(P) Lec 5: Analytical Techniques in CC Flashcards by Kchianna Lauren Coronel

The 4 analytical techniques MAINLY used in the laboratory, except:
A. Electrophoresis
B. Osmometry
C. Turbidity
D. Nephlometry
E. NOTA

colorimetry, volumetric, turbidimetry, nephelometry, and electrophoresis

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Transmitted via EM waves characterized by its frequency and wavelength

Energy

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Analytes when placed in a machine are converted into?

energy

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Wavelength

The distance between 2 successive peaks and is expressed in?

nanometer (nm)

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TOF. Some analytes require specific wavelengths for their measurement.

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3 regions where measurements are done

UV, Visible and Infrared region

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Wavelength

less than 400 nm

UV Region

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Wavelength

400 nm to 700 nm; majority of the analytes are measured here

Visible Region

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Wavelength

TOF. Infrared (IR) Region has more than 800 nm.

F (700)

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The Relationship Between Wavelength and Energy is Described by what formula?

E = hv

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No. of vibrations of waves per second created during analysis

Frequency

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TOF. Frequency is DIRECTLY proportional to wavelength and energy.

Frequency is inversely proportional to wavelength and energy

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Frequency

TOF. The lower the wave frequency, the longer the wavelength and energy.

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Frequency

TOF. Wavelength and energy are directly proportional with each other.

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Represent the wavelength in nm at peak transmittance of the analyt

Nominal Wavelength

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Light that completely passed through the sample is called?

Peak transmittance

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TOF. A slight error in adjustment can introduce significant errors in absorbance readings.

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wavelength indicated on the control dial being the actual wavelength of light that has passed through (transmittance) the monochromator

Wavelength Accuracy

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Wv accuracy

wavelength indicated on the control dial being the actual wavelength of light that has passed through (transmittance) the?

monochromator

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Used to check for wavelength accuracy and proper calibration (quality control)

Didymium or Holmium Oxide Filter

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Verifies the absorbance accuracy on linearity
Ensures correct readings on the samples in machines

Neutral Density Filters and Dichromate Solution

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→ the color of the solution has an effect on the reading of the results

Colorimetry

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This instruments measure light intensity without considering the wavelength (simple)

Photoelectric Colorimetry

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this uses the isolation of discreet portions of the spectrum or wavelength for measurement purposes

spectrophotometry or filter photometry

photoelectric Colorimetry

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# Photoelectric Colorimetry Two types of measurement:

1. Spectrophotometric Measurment 2. Photometric measurement

* The measurement of light intensity in a narrower wavelength (narrow range) * May be on the UV, visible, or IR region

Spectrophotometric Measurement

# methods of measurement Measurement of light intensity

Photometric measurement ## Footnote it's also actually applicable to spectro also

TOF. Photoelectric measures the amount of light transmitted by a solution to determine the concentration of the light-absorbing substance in the solution

F (Spectrophotometry)

# Spectrophotometry The light that passes through the sample

Light transmitted

# Spectrophotometry Follows what principle?

principle of Beer’s Law

# Beer’s Law TOF. The concentration of an unknown substance is Directly proportional to the absorbed light (absorbance or optical density.

# Spectrophotometry TOF. The concentration of the unkown substance is directly proportional to the amount of transmitted light (transmittance)

F (indirectly)

TOF. Beer's law mathematically established the relationship between CONCENTRATION and ABSORBANCE.

# Spectro TOF. a sample that is darkly colored/turbid, has higher concentration

# Spectrophotometry: TOF. If you introduce the light on it, the lightt absorbance is also?

increased

TOF. As light absorbance is increased, the transmitted light is low.

T ## Footnote If majority of the light is already absorbed, the amount of light that passes through it will be much lower

TOF. A sample that is lightly colored/clear has a high concentration and high light transmittance.

F (low concentration; high light transmisttance.

→ aka optical density → amount of light absorbed by the solution → proportional to the inverse logarithm of transmittance (reflected light)

Absorabnce A

TOF. Absorbance (A) is proportional to the DIRECT log of transmittance (reflected light)

F (inverse)

# Absorbance (A) mathematically derived from?

%T (Percent transmittance)

# Absorbance FORMULAE (3)

A = abc A = 2 - log%T A = -logT ## Footnote A - absorbance a - molar absorptivity (compound absorptivity under standard conditions) b - length of light through the solution c - concentration of absorbing molecules or solutions

Formula for the concentration of the unknown

Cu = Au/As x Cs

TOF. The absorbance portion is provided by the machine using the abc formula.

T ## Footnote Absorbance standard; ang need lang natin hanapin is Cu, la ka na pake sa iba

TOF. In the laboratory, we receive the concentration of the known derived from the absorbance formula.

