Instrumentation 1

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Chemical Specifications

Analytical Reagents/American Chemical Society Grade, Ultrapure Reagents, United States Pharmacopeia/National Formulary, Chemically Pure Reagents, Technical/Commercial Grade

Standard chemical that we use for analysis

American Chemical Society Grade

Higher form of analytical reagent. Use for test that require high sensitivity and specificity.

Ultrapure Reagents

Test that require high sensitivity and specificity.

Mass Spectrometry

Measured using mass spectrometry. Can be used as molecular techniques. Require highly purified reagents to purify contamination.

Liquid Chromatography/Mass Spectrometry, Gas Chromatography/Mass Spectrometry

Marked safe for human consumption

National Formulary

Used by pharmacists when they manufacture therapeutic drugs

United States Pharmacopeia/National Formulary

- Lower level of analytical Reagents - Cheaper - Require purification process

Chemically Pure Reagents

For daily consumption, not for analysis

Alcohol

For daily use of chemical specifications

Technical/Commercial Grade

Reference Materials

Primary Standard, Secondary Standard

Purity = 100 (+/-0.02%) -> Margin of error Marking: NIST (National Institute of Science and Technology) standard reference materials Ex. 100mg/dL

Primary Standard

- Lower Purity -Concentrations are determine based on primary Standard

Secondary Standard

Formula of Reference Materials: Concentration of secondary standard/Concentration of primary standard = Absorbance of secondary standard/ Absorbance of primary standard

True

Reagent Grade Water

Type I, Type II, Type III, Special Reagent Water

Production of Reagent Grade Water:

Filtration (Sieve), Reverse Osmosis (Semi-permeable Membrane), Distillation (Removal of Organic Compounds), Deionization, Restrictive Filtration

Regeant Grade Water: Highest purity (GCMS/LCMS)

Type I

Reagent Grade Water: routine procedures/analysis (Distilled Water)

Type II

Reagent Grade Water: Washing (Glasswares)

Type III

Two types of Distillation

Evaporation, Condensation

multiple spectrum/set of values (wavelengths)

Spectro

LIGHT (EM radiation) • Photons/energy travelling in WAVES

Photo

Measurement

Metry

Only visible light in spectrophotometry

Ultra Violet

Nm need for UV light

380nm

Visible UV light

400-700nm

Beyond red - Higher than 700

Infrared

Beyond violet - Lower than 300

Ultraviolet

The amount of the absorb light

Absorbance

Remaining light/ Not absorbed

Transmittance

Beer's Law

Absorbance, Transmittance

Absorbance: A= abc a - b - c -

Molar Absorptivity, Light Path, Concentration

Separate the light into different spectrum/wavelengths

Monochromator

Holds the sample

Cuvette

Light source: Both UV & Visible light

Mercury Arc, Sodium Vapor, Xenon

Ultraviolet (Light source)

Deiterium, Hydrogen Lamp

Visible light only

Tungsten

• Simplest • Inexpensive • Not precise wavelength • 500-600

Colored Filters

A glass is rotated to isolate the desired wavelength Grooves: 15,000 - 30,000

Prisms

• Most common • Use parallel grooves with sharp corners • bending of light • Prone to “STRAY LIGHTS

Diffraction Grating

Constructive interference of waves

Interference Filters

Bending of light

Diffraction

Monochromators

Colored Filters, Prisms, Diffraction Gratings, Interference Filters

Monochromator Sample Cell: Used for UV More scratch resistant

Quartz

Monochromator Sample Cell: • Aluminosilicate glass • Used for visible light • Prone to scratches

Glass

Monochromator Sample Cell Shape: Lens” effect (convex xhape)

Rounded

Monochromator Sample Cell Shape: More accurate light path

Square

To measure the amount of transmitted light and be converted to absorbance

Photo Detector

A light sensitive material (selenium/silver) will produce electrons upon exposure to light and conducted by silver

Barrier Layer Cell/Photo Cell

Photosensitive cathode (rubidium or lithium) with electron receiver anode enclosed in a glass tube

Phototube

Electrons are amplified as they pass through a series of anodes (dynodes) • Highest sensitivity • It can amplify even the smallest amount of light (dynodes) • Most common to use

