Automation (Clinical Chemistry) Flashcards
References: CC ni Hyacinth Yambao CC ni Rovie Vila (172 cards)
1
Q
4 analytical techniques
A
• Spectrometry
• Luminescence
• Electroanalytic methods
• Chromatography
2
Q
has provided scientists with a means to use both qualitative and quantitative methods of measuring analytes in body fluids. (McPherson & Pincus, 2017)
A
Absorption spectroscopy
3
Q
2 types of Spectroscopy
A
- Photometric measurement
- Spectrophotometric measurement
4
Q
measurement of light intensity without consideration of wavelength
A
Photometric measurement
5
Q
• measurement of light intensity in a narrower wavelength.
• Spectrum of light
A
Spectrophotometric measurement
6
Q
It is transmitted by via electromagnetic waves that are characterized by their frequency and waves
A
Energy
7
Q
It is characterized by waves and frequency
A
Electromagnetic waves
8
Q
is described as photons of energy traveling in waves—Electromagnetic waves.
A
Electromagnetic Radiation
9
Q
It states that the relationship between wavelength and energy.
A
Planck’s law
10
Q
It is the number of vibrations of wave motion per second.
A
Frequency
11
Q
distance between two peaks.
- Lower frequency = Longer wavelength
(Ex. Red)
- Higher frequency = Shorter wavelength (Ex. Violet)
A
Wavelength
12
Q
Visible: 400 – 700 nm =
A
visible spectrum
13
Q
<400 nm =
A
ultraviolet region (UV)
14
Q
> 700 nm =
A
infrared region (IR)
15
Q
Light source for spectrophotometry
A
Light Amplification by Stimulated Emission of Radiation (LASER)
16
Q
Who invented the Beer’s law?
A
Lambert and Beer
17
Q
Describes the relationship between absorption of light by a solution and the concentration of that solution.
A
Beer’s Law
18
Q
Beer’s law state that the concentration of a substance is _____ proportional to the amount of light absorbed
A
directly
19
Q
Beer’s law state that the concentration of a substance is _______ proportional to the logarithm of the transmitted light.
A
inversely
20
Q
It is the light absorbed by the solution
A
Absorbance
21
Q
Ratio of the incident light and light transmitted.
A
Transmittance
22
Q
- Characteristic of a substance to absorb a specific fraction of a specific wavelength
- Varies from one analyte to another
- Constant
A
Molar absorptivity
23
Q
- Length that the light needs to travel through the solution
- Dependent on the cuvet
- Constan
A
Path length
24
Q
used to measure the light transmitted by a solution to determine the concentration of the light-absorbing substance in the solution.
A
Spectrophotometer
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measured through the different components of the spectrophotometer
Radiant energy
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- provides the energy that the sample will modify or attenuate by absorption.
- Provides incident light for the system.
Light source
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2 types of light source in spectrophotometer
- Continuum source
- Line source
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emits radiation that changes in intensity very slowly as a function of wavelength.
Continuum source
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Provides a vast array of wavelength – visible to near IR region
Incandescent tungsten or tungsten iodide lamp
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Alternative for UV spectrum
▪ Mercury arc (+ visible)
▪ Deuterium lamp
▪ Hydrogen lamp
▪ Xenon lamp
31
Spectrum usually used for enzymatic reactions
UV spectrum
32
Alternatives for IR spectrum
▪ Tungsten lamp (+visible)
▪ Nernst/ Merst glower
▪ Globar lamp (Si Carbide)
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emit a limited number of discrete lines or bands of radiation, each of which spans a limited range of wavelengths
Line sources
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• minimizes stray light
• prevents the entrance of scattered light
• where light passes through
Entrance slit
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- refers to any wavelengths outside the band
- from the monochromator system
- absorbance error
Stray light
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Entrance slit that prevents stray light
Nickel sulfate
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It is an anti-stray light
Cutoff filter
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It is 1⁄2 peak transmittance
Bandpass
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Use to isolate an individual wavelength of light
Monochromator
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Degree of isolation of the wavelength is affected by the:
1) monochromator
2) the width of the entrance
3) exit slits
41
Produces a monochromatic light based on the principle of constructive interference of waves
Colored Glass Filters
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when 2 electromagnetic waves meet, synergism will occur
Constructive interference
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transmit multiples of the desired wavelengths, they require accessory filters to eliminate these harmonic wavelengths.
