Chromatography Techniques (5C) - Chem & Physics

1. A mixture of three neutral compounds is separated by normal-phase silica chromatography. The lab observes that increasing ethyl acetate in the mobile phase causes all compounds to elute earlier, with the most polar compound showing the largest decrease in retention time. Which statement is most consistent with this observation?
   Answer: A more polar mobile phase competes with analytes for silica adsorption sites, especially affecting polar analytes This question tests understanding of mobile phase competition in normal-phase chromatography. In normal-phase silica chromatography, the mobile phase and analytes compete for adsorption sites on the polar silica surface. Increasing ethyl acetate (polar component) in the mobile phase enhances this competition, reducing analyte retention. The effect is most pronounced for polar analytes because they rely most heavily on polar interactions with silica - when the mobile phase becomes more competitive for these same interactions, polar compounds show the largest decrease in retention. Choice B incorrectly suggests that polar mobile phase increases adsorption, missing the competitive nature of the interaction. The key principle is that in normal-phase chromatography, mobile phase polarity and analyte retention are inversely related due to competitive adsorption.
2. A mixture is separated by gas chromatography (GC) using an inert carrier gas and a nonpolar stationary phase. Two components have similar polarity but different boiling points: Compound S (bp 80°C) and Compound T (bp 140°C). The oven temperature is held constant at 100°C. Which component is expected to elute first, assuming both are stable and volatile enough for GC?
   Answer: Compound S, because lower boiling point generally corresponds to higher volatility and shorter retention This question tests understanding of gas chromatography separation based on volatility and boiling points. In GC, compounds are separated based on their partitioning between the gas phase and the stationary phase, which is strongly influenced by volatility. At a column temperature of 100°C, Compound S (bp 80°C) will be completely vaporized and highly volatile, spending more time in the gas phase and less time interacting with the stationary phase. Compound T (bp 140°C) will be less volatile at 100°C, condensing more on the stationary phase and being retained longer. Choice A incorrectly relates higher boiling point to higher vapor pressure - compounds with higher boiling points have lower vapor pressures at a given temperature. The key principle is that in GC, lower boiling point compounds are more volatile and elute before higher boiling point compounds.
3. A biochemist separates a mixture of nucleotides by reverse-phase HPLC using a C18 column (nonpolar stationary phase). The mobile phase starts as 95% water/5% acetonitrile (v/v) and is ramped to 60% water/40% acetonitrile over 10 minutes. Two analytes are injected: compound M (more hydrophobic) and compound N (more hydrophilic). Detection is by UV absorbance. Which outcome is most consistent with this method? Assume both compounds are stable and have similar UV response factors.
   Answer: Compound M elutes after compound N, and increasing acetonitrile promotes elution of M This question tests understanding of reverse-phase HPLC separation principles. In reverse-phase chromatography with a C18 column (nonpolar stationary phase), compounds are separated based on their hydrophobicity, with more hydrophobic compounds having stronger interactions with the nonpolar stationary phase. The mobile phase starts highly aqueous (95% water) and becomes more organic (40% acetonitrile), which increases its elution strength for hydrophobic compounds. Compound N, being more hydrophilic, has weaker interactions with the C18 stationary phase and will elute first when the mobile phase is still highly aqueous. Compound M, being more hydrophobic, binds more strongly to the C18 phase and requires a higher proportion of organic solvent (acetonitrile) to disrupt these hydrophobic interactions and promote elution. A common misconception is that hydrophilic compounds bind more strongly to C18 (choice A), which reverses the fundamental principle of reverse-phase chromatography. To predict reverse-phase elution order, remember: hydrophilic compounds elute first, hydrophobic compounds elute later, and increasing organic solvent promotes elution of hydrophobic compounds.
4. A clinical lab uses size-exclusion chromatography (SEC) to separate proteins in a serum sample using a column with porous beads. The sample contains Albumin (66 kDa), IgG (150 kDa), and a small peptide hormone (3 kDa). The mobile phase is aqueous buffer (pH 7.4) at constant flow rate. Which component is expected to elute first?
