A chem Ch 31-34
retardation factor
In thin-layer chromatography, ___ is the ratio of distance traveled by the solute to the distance traveled by the solvent front
Compare supercritical fluid chromatography with other column chromatographic methods.
Like GC, SFC is inherently faster than liquid chromatography because of the lower viscosity and higher diffusion rates in the mobile phase. Unlike GC, however, the intermediate diffusivities and viscosities of supercritical fluids reduce longitudinal band spreading, similar to the situation encountered in HPLC.
What variables must be controlled if satisfactory quan- titative data are to be obtained from chromatograms?
Sample injection volume, carrier gas flow rate and column condition are among the parameters which must be controlled for highest precision quantitative GC. The use of an internal standard can minimize the impact of variations in these parameters.
migration rate
The _____ v of an ion in an electric field is given by v=µeE=µe* V/L
zwitterion
The species that results from the transfer in solution of a proton from an acidic group to an acceptor site on the same molecule.
Describe two general methods for improving the resolution of two substances on a chromatographic column.
Two general methods for improving the resolution of a column are to increase the column length and to reduce the plate height.
paper chromatography
a technique for separating dissolved compounds such as chlorophyll, carotene, and xanthophyll
electrical FFF
an electric field is applied perpendicular to the flow direction. Retention and separation occur based on electrical charge.
isobaric
constant pressure
UV-vis absorbance detector
most widely used detectors for liquid chromatography are based on absorption of ultraviolet or visible radiation
liquid partition chromatography
useful for Molecular species that are nonvolatile and are thermally stable
For supercritical carbon dioxide, predict the effect that the following changes will have on the elution time in an SFC experiment. (a) Increase the flow rate (at constant temperature and pressure). (b) Increase the pressure (at constant temperature and flow rate). (c) Increase the temperature (at constant pressure and flow rate).
(a) An increase in flow rate results in a decrease in retention time. (b) An increase in pressure results in a decrease in retention time. (c) An increase in temperature results in a decrease in density of supercritical fluids and thus an increase in retention time.
WCOT columns
A ____ is simply a capillary tubing fashioned from fused silica, stainless steel, aluminum, copper, plastic or glass. Its inner walls are coated with a thin layer of the mobile phase.
ampholytes
Amphoteric compounds usually containing carboxylic acid and amino groups.
Describe a major advantage of micellar electrokinetic capillary chromatography over conventional liquid chromatography.
Describe a major advantage of micellar electrokinetic capillary chromatography over conventional liquid chromatography.
Describe a method for determining the number of plates in a column.
Determine the retention time tR for a solute and the width of the solute peak at its base, W. The number of plates N is then N=16(tR/W)^2
What types of flow meters are used in GC?
Electronic, bubble, bubble with digital readout, digital mass or volume flow meters are used.
What is the effect of stationary phase film thickness on gas chromatograms?
Film thickness influences the rate at which analytes are carried through the column, with the rate increasing as the thickness is decreased. Less band broadening is encountered with thin films.
What variables must be controlled if satisfactory qual- itative data are to be obtained from chromatograms?
For satisfactory qualitative data, carrier gas flow rate, column temperature, injection port temperature, and injection volume (sample size) are among the most important variables.
What are the advantages of fused-silica capillary columns compared with glass or metal columns?
Fused silica columns have greater physical strength and flexibility than glass open tubular columns and are less reactive toward analytes than either glass or metal columns.
permeation limit
In gel permeation chromatography, the ___ is the molecular weight below which the solute molecules can penetrate into the pores completely.
pseudostationary phase
In micellar electtokinetic chromatography, neutral species partition between the mobile phase and a __________, a component that moves along the length of the column but at a much slower rate than the mobile phase
on-line extraction
In the ___ method, the effluent from the restrictor, after depressurization, is transferred directly to a chromatographic system.
What types of species can be separated by HPLC but not by GC?
