1. Clinical Chemistry Chapter 4
Analytical Techniques and Instrumentation

2. Introduction
How do we actually measure the concentrations of molecules that are dissolved in the blood? In Star Trek, they get their medical tricorder. Until then we have to use other techniques.
Spectrophotometry ( mix chemicals together to produce colored products , shine a specific wavelength of light thru the solution and measure how much of the light gets “absorbed” )
Nephelometry and Turbidimetry ( mix chemicals together to produce cloudy or particulate matter , shine a light thru the suspension and measure how much light gets “ absorbed” or “refracted” )
pH Meters / Ion Selective Electrodes (ISE) ( Electrically charged ions effect potentials of electrochemical circuits )
Electrophoresis ( Charged molecules move at different rates when “pulled” through an electrical field )
Osmometers ( Dissolved molecules & ions are measured by freezing point depression and vapor pressure )

3. Key Terms Electrophoresis Fluorometry Ion Selective Electrodes ( ISE )
Spectrophotometry
Nephelometry
Turbidimetry
Atomic Absorption
Potentiometer
Osmometer
Densitometer
Beer’s Law
Absorbance ( A )
Transmittance ( T )
Wavelength
Monochomator
Bandpass
Photo detector
Linearity
Fluorescence
Electrical potential
Reference electrode
Measuring electrode

4. Objectives
Explain the basic principles and components of the following analytic techniques
Spectrophometry
Fluorometry
Atomic Absorption
Nephelometry
Turbidimetry
Electrophoresis
Osmometry
Ion Selective Electrodes ( Potentiometry )

5. Spectrophotometry
A narrow wavelength of light is isolated from the light source and directed
thru a curvet containing patient’s molecules and reagents. Chemical reactions
between the patient’s molecules and reagents produce new molecules that absorb this wavelength of light energy. Light that is not absorbed ( transmitted ) passes thru the curvet and strikes the photodetector and converted into an electrical signal. Absorbance (A) is a mathematical relationship between the initial light intensity and the transmitted light measured by the photodetector.

6. Spectrophotometry ( cont )
I = Transmitted light ( passes thru the curvet and hits the photodetector )
I0 = Incident light ( beginning light intensity, before passing thru curvet )
PS … This is just a little college algebra

7. Spectrophotometry ( cont )
If concentration is plotted against %T and A, two types of graphs are created.
Which one you do you think is easier to work with ???

8. Beer’s Law Beer’s Law : Absorbance = (ε) (b) (c)
ε = molar absorptivity ( constant for each type of molecule )
b = length of the lightpath ( curvet )
c = concentration of the molecule absorbing the light
Hopefully, you will use the same size curvets for all your measurements,
so b remains the same for all measurements.
Also, since molar absorptivity does not change either, it remains the same
for all measurements.
If ε and b are constants, then absorbance is directly related to
concentration !!!
It’s beautiful, baby !!!

9. Spectophotometry ( cont )
Because Beer’s Law states that absorbance (A) is directly proportional to concentration ( it’s a linear relationship ), the following mathematical relationships can be established:
Cunknown = Concentration of the Unknown
Cstandard = Concentration of the Standard
Aunknown = Absorbance of the Unknown
Astandard = Absorbance of the Standard
This can be rewritten as:

10. Spectrophotometry ( cont )
Example of
If you have a Standard Solution of known concentration, say Glucose = 100 mg/dl,
and you want to measure the glucose of an unknown solution, perform the glucose test
procedure on both the Standard and Unknown solutions and compare the Absorbance
values for each solution. If...
Absorbance (A) of the Standard = 0.8
Absorbance (A) of the Unknown = 1.0
This is a permissible technique that is often used in manual techniques that have only
one Calibrator, but it has the draw-back of only having one calibration point to compare
unknowns with.

11. Spectrophotometry ( cont )
Automated clinical analyzers will use multiple calibration points to create
a “good curve” and compare the absorbance values of unknown solutions
to determine the concentrations of unknown solutions.
Example:
If an unknown solution has an
absorbance of 0.5, then the
concentration of that solution is
approximately 40
2.0 A
1.0
Each star represents the
absorbance values of different
calibration solutions with known
concentrations. A “best line” is
mathematically constructed by the
computer.
Note that the points used to create
the line do not necessarily fall on the
line itself. 50
100
Concentration

12. Spectrophotometry ( cont )
2.0
Where’s the line?
When calibration errors produce
results that are inconsistent with
Beer’s Law, the calibration has
failed and will need to be
investigated.
Most clinical analyzers will not
allow results to be generated when
there is no current valid calibration. A
Concentration

