Measurement of Analytes

1. Measurement of Analytes Lab. 2

2. Introduction • Few of the chemical constituents of blood, plasma or urine can be measured directly. • Analytical chemists have, however, developed indirect means of detecting and measuring quantitatively many of the constituents of clinical interest. • Frequently the method involves adding to the sample a substance (the reagent) that chemically reacts specifically with the particular component to be quantitated (the analyte) to form a product that is measured relatively easily.

3. The analyte The reagent which reacts specifically with the analyte Product that is measured relatively easy. quantity is proportional to the original concentration of the analyte.

4. Properties of Light • Light can be: • Absorbed (sucked up) • Transmitted (passes through) • Reflected (bounces off)

5. Properties of Light • Light absorption is one of the many optical effects that have been most often exploited for analytical purposes. • The majority of quantitative measurements made in clinical chemistry laboratories are based on: • the production of coloured reaction products, • most frequently photoelectric absorbance devices such as spectrophotometers are used.

6. Spectrophotometer • An instrument that uses a photodetector to measure the amount of a specific wavelength of light transmitted through a test solution to determine concentration

8. Spectrophotometer

9. Wavelength Selector or Discriminator (Filters, Gratings) • Absorbance varies with wavelength; the wavelength discriminator provides the means to select or discriminate across wavelengths. • The simplest wavelength discriminator is an optical pass band filter. • This filter passes one wavelength while blocking the other regions of the spectra. • The user may insert the appropriate filter into the instrument, or may turn a wheel to select one of several filters mounted on the wheel. • The term colorimeter is often used to describe filter-based systems.

10. Wavelength Selector or Discriminator (Filters, Gratings) • Spectrophotometers usually use a diffraction grating for wavelength discrimination. • Diffraction-gratings are similar to prisms in that white light is spread into a spectrum by redirecting light at angles that are wavelength-dependent.

11. commonly used cuvettes (sample cells) The sample solution, held in the cuvette, absorbs a proportion of the incident radiation; the remainder is transmitted to a light detector, where it generates an electrical signal.

12. Beer’s law • The mathematical basis for quantitative measurements is based on the experimentally derived Beer–Lambert relationship. • Beer’s law states that the absorption of radiant energy is proportional to the total number of molecules in the light path or inversely proportional to the logarithm of the transmitted light.

13. Beer’s law • The values for I and Io cannot be measured in absolute terms and measurements are most conveniently made by expressing I as a percentage of Io. • Using these measurements, the Beer–Lambert law can be expressed as: • Light Absorbed (A) = - log(%T) e A= Log10 (I0/I) = ecl

14. where c is concentration of the substance in moles/litre, • l is the optical path length in centimetres • and e is the molar absorption coefficient for the substance, expressed as litres/mole/centimetre. • This reciprocal logarithmic function of I and Io, is known as absorbance (A).

15. Transmittance & concentration • As the cell thickness increases, the transmitted intensity of light I decreases. • Percentage transmittance (%T) versus concentration is a nonlinear function.

16. Absorbance & concentration • Absorbance versus concentration is a linear function. • The straight line obtained from this relationship can be used to determine the concentration of unknown samples

17. Test values are usually calculated by comparing the absorbance readings of the test samples with readings obtained from assaying a series of standards or calibrators of known value. A = ecl e = A/cl A1/c1l1= A2/c2l2 A1/c1= A2/c2

18. Three Types of Spectrophotometric Methods • The spectrophotometer measures the absorbance of light of one of the components of a chemical reaction. • Three examples of common types of chemical reactions that are measured by the spectrophotometer are: • Endpoint colorimetric • Eg. The Jaffe reaction for creatinine • Endpoint enzymatic • Eg. The hexokinase reaction with glucose (enzymes catalyze the reaction to measure the analyte). • Kinetic reactions • Eg. Alaninetransaminase (ALT).