Instrumentation and Analytical Principles
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What does Polarography employ?
Employs an electrochemical cell
What type of instrumentation gradually increases the voltage applied between two electrodes of the cell in contact with a solution containing the analyte?
How is the current measured in Polarography?
Voltage change versus current plotted to produce a polargram
What is true about the amount in the current and the concentration of the analyte in Polargraphy?
Amount of increase in current (i.e. the wave height) proportional to the concentration of analyte
What type of voltammetry is based on polargraphy?
Anodic Stripping Voltammetry
How does Anodic stripping voltammetry work?
- Negative potential applied to one of the electrodes
- Trace metal ions in the solution reduced and plated onto anodic electrode - preconcentrating step
- Plating electrode used as anode in polargraphic cell - metal stripped off anode
- Current flow during stripping provides polarogram that identifies and quantifies the analyte being measured (trace metals)
What is used to assay heavy metals such as lead in blood?
What is an instrument that uses the principle of charged particles moving through a magnetic or electric field, with ions being separated from other charged particles according to their mass-to-charge ratios?
How is a sample separated by their mass-to-charge ratios in Mass Spectrometry?
Electrons bombard a sample, ionizing the compound into fragment ions, which are separated by their mass-to-charge ratios
How is the identification and quantification of a sample produced by Mass Spectrometry?
- The mass spectrum produced is unique for a compound (identification)
- The number of ions produced relates proportionally to concentration (quantification)
What is a high-quality technique for identifying drugs or drug metabolites, amino acid composition of proteins, and steroids?
What also has applications in the field of proteomics?
How does the Mass Spectrometry work?
- Eluate gas from a gas chromatograph may be introduced into a mass spectrometer that functions as the detector system OR
- Liquid eluate may be introduced from a high-performance liquid chromatograph
Name the components of a Mass Spectrometer
- Ion source
- Vacuum system
What happens at the Ion Source in Mass Spectrometry?
- Samples enter the ion source and are bombarded by the ionization beam
- When the sample is in gas form and introduced from a gas chromatograph, the ion source may be electron or chemical ionization
What component of the Mass Spectrometry bombards the sample by the ionization beam?
What component in Mass Spectrometry prevents collision of ions with other molecules when electronic or magnetic separation is occurring?
What are the types of Analyzer in Mass Spectrometry?
Which type of analyzer is a destructive process, where ions pass through the analyzer one time and then strike the detector?
What is a beam-type analyzer, where mass-to-charge ratios are scanned during a prescribed time period to form a mass spectrum?
What component of a Mass Spectrometry usually detects ions using electron multipliers, such as discrete dynode and continuous dynode electron multipliers?
What component of the Mass Spectrometry converts the detector's signal to a digital form?
Computer and software
To further improve selectivity and sensitivity, what system can be used?
Tandem mass spectrometers
What type of spectrometer is used where a gas chromatograph or high-performance liquid chromatograph is connected to two mass spectrometers (GC/MS/MS) OR (HPLC/MS/MS)?
Tandem Mass Spectrometers
In Tandem Mass Spectrometers what happens?
Ions of a specific mass-to-charge ratio are allowed to continue to the second mass spectrometer, where additional fragmentation occurs and final analysis is done
Name the components of High-Performance liquid chromatograph (HPLC)
- Solvent reservoir(s)
- One or more pumps to propel the solvent(s)
- Chromatographic column
- Computer to process data and control the operation of the system
What is a type of liquid chromatography where the mobile phase is a liquid that is passed over the stationary phase of the column?
HPLC (High-performance liquid chromatography)
What is the separation of the solutes in a sample governed by in HPLC?
Governed by the selective distribution of the solutes between the mobile and stationary phases
What are commonly used for the mobile phase of HPLC?
Name the solvents commonly used for the mobile phase of HPLC
What type of elution in HPLC happens when the strength of solvent remains constant during separation?
What type of elution in HPLC happen when the strength of solvent continually increases (%/min) during separation?