   Answer: IgG, because the largest protein is excluded from pores and has the shortest path This question tests understanding of size-exclusion chromatography (SEC) separation principles. In SEC, separation is based solely on molecular size, not chemical interactions - larger molecules are excluded from the pores of the gel beads and travel through the column via the void volume between beads. The largest protein, IgG (150 kDa), cannot enter the pores and takes the shortest path through the column, eluting first. Albumin (66 kDa) can partially enter pores and elutes second, while the small peptide (3 kDa) can access most pore volume, taking the longest path and eluting last. Choice A incorrectly reverses the SEC principle - small molecules do not travel fastest but rather slowest due to their ability to explore pore volumes. A key concept for SEC is that elution order is always: large molecules first, small molecules last.
5. A mixture of two neutral compounds is separated on a normal-phase silica column using hexane/ethyl acetate. Compound U has one amide group; Compound V is a hydrocarbon of similar molecular weight. Which statement is most consistent with their elution behavior?
   Answer: Compound V elutes before U because it is less polar and interacts less with silica This question tests understanding of polarity effects in normal-phase chromatography. On silica gel, separation is based on polarity differences - more polar compounds are retained longer due to stronger interactions with the polar silica surface. Compound U with an amide group can form hydrogen bonds with silica and is more polar than Compound V (a hydrocarbon). Therefore, Compound V will interact less with silica and elute first, while Compound U will be retained longer. Choice A incorrectly suggests amides decrease interaction with silica, when they actually increase it through hydrogen bonding. The key principle in normal-phase chromatography is that compounds elute in order of increasing polarity: nonpolar compounds first, polar compounds last.
6. A chemist uses TLC on silica to monitor a reaction converting a nonpolar starting material (SM) to a more polar product (P). The developing solvent is hexane:ethyl acetate = 9:1 (v/v). After development, the solvent front traveled 8.0 cm. The SM spot is at 6.4 cm and the P spot is at 2.4 cm. Based on these results, which action is most consistent with improving TLC separation (greater distance between SM and P) if the spots are too close in a different trial? Assume silica is polar and increasing ethyl acetate increases mobile phase polarity.
   Answer: Decrease the ethyl acetate fraction to reduce overall elution strength and increase retention differences on silica This question tests understanding of how to optimize TLC separation by adjusting mobile phase polarity. The current separation shows the nonpolar starting material (SM) with Rf = 0.80 and the polar product (P) with Rf = 0.30, giving a ΔRf of 0.50. To improve separation (increase the distance between spots), we need to increase the difference in how strongly each compound interacts with the stationary phase. Decreasing the ethyl acetate fraction makes the mobile phase less polar, which reduces its ability to compete with silica for both compounds. This causes both compounds to be retained more strongly, lowering both Rf values, but the effect is more pronounced for the polar product, which already has strong interactions with silica. This increases the relative difference in retention between SM and P, improving their separation on the plate. A common misconception is that increasing mobile phase polarity improves separation (choice A), but this would cause both spots to move up the plate with potentially less differentiation. To optimize TLC separation, remember: decreasing mobile phase polarity in normal-phase TLC increases retention differences between compounds of different polarities.
7. A lab uses anion-exchange chromatography (positively charged stationary phase) to separate two peptides at pH 8.0. Peptide A has net charge −2 at pH 8.0; Peptide B has net charge +1 at pH 8.0. The column is washed with low-salt buffer, then eluted with increasing NaCl. Which outcome is most consistent with this setup?
   Answer: Peptide A binds more strongly and elutes at higher salt because it is negatively charged This question tests understanding of ion-exchange selectivity based on charge. An anion-exchange column has positively charged groups that attract and bind negatively charged species. At pH 8.0, Peptide A with net charge -2 will bind strongly to the positively charged resin, while Peptide B with net charge +1 will be repelled and flow through without binding. During salt gradient elution, Cl- ions compete with Peptide A for binding sites - higher salt concentration is needed to displace the strongly bound Peptide A. Choice A incorrectly identifies the positively charged peptide as binding to the anion exchanger, contradicting the principle of opposite charge attraction. The key principle is that ion-exchange chromatography separates based on charge - only species with charge opposite to the resin will bind.
