the triplet state to the singlet ground state.

 ELECTROPHORESIS

PRINCIPLE:
 Because of their varied electrophoretic mobility, molecules with different overall charges
will begin to separate when a potential difference is applied. Even molecules with
comparable charges will begin to separate if their molecular sizes differ, since frictional
forces would be different. As a result, some types of electrophoresis rely almost entirely
on the various charges on the molecules for separation, while others take advantage of
differences in molecule size (molecular size).
 Because the electric field is eliminated before the molecules in the samples reach the
electrode, electrophoresis is considered an incomplete type of electrolysis. However, the
molecules will have already been sorted according to their electrophoretic mobilities.
 The separated samples are subsequently located by staining with an appropriate dye or,
if the sample is radiolabeled, by autoradiography.

CONCEPT:
 Iontophoresis - tiny ion migration
 Depending on their charges, ionized species travel towards the anode or cathode.
 In a solution more acidic than its isoelectric point (pI), ampholytes become positively
charged, while in a more alkaline solution, they become negatively charged.
Rate of migration depends on:
 Net electrical charge of the molecule
 Size and shape of the molecule
 Electrical field strength
 Properties of the supporting medium
 Temperature of operation
 Electrophoretic mobility (µ) – rate of migration (cm/s) per unit field strength (volts/cm)
µ = Q/6∏rȠ
  Where:
 µ = electrophoretic mobility in cm2/(V)(s)
 Q = net charge on the ion
 r = ionic radius of the solute
 Ƞ = viscosity of the buffer soln

 Other factors affecting mobility:

 Endosmotic flow is the preferential passage of water through an electrophoresis
medium in one direction due to selective binding of one type of charge on the
medium's surface.
 Wick flow refers to the movement of water from a buffer reservoir to the center of an
electrophoresis gel or strip in order to replenish water lost due to evaporation.

 CHROMATOGRAPHY

PRINCIPLE:
 A mobile phase and a stationary phase are typically used in chromatography. The
mobile phase is a combination of separated components that is dissolved in a liquid or
gas. The stationary phase consists of a porous solid matrix through which the mobile
phase sample percolates. The chemical is separated from the mixture as a result of the
interaction between the mobile phase and the stationary phase.
 Chromatography is commonly based on the notion of solute partitioning between two
phases. A Mobile Phase and a Stationary Phase are generally present.
 The Mobile Phase is a combination of the compounds to be separated that has been
dissolved in a liquid or gas.
 The Stationary Phase is a porous solid matrix that allows the sample from the mobile
phase to seep through.

CONCEPT:
 Separate a combination into individual components by separating and resolving it
(solutes)
 Place a sample in a mobile phase -> MP passes via a support particle bed (stationary
phase)
 MP transports the sample into and out of SP.
 Solutes that are more soluble in the MP or
 Those that have a lower affinity for the SP travel faster than those who do not.
 Mobile phase 
 gas in gas chromatography (GC)
 liquid in liquid chromatography (LC)
 Stationary phase
 Planar chromatography: spread on a flat surface eg. TLC, paper
 column chromatography: contained in a column
  Other techniques:
 HPLC: high-performance column with LC
 GC-MS: combine GC with mass spectrometry
 LC-MS
 Column Chromatography 
 column effluent passed thru a detector
 detects the separated zones of solutes
 zones appear as peaks
 measure peak: height, width, area
 Planar Chromatography
 separated zones appear as individual spots

 ULTRACENTRIFUGATION

PRINCIPLE:
 A centrifuge is a device for separating particles from a solution according to their size,
shape, density, viscosity of the medium and rotor speed.
 In a solution, particles whose density is higher than that of the solvent sink (sediment),
and particles that are lighter than it float to the top.
 The greater the difference in density, the faster they move. If there is no difference in
density (isopyknic conditions), the particles stay steady.
 To take advantage of even tiny differences in density to separate various particles in a
solution, gravity can be replaced with the much more powerful “centrifugal force”
provided by a centrifuge.
 The homogenized sample is centrifuged in an ultracentrifuge
 An ultracentrifuge consists of a refrigerated, low-pressure chamber containing a rotor
which is driven by an electrical motor capable of high speed rotation.
 Samples are placed in tubes within or attached to the rotor. Rotational speed may reach
up to 100,000 rpm for floor model, 150,000 rpm for bench-top model (Beckman Optima
Max-XP or Sorvall MTX150), and creating centrifugal speed forces of 800,000g to
1,000,000g. This force causes sedimentation of macromolecules, and can even cause
non-uniform distributions of small molecules.