13. Spectrophotometry ( cont )
This is a linear relationship
Concentration is directly proportional
to Absorbance (A)
By taking standard solutions of known concentrations, a standard
( calibration ) curve can be constructed
The absorbance values of unknown solutions ( patient specimens ) can be
compared to the calibration curve to determine their own concentrations
Once established, calibrations can be “remembered” by analyzers for periods
of time ( days to weeks )

14. Spectrophotometry ( cont )
Beer’s Law may not last forever. As concentrations continue to increase, the
linear relationship between absorbance and concentration may break down
When this happens, we say that the methodology is “out of linearity” or
“out of range”. Instruments will usually report an “error code” or “error message”
when this occurs.
Specimens with extremely high concentrations will need to be diluted with
appropriate solutions and retested.
The final concentration from diluted specimens must be multiplied by the dilution factor to give the correct concentration in the undiluted specimen
( sometimes analyzers will do this for you automatically ).

15. Nephelometer Light is scattered from insoluble complexes
( often Ab-Ag complexes )
Light “bounces” off insoluble complexes and hits a photodetector that has
been placed at an angle off from the initial direction of the light. This is a
measurement of Transmitted light
Turbidimetry is similar, except that the photodetector is placed in the same angle of the initial light path. This is a measurement of Absorbance (A) - light that has been blocked by the insoluble complexes

16. Fluorometry Detection of fluorescent light emitted by fluorescent
molecules
The photodetector must be
placed at a 90° angle from
the initial light source
This eliminates any
interference from the initial
light source and detects only
fluorescent light.

17. Fluorometry ( cont ) Definition of fluorescence :
Certain molecules absorb light and a given frequency, and then re-emit that
light at a different and longer frequency
Advantages of fluorescence: Very specific and sensitive
Disadvantages of fluorescence: Few molecules fluoresce and very susceptible
to pH and temperature changes

18. Atomic Absorption
A cathode tube containing one given metal emits a light wavelength unique for that metal. Common metals are lead and mercury
The patient specimen is vaporized ( literally !!! ) into an acetylene flame and is dispersed into the light path.
Metal atoms in the ground state from patient specimen absorb the light energy from the cathode tube.
Because light is emitted from the metals in the cathode tube and the patient metals in the flame, the chopper breaks up the signal from the cathode tube.
The photo detector measures the difference in the signals to measure the light emitted from the flame only.
Atomic Absorption is only suited for metallic substances that are not destroyed by the flame - Everything else gets “fried”.

19. Atomic Absorption ( cont )
Illustration of Atomic Absorption

20. Electrophoresis
Molecules from patient specimens are treated with buffer solutions to give them electrical charge and are placed onto the surfaces of semi-solid supporting media
The media must be able to conduct electrical current and connects a cathode (=) to an anode (+)
When electrical current flows through the media, electrically charged molecules “migrate”, or move along the supporting media
The rate at which different molecules move along the supporting media ( strip ) will vary depending on the physical characteristics of the molecules and the methodology of the electrophoresis
After migration, the strip is removed and stained with an appropriate stain - “bands” of stained molecules will be visible
A densitometer scans the stained strip and reports a graphical representation of the bands

21. Electrophoresis ( cont )
Each peak represents a different band of molecules that migrated together during electrophoresis
Peaks with narrow bases reflect homogeneous molecules that migrated closely together
Peaks with wide bases reflect heterogeneous molecules that spread out during migration
Factors that affect migration rates of molecules:
Molecular weight
Molecular shape
Molecular electrical charge in the buffer ( buffer pH )
Supporting media
Temperature
Electrical voltage
Migration time

22. Electrophoresis
Illustration of Electrophoresis chamber

23. Electrophoresis ( continued )
The densitometer scans the
stained electrophoresis strip
and converts the intensity of
the stain into graphical form

24. Electrophoresis ( continued )
Graphical representation of the
densitometer’s scan of the electrophoresis
strip.
The densitometer calculates the area under
the curves and expresses each as a
percentage of the total

25. Ion Selective Electrodes ( ISE )
ISEs are modified electrochemical half cells
An electrochemical cell contains two components that have chemical reactions producing and consuming electrons. It is an electrical circuit completed by a salt bridge.
There are two main components to an electrochemical half cell
Reference Electrode ( generates a known, constant voltage )
Measuring Electrode ( generates a variable voltage )
Voltage ( or potential ) may be thought of as electrical “pressure” that forces electrons through the circuit
Voltage = Current x Resistance or V = I x R
Because the reference electrode contributes a constant voltage to the circuit, any measured change in the voltage of the circuit reflects a contribution from the measuring electrode

