Karteikarten
Module 6: Chromatographic and Electrophoretic Methods
Karten in diesem Stapel (107)
- 1what are some limitations with simple separations ?analyte in matrix may not be compatible with method, multiple analyses need multiple extractions (more time, money), when distribution ratio of all sp. are similar, hard to sep.
- 2to separate an analyte from its matrix, the distribution ratio, D, of the analyte must be greater than all other sp.fff
- 3more efficient way to separate mixturesby first extracting the analyte into the extracting phase and then extracting again into a fresh portion of the initial phase
- 4counter current extractionresults in more efficient extractions than the conventional approach. basis of modern chrom. techniques.
- 5mobile phasethe extracting phase that moves through the system
- 6stationary phasethe extracting phase that remains in a fixed position
- 7those components whose distribution ratio ____ the stationary phase require a ___ time to pass through the systemfavours, longer
- 8analytical separations can be classified in 3 ways:physical state of of M,S phase, method of contact bw MP and SP, c/p mechanism responsible for separation
- 9mobiles phase can be a...gas, liquid, or supercritical fluid
- 10stationary phase can be a....solid, liquid film coated on a solid surface
- 11explain column chromatographySP is placed in a narrow column through which the MP moves under the influence of gravity or pressure. SP is either solid or a thin, liquid film coating on a solid particulate packing material on column's wall
- 12explain planar chromatographySP coats a flat glass, metal of plastic plate which is placed in a developing chamber. reservoir containing mp placed in contact with sp and the mp moves up by capillary action
- 13adsorption chromatographysolutes separates based on their ability to adsorb on the surface of the solid particles; more stronger a solute is adsorbed, the slower it travels through column
- 14what are the difference between adsorption and absorption?adsorption is a surface phenomenon, exothermic process, favoured by low temp
- 15partition chromatographythin liquid film coating a solid surface serves as the SP; separation is based on a difference in equilibrium partitioning of solutes bw the liquid SP and MP
- 16ion exchange chromatographySP consisting of solid support with covalently attached anionic or cationic functional groups; ionic solutes of opposite charge are attracted to the SP by electrostatic forces
- 17molecular exclusion chromatographyporous gels are used as SP and separation based on size of solutes; large solutes unable to penetrate into porous sp = elute quicker than small ones.
- 18electrophoretic chromatographyno sp, charged solutes migrate under the influence of an applied potential field; difference in ion mobility of ion account for their separation
- 19theory of column chromatographythe strength of each solutes interaction w/ SP is sufficiently different and results in separation of the analyses onto 2 ind. bands
- 20what does a chromatogram do?displays a peak for each of the separated solute bands
- 21retention time (tr)time it takes for a solute to move from point of injection to the detector; can also be described as the retention volume.
- 22retention volume (Vr)volume of mobile phase needed to move a solute from its point of injection to the detector
- 23baseline widthwidth of solutes chromatographic band measured at the baseline;
- 24how is the baseline width determined?intersection with the baseline of tangent lines drawn through the inflection points on either side of the chromatographic peak; measured in units of time/vol.
- 25void time (Tm)time required for an unretained solute to move from the point of injection to the detector; implies that solute does not interact with sp; can also be described as void volume
- 26void volumevolume of mp needed to move an unretained solute from the point of injection to the detector
- 27goal of chromatographygoal of chromatography is to separate a sample into a series of chromatographic peaks, each one representing a single component of all the analytes in the sample
- 28chromatographic resolution (R)a quantitative measure of the degree of separation bw 2- chrom. peaks, A and B
- 29resolution equationR = 2(change in Tr)/ second - first width
- 30ways to improve Rincreasing change in tr; deacreasing w1 or w2 or both
- 31how to increase change in retention time?enhancing the interaction of the solutes with column; increasing the column's selectivity for one of the solutes
- 32how to decrease w1, w2?peak width is a kinetic effect associated with the solutes movement w/in and bw the MP and SP; governed by column efficiency.
- 33factors affecting widthcapacity factor, column selectivity and column efficiency
- 34capacity factor (k')provides a measure of how strongly a solute is retained on the SP. k' = time solute spends in SP/time solute spends in MP; if solute spends all of its time in the MP it would elute at time tm
- 35capacity selectivity (a)the relative selectivity of a chromatographic column for a pair of analytes is expressed by the selectivity factor, a; k'a/k'b
- 36what does it mean when capacity selectivity = 1then 2 solutes have identical elution times
- 37band broadeningincrease in a solute's baseline width as it moves from the point of injection to the detector
- 38column efficiencyused to quantitatively measure the extent of band broadening
- 39theoretical platepartitioning of solute bw MP and SP; do not actually exists but provides a quantitative measure of efficiency
- 40number of theoretical plates, N, =Length of column / Height of plate
- 41smaller plate height (mm/plates) means...more plates on column, narrower chromatographic peaks and better resolution
- 42peak capacitymaximum number of solutes that can be theoretically resolved (separated) on a particular column is referred to as peak capacity (nc)
- 43frontinga tail at the beginning of a peak, usually caused by injection of too much sample on the column; referred to as column overloading
- 44tailingtail at end of a peak arising when some sites on the SP retain the solutes more strongly than other sites; caused by active sites on the SP which means SP has deteriorated.