CONCEPT:
 A centrifugal force is applied to a particle when it is rotated at a high speed, whether it is
a precipitate, a macromolecule, or a cell organelle.
 Centrifugal force is defined as F=mwr
 Where, F= intensity of centrifugal force
m= effective mass of sedimenting particle
w=angular velocity of rotation
r=distance of migrating particles from central axis of rotation
  The induced gravitational field's magnitude is quantified in terms of the G value: a G
value of 1000 denotes an induced field that is a thousand times stronger than gravity's.
The G value, also known as the RCF (relative centrifugal force), is determined by the
rotor's rotation speed and how the centrifuge tubes are held by the rotor.

 CHEMILUMINESCENCE

PRINCIPLE:
 Chemiluminescence is the light emission produced during a chemical reaction
 Chemiluminescence molecule is used as an indicator label to detect and quantify
immunological reactions
 Isoluminol and acridinum esters are the most important example of Chemiluminescent
labels
  Emission of light with limited emission of heat (luminescence), as the result of a
chemical reaction.
[A] + [B] → [◊] → [Products] + light

[A], [B]: reactants

[◊]: excited intermediate

 For example, if [A] is luminol and [B] is hydrogen peroxide in the presence of a suitable
catalyst we have:
luminol + H2O2 →3-APA[◊] →3-APA + light
Where:
 3-APA is 3-aminophthalate
 3-APA [◊] is the excited state producing light as it decays to a lower energy level.

CONCEPT:
 Sufficient excitation energy provided (by the chemical reaction) for red emission (λ = 600
nm) 47.6 kcal/mol for blue emission (λ = 450 nm) 63.5 kcal/mol
 The liberation of this much energy usually comes from bond cleavage or electron
transfer.
 Formation of products capable of forming an excited state multiple bonds, conjugation,
aromatic systems.
 Presence of an emitter (doesn’t have to be a product) multiple bonds, conjugation,
aromatic systems
 Rapid kinetics (of the chemical reaction) rate is more important than high yield (φCL)
φCL = φrxn(rxn) × φf(fluor.) << 10% (typical)
 Reaction coordinate system must favor formation of an excited state over ground state
of product(s)
 Chemiluminescent reactions can be grouped into three types:
 Chemical reactions using synthetic compounds and usually involving a highly
oxidized species such as a peroxide are commonly termed chemiluminescent
reactions.
 Light-emitting reactions arising from a living organism, such as the firefly or jellyfish,
are commonly termed bioluminescent reactions.
 Light-emitting reactions which take place by the use of electrical current are
designated electrochemiluminescent reactions
  ELECTROCHEMISTRY

PRINCIPLE:
 All electrochemical reactions involve oxidation and reduction.
 Oxidation means the loss of electrons (it does not always involve oxygen).
 Reduction means the gain of electrons (gaining of negatives, that is electrons, reduces
the oxidation number of an atom. We will discuss oxidation number latter in this
program).
 Whenever electrons are lost by one substance they must be gained by another.
 The substance that loses electrons is referred to as a reducing agent (it lets another
substance be reduced, that is, gain the electrons).
 The substance that gains electrons is referred to as an oxidizing agent (it lets another
substance be oxidized, that is, lose the electrons).