Interference Filter
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- wedge-shaped glass, quartz or sodium chloride.
- separates white (visible) light into a
continuous spectrum
Prisms
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- most commonly used: better resolution than prism
- Parallel grooves or slits into an aluminized surface of a plat piece of a crown glass
- Wavelengths are bent as they pass a
sharp corner
Diffraction Gratings
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the separation of light into component wavelengths, is based on the principle that wavelengths bend as they pass a sharp corner
Diffraction
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- Controls the width of the light beam (band pass)
- Allows only a fraction of the spectrum to reach the sample cuvette.
Exit slit
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- Also known as analytical cell
- Holds the solution of which the absorption is to be measured
Sample cell/cuvet
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2 shapes of Sample cell
Round and rectangular cuvets
50
Why rectangular cuvets are the most common shape used in spectrophotometry?
it’s easier to maintain the length of light
or the measurement of the cuvet.
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Kinds of sample cell used for visible range
Glass cuvette
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Kind of cuvette used for UV range
Quartz or fused silica
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kind of cuvette that is used for 350-2000 nm wavelength
Borosilicate
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Converts transmitted radiant energy into an equivalent amount of electrical energy
Photodetector
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- Barrier layer cell/Photovoltaic cell
- Simplest and least expensive
- Low sensitivity and fatigue are two
distinct disadvantages of these cells.
Photocell
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- anode and cathode enclosed in a glass tube
- It gives off electron when light energy
strikes it.
Phototube
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- more sensitive than vacuum phototubes
but less sensitive than the PMTs
- multitude of wavelength
Phototransistors/photodiode
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- most common type (visible and UV)
- commonly used when radiant power is very low, which is characteristic of very low-analyte concentrations.
- highly sensitive to ultraviolet and visible
radiation
- amplifies radiant energy (200x sensitive)
Photomultiplier Tube (PMT)
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Multiplies the radiant energy
Dynode
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It displays the output of the detection system.
Read-out device
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implies that a photometer is measuring at the wavelength that it is set to.
Wavelength accuracy
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can be assessed easily using special glass-type optical filters.
Photometric accuracy
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performed using glass filters or solutions that have known absorbance values for a specific wavelength.
Absorbance check
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defined as the ability of a photometric system to yield a linear relationship between the radiant power incident upon its detector and the concentration
Linearity
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Used to measure concentration by detecting the absorption of electromagnetic radiation by atoms rather than by molecules
Atomic absorption spectrophotometry (AAS)
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Used to measure concentration by detecting the absorption of electromagnetic radiation by atoms rather than by molecules
Atomic absorption spectrophotometry (AAS)
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Things that are usually measure using AAS
- Al
- Ca
- Cu
- Pb
- Mg
- Li
- Zn
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2 types of light source in AAS
- Electrophoresis discharge lamp
- Hollow-cathode lamp
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Light source that consists of bulb filled with argon and the element to be tested
ELECTRODELESS DISCHARGE LAMP
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Light source consists of an evacuated gas-tight chamber containing an anode, a cylindrical cathode, and an inert gas, such as helium or argon
HOLLOW-CATHODE LAMP
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• Modulates the hollow cathode light beam
• Produces pulses of light
Beam chopper
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Delivers a fine spray of sample containing the metallic ion into the flame/cylinder (atomizer)
Nebulizer
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Sample cell of the instrument
Flame/Graphite furnace
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- Dissociates the solution into its neutral
and individual atoms
- produce an individual atom
out of those that are bound
together
Atomizer
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A fuel gas (acetylene) with an oxidizing
agent (compressed air) is burned to
produce the flame
FLAME ATOMIZER
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- Flameless Atomic Absorption Spectrophotometry
Doesn’t use flame but graphite furnace
ELECTROTHERMAL ATOMIZER –GRAPHITE FURNACE
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It will be subjected to increasing temperature whereby the samples will start to vaporize – bounded atoms will be liberated from whatever molecule it’s found, forming an atomized sample
Graphite cylinder
77
- Isolate desired emission line from other lamp emission lines
- Isolates a particular light from the sample that will be delivered to the photodetector
Monochromator in AAS
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Programmed only to detect the pulsating light coming from the atoms
Photodetector in AAS
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Principle of AAS
Dissociation (unionized, unexcited, ground state)
80
Preferred internal std Potent antidepressant in AAS
Lithium
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Based on an energy exchange process that occurs when certain compounds absorb electromagnetic radiation, become excited, and return to an energy level lower than or equal to their original level
Luminescence
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What are the different types of luminescence?
