Cc Rodriguez Analytical Method

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THINGS TO REMEMBER IN ANALYTICAL METHOD

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Principle of the method

Purpose of the method

Distinguishing Characteristic of the test

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Visible spectrum

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400-700nm

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Ultraviolet region (UV)

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<400nm

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Infrared Region (IR)

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>700nm

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Number of vibrations of wave per second

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Frequency

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Represents the wavelenght in nanometers at peak transmittance

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Nominal wavelenght

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Wavelenght indicated on the control dial and is the actual wavelenght of light passed by the monochromator

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wavelenght accuracy

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Used to check wavelength accuracy (wavelenght calibration)

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Didymium or holmium oxide filter

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Verify absorbance accuracy on linearity

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Neutral density filters and dicromate solution

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Involves measurement of the light transmitted by a solution to determine the concentration of the light-absorbing substances in the solution.

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Spectrophotometry

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Simplest type of absorption spectometer, designed to make one measurement at a time at one specific wave lenght.

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Single Beam spectrophotometer

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Part of spectrophotometer Provides polychromatic light and must generate sufficient radiant energy or power to measure the analyte of interest.

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Light/Radiant Source

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Commonly used light source in spectrophotometer

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Tungsten light bulbs

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Routineline used to provide UV radiation innanalytic spectrophotometers.

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Deuterium lamp

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Minimize unwanted or stray light and prevents the entrance of scattered light into the monochromator system.

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Entrance slit

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Unwanted light inside the spectrophotometer

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Stray light

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Most common cause of loss of linearity at high analytr concentration

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Stray light

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Causes absorbance error

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Stray light

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Isolate specific or individual wavelenght of light

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Monochromator

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Wedge-shaped piecew of glass, quartz or sodium chloride

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Prisms

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most commonly used monochromator

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Diffraction gratings

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Filters stray light and specific light

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Sharp-cut off filter

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Simple , least expensive not precise but useful.

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Filters

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Controls width of the light beam (bandpass)- allows only a narrow fraction of the spectrum to reach the sample cuvette

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Exit slit

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Is the total range of wavelengths transmitted

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Bandpass

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Also called absorption cell/analytical cell/sample cell

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cuvet

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Most commonly used cuvet (can be used in 350-2000nm)

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Alumina silica glass

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Used for measurement of solution requiring visible and ultraviolet spectra

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Quartz/plastic

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detects and converts transmitted light into photoelectric energy

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Photodetector

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Simpliest detector, least expensive; temperature-sensitive

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Barrier layer cell/ Photocell/ photovoltaic cell

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used to filter photometers with wide band pass

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Barrier layer cell/ Photocell/ photovoltaic cell

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kinds of detectors

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Barrier layer cell/ Photocell/ photovoltaic cell

phototube

photomultiplier tube (PMT)

photodiode

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Most sensitive detector and most commlnly used.

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Photomultiplier tube (PMT)

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displays output of the detection system

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meter or read out device

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Formula for absorbance

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A= abc = 2-log%T

A= absorbance

a= molar absorptivity

b= lenght of light through the solution

c= concentration of absorbig molecules/solution

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Fomula for percent transmittance

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%T= It/Io x 100

It = trasmitted light thru the sample

Io = intensity of light striking the sample

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Flame Emission Photometry

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- Measures the light emitted by a single atom burned in a flame

Principle : Excitation of electrons from lower to higher energy state

Light source: Flame

Used to measure excited ion (sodium and potassium

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Atomic Absorption Spectrophotometry (AAS)

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Principle: Element is not dissociated but by heat energy from its chemical bonds.

Light Source: Hallow-cathod lamp

Used for measurement of unexcited trace metals (calcium and magnesium)

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Reference method for measurement of Magnesium

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AAS

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Volumetric (Titrimetric)

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Principle: unknown sample is made to react with a known solution in the presence of an indicator

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Turbidimetry

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Principle : Amount of light blocked (reducing of light)

Measuring abundant large particles (proteins)

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In turbidimetry an increase in concentration means?

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Increase turbidity

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What is the analyte that uses turbidimetry because they are abundant in the serum plasma and whole blood, they also have large particle.