Nonvolatile and thermally unstable compounds.
pellicular particle
Packing used in HPLC.. Spherical, nonporous, glass or polymer beads with typical diameters of 30 to 40 mm. A thin, porous layer of silica, alumina, a polystyrene-divinyl benzene synthetic resin, or an ion-exchange resin was deposited on the surface of these beads
What determines the elution order in sedimentation FFF?
Particle size and mass.
exclusion limit
The ___ is the molecular weight above which compounds do not separate in gel permeation chromatography.
What properties should the stationary phase liquid for gas chromatography possess?
The stationary phase liquid should have low volatility, good thermal stability, chemical inertness and solvent characteristics that provide suitable retention factor and selectivity for the separation.
temperature programming
The systematic adjustment of column temperature in gas chromatography to optimize migration rates for solutes. .... involves increasing the temperature of a gas-chromatographic column as a function of time. This technique is particularly useful for samples that contain constituents whose boiling points differ significantly.
critical temperature
The temperature above which a substance can no longer exist in the liquid state, regardless of pressure.
capillary electrophoresis
a family of electrokinetic separation methods performed in submillimeter diameter capillaries and in micro- and nanofluidic channels
two-dimensional thin-layer chromatography
development is carried out with two solvents that are applied successively at right angles to one another.
retention time
for an analyte is the time interval between its injection onto a column and its appearance at the detector at the other end of the column.
plate height
of a chromatographic is defined by the relationship H = σ2 / Lwhere σ2 is the variance obtained from the Gaussian shaped chromatographic peak and Lis the length of the column packing in centimeters. A quantity describing the efficiency of a chromato- graphic column. The term comes from the height of a plate, or distil- lation stage, in a traditional distillation column.
joule healing
sets up a temperature gradient across the capillary, leading to convection and band broadening.
suppressor-column ion chromatography
the chromatographic column is followed by a column whose purpose is to convert the ions used for elution to molecular species that are largely nonionic and thus do not interfere with conductometric detection of the analyte species
critical pressure
the lowest pressure at which a substance can exist as a liquid at the critical temperature
affinity
useful for Molecules that selectively bind the affinity ligand usually biomolecules
How do instruments for supercritical fluid chromatography differ from those for (a) HPLC and (b) GC?
(a) Instruments for supercritical-fluid chromatography are very similar to those for HPLC except that in SFC there are provisions for controlling and measuring the column pressure. (b) SFC instruments differ substantially from those used for GC in that SFC instruments must be capable of operating at much higher mobile phase pressures than are typically encountered in GC'
supercritical fluid
A substance that is maintained above its critical temperature; its properties are intermediate between those of a liquid and those of a gas. a substance that is maintained above its critical temperature so that it cannot be condensed into a liquid no matter how great the pressure.
partition chromatography
A type of chromatography based on the distribution of solutes between a liquid mobile phase and a liquid stationary phase retained on the surface of a solid. eparations are based on distribution equilibria between two immiscible liquids.
Why are gas chromatographic stationary phases often bonded and cross-linked? What do these terms mean?
Currently, liquid stationary phases are generally bonded and/or cross-linked in order to provide thermal stability and a more permanent stationary phase that will not leach off the column. Bonding involves attaching a monomolecular layer of the stationary phase to the packing surface by means of chemical bonds. Cross linking involves treating the stationary phase while it is in the column with a chemical reagent that creates cross links between the molecules making up the stationary phase.
Describe the fundamental difference between adsorption and partition chromatography.
In adsorption chromatography, separations are based on adsorption equilibria between the components of the sample and a solid surface. In partition chromatography,separations are based on distribution equilibria between two immiscible liquids.
What are the major differences between gas-liquid and liquid-liquid chromatography?
In gas-liquid chromatography, the mobile phase is a gas, whereas in liquid-liquid chromatography, it is a liquid.
What is electroosmotic flow? Why does it occur?