CONCEPT:
OXIDATION & REDUCTION
 OXIDATION:
Zn(s)  Zn+2 (aq) + 2 e-
Metallic zinc is oxidized to zinc ion. Metallic zinc is serving as a reducing agent. (electron
loser)
 REDUCTION:
Cu+2 (aq) + 2 e- Cu(s)
Copper ion is reduced to copper metal. Copper ion is serving as an oxidizing agent (electron
gainer)
 In the overall reaction two electrons are transferred from the zinc metal to the copper
ion. Zn(s) + Cu+2 (aq) Zn+2 (aq) + Cu(s) 3
 Oxidation-Reduction
 The term redox stands for reduction-oxidation
 It refers to electrochemical processes involving electron transfer to or from a
molecule or iron changing its states.
 The atom or molecule which loses electrons is known as the reducing agent.
 The substance which accepts the electrons is called the oxidizing agent.
 Balancing redox reactions
 Acidic medium
 Example of manganese reacts with sodium bismuthate
 Unbalanced reaction: Mn2+ (aq) + NaBiO3(s) → Bi3+ (aq) + MnO4 – (aq)
 Oxidation: 4 H2O(l) + Mn2+ (aq) → MnO4 – (aq) + 8 H+ (aq) + 5 e–
 Reduction: 2 e– + 6 H+ (aq) + BiO3 – (s) → Bi3+ (aq) + 3 H2O(l) 8 H2O(l) + 2
Mn2+ (aq) → 2 MnO4 – (aq) + 16 H+ (aq) + 10 e– 10 e– + 30 H+ (aq) + 5 BiO3 –
(s) → 5 Bi3+ (aq) + 15 H2O(l)
 Reaction balanced: 14 H+ (aq) + 2 Mn2+ (aq) + 5 NaBiO3(s) → 7 H2O(l) + 2
MnO4 – (aq) + 5 Bi3+ (aq) + 5 Na+
  IMMUNOCHEMISTRY

PRINCIPLE:
 The localization of antigens in tissue sections by the use of labeled antibodies as a
specific reagents
 Antigen-antibody interactions that are visualized by a marker such as fluorescent dye,
enzyme, radioactive element or colloidal gold.
 A method for localizing antigen in tissues or cells based on antigen antibody reaction
 The site of antibody binding is identified either by tagging the antibody , directly or
indirectly with a visible label
 Fluorescent dye, colloidal metal, hapten, radioactive marker.

CONCEPT:
 It is the pivotal reagent common all IHC techniques.
They impart specificty.
Two types of antibodies are used:
 Polyclonal antibodies.
 Monoclonal antibodies.
 Polyclonal antibodies
 They are a heterogenous mixture of antibodies directed against various epitopes
of same antigen.
 Generated by different B-cell clones of the animals → immunochemically
dissimilar..
 Monoclonal antibodies
 They are a homogenous population of Ig directed against a single epitope.
 Generated by a single B-cell clone from one animal → immunochemically similar.
 Developed by Kohler and Milstein in 1975.

 DRY CHEMISTRY

PRINCIPLE:
 Reflectance spectrophotometry measures the reflectance of materials. Reflectance
measurements are of great value in providing a reference standard for the comparison of
the color of different samples.
 Reflectance measurements are made using both diffuse and specularly reflected light. In
diffuse reflectance, light is scattered in all directions from the sample. Provided that this
scattered light can be collected onto an optical detector, the surface reflectance may be
measured either at a given wavelength, or by performing a scan over a range of
wavelengths. Such a wavelength scan can then be used to characterize color.
 A reflectance spectrophotometer is similar to a standard UV/Visible spectrophotometer.
It should have a bandwidth narrow enough to provide well resolved visible spectra yet
wide enough to provide a good energy level for diffuse reflectance measurements. The
reflectance spectrophotometer must also have optics and electronics systems of high
sensitivity, and should be able to physically accommodate reflectance and transmission
accessories. The adapted spectrophotometer must be able to make measurements both
at selected fixed wavelengths or perform scans over the complete wavelength range.
CONCEPT:
 In dry chemistry, slides are dry, multilayered analytical elements coated on polyester
supports. A small amount of patient sample is deposited onto the slide and evenly
distributed to all of the layers. The spreading layer contains the appropriate substrate
and other components needed for the reaction.
 The analyte in the sample catalyzes the reaction sequence to yield products which
absorb light at wavelengths in various regions (340 – 680nm), diffuses into the
underlying layer, and is monitored by reflectance spectrophotometry. The test types are
colorimetric, enzymatic end point, two-point or multi-point rate, or potentiometric.
 The rate of change in reflection density in converted to enzymatic activity or the amount
of colored complex formed is proportional to the analyte concentration in the sample.
 2 ml of serum collected in a red top tube with a serum separator (gel barrier). Centrifuge
the specimen after it has clotted to prevent hemolysis. Send to the lab at room
temperature. If the blood is not sent to lab the same day it is drawn, centrifuge the
specimen and refrigerate. Serum that is hemolyzed and/or lipemic may interfere with
some chemistry and may be rejected.