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Serum protein

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2 distinct test for Proteins

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Turbidimetry

Nephelometry

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Nephelometry

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Principle : Determines amount of scattered light in a turibid soluiton

Measuring amount of antigen-antibody complexes (protein)

Measurement of gamma globulins

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Electrophoresis

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Principle : migration of charged particles in an electric field

Most important initial test for the identification and differentiation of isoenzyme

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Buffer for electrophoresis

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Barbital a.k.a Veronal

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Has a net charge that can be either positive or negative depending on pH conditions

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Amphoteric

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Factors affecting rate of migration

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Net electric charge of the molecule

Size and shape of the molecule

Electric field strength

Nature of the supporting medium

Temperature of operation

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Supporting Media for Electrophoresis

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Cellulose acetate

Agarose gel

Polyacrylamide Gel

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Supporting Media for electrophoresis that separates molecular size and is commonly used in SPE.

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Cellulose acetate

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Separates by electrical charge; it does not bind protein

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Agarose gel

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Best supporting media in electrophoresis and it is used to study isoenzymes

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Polyacrylamide Gel

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Fastest migrating protein in electrophoresis

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Albumin

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Slowest migrating protein in electrophoresi

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Gamma globulin

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Normal SPE

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5 bonds ( 1 albumin , 4 globulins )

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Stains for protein and enzymes

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Amido black

Ponceus S

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Stains for Lipoprotein

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Sudan Black

Oil Red O

Fat Red 7B

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Stain for CSF protein

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Coomassie Blue

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Measures the absorbance of stain concentration of the dye and protein fraction.

It scans and quantitates electrophoresis pattern

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Densitometry

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Separation of soluble components in a solution by a specific differences in physical-chemical characteristics of the different constituents

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Chromatography

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Semiquantitative drug screening test

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TLC (thin layer chromatography)

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Only test that has Rf value

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TLC

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Used for separation of steroids, barbiturates, blood, alcohol, and lipids.

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Gas Chromatography (GC)

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Based on the fragmentation and ionization of molecules using a suitable source of energy.

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Mass spectroscopy

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What is the most specific and sensitive method used in drug testing?

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GC-MS

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Which between GC and MS is responsible for identification of the drug whether therapeutic or illicit present on the sample?

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Mass spectroscopy

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Gold standard for Drug testing

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GC-MS

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Used for therapeutic drug monitoring and drug testing

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GC-MS

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Based on the distribution of solutes between a liquid mobile phase and a stationary phase

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Liquid Chromatography

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Complementary method to GC-MS

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LC-MS (liquid chromatography- MS)

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Fluorometry/Molecular Luminescence Spectrophotometry

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Principle : determines amount of light emitted by a molecule after excitation by electromagnetic radiation

Use: porphyrins, magnesium, Calcium, and cathecolamines

Measures amount of light intensity present over a zero backgorund

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Is the difference between the maximum wavelength, excitation, and emitted fluorescence

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Stoke Effect

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2 monochromators for Fluorometry

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Primary monochromator (excitation monochromator)

>Selects wavelength- isolates specific light

Secondary monochromator (emission monochromator)

> prevents incident light ( stray light)

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Chemiluminescence

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Measurement of light signal is against a dark background since no excitation of light is required

Has no monochromator

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ELECTROCHEMISTRY TECHNIQUES

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Potentiometry

Coulometry

Amperometry

Voltammetry

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Measurement of current or voltage generated by the activity of a specific ion

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Electrochemistry techniques

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Measure blood pH, blood gasses, glucose, ionize calcium, lead, and chloride

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Electrochemistry techniques

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Measurement of PCO2

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Potentiometry

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Nernst equation

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Potentiometry

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Potentiometry

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Measurement of differences in voltage (potential) at constant current

Reference electrode: Calomel (internal) and silver-silver chloride (external), glass pH electrode

Use: pH and PCO2 , electrolytes

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Instrument use in potentiometry

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Ion Selective Electrode (ISE)

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Measurement of the amount of electricity at fixed

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Coulometry

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Faraday's law

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Coulometry

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Chloride test

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Coulometry

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Amperometry

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Measurement of current flow produce by an oxidation-reaction

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Measures pO2 and Glucose

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Polarography

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Ilkovic equation

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Polarography

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Measurement of current after which a potential is applied to an electrochemical cell

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Voltammetry

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Measures heavy metals such as lead

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Anodic stripping voltametry