8. A researcher purifies a small-molecule inhibitor from a crude reaction mixture using silica gel column chromatography (polar stationary phase). The mixture contains four neutral compounds at room temperature: hexane (H), ethyl acetate (EA), acetophenone (AP), and aniline (AN). The column is initially eluted with 100% hexane, then switched to 30% ethyl acetate in hexane (v/v) after 5 column volumes. No acid/base modifiers are used. Which component is expected to elute first under the initial 100% hexane mobile phase conditions? Assume relative polarity increases in the order: H < EA < AP < AN, and that more polar compounds interact more strongly with silica.
   Answer: Hexane (H), because it is least polar and interacts weakest with the polar stationary phase This question tests understanding of normal-phase column chromatography principles, specifically how compound polarity affects elution order. In normal-phase chromatography with a polar stationary phase (silica gel) and nonpolar mobile phase (100% hexane), compounds are separated based on their differential interactions with the stationary phase. Since hexane is the least polar compound in the mixture, it has the weakest interaction with the polar silica gel and will spend the least time adsorbed to the stationary phase. The more polar compounds (EA, AP, and AN) will interact more strongly with silica through hydrogen bonding and dipole-dipole interactions, causing them to be retained longer on the column. A common misconception is that the mobile phase composition determines which compound elutes first (choice B), but in reality, it's the relative affinity for the stationary phase that matters. To predict elution order in normal-phase chromatography, remember: less polar compounds elute first with nonpolar mobile phases, while more polar compounds are retained longer due to stronger interactions with the polar stationary phase.
9. A lab uses TLC on silica to choose a solvent for a future column. In solvent A (hexanes:ethyl acetate 9:1), the target compound has $R_f = 0.05$. In solvent B (hexanes:ethyl acetate 6:4), the target has $R_f = 0.80$. Which choice is most consistent with selecting a solvent system for good column separation of the target from nearby impurities? Assume impurities have similar polarity to the target.
   Answer: Use an intermediate polarity solvent (between A and B) to target a moderate $R_f$ (e.g., ~0.2–0.4) on TLC This question tests solvent selection for column chromatography using TLC data. Optimal column separation requires a solvent where the target has moderate Rf (e.g., 0.2–0.4) to balance retention and resolution from impurities. Solvent A gives too low Rf (0.05, excessive retention), and B too high (0.80, minimal retention), so an intermediate polarity is best. This targets good separation without prolonged run times or co-elution. Choice A fails because very low Rf leads to poor resolution and long runs, not faster separations. For similar optimizations, use TLC Rf to guide column solvents. A strategy is to aim for Rf ~0.3 for the target, adjusting polarity accordingly.
10. A purification uses reverse-phase HPLC with a gradient from 90:10 water:acetonitrile to 10:90 over 15 minutes. A peptide elutes at 12 minutes. Which change is most consistent with causing the peptide to elute earlier (smaller retention time) on the same C18 column? Assume constant flow rate and temperature.
    Answer: Increase the fraction of acetonitrile earlier in the run (make the mobile phase less polar sooner) This question examines methods to adjust retention time in reverse-phase HPLC gradients. In reverse-phase HPLC on C18, increasing the organic fraction (acetonitrile) makes the mobile phase less polar, reducing retention of hydrophobic analytes like peptides. To elute the peptide earlier than 12 minutes, ramp up acetonitrile sooner in the gradient. This change strengthens elution power earlier, shortening retention. Choice B is incorrect because switching to silica (normal-phase) would increase retention for polar peptides, not decrease it, and requires different solvents. For similar optimization, consider how gradient steepness affects elution timing. A strategy is to adjust organic content inversely to desired retention: more organic for faster elution in reverse-phase.