F (unknown)

Formula of Percent Transmittance (%T)

%T = It/Io x 100 ## Footnote ratio of the radiant energy transmitted (T) divided by the radiant energy incident in the sample (I) kahit 'wag na kabisa kasi sabi galing lang rin naman sa machine 'to makukuha

# Percent Transmittance (%T) It

transmittance of light through the sample

# Percent Transmittance (%T) Io

transmittance of light striking the sample

TOF. if the sample is blank/clear, light can pass through it completely, assuring a 90% transmittance.

F (100%)

Single Beam A. the simplest type of absorption spectrometer B. designed to make one measurement at a time at one specified wavelength C. absorption maximum or wavelength of the analyte must be known in advance when used D. AOTA | correct where

# Double beam spectrophotometer * An instrument that splits the monochromatic light into 2 components: beam for a sample and a beam for a reference solution * the sample absorbance can be recorded directly as the electrical output of the sample beam

Regular double beam

# Regular Double Beams This beam corrects for the variation in the light source intensity

The second beam (that passes through the reference solution or blank)

→ uses 2 photodetectors for the sample beam and reference beam

Double Beam in Space

→ uses 1 photodetector and alternately passes the monochromatic light through the sample cuvette and then through the reference cuvette using a **chopper or rotating sector mirror**

Double Beam in Time

THE 6 COMPONENTS OF A SINGLE OR DOUBLE BEAM CONFIGURATION SPECTROPHOTOMETER

Stable source of radiant energy (light source) Filter that will isolate a specific region of the EM spectrum Sample holder Radiation detector Signal processor Read-out device

# PARTS OF THE SPECTROPHOTOMETER → provides polychromatic light and must generate sufficient power to measure the analyte of interest → an intense beam of light is directed through the monochromator in the sample

Light/Radiant Energy

# Light/Radiant Energy to give accurate absorbance measurements throughout its absorbance range, the response to a change in light intensity should always be?

linear

# Light/Radiant Energy TOF. If the light changes and the intensity continuously increase, it should decrease after some time.

F (kapag increase, increase lang, same for decreasing)

# Light/Radiant Energy → emits radiation that **changes in intensity (is flexible) **which should be linear → the most widely used in the laboratory

Continuum Source

most commonly used light source in the visible and near IR region

Tungsten Light Bulb

# Continuum Source → routinely used to provide UV radiation in analytic spectrometers

Deuterium Lamp

# Continuum Source → produces a continuous source of radiation which covers both the UV and visible range

Xenon Discharge Lamp

# ALTERNATIVES FOR TUNGSTEN Visible and UV

Mercury Arc

# ALTERNATIVES FOR TUNGSTEN UV

Deuterium Lamp (165 nm) Hydrogen Lamp Xenon Lamp

# ALTERNATIVES FOR TUNGSTEN IR

Merst Glower Globar (Silicone Carbide)

# Line Source An example used to measure UV and visible regions

Mercury Sodium Vapor Lamps

# Line source light source used for AAS

Hollow Cathode Lamps

FACTORS TO CONSIDER WHEN CHOOSING A LIGHT SOURCE

* Range * Spectral distribution within the range * Source of radiant production * Stability of the radiant energy * Temperature

# FACTORS TO CONSIDER WHEN CHOOSING A LIGHT SOURCE: - if it can measure the UV, visible, or IR region

range

# FACTORS TO CONSIDER WHEN CHOOSING A LIGHT SOURCE: For proper storage

Temperature

# FACTORS TO CONSIDER WHEN CHOOSING A LIGHT SOURCE: if it’s a continuum or line source

Source of radiant production

→ minimizes unwanted or stray light; prevents entrance of scattered light into the monochromator system

Entrance slit

# Stray Light TOF. Does not originate from the polychromatic light source.

# Stray Light TOF. Any wavelength INSIDE the band transmitted by the monochromator.

F (outside)

# Stray Light TOF. The most common cause of loss of linearity at a high-analyte concentration.

# Stray light TOF: It LIMITS the maximum absorbance that a spectrophotometer can achieve

this isolates specific or individual wavelengths of light; each one has their own band pass

Monochromator

* The total range of wavelengths transmitted * Defines the range of wavelengths transmitted and is calculated as width at more than half the maximum transmittance

Band Pass

# TYPES OF MONOCHROMATORS → simple, least expensive, not precise, but useful

Filters

# TYPES OF MONOCHROMATORS → usually passes a relatively wide band of radiant energy and has a low transmittance of the selected wavelength

Filters

# TYPES OF MONOCHROMATORS → made by placing semi-transparent silver films on both sides of a dielectric (magnesium fluoride)

