Clinical Chemistry Chapter 4 Analytical Techniques and Instrumentation

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 )

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

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

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.

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

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 ???

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 !!!

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:

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.

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

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

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 )

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 ).

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

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.

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

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”.

Atomic Absorption ( cont ) Illustration of Atomic Absorption

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

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

Electrophoresis Illustration of Electrophoresis chamber

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

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

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

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

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

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

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

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

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 - 0.53 ° C The osmolality of normal plasma is about 284 mOsmol / kg

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

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.

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

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