Chapter 6: Separation Methods Flashcards
What is normal phase chromatography? What types of substances are best separated this way?
In normal phase chromatography, the mobile phase is less polar than the stationary phase. The nonpolar components of the analyte will move more quickly through the column, but the polar component will move more slowly as it is attracted to the polar stationary phase.
Hydrophilic and ionic compounds are best separated through normal phase chromatography.
What does the term “stationary phase” mean? What type of stationary phases are used in gas chromatography?
The stationary phase is a finely divided solid material or viscous liquid contained within a long column. In gas chromatography, the stationary phase is a thick, high boiling, viscous liquid that has the consistency of molasses.
What type of substance is the mobile phase in gas chromatography?
In gas chromatography, the mobile phase is an inert gas. The most commonly used gas is helium. The best gas to use is hydrogen, but it is expensive and flammable. Nitrogen is also used, but gives inferior results.
List two advantages of HPLC over GC.
The stationary phase is always more polar than the mobile phase in GC. In HPLC, stationary phases and mobile phases can be designed so that the stationary phase is less polar than the mobile phase. This is helpful in separating nonpolar substances that would not separate well using a polar stationary phase.
Another advantage is that HPLC allows the composition of the mobile phase to be altered during the run. This is called gradient chromatography.
Explain how a flame ionization detector works. What other types of detectors are used in gas chromatography?
A flame ionization detector produces a small flame from the reaction of hydrogen (tank) and oxygen (air). When the analyte reaches the flame, it loses an electron and becomes ionized. The ions create an electric current that is amplified and sent to the computer.
Other detectors are a mass spectrometer detector, nitrogen-phosphorus detector, thermal conductivity detector, and electron capture detector.
What is reverse phase liquid chromatography? What types of substances are best separated using this technique?
In reverse phase liquid chromatography, the mobile phase is more polar than the stationary phase. It can be used in liquid chromatography and thin-layer chromatography. Reverse phase may be preferred if you want to use a polar solvent, and thus the mobile phase can be polar and the stationary phase can be nonpolar.
Polar substances are best separated using this technique.
In HPLC, why would one want to use two pumps for the mobile phase?
Two pumps can be used in HPLC mobile phase to allow high-pressure mixing of two individual solvents.
List and describe three types of detectors used in HPLC.
Diode array detector: measures UV and visible spectrum of the solution as it flows through.
Fluorescence: detects only substances that exhibit fluorescence, e.g. LSD
Conductivity: electric current can tell the conductivity of the substance, which is different for each substance.
Describe three advantages of thin-layer chromatography over other types.
TLC is much cheaper and no instrument is required than GC and HPLC. Multiple samples can be run at one time with TLC.
Give at least two advantages of HPLC over TLC.
HPLC is much more sensitive and less samples are required than in TLC. Quantitative analysis is much more easily performed with HPLC and GC rather than TLC.
What are the two general requirements for an analyte in order for it to be separated by GC?
The analytes need to be highly volatile: they must vaporize when heated to at most 350ºC. Analytes must also be thermally stable at such temperatures.
In electrophoresis, what are the stationary phase and mobile phase?
In gel electrophoresis, the stationary phase is a slab of gel material (e.g. agarose or polyacrylamide). The mobile phase is an electric current.
In capillary electrophoresis, the stationary phase is a thin column and the mobile phase is electric current.
What is the major advantage of electrophoresis over other types of chromatography?
Capillary electrophoresis is an extremely sensitive technique, more sensitive than other methods. It is also the only method available for separating DNA fragments for forensic DNA typing.
Define pH. Why is it important in liquid extractions?
pH is a property of chemical compounds and is described by their acidity or alkalinity relative to water. Liquid phase extractions involve separating substances based on their polarity and pH.
How do extractions differ from chromatography?
Extractions use two immiscible liquids, and chromatography uses a stationary and mobile phase to separate substances.