Electroosmotic flow is the migration of the solvent towards the cathode in an electrophoretic separation. This flow is due to the electrical double layer that develops at the silica/solution interface. At pH values higher than 3 the inside wall of the silica capillary becomes negatively charged leading to a build-up of buffer cations in the electrical double layer adjacent to the wall. The cations in this double layer are attracted to the cathode and, since they are solvated they drag the bulk solvent along with them.
List the types of substances to which each of the following separation methods is most applicable: (a) supercritical fluid chromatography. (b) thin-layer chromatography. (c) capillary zone electrophoresis. (d) sedimentation FFF. (e) flow FFF. (f) micellar electrokinetic capillary chromatography.
(a) Nonvolatile or thermally unstable species that contain no chromophoric groups. (b) Particularly important for drug purity determination and in clinical laboratories. (c)Inorganic anions and cations, amino acids, catecholamines, drugs, vitamins, carbohydrates, peptides, proteins, nucleic acids, nucleotides, and polynucleotides. (d) Molecules with molecular masses exceeding 10^6 like polymers, biological macromolecules, natural and industrial colloids, emulsions and subcelluar particles. (e) Proteins, synthetic polymers, and colloidal particles. (f) Low molecular mass aromatic phenols and nitro compounds.
What are the principal advantages and the prin- cipal limitations of each of the detectors listed in Problem 32-10?
(a) Advantages of thermal conductivity: general applicability, large linear range, simplicity, nondestructive.Disadvantage: low sensitivity. (b) Advantages of flame ionization: high sensitivity, large linear range, low noise, ruggedness, ease of use, and response that is largely independent of flow rate. Disadvantage: destructive. (c) Advantages of electron capture: high sensitivity selectivity towards halogen-containing compounds and several others, nondestructive. Disadvantage: small linear range. (d) Advantages of thermionic detector: high sensitivity for compounds containing nitrogen and phosphorus, good linear range.Disadvantages: destructive, not applicable for many analytes. (e) Advantages of photoionization: versatility, nondestructive, large linear range.Disadvantages: not widely available, expensive.
List the variables that lead to (a) band broadening and (b) band separation in gas-liquid chromatography.
(a) Band broadening arises from very high or very low flow rates, large particles making up packing, thick layers of stationary phase, low temperature, and slow injection rates. (b) Band separation is enhanced by maintaining conditions so that k lies in the range of 1 to 10, using small particles for packing, limiting the amount of stationary phase so that particle coatings are thin, and injecting the sample rapidly.
adsorption chromatography
A separation technique in which a solute equilibrates between the eluent and the surface of a finely divided adsorbed solid. separations are based on adsorption equilibria between the components of the sample and a solid surface.
amphiprotic compound
A species that in solution is capable of both donating and accepting a proton.
reversed-phase chromatography
A type of liquid-liquid partition chromatography that uses a nonpolar stationary phase and a polar mobile phase; compare with normal-phase chromatography.
Normal phase chromatography
A type of partition chromatog- raphy that involves a polar stationary phase and a nonpolar mobile phase; compare with reversed-phase chromatography.
gel permeation chromatography
A type of size exclusion chroma- tography that uses a hydrophobic packing. Used to separate nonpolar species.
gel permeation
A type of size exclusion chroma- tography that uses a hydrophobic packing. Used to separate nonpolar species. useful for High molecular mass compounds that are soluble in nonpolar solvents
gel filtration chromatography
A type of size exclusion chromatog- raphy that uses a hydrophilic packing. Used to separate polar species.
ion chromatography
An HPLC technique based on the partition- ing of ionic species between a liquid mobile phase and a solid poly- meric ionic exchanger; also called ion-exchange chromatography. useful for Substances that are ionic
Three large proteins are ionized at the pH at which anelectrical FFF separation is carried out. If the ions are designated A^2+, B^+, and C^3+, predict the order of elution.
B+ followed by A^2+ followed by C^3+.
How could electroosmotic flow be repressed? Why would one want to repress it?
