Flashcards
Chapter 28: liquid chromatography
Cards in this deck (78)
- 1LC is different from GC in the way that the solute interacts withboth stationary and mobile phase
- 2affinity chromatography is widely used for isolation and preparation ofbiomolecules
- 3chiral chromatography is used for separatingenantiomers.
- 4Based on Cm, the efficiency of an LC column should improve dramatically as the particle sizedecreases
- 5In LC, significant band broadening sometimes occurs outside the column packing itself. This extracolumn band broadening occurs asthe solute is carried through open tubes such as those found in the injection system, the detector region, and the piping connect- ing the various components of the system.
- 6extracolumn band broadening arises from differences in flow rates between layers of liquid adjacent to the wall and the center of the tube. As a result,the center part of a solute band moves more rapidly than the peripheral part.
- 7Extracolumn broadening can become quite serious whensmall-bore columns are used.
- 8Because of these high pres- sures, the equipment for HPLC tends to be more __________________ than equipment for other types of chromatography.elaborate and expensive
- 9Dissolved gases can lead toirreproducible flow rates and band spreading; in addition, both bubbles and dust interfere with the performance of most detectors.
- 10Degassers may consist of a vacuum pumping system, a distillation system, a device for heating and stirring, or a system for sparging, in whichthe dissolved gases are swept out of solution by fine bubbles of an inert gas that is not soluble in the mobile phase.
- 11isocratic elution.An elution with a single solvent or solvent mixture of constant composition
- 12gradient elution,two (and sometimes more) solvent systems that differ significantly in polarity are used and varied in composition during the separation.
- 13gradient elution shortened the time of separation significantly without sacrificingthe resolution of the early peaks.
- 14The requirements for liquid chromatographic pumps include(1) the generation of pressures of up to 6000 psi, (2) pulse-free output, (3) flow rates ranging from 0.1 to 10 mL/min, (4) flow reproducibilities of 0.5% relative or better, and (5) resistance to corrosion by a variety of solvents.
- 15Two major types of pumps are used in LC:the screw-driven syringe type and the reciprocating pump. Reciprocating pumps are used in almost all modern commercial chromatographs.
- 16Reciprocating pumpsconsist of a small chamber in which the solvent is pumped by the back and forth motion of a motor- driven piston . Two ball check valves, which open and close alternately, control the flow of solvent into and out of a cylinder. The solvent is in direct contact with the piston.
- 17Reciprocating pumps have the disadvantage ofproducing a pulsed flow, which must be damped because the pulses appear as baseline noise on the chromatogram.
- 18Modern LC instruments use dual (or triple) pump heads or elliptical cams to minimizepulsations in flow caused by pumps
- 19The advantages of reciprocating pumps includetheir small internal volume (35–400 μL), their high out- put pressures (up to 10,000 psi, 20,000 psi in UHPLC systems), their adaptability to gradient elution, their large solvent capacities, and their relatively constant flow rates, which are largely independent of column back pressure and solvent viscosity.
- 20Displacement pumpsconsist of large, syringe-like chambers equipped with a plunger activated by a screw-driven mechanism powered by a stepping motor.
- 21Displacement pumps also produce a flow that tends to beindependent of viscosity and back pressure. In addition, the output is pulse free.
- 22disadvantage of displacement pumpsDisadvantages include limited solvent capacity (,250 mL), relatively low pressure limits (,10,000 psi), and considerable inconvenience when solvents must be changed.
- 23Often, the limiting factor in the precision of liquid chromatographic measurements is thereproducibility with which samples can be introduced onto the column packing. sample volumes must be very small—a few tenths of a microliter to perhaps 500 μL.
- 24The most widely used method of sample introduction in LC is based onsampling loops
- 25sampling loopshave interchangeable loops providing a choice of sample sizes from 1 μL to 100 μL or more.
- 26auto-injectorsunits are capable of injecting samples into the LC from vials on a sample carousel or from microtiter plates. They usually contain sampling loops and a syringe pump for injection volumes from less than 1 μL to more than 1 mL. Some have controlled-temperature environments that allow for sample storage and for carrying out derivatization reactions prior to injection.
