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Analytical Chemistry > HPLC > Flashcards

HPLC Flashcards

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1
Q

What does HPLC stand for?

A

high performance liquid chromatography

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2
Q

What does UHPLC stand for?

A

Ultra high pressure liquid chromatography

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3
Q

What is HPLC typically used for?

A

Typically used for separating the 95% of all compounds that are not sufficiently volatile to be analyzed directly by GC

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4
Q

When does LC give the narrowest peaks?

A

when the stationary phase particles inside the column are the smallest, but it takes more pressure to push liquid through a column of small particles

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5
Q

What is UHPLC?

A
  • Liquid chromatography using very small (1-2 micrometer) diameter particles and high mobile phase pressure
  • It is one of the modern trends in LC
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6
Q

Are two mobile phases and reservoirs mandatory in HPLC? What is this called?

A
  • Two mobile phases and revervoirs are not mandatory
  • Running with a single constant mobile phase composition is called isocratic mode and is the best way to run provided all the analytes elute from the column in a reasonable time
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7
Q

What is it called when HPLC has two mobile phases, reservoirs, and pumps?

A
  • If the instrument is equipped with two (or more) different mobile phases, reservoirs, and pumps, then you have the option to change the mobile phase composition during the run (called a mobile phase gradient) to shorten the retention times of more tightly bound analytes.
  • That can save time during the run, but then there is a delay since the system must be re- equilibrated at the initial mobile phase composition for ~15 minutes before the next run can be started.
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8
Q

What is the function of the pulse damper and why is it needed?

A
  • The pulse damper helps maintain a very constant pressure, even when piston pumps are used.
  • Reason the pulse damper is needed: most detectors can sense pressure variations that would otherwise produce an undesirable small, periodic signal in the baseline of the chromatogram.
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9
Q

Why use a guard column? What two things is it guarding against?

A

A guard column is a protective column or cartridge installed between the injector and the analytical column. It serves to remove the impurities and suspended solids from reaching the analytical column. Typically it has a length of about 2 cm and internal diameter of 4.6 mm. Guard columns are packed with pelicullar particles of around 40 μm size to offer negligible pressure drop.

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10
Q

What is an optional component of an HPLC?

A

HPLC’s can be equipped with a column oven, but many are not; most HPLC is performed at room
temperature anyway so the oven isn’t always needed.

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11
Q

What is a key difference between HPLC and GC?

A

Unlike GC, the analyte boiling points are not important in HPLC.

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12
Q

Describe HPLC detectors:

A
  • most common is the spectrophotometric detector operating at UV or visible wavelengths
  • photodiode array spectrophotometers are also widely used and produce a complete UV- visible spectrum at each time point along the chromatogram.
  • Use a conductivity detector if the analytes are ionic
  • a mass spectrometer is an excellent detector if one can afford the cost.
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13
Q

What are normal and reverse phase HPLC?

A
  • in “normal phase” HPLC, the stationary phase is polar & mobile phase is nonpolar
  • in “reversed phase” HPLC, the stationary phase is nonpolar & mobile phase is polar
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14
Q

What are common stationary phase materials for normal-phase HPLC?

A
  • silica, alumina, activated charcoal
  • all of these are polar and the separation mechanism for them is adsorption
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15
Q

What are common mobile phases for normal-phase HPLC?

A
  • The mobile phase for normal-phase HPLC is typically n-hexane, n-pentane, or other fairly nonpolar solvent.
  • That can be mixed with a somewhat more polar solvent (such as isopropanol or ethyl acetate) to increase the polarity, but the mixture must be mainly nonpolar overall.
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16
Q

What is the stationary phase for reverse phase HPLC?

A
  • small (2-5 μm) beads of silica or organic resin are usually used
  • The beads’ surfaces are covered with vast numbers of covalently-bonded organic groups, which makes their surfaces much more nonpolar than the silica, etc. used in normal-phase HPLC.
17
Q

What is the most widely used reversed phase stationary phase in HPLC? How does it work?

A
  • the most widely-used reversed-phase stationary phase is the “C18” phase, where –(CH2)17CH3 chains are
    attached to the beads through -O-Si- (siloxane) linkages, as shown in class
  • To an analyte, the C18 phase looks like a very nonpolar bed of octadecane.
  • Nonpolar analytes have a strong affinity for this phase and therefore long retention times.
  • Polar analytes are less attracted to the stationary phase but are attracted to the relatively polar mobile phase and, so, have shorter retention times.
18
Q

What can be done to make a more polar reverse phase stationary phase?