Electroosmotic flow can be repressed by reducing the charge on the interior of the capillary by chemical treatment of the surface. Sometimes, species elute from the capillary before they are resolved because of electroosmotic flow. Flow rates can also be erratic due to surface capillary conditions such non-uniform charge distributions and adsorption of ions.
List the major advantages and limitations of FFF compared to chromatographic methods.
FFF methods are particularly well suited to high molecular mass materials such as polymers, large particles and colloids. In addition, no packing material or stationary phase is needed for separation to occur avoiding undesirable interactions between the packing material and the sample constituents. Finally, the geometry and flow profiles in FFF are well characterized allowing fairly exact theoretical predictions of retention and plate height to be made.
Which of the GC detectors in Table 32-1 are suitable for HPLC? Why are some of these unsuitable for HPLC?
GC detectors that are suitable for HPLC are the mass spectrometer, FTIR and possible photoionization. Many of the GC detectors are unsuitable for HPLC because they require the eluting analyte components to be in the gas-phase.
Why is gas-solid chromatography not used as exten- sively as gas-liquid chromatography?
Gas-solid chromatography has limited application because active or polar compounds are retained more or less permanently on the packings. In addition severe tailing is often observed owing to the nonlinear character of the physical adsorption process.
What kind of mixtures are separated by gas-solid chromatography?
Gas-solid chromatography is used primarily for separating low molecular mass gaseous species, such as carbon dioxide, carbon monoxide and oxides of nitrogen.
Describe the difference between gel-filtration and gel- permeation chromatography.
Gel filtration is a type of size-exclusion chromatography in which the packings are hydrophilic and eluents are aqueous. It is used for separating high molecular mass polar compounds. Gel permeation chromatography is a type of size-exclusion chromatography in which the packings are hydrophobic and the eluents are nonaqueous. It is used for separating high molecular mass nonpolar species.
HPLC
High performance liquid chromatography-Column chromatography in which the mobile phase is a liquid, often forced through a stationary phase by pressure.
gas-liquid chromatography
In ..., the stationary phase is a liquid that is immobilized on a solid. Retention of sample constituents involves equilibria between a gaseous and a liquid phase.
gas-solid chromatography
In ....., the stationary phase is a solid surface that retains analytes by physical adsorption. Here separation involves adsorption equilibria.
chiral chromatography
In ___, enantiomers are separated based on their different interactions with a chiral resolving agent. useful for chiral compounds.
CIEF
In ____, amphiprotic compounds move through a pH gradient to the pH at which no net migration occurs.(, called the isoelectric point.)
capillary gel electrophoresis
In ____, obstructing strands of material occupy the column space causing large ions to move slower than small ions in an electric field.
off-line extraction
In _____, the analytes are collected by immersing the restrictor in a few milliliters of solvent and allowing the gaseous supercritical fluid to escape into the atmosphere. Separated analytes are then identified by any of several other methods.
bonded-phase packing
In a _____, the stationary phase liquid is held in place by chemically bonding it to the solid support.
gradient elution
In a ______, two or more solvents are employed and the composition of the eluent is changed continuously or in steps as the separation proceeds.
List some of the advantageous properties of super- critical CO2 as a mobile phase for chromatographic separations.
It is an excellent solvent for a variety of nonpolar organic molecules. It transmits in the UV range and is odorless, nontoxic, readily available, and remarkably inexpensive relative to other chromatographic solvents. In addition, its critical temperature and pressure are relatively low allowing a wide selection of temperatures and pressures to be used without exceeding the operating limits of modern instrumentation.
The ideal detector for GC is described in Section 32A-4. Which of the eight characteristics of an ideal GC detector are applicable to HPLC detectors? What additional characteristics would be added to describe the ideal HPLC detector?
The ideal HPLC detector would have all the same characteristics as listed for the ideal GC detector. In addition, the HPLC detector should have low dead volume and be compatible with the liquid flows and pressures encountered in HPLC.
electroosmotic flow
The net flow of bulk liquid in an applied electric field through a porous material, capillary tube, membrane, or microchannel.
isoelectric point
The pH at which an amino acid has no tendency to migrate under the influence of an electric field.