1) Fluorescence
2) Phosphorescence
3) Chemiluminescence
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What is the principle of Fluorescence?
Photoluminescence
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The emission is basically immediate and therefore generally only visible, if the light source is continuously on
Fluorescence
85
What is the principle of Phosphorescence?
Photoluminescence
86
characterized when materials can store the absorbed light energy for some time and release light later
Phosphorescence
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emission of light is created from a chemical or electro-chemical reaction and not from absorption of electromagnetic energy
Chemiluminescence
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- Measures the amount of light emitted by a molecule after
excitation of electromagnetic radiation
- Aka Molecular Luminescence Spectroscopy
FLUOROMETRY
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Source of the SHORT WAVE and HIGH ENERGY LIGHT
Light source of Fluorometry
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Intensifies the light from the light source
Attenuator
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Selects the wavelength that will best absorbed by the solution to be measured
Monochromator of Fluorometry
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- Contains the sample/solution
- More square-shaped than round
Cuvette of Fluorometry
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Placed on a right angle from the cuvette to avoid incidence light from reaching the detector
Secondary/Emission Monochromator
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- Converts light energy to its equivalent electrical energy
- detects the fluorescence light
Photodetector of Fluorometry
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The most common photodetector in Fluorometry
Photomultiplier tube
96
This phenomenon when the excited state of the molecule loses some of its energy by interaction to other components of the reaction system
QUENCHING PHENOMENON
97
- Process of separating the charged constituents of a sample by
means of an electrical current
- Important instrument for Clinical Chemistry and Molecular
Biology
Electrophoresis
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2 common methods of Electrophoresis
1) Iontophoresis
2) Zone electrophoresis
99
- Migration of small ions
- Used in sweat test, Cystic Fibrosis
Iontophoresis
100
▪ Migration of charged macromolecules in a
porous support medium
▪ DNA, Proteins, lipoproteins
Zone electrophoresis
101
can be used for quantifying the concentrations of the substance that was subjected into the electrophoresis
- device that measures the degree of darkness (optical density) of a photographic or semitransparent material or of a reflecting surface
Densitometer
102
Ions with charge
Analytes
103
Substance that can have a negative, zero or
positive charge depending on conditions
- Positive and negative charge – one at a time
Amphoteric
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molecule containing both acid and base functionality
Ampholyte
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Molecule that has both positive and negative charges at the same time
Zwitterion
106
• Negatively charged
• Migrates to the anode
Anion
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positively charged electrode
anode
108
• Positively charged
• Migrates to the cathode
Cation
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negatively charged electrode
cathode
110
Supplies constant current or voltage in the system
Power supply
111
2 types power supplies used in Electrophoresis
- UPS
- AVR
112
helps the ions to move through the support medium
Driving force
113
Used to provide ions that carry a current and to maintain the pH at a relatively constant value
Buffer
114
pH level of Barbital (veronal)
pH 8.6
114
pH level of Tris-boric EDTA
pH 8.7
115
A network of interacting fibers or a polymer that is solid but traps large amount of solvent in pores or channel inside
Support Medium
116
- Movement of buffer ions and solvent relative to the fixed supports
- Forms an ionic cloud, preventing the particles that you really want to detect from entering into the support medium
Electroendosmosis
117
3 types of support media
1. Cellulose Acetate
2. Agarose Gel
3. Polyacrilamide Gel
118
Support media that separates serum proteins into 5 bands
Cellulose Acetate
119
- Support media that separates serum proteins into 10-15 bands
- Used a purified fraction of agar from the red algae
Agarose Gel
120
- Support media that separates serum proteins into 20 or more
- Have better resolution
Polyacrilamide Gel
121
Wells in the support media where samples are dispensed