A
  • To make a more polar reversed-phase stationary phase, shorten the chain length (making C8, C4, etc. instead of C18).
  • To make it still more polar, add phenyl groups (making a Ph or “phenyl” phase.)
  • Adding a cyano group (–CN) instead of phenyl makes the phase even more polar.
  • One of the most polar of the common reversed phases is the “amino” phase (NH2), which contains amino groups.
  • If you need a more polar stationary phase than amino, then you should be doing normal- (not reversed) phase HPLC.
19
Q

What is the polarity order from least to most polar of stationary phase molecules?

A

The polarity order is: C18<C8<C4<Ph<CN<NH2<silica, alumina, charcoal.

20
Q

What is the mobile phase for reversed phase HPLC?

A
  • must be rather polar: typically methanol, acetonitrile, or a mixture of one of those with water
  • Pure water is virtually never used as the mobile phase since it can damage some brands of C18 columns.
21
Q

What can be controlled in HPLC that is not possible in GC?

A

Unlike GC, it is possible to also change the polarity of the mobile phase in HPLC, providing more control for fine-tuning separations

22
Q

What are strong vs weak mobile phases?

A

“Strong” mobile phases are ones that cause analytes to elute quickly from the column. “Weak” mobile phases give longer retention times.

23
Q

How do you change the mobile phase in reversed-phase? Normal phase?

A
  • For reversed-phase (which has a relatively nonpolar stationary phase), a stronger mobile phase is made by increasing the fraction of the organic component (methanol, acetonitrile, etc.) and decreasing the fraction of highly polar water. That way, the more non-polar mobile phase will tend to strip the analytes off the nonpolar stationary phase, reducing their retention times.
  • For normal-phase HPLC (which uses a polar stationary phase), the mobile phase can be made stronger by increasing its polarity to help it strip the analytes off the polar stationary phase.
24
Q

How should you change the mobile phase during the run?

A
  • the mobile phase begins as weak and then is changed to strong to drive tightly-bound analytes off the column.
  • called running a mobile phase gradient (like a temperature program in GC)
  • temperature programs are not used in HPLC
25
Q

What is often used for ion analytes?

A
  • For analytes that are ions, ion-exchange chromatography is often used.
  • You separated anions in the lab by using an ion-exchange column with positively-charged binding sites. The method can also be used to separate cationic analytes provided the stationary phase has negatively- (instead of positively-) charged binding sites.
  • An analyte ion binds to the column and remains “stuck” there until an ion in the mobile phase bumps it off, placing it back in the mobile phase where it is carried further down the column.
  • It moves down the column in a series of “hops.” The faster the ion hops, the shorter will be its retention time. The ions that displace the analytes from their binding sites are contained in the mobile phase, and the higher their concentrations the shorter the analyte ion retention times will be.
26
Q

What is size-exclusion chromatography?

A
  • Size-exclusion chromatography, sometimes called “gel permeation” or “gel filtration,” uses stationary phase particles that contain pores of, typically, 10-100 nanometers in diameter. Small molecules get trapped in these pores and have long retention times. Large molecules, including biopolymers like proteins, nucleic acids, and carbohydrates, are too big to fit in the pores and have shorter retention times that depend on their molecular sizes and shapes.
  • By using biopolymers of known molecular weights to make a calibration curve (MW vs. retention time), size-exclusion chromatography can be used to estimate molecular weights of unknown molecules.
27
Q

Describe what mode of HPLC to try initially for separating a mixture of analytes.

A
  • For large analyte molecules (>30 nm diam.), try size exclusion.
  • For small analytes that are nonpolar, try normal-phase with silica or alumina, or use reversed phase with one of the relatively polar stationary phases like CN or NH2.
  • For small analytes that are moderately polar (alcohols, esters, etc.), try reversed-phase with Ph, CN, or C18 stationary phases.
  • For analytes that dissolve in polar solvents but are not ionic, try reversed-phase HPLC using C18, C8, etc.
  • For ionic analytes, use ion- exchange HPLC.

Analytical Chemistry (4 decks)

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