What properties of a supercritical fluid are important in chromatography?
The properties of a supercritical fluid that are important in chromatography include its density, its viscosity, and the rates at which solutes diffuse in it. The magnitude of each of these lies intermediate between a typical gas and a typical liquid.
SCOT columns
a support coated open-tubular column.
gel filtration
a type of size-exclusion chromatography in which the packings are hydrophilic, and eluents are aqueous. It is used for separating high molecular mass polar compounds.
gel-permeation chromatography
a type of size-exclusion chromatography in which the packings are hydrophobic and the eluents are nonaqueous. It is used for separating high molecular mass nonpolar species.
What is the principle of separation by capillary zone electrophoresis?
Under the influence of an electric field, mobile ions in solution are attracted or repelled by the negative potential of one of the electrodes. The rate of movement toward or away from a negative electrode is dependent on the net charge on the analyte and the size and shape of analyte molecules. These properties vary from species to species. Hence, the rate at which molecules migrate under the influence of the electric field vary, and the time it takes them to traverse the capillary varies, making separations possible.
ion-pair chromatography
a large organic counter-ion is added to the mobile phase as an ion-pairing reagent. Separation is achieved either through partitioning of the neutral ion-pair or as a result of electrostatic interactions between the ions in solution and charges on the stationary phase resulting from adsorption of the organic counter-ion.
reversed-phase packing
a nonpolar packing that is used in partition chromatography with a relatively polar mobile phase.
electropherogram
a plot of results from separation done by electrophoresis looks like a chromatogram but with narrower peaks.
elution
a process in which species are washed through a chromatographic column by additions of fresh mobile phase
critical point
he point on a temperature / pressure phase diagram above which the gas acquires supercritical fluid properties.
retention factor
defined by the equation k = KAVS / VM where KA is the partition ratio for the species A and VS and VM are the volumes of the stationary and mobile phases, respectively a term used to describe the migration of a species through a chromatographic column. Its numerical value is given by k=(tR- tM)/tM, where tR is the retention time for a peak and tM is the dead time; also called the capacity factor.
Describe the principle on which each of the following GC detectors are based: (a) thermal conductivity, (b) flame ionization, (c) electron capture, (d) therm- ionic, and (e) photoionization.
(a) The thermal conductivitydetector is based on the decrease in thermal conductivity of the helium or hydrogen carrier gas brought about by the presence of analyte molecules. (b) The flame ionization detector is based on measuring the current that results from ions and electrons produced when organic compounds are combusted in a small air/hydrogen flame. (c) The electron capture detector is based on the affinity of halogen-containing organic compounds for electrons emitted by nickel-63 leading to a reduction in the monitored current. (d) The thermionic detector is based on the ion currents produced when the mobile phase is combusted in a hydrogen flame and then passed over a heated rubidium silicate bead. It is used primarily for detecting analytes that contain phosphorus or nitrogen. (e) The photoionization detector is based on the ion currents that develop when analyte molecules are irradiated with an intense beam of far-ultraviolet radiation.
Indicate the order in which the following compounds would be eluted from an HPLC column containing a reversed-phase packing: (a) benzene, diethyl ether, n-hexane. (b) acetone, dichloroethane, acetamide.
(a) diethyl ether, benzene, n-hexane. (b) acetamide, acetone, dichloroethane.
Indicate the order of elution of the following com- pounds from a normal-phase packed HPLC column: (a) ethyl acetate, acetic acid, dimethylamine.(b) propylene, hexane, benzene, dichlorobenzene.
(a) ethyl acetate, dimethylamine, acetic acid. (b) hexane, propylene, benzene, dichlorobenzene.