- 45gas chromatographysample (gas/liquid) is injected into a stream of an inert gaseous MP; sample is carried through a column where sample components are separated based on their ability to distribute bw MP and SP
- 46the type of column used influences:amount of sample can be handled, efficiency of separation, number of analytes that can be separated, amount of time required for separation.
- 47packed columns for GCfilled with a particulate solid support.
- 48capillary columnsconstructed from silica coated with a protected polymer; two types: wall-coated and support-coated open tubular columns
- 49wall-coated open tubular columns (WCOT)SP typically 0.25 um thick, coated on capillary's inner walls
- 50SCOTthin film layer of a solid support such as diatomaceous earth is coated with a liquid SP is attached to the capillary's inner wall
- 51explain the reasons why capillary columns has a greater separation efficiency relative to packedshorter analysis times, increased sensitivity, lower sample capacity (vol of sample)
- 52main factor that drives GC is ___boiling point
- 532 solutes with similar boiling points can be separated only if the SPselectively interacts with one of the solutes. in general, non polar solutes are more easily separated with a non polar SP and polar solutes easier with polar SPs
- 54main criteria for selecting a SP:chemically inert, thermally stable, low volatility, similar polarity to solutes being separated
- 55how to increase/decrease polarity of SPs?replacing some of the methyl groups with other substituents increases the SP polarity.
- 56why is the injection port temperature purposefully elevated to > 250 degrees C?to promote flash vaporization.
- 57three ways to inject samples onto an open tubular columnsplit, splitless, on-column
- 58split injectioninjecting samples onto capillary column in which only a small portion of the sample enters the column; 0.2 - 2% actually gets onto column. rest out of split vent; septum vent always off
- 59splitless injectionallows higher percentage of sample to enter the column; ~80%; septum and split vents always shut
- 60on-column injectiondirect injection of thermally unstable samples onto capillary column; 100%; should not be used all the time, can clog head of column; can get stuck and serve as SP
- 61what are the 5 main detectors for GCsthermal conductivity, flame ionization, electron capture, nitrogen-phosphorous, mass spec.
- 62thermal conductivity detector (TCD)measures ability of a substance to transport heat from a hot region to a cold one;
- 63why does a GC column not need to be packed?gases have different rates of diffusion than liquids, gases are faster.
- 64if we wanted to make the process of partitioning quick in a WCOT GC, how would we do that?make SP thinner, less space to partition into and out of. decrease diameter of column = less space.
- 65if we wanted WCOt to be faster at partitioning, why not make the diameter infinitely smaller and the SP to be infinitely thinner?analyte amount that can enter decreases, takes longer time to process all of sample.
- 66Thermal conductivity detector (TCD)Measures ability of substance to transport heat from a hot region to a cold one
- 67why is He used in TCD?heat transfer takes place at slower rate for materials with low thermal conductivity; He has has 2nd highest thermal conductivity (great ability to remove heat), so any analyte mixed with He lowers the conductivity of the gas stream.