Filters

→ produces monochromatic light based on the principle of constructive interference of waves wherein light waves enter one side of the filter and are reflected at the second surface

Interference Filters

# TYPES OF MONOCHROMATORS → wedge-shaped pieces of glass, quartz, or sodium chloride → a narrow beam focused on a prism is refracted as it enters the more dense glass (advantage over filters) → the prism can be rotated, allowing only the desired wavelength to pass through an exit slit

Prisms

refracted more than long wavelengths, resulting in a dispersion of white light into a continuous spectrum

short wavelengths

# TYPES OF MONOCHROMATORS → most commonly used; gives a better resolution compared to prisms → consists of many parallel/cutting grooves (15k or 30k per inch) or slits into an aluminized surface of a flat piece of crown glass etched onto a polished surface

Diffraction Gratings

separation of light into component wavelengths based on the principle that wavelengths bend as they pass a sharp corner

Diffraction

# TYPES OF MONOCHROMATORS → much more expensive than diffraction gratings

Holographic Gratings

→ controls the width of the light beam (bandpass) → allows only a narrow fraction of the spectrum to reach the sample cuvette (becomes more select on the fraction that is needed) → accurate absorbance measurement requires a bandpass of less than ⅕ of the natural bandpass of the spectrophotometer

Exit Slit

# Exit slit → accurate absorbance measurement requires a bandpass of less than _____ of the natural bandpass of the spectrophotometer

⅕

# Exit slit TOF. spectral purity is reflected by the bandpass wherein: the NARROWER the bandpass, the greater the resolution

→ aka absorption, analytical, or sample cell → it holds the solution whose concentration is to be measured later on

Cuvette/Cuvet

# Cuvette → most commonly used in 350-2,000 nm (wide range)

Alumina Silica Glass

# Cuvette → for measuring solutions requiring visible and UV spectra

Quartz/Plastic

Types of Cuvette

* Alumina Silica Glass * Quartz/Plastic * Borosilicate Glass * Soft Glass

TOF. Cuvettes with scratches on their optical surface produce scattered light (should be discarded).

Silica cuvettes can transmit light effectively at a wavelength of?

more than 220 nm

TOF. Alkaline solutions can be left in cuvettes for prolonged periods.

F ## Footnote it can damage the cuvette along with the readings

The path of length of the cuvette is

1 cm

TOF. Much LONGER path lengths are used in automated systems

TOF. To increase sensitivity, some are designed to have a path length of 20cm.

10cm

→ detects and converts transmitted light into photoelectric energy → detects the amount of light that passes through the sample in the cuvette

Photodetector

Kinds of photodetector:

* Photocell/Barrier Cell/Photovoltaic Cell * Phototube * Photomultiplier Tube (PMT) * Photodiode

# Photodetector → the simplest and least expensive → is temperature sensitive (disadvantage) → used in filter photometers with a wide bandpass → a basic phototransducer used for detecting and measuring radiation in the visible region → composed of selenium on a plate of iron covered with a transparent layer of silver → advantage: it requires no external voltage source but utilizes internal electron transfer for the production of a low internal resistance current (advantage)

Photocell/Barrier Cell/Photovoltaic Cell

# Photodetector → contains a cathode and anode enclosed in a glass case (positive and negative poles) → has a photosensitive material that gives off electrons when light energy strikes it which is converted into energy → requires an external voltage for operation

Phototube

# Photodetector → most commonly used and most sensitive; has a rapid response and can detect very low levels of light (highly concentrated solution) → measures both the visible and UV region → the response begins when incoming photons strike a photocathode → prone to breakage when exposed to room light (disadvantage) → are limited to measuring low power radiation because intense light can cause irreversible damage to the surface (another disadvantage)

Photomultiplier Tube (PMT)

→ not as sensitive as the PMT but has excellent linearity → measures light at a multitude of wavelengths (lesser amounts of light) → has a lower dynamic range and higher noise compared to PMT → most useful as a simultaneous multi channel detector

Photodiode

→ displays the amount of the detection system → examples: galvanometers, ammeters, and LED displays

Meter or Read-Out Device

# Color absorbed and complementary color 350-430

Violet, Yellow-Blue

# Color absorbed and complementary color 431-475

Blue, Yellow

# Color absorbed and complementary color 476-495

green-blue, orange

# Color absorbed and complementary color 496-505

blue green, red

# Color absorbed and complementary color 506-555

Green, purple

# Color absorbed and complementary color 556-575

yellow-green, violet

# Color absorbed and complementary color 576-600

yellow, blue

# Color absorbed and complementary color 601-650

orange, green-blue

# Color absorbed and complementary color 651-700

red, blue-green

# Read-Out Device color detected by our naked eye

Color absorbed

# Read-Out Device how the machine reads the sample

Complementary color

Amount of light absorbed at a particular wavelength depends on?

molecular and ion type present ## Footnote This amount may also vary with concentration, pH, and temperature

TOF. The light path must be kept constant to have an absorbance proportional to the concentration.