PLOT columns
A ____ is a porous layer open tubular column, which is also called a support coated open-tubular (SCOT) column. The inner surface of a ____ is lined with a thin film of a support material, such as a diatomaceous earth. This type of column holds several times as much stationary phase as does a wall-coated column.
chiral stationary phase
A chiral agent is immobilized on the surface of a solid support
Describe a chromatogram and explain what type of information it contains.
A chromatogram is a plot of detector response versus time. The peak position, retention time, can reveal the identity of the compound eluting. The peak area is related to the concentration of the compound.
suppressor based chromatography
A chromatographic technique involving a column or a membrane located between the analytical col- umn and a conductivity detector; its purpose is to convert ions of the eluting solvent into nonconducting species while passing ions of the sample.
Slab electrophoresis
A classic method used for many years to separate complex, high-molecular-weight species of biological and biochemical interest on a thin flat layer of porous semisolid gel in the presence of an electric field.
Mass spectrometry is an extremely versatile detection system for gas chromatography. Describe the major rea- sons why it is more difficult to combine HPLC with mass spectrometry than it is to combine GC with mass spectrometry.
A gas-phase sample is needed for mass spectrometry. The output of the LC column is a solute dissolved in a solvent, whereas the output of the GC column is a gas and thus directly compatible. As a first step in LC/MS, the solvent must be vaporized. When vaporized, however, the LC solvent produces a gas volume that is 10-1000 times greater than the carrier gas in GC. Hence, most of the solvent must also be removed.
What are hyphenated gas-chromatographic methods? Briefly describe three hyphenated methods.
A hyphenated gas chromatographic method is a method in which the analytes exiting from a column are identified by one of the selective techniques such as mass spectrometry, absorption or emission spectroscopy or voltammetry.
Although temperature does not have nearly the effect on HPLC separations that it has on GC separations, it nonetheless can play an important role. Discuss how and why temperature might or might not influence the following separations: (a) a reversed-phase chromatographic separation of a steroid mixture. (b) an adsorption chromatographic separation of a mixture of closely related isomers.
A number of factors that influence separation are clearly temperature dependent including distribution constants and diffusion rates. In addition, temperature changes can influence selectivity if components A and B are influenced differently by changes in temperature. Because resolution depends on all these factors, resolution will also be temperature dependent.(a) For a reversed phase chromatographic separation of a steroid mixture, selectivity and, as a consequence, separation could be influenced by temperature dependent changes in distribution coefficients.(b) For an adsorption chromatographic separation of a mixture of isomers, selectivity and, as a consequence, separation could be influenced by temperature dependent changes in distribution coefficients.
guard column
A precolumn located ahead of an HPLC column. The composition of the packing in the guard column is selected to extend the useful lifetime of the analytical column by removing par- ticulate matter and contaminants and by saturating the eluent with the stationary phase.
normal-phase packing
In a_____, the stationary phase is quite polar and the mobile phase is relatively nonpolar.
isocratic elution
In an ____, the solvent composition is held constant throughout the elution. Elution with a single solvent; compare with gradient elution
What is the major difference between isocratic elu- tion and gradient elution? For what types of com- pounds are these two elution methods most suited?
In an isocratic elution, the solvent composition is held constant throughout the elution. Isocratic elution works well for many types of samples and is simplest to implement. In a gradient elution, two or more solvents are employed and the composition of the eluent is changed continuously or in steps as the separation proceeds. Gradient elution is best used for samples in which there are some compounds separated well and others with inordinately long retention times.
How do gas-liquid and gas-solid chromatography differ?
In gas-liquid chromatography, the stationary phase is a liquid that is immobilized on a solid. Retention of sample constituents involves equilibria between a gaseous and a liquid phase. In gas-solid chromatography, the stationary phase is a solid surface that retains analytes by physical adsorption. Here separation involves adsorption equilibria.
What is the principle of micellar electrokinetic capillary chromatography? How does it differ from capillary zone electrophoresis?