Name the types of elutions in the mobile phase of HPLC
- Isocratic elution
- Gradient elution
What phase in HPLC is an organic material covalently bonded to silica that may be polar or nonpolar in composition?
What are the types of phases in the Stationary phase of HPLC?
- Normal phase
- Reversed phase
What phase of HPLC occurs when polar stationary phase and nonpolar mobile phase?
Normal phase liquid chromatography
What phase of HPCL occurs when Nonpolar stationary phase and polar mobile phase?
Reversed phase liquid chromatography
What type of delivery system in HPLC uses a solvent reservoir from which the pump can push the mobile phase through the column?
What component of HPLC produces a signal for ID and quantification of the solutes?
What are the commonly used detectors in HPLC?
- Photodiode array
- Mass Spectrometer
How is the concentration of a light-absorbing analyte in solution determined?
- A spectrophotmeter measures light transmitted by that analyte in solution
- Such an analyte may absorb, transmit, and reflect light to varying degrees, but always of a characteristic nature for the analyte
Name the components of a spectrophotometer
- Power supply
- Light source
- Entrance slit
- Exit slit
- Cuvet/sample cell
- Readout device
What component of a spectrophotometer produces an intense, reproducible, constant beam of light?
Light source or exciter lamp
Name the types of incandescent lamps
What is the Tungsten lamp used for?
Most common, used in visible and infrared regions
What is the Deuterium lamp used for?
Used in the ultraviolet region
When a lamp is changed in a spectrophotometer, what must happen and why?
- The instrument must be recalibrated
- Changing the light source changes the angle of the light striking the monochromator
What type of monochromators are used in photometers?
- Glass filters
- Interference filters
What type of monochromators are used in spectrophotometers?
- Diffraction gratings
What is the range of wavelengths in nanometers that is transmitted by the monochromator and exit slit between two points of a spectral scan where light transmitted is one-half of the peak (maximum) transmittance?
Bandpass or spectral bandwidth
What allows lamp light to enter, and is fixed in position and size?
What disperses the light into wavelengths?
What selects the bandpass of the monochromator that allows light of the selected wavelength to pass throught the cuvet onto the detector?
What part of the spectrophotometer converts the elctromagnetic radiation (light energy) transmitted by a solution into an electrical signal that is measured?
With photodetectors, what is true about the more light transmitted?
The more light transmitted, the more energy, and the greater the electrical signal that is measured
What part of the spectrophotometer is when the electrical energy from a detector is displayed on some type of digital display or readout system (which may be a chart recorder or computer printout)?
What kind of properties does Electromagnetic radiation have?
Wave-like and particle-like properties
Describe radiant energy
Characterized as a spectrum from short wavelength to long wavelength - cosmic, gammy rays, X-rays, ultraviolet, visible, infrared, microwaves, radiowaves
The distance traveled by one complete wave cycle (distance between two successive crests) measured in nanometers (nm)
If the wavelength is short, then what does that also mean?
The shorter the wavelength, the greater the energy contained in the light, and the greater the number of photons
How is light classified?
Light is classified according to its wavelength
What light classification has very short wavelengths?
Ultraviolet (UV) light
What light classification has very long wavelengths?
Infrared (IR) light
What has to happen for white light to occur?
All visible wavelengths of light (400-700 nm) are combined
What color shows when a wavelength of light transmitted (not absorbed) by an object?
What are particles of light called?
When an atom absorbs a photon, what happens to the atom?
The atom becomes excited
How do atoms become excited when an atom absorbs a photon?
- Occurs 3 ways:
- Electron is moved to a higher energy level
- Mode of the covalent bond vibration is changed
- Rotation around its covalent bonds is changed
What happens when energy is absorbed as a photon, an electron is moved to a higher energy level where it is unstable?
- An excited electron is not stable and will return to ground state
- An electron will emit energy in the form of light (radiant energy) of a characteristic wavelength
- Absorption or emission of energy forms a line spectrum that is characteristic of a molecule and can help identify a molecule
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