26. ISE ( cont )
Different membranes that are selectively sensitive to the electrical effects of different electrolytes can be placed over the tips of the measuring electrodes, making them susceptible only to the effects of these particular electrolytes
Differences in the measured potential of the circuit can be calibrated with known concentrations of electrolytes
Because ISE measures electrical potential, it is an example of potentiometry
Common substances measured by ISE
Sodium (Na)
Potassium (K)
Chloride (Cl)
Ionized Calcium (Ca)
Hydrogen ions (H) … Also known as a pH meter

27. ISE ( cont )
Illustration of an electrochemical cell - Electrons are produced
at the anode and consumed at the cathode

28. Osmometry Colligative Properties
Properties of solutions determined by the number of dissolved molecules
It doesn’t matter what the molecules are, they all contribute equally
Freezing point
Dissolved molecules lower the melting point of solutions
1 mole of dissolved molecules lowers the freezing point of deionized water by 1.86 ° C
Vapor pressure
Dissolved molecules in a solution decrease the rate of evaporation the solution
Osmotic Pressure
The numbers of dissolved molecules in a solution regulates the the flow of water across semi-permeable membranes
Water moves from areas of low to high solute concentration

29. Osmometry ( cont )
Osmometers are analytical instruments used to measure the concentrations of total dissolved substances that contribute to the osmotic pressure of solutions
There are two common types of osmometers
Freezing Point Depression analyzers
Vapor Pressure analyzers

30. Osmometry ( cont ) Freezing Point Analyzers
Sample is quickly cooled below its freezing point to about ° C
Vigorous agitation ( usually by a metal probe ) of the specimen causes crystallization of ice in the specimen
The rapid crystallization of water molecules releases heat
A temperature equilibrium is established when the heat from crystallization equals the heat absorbed in the melting process
The temperature at which the equilibrium occurs is inversely proportional to the osmolality

31. Osmolality ( cont )
Calculation of osmolality using freezing point depression
Remember that 1 mole of dissolved molecules per kg depresses the freezing point
of water by 1.86 ° C
Normal plasma freezes at around ° C
The osmolality of normal plasma is about 284 mOsmol / kg

32. Osmometry ( cont ) Vapor Pressure Analyzers
Sample is sealed within a small chamber
Humidity equalizes between the air in the chamber and the specimen
Electrical potential is passed through a wire in the chamber by a thermocouple which measures the temperature at which the chamber is saturated with vapor from the specimen
The thermocouple also transfers heat from the chamber and cools it below the dew point
As the current from the thermocouple is turned off, the temperature in the chamber rises and vapor condenses on the wire
A temperature equilibrium occurs when evaporation equals condensation on the wire , affecting its voltage
The vapor pressure is directly proportional to the thermocouple voltage

33. Osmometry ( cont )
A thermocouple is a type of thermometer made from a wire loop consisting of different metals connected together. One part of the wire connection is at a known “reference” temperature – the other end of the connection is in contact with the item to be measured. When the two connections are at different temperatures, an electrical potential is generated thru the loop. The measured potential is related to the temperature of the item being measured.

34. Oximetry Oximetry is a variation of spectrophotometry
A solution can be scanned with multiple wavelengths, producing absorption spectrums consistent with molecules
Oximetry is commonly performed during Blood Gas analyses to measure different forms of hemoglobin
Selective wavelengths are used for the analysis of hemoglobins ( oxygenated hemoglobin, deoxygenated hemoglobin, carboxyhemoglobin )
In the case of oxygenated hemoglobin, there is a direct relationship between the ratios of absorbances taken at two different wavelengths ( 650 and 805 nm ) and the per cent Oxygen Saturation

35. Top 10 Wavelength and energy of light are inversely proportional
Beer’s Law : Absorbance is directly proportional to concentration
Absorbance = ε x b x c
Absorbance = 2 – log %T
Nephelometry and Turbidimetry measure insoluble complexes
Fluorescence : Emitted light at a longer wavelength that initial wavelength
Atomic Absorption : Ground level energy, metal atoms absorb light
Electrophoresis : Charges molecules migrate through an electrical field
ISE : Electrolytes alter electrical potential in an electrochemical cell
Osmometers measure total dissolved molecules and atoms by freezing point depression or vapor pressure