- 27Liquid-chromatographic columns are usually constructed fromsmooth-bore stainless steel tubing or heavy-walled glass tubing and polymer tubing, such as polyetheretherketone (PEEK)
- 28There are two types of precolumns.A precolumn between the mobile phase reservoir and the injector is used for mobile-phase conditioning and is termed a scavenger column. A second type of precolumn is a guard column, positioned between the injector and the analytical column.
- 29scavenger columnThe solvent par- tially dissolves the silica packing and ensures that the mobile phase is saturated with silicic acid prior to entering the analyt- ical column. This saturation minimizes losses of the stationary phase from the analytical column.
- 30A guard columnis a short column packed with a similar stationary phase as the analytical column. The purpose of the guard column is to pre- vent impurities, such as highly retained compounds and particulate matter, from reaching and contaminating the analytical column. The guard column is replaced regularly and serves to increase the lifetime of the analytical column.
- 31For some applications, close control of column temperature is not necessary and columns are operated at room temperature. Often, however,more reproducible chromatograms are obtained by maintaining constant column temperature.
- 32frictional heating resulting from ultra-high pressures creates temperature gradients in the column. These gradients may be minimized bygood temperature control, which leads to column stability and high-efficiency separations.
- 33Two basic types of packings have been used in LC,pellicular and porous particle.
- 34pellicular particlesThe original pellicular particles were spherical, nonporous, glass or polymer beads with typical diameters of 30–40 μm. A thin, porous layer of silica, alumina, a poly-styrene-divinylbenzene synthetic resin, or an ion-exchange resin was deposited on the surface of these beads. Small porous microparticles have completely replaced these large pellicular particles.
- 35The typical porous particle packing for LC consists ofporous microparticles having diameters ranging from 3 to 10 μm
- 36LC detectors have often been traditional analytical instruments adapted withflow cells to measure low concentrations of solutes in liquid streams.
- 37ideal LC detectorLC detector need not be responsive over as great a temperature range. In addition, an HPLC detector should have minimal internal volume to reduce zone broadening and should be compatible with liquid flow.
- 38There are two basic types of detectors in LC,Bulk-property detectors and solute-property detectors
- 39Bulk-property detectorsrespond to a mobile-phase bulk property, such as refractive index, dielectric constant, or density, that is modulated by the presence of solutes.
- 40solute-property detectorsrespond to some property of solutes, such as UV absorbance, fluorescence, or diffusion current, that is not possessed by the mobile phase.
- 41in adsorption chromatography, describe the stationary and mobile phaseStationary: silica and alumina
mobile: same as normal phase, non polar like hexane
Also used eluent strength index, which is important for the separation of isomeric mixtures - 42Normal-phase chromatographystationary phase= polar silica
mobile phase polarity determines the strength. More polar solvent= stronger mobile phase - 43Eluent strength is a measure ofsolvent adsorption energy on bare silica, pentane is 0.