11. A biochemist separates a mixture of proteins using size-exclusion chromatography (SEC) to remove a small-molecule inhibitor from a 150 kDa enzyme. The SEC resin has pores that exclude molecules larger than ~100 kDa. The sample contains: 150 kDa enzyme, 60 kDa contaminant protein, and 300 Da inhibitor. Which component is expected to elute first? Assume no specific binding to the resin and identical buffer conditions for all components.
    Answer: 150 kDa enzyme, because it is excluded from pores and travels the shortest path This question examines separation principles in size-exclusion chromatography based on molecular size. Size-exclusion chromatography separates by size, with larger molecules excluded from resin pores eluting first at the void volume, while smaller ones enter pores and elute later. The resin here excludes >100 kDa, so the 150 kDa enzyme is fully excluded, the 60 kDa protein partially enters, and the 300 Da inhibitor fully enters. Thus, the 150 kDa enzyme elutes first, traveling the shortest path without pore entry. Choice A fails because small molecules like the 300 Da inhibitor elute last in SEC, not first, due to longer paths through pores, not faster diffusion. In similar SEC problems, compare analyte sizes to the exclusion limit. A useful strategy is to visualize elution as inverse to pore accessibility: excluded = early, fully included = late.
12. A student performs normal-phase column chromatography on silica and notices that a band becomes wider (more dispersed) as it travels down the column, reducing resolution between two nearby components. Which explanation is most consistent with chromatographic band broadening? Assume steady flow and no chemical reaction.
    Answer: Longitudinal diffusion and non-ideal mass transfer cause spreading of analyte bands during migration This question explores causes of band broadening in column chromatography. In normal-phase column chromatography, bands broaden due to longitudinal diffusion and non-ideal mass transfer, dispersing analytes and reducing resolution. As the band travels, these effects widen it on the silica column. This explains the observed spreading and poorer separation. Choice D is misleading because broadening occurs in any mobile phase, not just aqueous, from fundamental chromatographic processes. In similar scenarios, identify broadening sources like diffusion. A strategy is to minimize broadening by optimizing flow rate for better efficiency.
13. A lab is purifying a plant alkaloid extract using normal-phase silica gel column chromatography (stationary phase: silica; mobile phase: hexane/ethyl acetate, 80:20 v/v). The mixture contains Compound P (neutral, one phenyl ring), Compound Q (neutral, one phenyl ring + one hydroxyl group), and Compound R (neutral, two hydroxyl groups). The column is run isocratically at room temperature and collected in equal-volume fractions. Which component is expected to elute first under these conditions?
    Answer: Compound P, because it is least polar and is retained least on silica This question tests understanding of normal-phase chromatography principles and compound polarity effects on retention. In normal-phase chromatography, the stationary phase (silica) is polar while the mobile phase (hexane/ethyl acetate) is relatively nonpolar. Compounds interact with silica through hydrogen bonding and dipole interactions - more polar compounds bind more strongly to silica and are retained longer. Since Compound P has no hydroxyl groups, it is the least polar and will have the weakest interactions with silica, causing it to elute first. Compound R with two hydroxyl groups would be most strongly retained and elute last, making choice A incorrect as it contradicts normal-phase principles. A key strategy is to remember that in normal-phase chromatography, elution order follows: least polar (elutes first) to most polar (elutes last).
14. A chemist uses TLC to choose a solvent system for a preparative silica column. In Solvent A, the target compound has $R_f = 0.95$; in Solvent B, $R_f = 0.02$; in Solvent C, $R_f = 0.35$. Which choice is most consistent with selecting a solvent system that will give practical column separation for the target?