Photomultiplier Tube

Measures light at a multitude of wavelengths • All points of the spectrum are assayed • Low sensitivity but high linearity

Photodiode

Removes STRAY light

Entrance Slit

selects a specific wavelength of light

Exit Slit

Types of Slit

Entrance Slit, Exit Slit

Apertures

Slit

Used to eliminate or subtract the effects of reagent or specimen colors that would interfere with accurately measuring an analyte. - Unusual color - Tare

Blanks

Use of Blanks

Water Blank/Air Blank, Reagent Blank 0.001, Patient Blank/Sample Bank 0.003

Quality Assurance of Spectrophotometers

Wavelength QA (glass filters), Stray lights, Linearity

Wavelength QA (Glass Filters)

Didymium (600nm) Visible - 0%, Holmium Oxide (360nm) - UV light

Stray Lights

Nickel Sulfate, Sodium Sulfate, Acetone

Linearity

Colored solutions with varying dilutions, Dichromate and neural density filter

Used on trace metals (metals with low concentration in the sample) • Light source: Hollow cathode lamp

Atomic Absorption Spectrophotometry

The cathode lamp is coded with?

Lead

Emit atoms together with the light

Inert Gas

Light source: Hollow cathode lamp

Anode, Cylindrical Cathode (Metal), Inert Gas

Flame Emission Photometry: Sodium:

Yellow Flame

Flame Emission Photometry: Potassium:

Purple Flame

Flame Emission Photometry: Lithium

Red Flame

Instrument: Spectrophotometer What is detected: Reduction of transmitted light Ideal for: Urine and CSF Proteins Photodetector Placement: 180 degrees

Turbidimetry

What is detected? Scattered light Ideal: Antigen-Antibody Interactions Photodetector Placement: Specific Angle

Nephelometry

• Uses UV light to excite fluorescent dyes in the sample. • Fluorescence is inversely proportional to temperature • Excitation vs Fluorescence energy • This technique is widely used for the detection of therapeutic and abused drugs

Fluoremetry

• 1,000 times more sensitive than most spectrophotometric methods • Fluorescence measures the amount of light intensity present over a zero background

Advantages of Fluoremetry

• QUENCHING • Highly sensitive to environmental changes • Contaminating chemicals or a change of solvents may change the structure of the analyte • UV light

Disadvantages of Fluoremetry

Used to separate fractions using a solid support media and electricity/current. • Proteins – protein fractions, immunoglobulin specific fractions, hemoglobin types, etc. • Migration from CATHODE to ANODE

Electrophoresis

Movement of buffer ions and solvent relative to the fixed support • paper, cellulose, acetate, and agar gel

Electroendosmosis

Components of Electrophoresis Support Media:

Cellulose acetate, Agarose gel, Polyacrylamide gel, Starch gel

Detector of Electrophoresis

Densitometer

• Dry brittle film • Used for isoelectric focusing

Cellulose Acetate

• Purified fraction of agar • Measured using densitometer • Does not bind protein

Agarose Gel

• Separation based on charge and size • Separation of isoenzymes

Polyacrylamide Gel

Separation based on surface charge and size

Starch Gel

Modifications of Electrophoresis

Isoelectric Focusing, Capillary Electrophoresis

Charged proteins migrate through a support medium that has a continuous pH gradient

Isoelectric Focusing

Separation is performed in narrow-bore fused silica capillaries

Capillary Electrophoresis

Separation based on affinity to phases (mobile vs. stationary)

Chromatography

Chromatography • Separated components: ______________

Mobile, Stationary

liquid-solid chromatography (affinity)

Adsorption

liquid-liquid chromatography (polar vs. nonpolar)

Partition

• liquid-solid chromatography (size and shape) • hydrophilic beads of cross-linked dextran, polyacrylamide, or agarose

Steric Exclusion

stationary phase is a resin capable of extracting ions

Ion Exchange

Uses pressure for fast separations, controlled temperature, in-line detectors, and gradient elution techniques

High Performance Liquid Chromatography (HPLC)

uses nonpolar stationary phase

Reverse High Performance Liquid Chromatography (RHPLC)

Gas Chromatography • Stationary phases: _________ or _________

Solid, Liquid

Gas Chromatography Mobile phase: ____________

Gas

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