Reflectance Spectrophotometry

Reflectance Spectrophotometry

Used in POCT (glucose) testing and urine analyzers

intensity

It measures the ________ of light reflected from a surface to calculate the concentration of an analyte.

detector

A light source illuminates the sample, and a _______ measures the bounced light.

increase; decrease

increased concentration = _______ intensity = ________ light reflected

Flame Emission Photometry

Measures light emitted by a single atom burned in a flame

Flame Emission Photometry

REFERENCE METHOD for the measurement of excited ions (Na, K, Li)

Flame

in Flame Emission Photometry, it serves as the light source and the cuvette

Lithium/Cesium

component of Flame Emission Photometry that corrects variation in flame and atomizer characteristics

Source (Burner)

component of Flame Emission Photometry: Flame maintained in constant form

Nebulizer and mixing chamber

component of Flame Emission Photometry that transports homogeneous solution to the flame steadily

viscosity

Samples be diluted 1:100 or 1:200 with a detergent containing lithium to reduce ____________

Simple color filters

component of Flame Emission Photometry: interference filters

Photodetector

component of Flame Emission Photometry that measures intensity of radiation emitted by flame

Atomic Absorption (Spectrophotometry)

Measures light absorbed by atoms in neutral, ground state dissociated by heat

Atomic Absorption (Spectrophotometry)

Measurement of UNEXCITED trace elements Aluminum, Calcium, Copper, Lead, Magnesium, Zinc, Lead

dissociation

principle of Atomic Absorption (Spectrophotometry): _______ of chemical bonds as free atoms in unexcited, neutral ground state

flame photometry

Atomic Absorption is more sensitive than ________ _________

Lanthanum and Strontium Chloride

__________ and _______ _________ is added to samples to form stable complexes with phosphate in Atomic Absorption

Hollow Cathode Lamp

Atomic Absorption Light source

Chopper

Atomic Absorption component that modulates the amount of light

Atomizer-Burner Assembly

Atomic Absorption component that serves as the SAMPLE CUVETTE that converts ions to atoms

Monochromator

Atomic Absorption component that eliminates light emitted by the ideal gas

Photodetector

Atomic Absorption component that converts radiant energy to electrical impulses

Turbidimetry

measures amount of light BLOCKED (reduction of light) by particle formation in a turbid solution

concentration

Turbidimetry depends on specimen _________ and particle size

INVERSELY

in Turbidimetry the light transmitted is ________ PROPORTIONAL to concentration

Uses of Turbidimetry

Protein measurements, bacterial growth and clot formation

Nephelometry

determines amount of light SCATTERED by particulate matter in a turbid solution

wavelength; size

in Nephelometry light scatter depends on _________ and particle ____

15-90

in Nephelometry light is measured at an angle _____ - ____ degrees to the incident on the cuvette

DIRECTLY

Detector output (PM tube) of Nephelometry is ________ PROPORTIONAL to concentration, number and size

Nephelometry

Used for measurement of antigen-antibody complexes

Volumetric/Titrimetric

The unknown sample is made to react with known solution in the presence of an indicator.

Chloride testing

Schales and Schales method

Calcium testing

EDTA titration method

Fluorometry (Molecular Luminescence Spectrophotometry)

measures light emitted by a molecule after excitation by electromagnetic radiation

Fluorescence

happens when atoms absorb light of particular wavelength and emit light of a longer wavelength and lower energy than the excited wavelength

Advantage (Fluorometry)

1000x more sensitive than spectrophotometer

Quenching

Disadvantage of flourometry

Quenching

any process that decreases the fluorescence intensity of a sample (transfer of energy to solvent)

LASER or mercury arc or xenon lam

Fluorometry Light source

PMT or phototube

Fluorometry Detector

Primary filter

component of Fluorometry that selects wavelength to be absorbed by solution

Secondary filter

component of Fluorometry that prevents incident light from striking the detector

Chemiluminescence

 the chemical reaction yields an electronically excited compound that emits light as it returns to its ground state, or that transfers its energy to another compound, which then produces emission

oxidant

Chemiluminescence excitation is caused by an ______

immunoassays (CLIA)

Chemiluminescence use

fluorescence

Chemiluminescence is more sensitive than __________

Chemiluminescence

Excitation is caused by oxidation of organic compounds that produces light without producing heat

Chemiluminescence Reagent

-Luminol

-Adamantyl 1,2-dioxetane

aryl phosphate

-Acridinium esters

Bioluminescence

Chemiluminescence found in biological systems (fireflies)

Bioluminescence reagent

Luciferase and Aequori

Electrochemiluminescence

Uses elements/compounds

Electrochemiluminescence reagent

Ruthenium chelate

Refractometry

Based on light refraction

refractivity

The ability of a substance to bend light is called

incidence-refractive

Refractivity = _______ angle - _______ angle

Refractometry

"Light bending" Passes light through a liquid medium

Reflectance Spectroprotometry

"Light bouncing" Shines light onto an opaque pad

Osmometry

An osmometer is used to measure the concentration of DISSOLVED SOLUTE particles in a solution.

osmotic pressure, vapor pressure, boiling point, and freezing point.