In micellar electrokinetic capillary chromatography surfactants are added to the operating buffer in amounts that exceed the critical micelle concentration. The components of a sample added to this system distribute themselves between the aqueous phase and the hydrocarbon phase in the interior of the micelles according to their partition coefficients, similar to partition chromatography. The negatively charged micelles do exhibit electrophoretic flow but at a much slower rate than the buffer. Thus, MEKC is a true chromatography, where CZE separates sample components simply on the basis of ion mobility in a homogeneous buffer solution.
Describe the physical differences between capillary and packed columns. What are the advantages and disadvantages of each?
In open tubular or capillary columns, the stationary phase is held on the inner surface of a capillary, whereas in packed columns, the stationary phase is supported on particles that are contained in a glass or metal tube. Open tubular columns contain an enormous number of plates that permit rapid separations of closely related species. They suffer from small sample capacities.
Describe the fundamental difference between ion- exchange and size-exclusion chromatography.
In size-exclusion chromatography separations are based on the size, and to some extent the shape, of molecules with little interactions between the stationary phase and the sample components occurring. In ion-exchange chromatography, in contrast, separations are based on ion-exchange reactions between the stationary phase and the components of the sample in the mobile phase.
What is the effect of pH on the separation of amino acids by electrophoresis? Why?
In solution, amino acids exist as zwitterions that bear both a positive and a negative charge. At low pH values, the net effective charge is positive owing to the interaction of hydrogen ions with the amine groups of the amino acid, whereas at high pH values the net charge is negative due to the dissociation of the carboxylic acid groups. Thus, at low pH values the protein molecules will be strongly attracted toward the negative electrode, while in basic solutions the reverse will be the case.
Describe the differences between single-column and suppressor-column ion chromatography.
In suppressor-column ion chromatography the chromatographic column is followed by a column whose purpose is to convert the ions used for elution to molecular species that are largely nonionic and thus do not interfere with conductometric detection of the analyte species. In single-column ion chromatography, low capacity ion exchangers are used so that the concentrations of ions in the eluting solution can be kept low. Detection then is based on the small differences in conductivity caused by the presence of eluted sample components.
dynamic extraction mode
In the ___ mode of supercritical fluid extraction, the valve between the extraction cell and the restrictor remains open so that the sample is continuously supplied with fresh supercritical fluid and the extraction material flows into the collection vessel where depressurization occurs.
static extraction mode
In the ___ mode, the valve between the extraction cell and the restrictor is closed and the extraction cell is pressurized. After a suitable period, the exit valve is opened and the cell contents are transferred through the restrictor by a dynamic flow of fluid from the pump.
packed-column electrochromatography
In this method, a polar solvent is usually driven by electroosmotic flow through a capillary packed with a reversed-phase HPLC packing.
What are megabore open tubular columns? Why are they used?
Megabore columns are open tubular columns that have a greater inside diameter (530 μm) than typical open tubular columns (150 to 320 μm). Megabore columns can tolerate sample sizes similar to those for packed columns, but with significantly improved performance characteristics. Thus, megabore columns can be used for preparative scale GC purification of mixtures where the compound of interest is to be collected and further analyzed using other analytical techniques.
Describe two types of pumps used in high- performance liquid chromatography. What are the advantages and disadvantages of each?
Pneumatic pumps are simple, inexpensive and pulse free. They consist of a collapsible solvent container housed in a vessel that can be pressurized by a compressed gas. This pump has limited capacity and pressure output and is not adaptable to gradient elution. The pumping rate depends on solvent viscosity. Screw-driven syringe pumps consist of a large syringe in which the piston is moved by a motor-driven screw. They are pulse free and the rate of delivery is easily varied. They suffer from lack of capacity and are inconvenient when solvents must be changed. Reciprocating pumps are versatile and widely used. They consist of a small cylindrical chamber that is filled and then emptied by the back-and-forth motion of a piston. Advantages include small internal volume, high output pressures, adaptability to gradient elution, and constant flow rates that are independent of viscosity and back pressure. The pulsed output must be damped.
