D. Harvey - Modern Analytical Chemistry (794078), страница 61
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Filtration also can be used to isolate analytespresent as solid particulates from dissolved ions in thesample matrix. For example, this is a necessary step ingravimetry, in which the analyte is isolated as a precipitate. A more detailed description of the types of availablefilters is found in the discussion of precipitationgravimetry and particulate gravimetry in Chapter 8.Table 7.4Classification of SeparationTechniquesBasis of SeparationSeparation Techniquesizefiltrationdialysissize-exclusion chromatographycentrifugationmaskingdistillationsublimationrecrystallizationprecipitationion exchangeelectrodepositionvolatilizationextractionchromatographymass and densitycomplex formationchange in physical statechange in chemical statepartitioning between phases1400-CH07 9/8/99 4:03 PM Page 206206Modern Analytical ChemistryFigure 7.11Illustration of a dialysis membrane in action.In (a) the sample solution is placed in thedialysis tube and submerged in the solvent.(b) Smaller particles pass through themembrane, but larger particles remainwithin the dialysis tube.dialysisA method of separation that uses a semipermeable membrane.size-exclusion chromatographyA separation method in which a mixturepasses through a bed of porous particles,with smaller particles taking longer topass through the bed due to their abilityto move into the porous structure.(a)(b)Another example of a separation technique based on size is dialysis, in which asemipermeable membrane is used to separate the analyte and interferent.
Dialysismembranes are usually constructed from cellulose, with pore sizes of 1–5 nm. Thesample is placed inside a bag or tube constructed from the membrane. The dialysismembrane and sample are then placed in a container filled with a solution whosecomposition differs from that of the sample. If the concentration of a particularspecies is not the same on the two sides of the membrane, the resulting concentration gradient provides a driving force for its diffusion across the membrane.
Although small particles may freely pass through the membrane, larger particles areunable to pass (Figure 7.11). Dialysis is frequently used to purify proteins, hormones,and enzymes. During kidney dialysis, metabolic waste products, such as urea, uricacid, and creatinine, are removed from blood by passing it over a dialysis membrane.Size-exclusion chromatography, which also is called gel permeation or molecularexclusion chromatography, is a third example of a separation technique based onsize. In this technique a column is packed with small, approximately 10-µm, porousparticles of cross-linked dextrin or polyacrylamide. The pore size of the particles iscontrolled by the degree of cross-linking, with greater cross-linking resulting insmaller pore sizes. The sample to be separated is placed into a stream of solvent thatis pumped through the column at a fixed flow rate.
Particles too large to enter thepores are not retained and pass through the column at the same rate as the solvent.Those particles capable of entering into the pore structure take longer to passthrough the column. Smaller particles, which penetrate more deeply into the porestructure, take the longest time to pass through the column. Size-exclusion chromatography is widely used in the analysis of polymers and in biochemistry, where itis used for the separation of proteins.7F.2 Separations Based on Mass or DensityIf there is a difference in the mass or density of the analyte and interferent, then aseparation using centrifugation may be possible. The sample, as a suspension, isplaced in a centrifuge tube and spun at a high angular velocity (high numbers ofrevolutions per minute, rpm).
Particles experiencing a greater centrifugal force havefaster sedimentation rates and are preferentially pulled toward the bottom of the1400-CH07 9/8/99 4:03 PM Page 207Chapter 7 Obtaining and Preparing Samples for AnalysisTable 7.5207Conditions for the Separation of Selected CellularComponents by CentrifugationComponentseukaryotic cellcell membranes, nucleimitochondria, bacterial cellslysosomes, bacterial membranesribosomesCentrifugal Force(× g)1000400015,00030,000100,000Time(min)5102030180Source: Adapted from Zubay G.