The four physical properties of a solution that change with variations in the number of dissolved particles in the solvent are ______________, _______________, ______________, ___________,

increase; increase

_______ osmolality = _______ osmotic pressure and boiling point

increase; decrease

__________ osmolality = ______ vapor pressure and freezing point

Freezing Point Depression

Is the most commonly used method for measuring the changes in colligative properties of a solution.

temperature

It is based on the principle that addition of solute molecules lowers the at which _________ a solution freezes.

-0.53 C

Blood plasma, with an osmolality of about 285 mOsm/kg, has a freezing point of about

Potentiometry

Measures POTENTIAL DIFFERENCE in an electrochemical cell

calomel and silver-silver chloride

Potentiometry reference electrode

Direct ISE (without sample dilution)

Not subject to pseudohyponatremia caused by hyperlipidemic or hyperproteinemic samples

 Indirect ISE (with sample dilution)

Subject to pseudohyponetremia (due to dilution)

potentiometry

measures potential difference between electrodes. pH, PCO2

Amperometry

measures current produced by REDOX reactions. po2, GLUCOSE, AND PERIDOXASE DETERMINATION

Coulometry

measures total electric charge during electrolysis ata fixed potential. Chloride test (sweat, CSF, serum)

Voltammetry

measures current after a potential is applied to an electrochemical cell. anodic stripping voltammtery

conductometry

measures solution conductivity. titrations

impedance

measures response to AC voltage

Electrophoresis

Is the migration of charged particles in an electric field. Used clinically to separate and identify proteins, including serum, urine and cerebrospinal fluid (CSF) proteins, lipoproteins, isoenzymes, and so on

Barbital

pH 8.6 for alkaline electrophoresis

Citrate

pH 3 to 6.2 for acid electrophoresis

Cellulose acetate

separates by molecular size (serum protein electrophoresis)

Agarose gel

 separates by electric charge; it does not bind protein

Polyacrylamide Gel

separates on the basis of charge and molecular size sepárates proteins into 20 fractions; used to study isoenzymes

Protein

Amido black, Ponceau S, Coomasie Blue

Densitometry

Obtains a quantitative profile of the separated fractions by measuring the absorbance of the stain on a support medium

 Lipids

Oil Red O, Sudan Black, Fat Red 7B

Capillary electrophoresis

Sample molecules separated by electro-osmotic flow (EOF) based on differences in solute size.

Capillary electrophoresis

Carried out in a small bore (10-100 um) fused silica capillary tub. Uses nanoliter of specimen. For Drugs, forensic DNA

Chromatography

Is a technique where solutes in a sample are separated for identification based on physical differences that allow their differential distribution between a mobile phase and a stationary phase.

Mobile phase

May be an inert gas or a liquid

Stationary phase

May be silica gel bound to the surface of a glass plate or plastic sheet; may be silica or a polymer that is coated or bonded within a column

High Solvent Affinity (Soluble)

Molecules that dissolve easily in the moving solvent-and dOn't stick strongly to the paper fibers-travel quickly and move far up the paper.

High Paper Affinity (Adsorbed)

Molecules that bind tightly to the cellulose paper-or dissolve poorly in the solvent-move slowly and stay near the origin line.

Paper Chromatography

Used for fractionation of sugar and amino acid

Sorbent (stationary phase): Whatman paper

Thin Layer Chromatography

Semi-quantitative drug screening test

pH

Extraction of the drug is ___-dependent

Thin Layer Chromatography sorbent

Thin plastic plates impregnated with a layer of silica gel or alumina

Retention Factor (Rf)

is a unitless ratio that measures how far a specific compound travels up a chromatography plate or paper relative to the solvent front.

drug standard

Each drug has a characteristic Rf value and it must match the Rf of the _____ ________

highly soluble

A higher Rf value means the compound is _________ _______ in the mobile solvent and has low affinity for the stationary phase.

stationary

A lower Rf means the compound binds tightly to the ________ phase and dissolves poorly in the solvent.