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Clinical ChemistryChapter 4

Analytical Techniques and Instrumentation

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• 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 )

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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

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• Objectives

– Explain the basic principles and components of the following analytic techniques

• Spectrophometry• Fluorometry• Atomic Absorption• Nephelometry• Turbidimetry• Electrophoresis• Osmometry• Ion Selective Electrodes ( Potentiometry )

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Spectrophotometry

A narrow wavelength of light is isolated from the light source and directedthru a curvet containing patient’s molecules and reagents. Chemical reactionsbetween 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.

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Spectrophotometry ( cont )

% tan ( )Transm it ce TII

x0

1 0 0

I = Transmitted light ( passes thru the curvet and hits the photodetector )

I0 = Incident light ( beginning light intensity, before passing thru curvet )

Absorbance AII

T T( ) lo g lo g lo g % lo g %

01 00 % 2

PS … This is just a little college algebra

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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 ???

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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 !!!

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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 UnknownCstandard = Concentration of the StandardAunknown = Absorbance of the UnknownAstandard = Absorbance of the Standard

std

unk

std

unk

AA

CC

This can be rewritten as:

stdstd

unkunk Cx

AAC

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Spectrophotometry ( cont )

Example of stdstd

unkunk Cx

AAC

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 testprocedure on both the Standard and Unknown solutions and compare the Absorbance values for each solution. If...

Absorbance (A) of the Standard = 0.8Absorbance (A) of the Unknown = 1.0

dlmgdlmgxCunk /125/1008.00.1

This is a permissible technique that is often used in manual techniques that have onlyone Calibrator, but it has the draw-back of only having one calibration point to compareunknowns with.

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Spectrophotometry ( cont )

Automated clinical analyzers will use multiple calibration points to createa “good curve” and compare the absorbance values of unknown solutionsto determine the concentrations of unknown solutions.

Concentration

0

0

2.0

1.0

50 100

Example:

If an unknown solution has an absorbance of 0.5, then the concentration of that solution is approximately 40

Each star represents theabsorbance values of differentcalibration solutions with knownconcentrations. A “best line” ismathematically constructed by the computer.

Note that the points used to createthe line do not necessarily fall on theline itself.

A

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Spectrophotometry ( cont )

Concentration

A

Where’s the line?

When calibration errors produceresults that are inconsistent withBeer’s Law, the calibration hasfailed and will need to be investigated.

Most clinical analyzers will notallow results to be generated whenthere is no current valid calibration.

00

2.0

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Spectrophotometry ( cont )

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 periodsof time ( days to weeks )

This is a linear relationship

Concentration is directly proportionalto Absorbance (A)

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Spectrophotometry ( cont )Beer’s Law may not last forever. As concentrations continue to increase, thelinear 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 ).

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Nephelometer

Light “bounces” off insoluble complexes and hits a photodetector that hasbeen 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

Light is scattered from insoluble complexes( often Ab-Ag complexes )

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Fluorometry

Detection of fluorescentlight emitted by fluorescentmolecules

The photodetector must beplaced at a 90° angle fromthe initial light source

This eliminates any interference from the initiallight source and detects onlyfluorescent light.

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Fluorometry ( cont )

Definition of fluorescence :

Certain molecules absorb light and a given frequency, and then re-emit thatlight 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

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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”.

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Atomic Absorption ( cont )

Illustration of Atomic Absorption

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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

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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

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Electrophoresis

Illustration of Electrophoresis chamber

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Electrophoresis ( continued )

The densitometer scans the stained electrophoresis stripand converts the intensity ofthe stain into graphical form

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Electrophoresis ( continued )

Graphical representation of the densitometer’s scan of the electrophoresisstrip.

The densitometer calculates the area underthe curves and expresses each as a percentage of the total

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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

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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

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ISE ( cont )

Illustration of an electrochemical cell - Electrons are produced at the anode and consumed at the cathode

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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

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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

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Osmometry ( cont )

• Freezing Point Analyzers

– Sample is quickly cooled below its freezing point to about - 5.0 ° 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

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Osmolality ( cont )

86.1int)/( DepressionPoFreezingkgOsmolOsmolality

Remember that 1 mole of dissolved molecules per kg depresses the freezing pointof water by 1.86 ° C

Normal plasma freezes at around - 0.53 ° C

kgmOsmolkgOsmol /284/284.086.153.0

The osmolality of normal plasma is about 284 mOsmol / kg

Calculation of osmolality using freezing point depression

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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

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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.

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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

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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