A change in instrument or chemical reactions, will cause a deviation in?

Beer's law

commonly a result of a finite bandpass of the filter or monochromator

Instrument deviations

Turbidity readings on spectrophotometers are greater in the?

blue region

TOF. The linearity of the spectrophotometer is determined using **UV filters or solutions **that have known absorbance values for a given wavelength

F (optical filters or solution)

a blank solution contains serum WITHOUT a reagent to complete the assay

BLANKING TECHNIQUE

Which is false about BLANKING TECHNIQUE A. Effective for high turbidity serum B. Helps to check in absorbance C. Correct for artifactual absorbance readings or dual-wavelength methods may be used

# Blanking technique what is necessary to clear the serum of plasma and chylomicrons

ultracentrifugation

# Blanking Technique Scattering light (turbidity) is increased because of this interference

Lipids

measures light emitted by a single atom burned in a flame

Flame Emission Photometry (FEP) or Spectrophotometry

# Flame Emission Photometry (FEP) or Spectrophotometry Which is false: A. measurement of excited ions (e.g. electrolytes such as Na, K, Cl, Ca, and Mg) B. excitation of electrons from their higher state to lower energy state C. corrects variations in flame and atomizer characteristics D. requires an indirect internal standard method

# FEP this requires an indirect internal standard method

Lithium/cesium

Migration of charged particles in an electric field

Electrophoresis

# Atomic Absorption Spectrophotometry (AAS) measures light absorbed by atoms dissociated by?

heat

# AAS TOF. the element is not excited but dissociated from its chemical bond and placed in an unionized, unexcited, and ground state.

the light source ni AAS

hollow-cathode lamp

# Atomic Absorption Spectrophotometry (AAS) Which is false: A. measure of unexcited ions B. less sensitive, accurate, and specific compared to FEP C. internal standard is not needed

# ADDITIONAL PARTS OF THE AAS this converts ions into atoms

Atomizer/Nebulizer/Graphite Furnace

# ADDITIONAL PARTS OF THE AAS → modulates and controls the light source

Chopper

# ADDITIONAL PARTS OF THE AAS → added to samples to form stable complexes with phosphate → this reduces interferences because of the chemical bond created with phosphate

Anthanum/Strontium Chloride

# VOLUMETRIC (TITRIMETRIC) A. It determines the amount of scattered light by a particulate matter suspended in a turbid solution B. It determines the amount of light blocked (how much light is reduced) by a particulate matter in a turbid solution C. The unknown sample is made to react with a known solution is the presence of an indicator

The volumetric is mainly used for electrolyte measurement of?

chloride and calcium

# Turbidmetry A. It determines the amount of scattered light by a particulate matter suspended in a turbid solution B. It determines the amount of light blocked (how much light is reduced) by a particulate matter C. The unknown sample is made to react with a known solution is the presence of an indicator

# Turbidmetry TOF. Measurement of abundant small particles (proteins) and bacterial suspensions.

F LARGE PARYICLES

# Turbidimetry TOF. Is not independent on the specimen concentration and particle size.

# Turbidimetry applications include, except: A. Protein measurement B. Detects bacterial growth in broth cultures C. Antimicrobial test D. Detects clot formations E. A, B C F. A, D G. ABCD

All are correct

# NEPHELOMETRY A. It determines the amount of scattered light by a particulate matter suspended in a turbid solution B. It determines the amount of light blocked (how much light is reduced) by a particulate matter C. The unknown sample is made to react with a known solution is the presence of an indicator

# NEPHELOMETRY TOF. Light scattering is dependent on the specimen concentration and particle size.

F (that's for turbidimetry, nephelometry is dependent on the wavelength and particle size)

# NEPHELOMETRY WHICH IS MALI: A. Light is scattered forward B. for measuring the amount of antigen-antibody complexes C. the wavelengths used are 320-650 nm D. detector (PMT) output is inversely proportional to the concentration

THE 6 COMPONENTS OF A NEPHELOMETER

Light source Collimator Monochromator Sample cuvette Stray light trap Photodetector

# COMPONENTS OF A NEPHELOMETER narrows the beam of particles with waves (sample) for easy measurement; direction of scattered light becomes more aligned

Collimator

(P) Lec 5: Analytical Techniques in CC Flashcards

(152 cards)