Analytical methods Chapter 5 Flashcards by Shara Lim

What is Spectrophotometric measurement?

It is a measurement of light intensity in a narrower wavelength

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What is Photometric measurement?

It is a measurement of light intensity without consideration of its wavelength

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Is described as photons of energy traveling in waves

Electromagnetic radiation

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What is the formula of the Beer’s law?

Concentration of a substance is directly proportional to the amount of light absorbed or inversely proportional to the logarithm of the transmitted light

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What is the use of Light source?

Incident light for the system

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What are the common source of light used near infrared regions?

Incandescent tungsten and Tungsten light bulb

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What are the alternatives used for UV spectrum?

Deuterium discharge lamp and mercury arc lamp

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What is the use of Entrace slit?

It minimizes the unwanted straylight

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What is Straylight?

It is a wavelength outside the band which can affect the measurement and cause absorbance error

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What is the use of monochromator?

It isolates specific or individual wavelength of light

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Kinds of monochromator

Prisms, Diffraction gratings and Interference Filters

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The principle of prism

A narrow beam of light focused on a prism is refracted as it enters the more dense glass

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Most commonly used monochromator that consists of many parallel grooves

Diffraction gratings

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Principle of constructive interference

Light waves enter one side of the filter and are refracted at the 2nd surface

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What is the use of Exit Slit?

It controls the width of the light beam or band pass

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The narrower the light beam

the greater the resolution

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sample cell/ absorption cell/ analytical cell

cuvet

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kinds of cuvet and some definition

Alumina Silica glass- simple/ older
Quartz/ Plastic- used for UV spectrum
Borosilicate glass- can stand increase temperature
Soft glass- Common glass

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scatter light and should be discarded

scratches

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Produces etching and dissolves glass

Alkaline solutions

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What is a Photodetector?

It converts Light energy to Photoelectric Energy

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Kinds of Photodetectors:

Photocell, Phototube and Photomultiplier tube

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Requires external voltage and it gives off electron when light energy strikes it

Phototube

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Simplest detector and no external voltage

Photocell

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On a plate of iron covered with transparent layer of silver

Selenium

Most common type which can detect low level of light and amplifies radiant energy 200x

Photomultiplier tube

What is Sensitivity and Specificity?

Sensitivity is a term that can measure even the smallest movement of light while Specificity is a term that can only detect a specific light

It detects less amount of light and not as sensitive as photomultiplier tube

Phototransistors and Photodiode

moving needle on a dial or a digital display that indicates the amount of light passing

Galvanometer/ Ammeter

Splits the monochromator light into two components

Double-beam Spectrophotometer

Beam passes through:

1st-sample | 2nd- reference solution or blank

Used to check wavelength accuracy

Didychium or holmium oxide filter

Reagent blank

Blanking technique

light emitted by a single atom burned in flame and measurement of excited atoms (Na, K, Li)

Flame-emission spectrophotometry

Principle of Flame-emission photometry

Excitation of electrons from lower to higher energy state

Serves as a light source and cuvette for FEP?

Flame

Serves as a monochromator in FEP?

Filter

Components of FEP

Nebulizer, Burner and Monochromator system

Deliver a fine spray of sample

Nebulizer

Strikes the photomultiplier tube

Monochromator system

What is the light source used in Atomic absorption spectrophotometry?

Hollow cathode lamp

Light is not excited, unionized and at a ground state?

Atomic absorption spectrophotometry

Unexcited trace metals

Ca++ and Mg++

Converts Ions to atoms

Atomizer (Nebulizer grapite furnace)

Used to modulate light source

Chopper

Principle of Fluorometry

amount of light emitted by a molecule after excitation by electromagnetic radiation and uses 2 monochromator

light source used in fluorometry

mercury arc and xenon lamp

light detector used in fluorometry

Photomultiplier tube

difference between primary filter and secondary filter

The primary filter is the selection of wavelength that is best absorbed by the solution while the secondary filter prevent incident light from striking the photodetector

Advantage of Fluorometry

1000x more sensitive than most spectophotometry

Disadvantage of Fluorometry

Quenching - sudden change in temperature and reduces intensity of fluorescence

Principle of Chemiluminescence

Exciting molecules by chemicals and requires no excitation radiation and monochromator (MOLECULES IN UNEXCITED STATE)

Advantage of Chemiluminescence

Speed (10s) - Picomdar detection | Simple instrumentation

Principle of Turbidimetry

Amount of Light blocked by suspension particles and is dependent on particle size and concentration

Abundant in blood sample

Albumin

Principle of Nephelometry

Light scattered by the small particles is measured at an angle forward to the incident light

the higher the scattered light equals the no. of particle

the increase in concentration

Process of separating by electric current

Electrophoresis

2 types of Electrophoresis

Iontophoresis and Zone Electrophoresis

Migration of small ions

Iontophoresis

Migration of charged macromolecules in porous support

Zone electrophoresis

Protein are negatively charged and they moved toward the _______

anode

The 2 Buffers and their corresponding pH

Barbital (veronal) pH 8.6 | Tris-boric EDTA pH 8.7

Acidic and basic, determine the net change on a protein (electrophoretic mobility)

Electrophoresis

3 supporting media

Cellulose acetate, Agarose gel, Polyacrylamide gel

separates serum proteins into 5 bands (no. of fractions)

Cellulose acetate

Agarose gel has how many bands?

10-15 bands

Polyacrylamide gel has how many bands?

720 bands

Migration is controlled by

net change of the partilly, Particle size and shape, Electric fields, supporting media and Temperature

movement of buffer and solvent relative to their fixed support

Electroendosmosis

separating molecules migrate(PH gradient) and proteins of identical size but with different net changes

Isoelectric Focusing

Separation is performed in narrow-bore fuse silica capillaries

capillary electrophoresis

Separation sample of mixtures between mobile and stationary phase

Chromatography

Modes of separation

Adsorption (Liquid-Solid Chromatography), Partition ( Liquid-Liquid Chromatography), Steric Exclusion, Ion exchange

Based on the competition of adsorptive sites (stationary phase)

Adsorption (Liquid-solid Chromatography)

Separation of solute based on the relative solubility of the compound

Partition

Separation by magnitude and charge of ionic species

Ion exchange

Gel filtration, Gel permeation or molecular chromatography and separates solutes

Steric Exclusion

2 stationary phase

cation- exchange resin | Anion- exchange resin

Side chains of cation

H+ ions

Side chains of Anion exchange

OH-

2 Types of Chromatography

Planar and Columnar

2 types of Planar Chromatography

:Paper and Thin layer Chromatography (drug screening)

Intube or coated column separation

Columnar Chromatography

distribution of solute between a liquid mobile phase and a stationary phase

Liquid Chromatography

used for fraction of drugs, hormones, lipids, carbohydrates and proteins

HPLC (High performance liquid chromatography)

pressure for fast separation of thermos substance

HPLC

used to separate mixture of compounds that are volatile

Gas chromatography