- 44Partition chromatography includesliq-liq and bonded phase chromatography
- 45in liquid-liquid chromatographystationary phase retained on surface of support by adsorption. Disadvantage is the loss of stationary phase through dissolution into the mobile phase
- 46in bonded phase chromatographystationary phase physically bonded to the surface of support (silica). Prvents dissolution and increases life of column
- 47Reverse phase chromatographynon-polar stationary phase
polar mobile phase (water)
most polar solvents elute first. Increasing polarity of the mobile phase increases the elution time - 48Increasing the rate at which the solute equilibrates between the stationary phase and mobile phase increasesthe efficiency of chromatography
- 49For gas chromatography with an open tubular column, rapid mass transfer is accomplished byreducing the diameter of the column so that the molecules can diffuse quickly between the channel and the stationary phase on the wall
- 50why are open open tubular column not used with liquid?diffusion coefficient is higher, and the diameter of the column is too great
- 51Liquid chromatography is conducted with packed columns so that a solute molecule does not have to diffuse very far toencounter the stationary phase
- 52_______-phase partition chromatography is the most common mode of HPLCreverse
- 53retention is tuned by changing the polarity of themobile phase
- 54changes in retention factor are generally made by usingsolvent modifers, to change the polarity of the solvent mixture
- 55changes in alpha, selectivity factor, are generally made throughthe selection of a different column
- 56what are the bases of the mobile phase in normal and reverse phasenormal=hexane
reverse=water - 57the ideal range for the retention factor in LC is2
- 58four solvent system for normal phaseethyl ether, methylene chloride, chloroform, hexane
- 59four solvent system for reverse phasemethanol, acetonitrile, tetrahydrofuran, water
- 60In liquid-liquid partition chromatography, the stationary phase iscovalently bonded to the silica surface
- 61Although bare silica is not used for partition chromatography, it can be used foradsorption chromatography
- 62why can only half the silica in a bonded column be functionalized, and how does this effect itSteric effects prevent all the silica from being functionalized. Unreacted silica lead to tailing. To prevent this, unreacted silanols are capped with chlorotrimethyl silane
- 63The polarity index measures the intermolecular attraction betweena solute and a solvent
- 64Snyder calculated the partitioning coefficient, 𝐾, of a n- alkane in the same molar volume of a probe solvent. Assumed that there were three additive molecular interactions:proton donor, proton acceptance, strong dipole
- 65Columns are expensive and easily degraded by dust or particles in the sample or solvent. So a ____ must be used and sample prepguard must be used to protect the main chromatography column and samples must the filtered and centrifuged
- 66how does using smaller particles effect the columnThey provide more uniform flow through the column, thereby reducing the multiple path term, A. The smaller the particles, the
less distance solute must diffuse. The effect decreases the C term for mass transfer. - 67how does pH affect the retention timeif you lower the pH lower than that of the pka of your analyte, you can make sure the molecule is not so polar (when unprotonated) and have a large affinity for water and not be retained
- 68disadvantage of using small particlesThe penalty for small particle size is resistance to solvent flow.
- 69advantages of heated columnlowers solvent viscosity, decreases retention time
- 70Refractive index detectorclosest to a universal detector. the solvent passes through one half of the cell on its way to the column; the eluate then flows through the other chamber. The two compartments are separated by a glass plate mounted at an angle such that bending of the incident beam occurs if the two solutions differ in refractive index. The resulting displacement of the beam with respect to the photosensitive surface of a detector causes variation in the output signal, which, when amplified and recorded, provides the chromatogram.
- 71Advantages and disadvantages of refractive index detectorad: responds to all analytes, reliable, unaffected by flow rate
Dis: temperature sensitive, not sensitive, affected by rapid changes in solvent composition - 72UV detectorUses Z-shaped flow-through cell. To minimize extracolumn band broadening, the volume of such a cell should be kept as small as possible. Uses a wheel filter w select wavenelngths, or double/single beam monochromators. Used phototube, photo multiplier tube, or photodiode array as detector.
- 73define amperometry, voltammetry, coulometry, and conductometryAmperometry – measure voltage
vs. current
• Voltammetry – measure current vs.
voltage
• Coulometry – measure charge vs.
time
• Conductometry – measure voltage vs. time - 74Electrochemical detectorRequires analyte to undergo a reversible oxidation-reduction reaction
- 75Why is it not ideal to pair MS detection with LCThe sample is dissolved in a solvent, and a gas form is needed, with most of the solvent removed.
- 76what ionization is used for MS detectors in GCThe most common ionization sources are electrospray ionization and atmospheric pressure chemical ionization
- 77Electrospray ionization-Eluent is vaporized and ionized as it passes through a stainless steel capillary.
‒ Capillary has an applied high voltage potential.
‒ Mobile phase pumped away by skimmer, which do produce build up - 78chiral chromatographyEither chiral mobile-phase additives or chiral stationary phases are required for these separations. Preferential complexation between the chiral resolving agent and one of the isomers results in a separation of the enantiomers. The chiral resolving agent must have chiral character itself to recognize the chiral nature of the solute.
Information
- Description
- GRE instrumental analysis Flashcards on Chapter 28: liquid chromatography, created by Ashlin Deatherage on 30/04/2023.
- Created by
- Ashlin Deatherage
- Views
- 1
- Rating