CLINICAL CHEM EXAM NA ATA ITO (LEC)

Centrifugation

Consist of head/rotor (attached to the shaft of the motor), carrier and shields.

Revolutions per minute and Relative Centrifugal force

The speed/centrifugal force is expresses by

Balanced, Vibration

Centrifuged must be properly ———- and free from excess ———-

Filtration

Paper, cellulose, polyester fibers and column materials

Spectrometry

I - Instruments that measure light energy

II - Based on the property of colored solutions to absorb light of specific wavelength.

Light

is a form of electromagnetic energy

̶ Transmitted via electromagnetic waves

̶ Waves is measured in nanometer (wavelength).

Monochromator

Slit

Source

Slit

Cuvette

Photocell

Beer’s Law

The concentration of a substance is: Directly proportional to the amount of light absorbed, Inversely proportional to the amt. of transmitted light

2 – Log %T

A=

Spectrophotometry

Measures the light transmitted by a solution to determine the concentration of the substance in the solution

Light Source

? → Entrance Slit → Monochromator → Exit Slit → Sample cell → Photodetector

Entrance Slit

Light Source → ? → Monochromator → Exit Slit → Sample cell → Photodetector

Monochromator

Light Source → Entrance Slit → ? → Exit Slit → Sample cell → Photodetector

Exit Slit

Light Source → Entrance Slit → Monochromator → ?→ Sample cell → Photodetector

Sample cell

Light Source → Entrance Slit → Monochromator → Exit Slit → ? → Photodetector

Photodetector

Light Source → Entrance Slit → Monochromator → Exit Slit → Sample cell → ?

Light Source

Provide incident light for the system

Incandescent Tungsten or Tungsten iodide lamp

For visible and near infrared spectrum

Deuterium-discharge lamp and Mercury arc lamp

For UV spectrum

Silicone carbide

For infrared spectrum

Monochromator

Isolates specific wavelength from the light source

Interference Filter

Based on constructive interference of waves

Prism

Separates white light into a continuous spectrum.

Diffraction grating

Separates light into component wavelengths

Entrance Slit

Exclude unwanted or “stray light

Exit Slit

Controls the width of the light beam

Sample Cell

Cuvette or analytical cell

Holds the solution of which the absorption is to be measured

Glass cuvette

for visible range

Quartz or fused silica

for UV range

Photodetector

Converts transmitted radiant energy into an equivalent amount of electrical energy.

Photocell (Barrier layer cell, selenide cell)

Generates electromotive force (no external voltage) Output is not amplified

Phototube

Similar to photocell but requires external voltage

Photomultiplier tube

Amplifies radiant energy (200x sensitive)

Phototransistors and Photo iodide

Uses a photosensitive positive-negative junction diode to produce a photocurrent.

Read-out Device

A moving needle on a dial or a digital display which indicates the amount of light passing through a sample.

Single- beam

Light Source

Monochromator

Slit

Sample

Detector

Amp

Recorder

Double Beam

Prism

Slits

Light Source

Beam Splitter

Mirror

Reference Cell

Ratio

Detector

Sample Cell

Flame Emission

Measures light emitted by excited atoms

̶ Measure sodium and potassium because they are easy to excite.

Principle:

A. Flame using propane is used to excite the atoms (higher energy state)

B. Excited atoms return to the ground state by emitting light energy.

Nebulizer (atomizer)

o Deliver a fine spray of sample containing the metallic ion to the burner.

Burner

A fuel gas (propane) with an oxidizing agent (compressed air) burned to produce the flame

Monochromator system

Allow only emitted line spectrum of specific element to strike the PMT.

Atomic Absorption

i. Measures light absorbed by ground state atoms

ii. Used to measure concentration of calcium atom

iii. 100 times more sensitive than FES

Light Source

Provide incident light for the system

Hallow cathode lamp

Evacuated gas tight chamber filled with inert gas

Electrodeless discharge lamp

Bulb filled with argon and the analyte Radiofrequency generator excites the element

Beam Chopper

Modulates the hollow cathode light beam.

Nebulizer

Deliver a fine spray of sample

Burner

▪ Long narrow slit (permits absorption of incident light)

▪ A fuel gas (acetylene) is burned to produce the flame.

Light Source

Components of AAS

1. ?

2. Beam Chopper

3. Nebulizer

4. Burner

5. Monochromator

6. Photodetector

Beam Chopper

Components of AAS

1. Light Source

2. ?

3. Nebulizer

4. Burner

5. Monochromator

6. Photodetector

Nebulizer

Components of AAS

1. Light Source

2. Beam Chopper

3. ?

4. Burner

5. Monochromator

6. Photodetector

Burner

Components of AAS

1. Light Source

2. Beam Chopper

3. Nebulizer

4. ?

5. Monochromator

6. Photodetector

Monochromator

Components of AAS

1. Light Source

2. Beam Chopper

3. Nebulizer

4. Burner

5. ?

6. Photodetector

Photodetector

Components of AAS

1. Light Source

2. Beam Chopper

3. Nebulizer

4. Burner

5. Monochromator

6. ?

Nebulizer (atomizer)

Components of FES

1. ?

2. Burner

3. Monochromator system

4. Photosensitive detector (photomultiplier tube)

Burner

Components of FES

1. Nebulizer (atomizer)

2. ?

3. Monochromator system

4. Photosensitive detector (photomultiplier tube)

Monochromator system

Components of FES

1. Nebulizer (atomizer)

2. Burner

3. ?

4. Photosensitive detector (photomultiplier tube)

Photosensitive detector (photomultiplier tube)

Components of FES

1. Nebulizer (atomizer)

2. Burner

3. Monochromator system

4. ?

Electrophoresis

̶ The process of separating the charged constituents of a sample by means of an electrical current.

Iontophoresis

o Migration of small ions

Zone electrophoresis

o Migration of charged macromolecules in a porous support

Driving force (power supply)

Electrophoresis Components:

i. ?

ii. Buffer

ii. Support medium

iv. Sample

v. Detecting System

Buffer

Electrophoresis Components:

i. Driving force (power supply)

ii. ?

ii. Support medium

iv. Sample

v. Detecting System

Support medium

Electrophoresis Components:

i. Driving force (power supply)

ii. Buffer

ii. ?

iv. Sample

v. Detecting System

Sample

Electrophoresis Components:

i. Driving force (power supply)

ii. Buffer

ii. Support medium

iv. ?

v. Detecting System

Detecting System

Electrophoresis Components:

i. Driving force (power supply)

ii. Buffer

ii. Support medium

iv. Sample

v. ?

Cellulose acetate

Separates serum proteins into 5 bands

Agarose Gel

10 -15 bands

Polyacrylamide gel

>20 bands

Electrophoretogram

̶ Result of electrophoresis consisting of separated strands of a macromolecule

Electroendosmosis

Movement of buffer and solvent relative to their fixed support.

Isoelectric focusing

Movement of buffer and solvent relative to their fixed support

Capillary electrophoresis

Separation is performed in narrow-bore fuse silica capillaries

Chromatography

Separate complex mixtures between mobile and stationary phase

Mobile Phase

carries the complex mixture

Stationary phase

through which mobile phase flows

Column

holds the stationary phase

Eluate

separated components

Body fluids

Blood, CSF, urine, sweat, gastric juices, etc.