- 68explain the method of TCDcarrier gas containing solute flows over hot tungsten-rhenium filament (3000C)
When a solute flows past filament, the conductivity of the gas stream decreases and so the voltage across filament changes = detector measures this change;
As temp increases = resistance increases = voltage increases - 69pros and cons of TCDUniversal in nature
Large linear dynamic range
Non-destructive
Poor detector limits compared to other detectors - 70How is flame ionization detector used?Combustion of organic compound in H2/air flame results in a flame rich in e and ions
In absence of solute, if a potential of 300V is appliedacross the flame, a small current of 10^-14 A develops
Analytes produce a current ~10^-12 A, which is converted to V and then amplified and converted to an analytical signal - 71pros and cons of FIDDetection limits ~1000x smaller than TCD
Large linear dynamic range
Destructive - 72Electron Capture Detector (ECD)Responds only to compounds with electronegative atoms Consists of a beta-emitter (high energy e-) such as radioactive Ni
The emitted e ionize the MP giving rise to a small electric current; when a solute with a high affinity passes by, they can capture electrons and decrease the electric current → serves as a signal - 73pros and cons of ECDSelective for electronegative compounds, detection limits small for these compounds, poor linear dynamic range, radioactive
- 74nitrogen-Phosphorus Detector (NPD)Modified FID but especially sensitive to compounds containing N and P; its response factor is 10^4 - 10^6 times greater for N/P than its response factor for C
Also a selective detector - 75high performance liquid chromatography (HPLC)uses high pressure to force solvent through closed columns containing fine particles that lead to high-resolution separations
- 76how does particle size affect HPLCdecreasing particle size allows us to improve resolution or to maintain the same resolution but decrease run time
- 77SP of HPLCchemically bonded to the silica surface; a liquid film coated silica packing material can sometimes be slightly soluble in the MP. therefore, should be covalently bonded to liquid film = bonded SP
- 78the properties of the SP can be determined by the nature of the ___organosilane's alkyl group, R
- 79the elution order of solutes in reverse phase chromatography is driven bypolarity with the order of elution starting with the most polar solute. by decreasing the polarity of the MP we can elute more tightly retained solutes
- 80how elution works with reverse phase chromatographyadding more organic solvent which reduced hydrophobic interaction bw the solute and the SP, this results in desorption. the more hydrophobic the solute, the more strongly it will be retained by the sp and the higher amount of organic solvent required to promote desorption
- 81the elution processinvolves solute transfer from a MP into or onto a SP; association of solute with the SP can involve partitioning, adsorption or both;
- 82reverse phase exploitsthe differences in solutes adsorption/desorption and partitioning onto a hydrophobic SP
- 83isocraticperformed with a single solvent (or constant solvent mixture) whose composition remains constant throughout the separation
- 84gradientsteady changes of the solvent composition to increase its non polar strength; analogous to temp. programming in GC
- 85HPLC column dimensions that affect elution processincrease length, increase efficiency; increasing particle size, decreases efficiency; decreasing internal diameter, increases efficiency
- 86smaller particles leads tohigher plate number, higher pressures, shorter run times, lower detection limits
- 87longer columns lead tohigher plate number, longer run times
- 88electrophoresisanalytes are separated based on their ability to move through a conductive medium under the influence of an applied electric field
- 89how does electrophoresis work?cations migrate through a conductive medium toward the electric fields negatively charged cathode. more highly charged and smaller sized ions migrate faster
- 90theory of electrophoresiswhen an EF is applied to a tube ions migrate as a result of two types of mobilities: electrophoretic mobility and electroosmotic mobility
- 91electrophoretic mobilitya measure of a solutes ability to move through a conductive medium in response to an applied EF
- 92electroosmotic mobilityoccurs when the conductive medium moves through a tube in response to applied EF; typically buffered solns move toward the cathode sweeping most analytes even anions toward the -ve charged cathode
- 93electrophoretic mobility equationVep = uep x E
where, uep = solutes electrophoretic mobility; E = magnitude of EF - 94a solutes total ion mobility is the sum of the ____ and _____electrophoretic, electroosmotic flow velocities
- 95why does the peak area made by electrophoresis need to be fixed?analytes w/ different mobilities pass through the detector at different rates; the greater the mobility, the shorter migration time and less time analyte spends in detector. peak area/migration time
- 96explain what happens in the capillary of capillary electrophoresisheat produced which can affect conductivity of medium. solution near centre becomes less viscous than near the walls; results in band broadening that can decrease separation efficiency.
- 97what size of capillary is desirable for CEthe smaller the better bc they generate less heat
- 98injection types of CEhydrodynamic and electrokinetic
- 99hydrodynamic injection type (CE)uses pressure to force a small portion of the sample into the capillary tube
- 100electrokinetic injection type (CE)done by placing both the capillary and the anode into the sample vial and briefly applying an electric field
- 101what kind of power source n capillary electrophoresisneed source that delivers a large EF; ensures shorter analysis times and more efficient separations; voltage up to 40kV can be applied
- 102what type of detectors are used for CEdetectors used for HPLC can be used in capillary electrophoresis
- 103micellar Electrokinetic Capillary Chromatography (MEKC)a form of CE in which neutral analytes are separated based on their ability to partition into a charge micelle
- 104in MEKC, how are the micelles formed?a surfactant like sodium dodecylsulfate is added to buffer solution; at high concentrations this creates micelles; agglomeration of molecules containing ionic heads and hydrophobic tails
- 105capillary cell electrophoresisa form of CE in which the capillary column contains a gel enabling separations based on size
- 106capillary electrochromatographya form of CE in which a SP is included within the capillary column; SP consists of silica particles coated with a bonded non-polar liquid film; partitioning bw SP and buffered solns. occurs resulting in HPLC like separation
- 107how is CE different from HPLC and GCsimilar levels of sensitivity and comparable degree of specificity; smaller injection volumes; higher separation efficiencies, fast analyses time and smaller instrument costs, higher detection limits and poor run-to-run repeatability as drifts in migration times can occur
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- Karteikarten am Module 6: Chromatographic and Electrophoretic Methods, erstellt von angeli Fernandez am 07/11/2022.
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