Describe the effect of pressure on supercritical fluid chromatography.
Pressure increases the density of a supercritical fluid, which causes the retention factor kfor analytes to change. Generally, increases in pressure reduce the retention times of solutes.
micelles
Spherical aggregates formed from 40-100 surfactant molecules with their hydrocarbon tails in the interior of the aggregate and their charged ends exposed to water on the outside
thin layer chromatography
TLC is a solid-liquid partitioning technique in which the mobile liquid phase ascends a thin layer of absorbent (generally silica, SiO2). The thin layer absorbent acts as a polar stationary phase for the sample to interact with. A very small sample is spotted near the base of the plate and via capillary action, the components of the spotted sample are partitioned between the mobile phase and the stationary phase.
List the variables that lead to band broadening in chromatography.
The variables that lead to band broadening include: (1) large particle diameters for stationary phases; (2) large column diameters; (3) high temperatures (important only in gas chromatography); (4) for liquid stationary phases, thick layers of the immobilized liquid; and (5) very rapid or very slow flow rates.
What important property of supercritical fluids is related to their densities?
Their ability to dissolve large nonvolatile molecules, such as large n-alkanes and polycyclic aromatic hydrocarbons.
How do the following capillary columns differ? (a) PLOT columns (b) WCOT columns(c) SCOT columns
a) A PLOT column is a porous layer open tubular column, which is also called a support coated open-tubular (SCOT) column. The inner surface of a PLOT column is lined with a thin film of a support material, such as a diatomaceous earth. This type of column holds several times as much stationary phase as does a wall-coated column. (b) A WCOT column is simply a capillary tubing fashioned from fused silica, stainless steel, aluminum, copper, plastic or glass. Its inner walls are coated with a thin layer of the mobile phase.
distribution constant
an equilibrium constant for the distribution of a species between two phases. the equilibrium constant for the distribution of an analyte between two immiscible solvents. It is approximately equal to the ratio of the equilibrium molar concentrations in the two solvents.
RI detector
based on the changes in the refractive index of the solvent that is caused by analyte molecules. the refractive index detector is general rather than selective and responds to the presence of all solutes.
conductivity detector
can be highly sensitive, they are universal for charged species, and as a general rule, they respond in a predictable way to concentration changes. Furthermore, such detectors are simple to operate, inexpensive to construct and maintain, easy to miniaturize, and usually give prolonged, trouble-free service.
stationary phase
in chromatography is a solid or liquid phase that is fixed in place. The mobile phase then passes over or through the stationary phase.
mobile phase
in chromatography is the one that moves over or through an immobilized phase that is fixed in place in a column or on the surface of a flat plate.
eluent-suppressor column
located after the ion-exchange column in ion chromatography. It converts the ionized species used to elute analyte ions to largely undissociated molecules that do not interfere with conductometric detection.
single-column chromatography
low capacity ion exchangers are used so that the concentrations of ions in the eluting solution can be kept low. Detection then is based on the small differences in conductivity caused by the presence of eluted sample components.
selectivity factor
of a column toward two species is given by the equation α = KB/KA, where KB is the distribution constant for the more strongly retained species B and KA is the constant for the less strongly held or more rapidly eluting species A. In chromatography, a=KB/KA, where KB is the distribution constant for a less strongly retained species and KA is the constant for a more strongly retained species.
critical micelle concentration
the level above which surfactant molecules begin to form spherical aggregates made up to 40 to 100 ions with their hydrocarbon tails in the interior of the aggregate and their charged ends exposed to water on the outside.
plate development
the process by which a sample is carried through the station- ary phase by a mobile phase.
electrophoretic mobility
the ratio of the migration rate of an ion to the applied electric field.
capillary isotachophoresis
the sample is injected between two buffers, one containing ions with higher mobility than the analyte ions and the other containing ions with lower mobility than analyte ions.
gas-liquid chromatography
useful for Substances that are somewhat volatile and are thermally stable.
