Instrumentation and Analytical Principles
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Name the components of Gas-liquid chromatography
- Carrier gas with flow-control to regulate gas flow
- Heated injector
- Chromatographic column to separate solutes
- Heated column oven
- Computer to process data and control the operation system
What is a technique used to separate volatile solutes?
Gas-liquid chromatography (GLC)
Describe what the Heated injector of GLC (Gas-Liquid chromatography) does
Sample is injected into the injector component of the instrument where the sample is vaporized because the injector is maintained approximately 50 degrees celcius higher than the column temp
Describe the Carrier gas flow of GLC (Gas-Liquid chromatography)
An inert carrier gas (mobile phase) carries the vaporized sample into the column
What are the commonly used carrier gases in the Mobile phase of GLC (Gas-Liquid Chromatography)?
What is critical to maintaining column efficiency and reproducibility of elution times with GLC (Gas-Liquid Chromatography)?
Carrier gas flow rate
How are the types of columns (stationary phase) used in GLC (Gas-Liquid Chromatography) designated?
Designated as packed or capillary
What produces a signal for identification and quantification of the solutes in GLC?
Name commonly used detectors for GLC
- Flame ionization
- Thermal conductivity
- Electron capture
- Mass spectrometer
What is true about the separation of solutes in GLC?
- The more volatile a solute, the faster it will elute from the column
- The less interaction of the solute with the column, the faster it will elute
What is the identification and quantification of a solute based on with GLC?
- Identification is based on retention time
- Quantification is based on peak size
What is a technique where solutes in a sample are separated for identification based on physical differences that allow their differential distribution between a mobile phase and a stationary phase?
Describe what the Mobile phase of Chromatography involves
May be inert gas or a liquid
Describe what the Secondary phase of Chromatography involves
- May be silica gel bound to the surface of a glass plate or plastic sheet
- May be silica or a polymer that is coated or bonded within a column
What type of chromatography is TLC (Thin-Layer Chromatography)?
Describe the Stationary phase of TLC (Thin-Layer Chromatography)
May be silica gel that is coated onto a solid surface such as a glass plate or sheet
Describe the Mobile phase of TLC (Thin-Layer Chromatography)
A solvent, where solvent polarity should be just enough to achieve clear separation of the solutes in the sample
What technique used clinically for Urine Drug Screens?
TLC (Thin-Layer Chromatography)
What action moves the TLC (Thin-Layer Chromatography) from the mobile phase through the stationary phase?
Absorption and Capillary action
What two phases of TLC (Thin-Layer Chromatography) work together to provide solute resolution and separation?
Mobile phase and Stationary phase
How do the Mobile phase and Stationary phase of TLC (Thin-Layer Chromatography) work together to provide solute resolution and separation?
- Solute will stay with the solvent front if solvent is too polar for the solute
- Solute will remain at origin if solvent is insufficiently polar
What values are affected by chamber saturation, temp, humidity, and composition of the solvent in TLC (Thin-Layer Chromatography)?
Name the types of luminescence where excitation requires absorption of radiant energy
What type of luminescence is the a process where atoms absorb energy at a particular wavelength (excitation), electrons are raised to higher energy orbitals, and the electrons release energy as they return to ground state by emitting light energy of a longer wavelength and lower energy than the exciting wavelength
What type of light is frequently used for the excitation and is passed through a primary filter for proper wavelength selection for the analyte being measured (when dealing with Fluorescence)?
What are the advantages of using fluorometry?
- 1000 times more sensitive than absorption techniques
- Increased specificity because optimal wavelengths are chosen both for absorption (excitation) and for monitoring emitted fluorescence
What are the limitations of using fluorometry?
- Changes from the established protocol that affect pH, temp, and solvent quality
What type of luminescence is the emission of light produced by certain substances after they absorb energy?
What type of luminescence is similar to fluorescence except that the time delay is longer (greater than 10-4 sec) between absorption of radiant energy and release of energy as photons of light?
Name the types of luminescence where excitation does NOT require absorption of radiant energy
What type of luminescence is the process where the chemical energy of a reaction produces excited atoms, and upon electron return to ground state, photons of light are emitted?
What type of luminescence is the process where and enzyme-catalyzed chemical reaction produces light emission?
What is a generic term for the type of instrument that is used to measure chemiluminescence and bioluminescence?
What is the measurement of light scattered by a particulate solution?
Generally, how is scattered light measured?
- Measured at an angle to the incident light when small particles are involved
- For large molecules, forward light scatter can be measured
For nephelometry, what is true about the amount of light scatter?
The amount of light scatter is directly proportional to the number and size of particles present in the solution
What is dependent upon the sensitivity of the nephelometry?
Depends on the absence of background scatter from scratched cuvets and particulate matter in reagents
What measures light blocked as a decrease in the light transmitted through the solution and is dependent on particle size and concentration?
What is used for measurement for turbidimetry?
Turbidimetry uses a spectrophotometer for measurement
What is limited in turbidimetry?
Limited by the photometric accuracy and sensitivity of the instrument
What is the principle of Atomic Absorption Spectrophotometry?
Ground-state atoms absorb light at defined wavelengths
What refers to the wavelengths at which an atom absorbs light?
What is true about metals and line spectrum?
Each metal exhibits a specific line spectrum
How does Atomic Absorption Spectrophotmetry work?
- Sample is atomized in a flame where the atoms of the metal to be quantified are maintained at ground state
- A beam of light from a hollow-cathode lamp (HCL) is passed through a chopper to the flame
- The ground-state atoms in the flame absorb the same wavelengths of light when excited
- The light NOT absorbed by the atoms is measured as a decrease in light intensity by the detector
- The difference in the amount of light leaving the HCL and the amound of light measured by the detector is indirectly proportional to the concentration of the metal analyte in the sample
Name the components - in order - of an Atomic Absorption Spectrophotmetry
- Hollow-cathode lamp
- Burner head for flame
- Readout device
What does a Hollow-cathode lamp contain?
- Anode - a cylindrical cathode made of metal being analyzed
- Inert gas - such as helium or argon
What is the principle of the Hollow-cathode lamp?
- Applied voltage causes ionization of the gas, and these excited ions are attracted to the cathode, where they collide with the metal coating on the cathode, knocking off atoms and causing atomic electrons to become excited
- When the electrons of the metal atoms from the cathode return to ground state, the characteristic light energy of that metal is emitted
How does the Hollow-cathode lamp work?
- Vaporized metal atoms from the sample can be found in the flame
- The flame serves as a cuvet in this instrument
- The light produced in the HCL passes through a chopper and then to the flame, and the light is absorbed by the metal in the sample
- The light NOT absorbed will be read by the photomultiplier tube
How does the flameless system of the Hollow-cathode lamp work?
- A carbon rod (graphite furnace), tantalum, or platinum to hold the sample in a chamber
- The temp is raised to vaporize the sample being analyzed
- The atomized sample then absorbs the light energy from the HCL
- More sensitive than the flame method
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