Sample
122
Detecting system used in electrophoresis
Electrophoretogram
123
types of detecting system
1) Direct observation
2) Staining
3) Radioactive dye
4) UV visualization
5) Densitometer
124
Detecting system that is useful for diagnosis of wide array of diseases – just look at the medium and you’ll be able to identify the molecules already separated
Direct observation
125
Detecting system that is specific for one chemical group
Staining
126
Detecting system that used Iodine-125 and very sensitive
Radioactive dye
127
It is the simplest detecting system
UV visualization
128
- Detects light that is scattered at various angles
- Scattered light yields a small signal that must be amplified
Nephelometry
129
Instrument used to detects light that is scattered at various angles
NEPHELOMETER
130
Angle used in nephelometer to measure the particles
15-90°
131
act as the monochromator in nephelometer– concentrate the light to
the cuvet
Lens
132
Agle of Linear process of turbidimetry similar to spectrophotometry
180 degrees angle
132
Measures a reduction in light transmission due to particle
formation
TURBIDIMETRY
133
Measurement of the osmolality of an aqueous solution such as
serum, plasma, or urine
OSMOMETRY
134
What are colligative properties in osmolality
1. Osmotic pressure
2. Boiling point
3. Freezing point
4. Vapor pressure
135
most commonly used method in measuring the changes in colligative properties of a solution
Freezing-point depression osmometry
136
An electrochemical transducer capable of responding to one
specific ion
ION SELECTIVE ELECTRODE (ISE)
137
Used to separate complex mixtures on the basis of different
physical interactions between the individual compounds and
the stationary phase of the system
CHROMATOGRAPHY
138
• Solvent or mixture where the sample is added
• Carries the complex mixture (sample)
• Usually solids
Mobile phase
139
Where mobile phase flows
Stationary phase
140
Holds the stationary phase
Column
141
Separated components through chromatography
Eluate
142
2 forms of chromatography
1) Planar
2) column
143
Used for fractionation of sugar (monosaccharide) and amino acid
Paper Chromatography
144
Common of fructose
fruit sugar/grape sugar
145
Common name of maltose
Malt sugar
146
Common name of lactose
Milk sugar
147
-Used for drug screening
Thin layer Chromatography
148
2 analytes in urine used for drug testing in the Philippines
- Tetrahydrocannabinol – metabolite for cannabis
- Methamphetamine – common name: Shabu
149
Gold standard for drug testing especially coupled with GC-MS
Gas Chromatography
150
Gas solid chromatography (GSC)
Differences in absorption at the solid phase surfaces
151
Preparation occurs by differences in solute partitioning between the gaseous mobile phase and the liquid stationary phase
Gas liquid chromatography (GLC)
152
based on the distribution of solutes between liquid mobile
phase and stationary phase
Liquid Chromatography
153
- Used for fractionation of drugs, hormones,
lipids, carbohydrates and proteins
- Commonly used in HbA1c
High performance liquid chromatography
154
mobile phase is more polar than stationary phase
Reverse phase HPLC
155
means the duly recorded, authorized movements, and custody of the seized drugs at each stage, from the moment of confiscation to the receipt in the forensic laboratory for examination until it is presented to the court.
Chain of Custody
156
Fragmentation and ionization
Mass Chromatography
157
long term monitoring of glucose (3-4 months)
HbA1c
158
Preferred test for HbA1c
affinity chromatography
159
Preferred test for HbA1c
affinity chromatography
160
Preferred specimen for HbA1c
Whole blood (EDTA tube)
161
Hemolyzed sample ______ (decreased,increased) HbA1c
decreased
162
Cutoff value of HbA1c
≤6.5%
163
Separates molecules based on differences in their size and shape
Gel/Gel permeation/Gel filtration/Size exclusion/Molecular Sieve
chromatography
164
- Separation of enzymes, antibodies, and proteins
- Examples: Dextran and agarose
Hydrophilic Gel (gel filtration)
165
- Separation of triglyceride and fatty acid
- Example: sephadex
Hydrophobic gel (gel permeation)
166
o Separation of nucleic acids and proteins depends primarily on the sign and ionic charge density
o Separation of amino acids, proteins and nucleic acid
Ion Exchange chromatography
167
o Based on relative solubility in an organic solvent (nonpolar) and an aqueous solvent (polar)
o Separation of therapeutic drugs and their metabolites
Partition Chromatography (liquid-liquid chromatography)
168
o For lipoproteins, CHO and glycated hemoglobins
o Separate and prepare larger quantities of proteins and Ab for study
Affinity Chromatography
169
Based on differences between the adsorption and desorption of
solutes at the surfaces of a solid particle
Adsorption Chromatography
