Clinical Chemistry (CHE221) Professor Hicks Week 7. Endpoint vs Kinetic Techniques. Instrumentation and Assay techniques

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1 Clinical Chemistry (CHE221) Professor Hicks Week 7 Instrumentation and Assay techniques Endpoint vs Kinetic Techniques two different approaches to analyzing for a substance Endpoint methods use all the stuff up in a process and measure how much of a product you get - larger A more colored product formed more analyte present - all the methods we have done so far are endpoint methods Kinetic methods if more of substance is present faster initial rate - faster initial rate more analyte is present Absorbance at 510 nm Time (min) Why does maintaining constant temperature matter (sometimes)? endpoint assays are based on using up all of the substance being assayed - reaction faster/slower endpoint is same kinetic assays do not use up all substrate - based on the initial rate of reaction higher temperature increases rate higher temp overestimate of amount/activity of substance being assayed

2 Kinetic Techniques Advantages: data is collected more quickly avoids interferences from slow reacting substances Disadvantages sensitive to temperature measurements must be done on a time schedule random error tends to be more than endpoint techniques Calculations with Kinetic Assays Instead of measuring endpoints for known and unknown solutions their rates measured Proportions setup assuming rate is proportional to the amount of analyte Rate patient sample Rate standard sample = Concentration patient sample Concentration standard sample Concentration Rate patient sample Concentration patient sample Rate standard sample standard sample Fluorescence Spectroscopy Some substances absorb light energy and emit a different color of light The color emitted must always be lower energy (longer wavelength) than the light absorbed - If green light is absorbed red light could be released, but not blue light Fluorescence can be used to quantify substances Fluorescence is proportional to concentration only for solutions with %T > 98%

3 Fluorescence assays Advantages Very sensitive Fast data collection Disadvantages Can be non-specific impurities can emit fluorescence No reference/background like absorbance - must always be calibrated with standards Fluorometers Light source monochromator sample Fluorescence monochromator detector Fluorometers have two monochromators - One to select the color of light the sample is exposed to the Excitation Monochromator - One to select the color of light coming from the sample - the Emission Monochromator Fluorometer Excitation monochromator Excitation slit Sample cuvette Detector Emission monochromator emission slit

4 Fluorometers The fluorescence light collected at a 90 degree angle to the excitation light transmitted light goes straight through so weak fluorescence signals can be measured with very bright light sources Emitted light is not one color but a range of colors (wavelengths) A Fluorescence Spectrum is a graph of the fluorescence released over a range of wavelengths Plate readers Absorbance, fluorescence spectrophotometers designed to read plates containing numerous wells Each well different sample, or Replicates can be run to improve data by averaging Most common plate sizes is 96 well (12 8 rectangle) Rayleigh Scattering When light encounters particles that are close to the size of the wavelength the light can be undergo Rayleigh Scattering I o =14 light rays I = 8 light rays get through Transmittance = I/Io = 8/ A = -log(t) = 0.24 In Rayleigh Scattering the wavelength of the light is not changed it is just redirected

5 Rayleigh Scattering Because light is redirected in all directions scattering results in less light reaching the detector If a sample that scatters light its % transmittance will be less than 100% its absorbance it would be larger than 0 The light was not absorbed by the sample though, it was scattered of the optical path did not make it to the detector Nephelometers/Turbidimeters Devices used to measure concentration of scattering particles Turbidimeter measures the light that gets through the sample as an absorbance log(i/i o ) - Absorption spectrophotometers are often used as turbidimeters Nephelometer measures the scattered light - Like a fluorometers measures light not along the path of the excitation beam - can be 90 degrees or other angles Fluorometers can be used as nephelometers assays using nephelometry/turbidimetry measurement of cell concentrations in growing cultures determination of clinical analytes by treatment with antibodies immunoprecipitation form insoluble complex that scatters light

6 prism separates white light into Roy G. Biv white light includes sunlight, fluorescent, and incandescent bulbs. V I B G Y O R R ed O range Y ellow G reen B lue I ndigo V iolet white light source white light is combination of all colors of the rainbow 16 Line Spectra line spectrum Robert Bunsen light different line spectra for every element like a unique fingerprint 17 more examples of line spectra line spectra can be detected from space telling us about the elements in stars 18

7 Atomic Absorption Spectrophotometry method of choice for trace metals atoms are formed from a sample by heating the line spectrum of the element is used as the light source I and I o are measured absorbance Hollow Cathode Lamp I or I o line spectum sample chamber analyte atoms or empty space monochromator detector The Hollow Cathode Lamp is a light source designed to emit the line spectrum of a single element Atomic Absorption highly specific each element s line spectrum is very unique extremely small interference from other elements most significant problem is in forming free atoms atoms must be in the ground state so too much energy (heat) cannot be added some substances held together very strongly do not fully separate Example Ca 2+ with PO 4 3- tend to stick together solution pretreatment of samples with ions to replace Ca 2+ in the compound such as Sr 2+ Expensive instrumentation every element requires its own Hollow Cathode Lamp In a fluorometer the excitation monochromator A) Selects the wavelength(s) of light that the sample is exposed to B) Scans through the wavelengths of light the samples emits to create a fluorescence spectrum C) Is used to collect the reference data D) Is positioned at 90 degrees to the minimize the amount of scattered light that reaches the detector

8 A nephelometer is most similar in its physical configuration to a A) fluorometer B) absorption spectrophotometer C) turbidimeter D) monochromoator If your labs dedicated turbidimeter was not functioning samples could be screened using a A) nephelometer B) absorption spectrophotometer C) fluorometer D) monochromator