Biochemistry, 2nd ed. Macmillan: New York, 1988, p. 120.centrifuge tube. For particles of equal density the separation is based on mass, withheavier particles having greater sedimentation rates. When the particles are of equalmass, those with the highest density have the greatest sedimentation rate.Centrifugation is of particular importance as a separation technique in biochemistry. As shown in Table 7.5, cellular components can be separated by centrifugation.12For example, lysosomes can be separated from other cellular components by repeateddifferential centrifugation, in which the sample is divided into a solid residue and a solution called the supernatant.
After destroying the cell membranes, the solutionis centrifuged at 15,000 × g (a centrifugal field strength that is 15,000 times thatof the Earth’s gravitational field) for 20 min, leaving a residue of cell membranesand mitochondria. The supernatant is isolated by decanting from the residueand is centrifuged at 30,000 × g for 30 min, leaving a residue of lysosomes.An alternative approach to differential centrifugation is equilibrium–density–gradient centrifugation. The sample is either placed in a solutionwith a preformed density gradient or in a solution that, when centrifuged,forms a density gradient. For example, density gradients can be establishedwith solutions of sucrose or CsCl.
During centrifugation, the sample’s components undergo sedimentation at a rate determined by their centrifugalforce. Because the solution’s density increases toward the bottom of the centrifuge tube, the sedimentation rate for each component decreases as it movestoward the bottom of the centrifuge tube. When a component reaches a position where its density is equal to that of the solution, the centrifugal forcedrops to zero and sedimentation stops. Each component, therefore, is isolatedas a separate band positioned where the density of the component is equal tothe density of the solution.
For example, a mixture of proteins, RNA, andDNA can be separated in this way since their densities are different. A densitygradient from 1.65 g/cm3 to 1.80 g/cm3 is established using CsCl. Proteins, with adensity of less than 1.3 g/cm3 experience no sedimentation, whereas RNA, with adensity of greater than 1.8 g/cm3 collects as a residue at the bottom of the centrifugetube. The DNA, which has a density of approximately 1.7 g/cm3 separates as a bandnear the middle of the centrifuge tube (Figure 7.12).7F.3 Separations Based on Complexation Reactions (Masking)One of the most widely used techniques for preventing an interference is to bind theinterferent as a soluble complex, preventing it from interfering in the analyte’s determination.
This process is known as masking. Technically, masking is not a separationProteinDNARNA(a)(b)Figure 7.12Illustration showing separation byequilibrium–density–gradient centrifugation.The homogeneous mixture in (a) separatesinto three bands (b) after applyingcentrifugal force.maskingA pseudo-separation method in which aspecies is prevented from participating ina chemical reaction by binding it with amasking agent in an unreactive complex.1400-CH07 9/8/99 4:03 PM Page 208208Modern Analytical ChemistryTable 7.6Selected Masking AgentsMasking AgentSpecies Which Can Be MaskedCN–SCN–NH3F–S2O32–tartrateoxalatethioglycolic acidAg, Au, Cd, Co, Cu, Fe, Hg, Mn, Ni, Pd, Pt, ZnAg, Cd, Co, Cu, Fe, Ni, Pd, Pt, ZnAg, Co, Cu, Fe, Pd, PtAl, Co, Cr, Mg, Mn, Sn, ZnAu, Cd, Co, Cu, Fe, Pb, Pd, Pt, SbAl, Ba, Bi, Ca, Ce, Co, Cr, Cu, Fe, Hg, Mn, Pb, Pd, Pt, Sb, Sn, ZnAl, Fe, Mg, Mn, SnCu, Fe, SnSource: Adapted from Meites, L.
Handbook of Analytical Chemistry, McGraw-Hill: New York, 1963.masking agentThe reagent used to bind the species tobe masked in an unreactive complex.technique because the analyte and interferent are never physically separated from eachother. Masking can, however, be considered a pseudo-separation technique, and is included here for that reason. A wide variety of ions and molecules have been used asmasking agents (Table 7.6), and, as a result, selectivity is usually not a problem.13EXAMPLE 7.12Suggest a masking agent for the analysis of Al in the presence of Fe.
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