    Answer: Solvent C, because a moderate $R_f$ often corresponds to manageable retention and separability This question tests understanding of using TLC to predict column chromatography conditions. An Rf of 0.95 indicates the compound barely interacts with silica and would elute too quickly on a column for practical collection. An Rf of 0.02 indicates excessive retention that would require large volumes of solvent and long elution times. An Rf around 0.35 suggests moderate retention - the compound interacts sufficiently with silica for separation but not so strongly that elution becomes impractical. This moderate Rf typically translates to reasonable retention times and good separation potential on a preparative column. Choice A incorrectly interprets high Rf as strong retention, when it actually indicates weak retention. The key strategy is that TLC Rf values between 0.2-0.4 generally predict optimal column chromatography conditions.
15. A forensic lab uses gas chromatography (GC) to analyze a mixture of volatile solvents on a nonpolar stationary phase. The oven temperature is held constant. Component P has a lower boiling point and weaker intermolecular forces than component Q. Which outcome is most consistent with GC under these conditions? Assume both components are chemically inert and similarly sized.
    Answer: P elutes before Q because it spends more time in the gas phase This question tests retention principles in gas chromatography based on volatility. In GC with a nonpolar stationary phase, retention depends on analyte volatility and interactions; lower boiling point compounds spend more time in the gas phase and elute earlier under isothermal conditions. Component P, with lower boiling point and weaker forces, interacts less with the stationary phase than Q. Therefore, P elutes before Q, as it partitions less into the liquid phase. Choice B is incorrect because low boiling point compounds actually elute earlier, not later, due to higher volatility, not stronger condensation. For similar GC problems, correlate retention with boiling points for nonpolar phases. A key strategy is to consider time in mobile (gas) vs. stationary phase: more volatile = more gas time = earlier elution.
16. A purification uses ion-exchange chromatography to isolate a DNA-binding protein (Protein Z). The column is cation-exchange resin (negatively charged groups on the stationary phase) equilibrated in buffer at pH 7.4. Protein Z has an isoelectric point $pI = 9.2$. A mixture also contains Protein Y with $pI = 5.1$. Which outcome is most consistent with loading the mixture at pH 7.4 and then eluting with increasing NaCl concentration?
    Answer: Protein Z binds to the cation exchanger and elutes at higher salt than Protein Y This question tests understanding of ion-exchange chromatography and the relationship between protein pI and charge. At pH 7.4, proteins with pI > 7.4 carry net positive charge, while those with pI < 7.4 carry net negative charge. Since Protein Z has pI = 9.2, it is positively charged at pH 7.4 and will bind to the negatively charged cation exchanger. Protein Y with pI = 5.1 is negatively charged at pH 7.4 and will not bind, flowing through immediately. When NaCl concentration increases, the salt ions compete with Protein Z for binding sites, eventually eluting it. Choice D incorrectly states that higher pI means more negative charge, which is the opposite of the correct relationship. The key principle is that proteins bind to ion exchangers when their net charge is opposite to the resin's charge.
17. A lab uses TLC on silica to monitor an esterification. The starting alcohol is polar; the ester product is less polar. Using a solvent system of 70:30 hexane/ethyl acetate, which observation is most consistent with successful product formation?
    Answer: A new spot appears with higher $R_f$ than the starting alcohol spot This question tests understanding of TLC monitoring for reaction progress based on polarity changes. In normal-phase TLC on silica, less polar compounds have higher Rf values because they interact less with the polar stationary phase. The esterification converts a polar alcohol (with -OH group) to a less polar ester (with -COOR group). Therefore, successful product formation would show a new spot with higher Rf than the starting alcohol. Choice B incorrectly predicts lower Rf for the less polar product, contradicting normal-phase principles. The key concept is that in normal-phase TLC, decreasing polarity leads to increasing Rf values, making this technique ideal for monitoring reactions that change compound polarity.
18. A student runs paper chromatography of plant pigments. In solvent system 1, chlorophyll b travels 3.0 cm while the solvent front travels 10.0 cm. In solvent system 2 (less polar than system 1), chlorophyll b travels 1.5 cm while the solvent front still travels 10.0 cm. Which statement is most consistent with these results? Assume the stationary phase (paper) is unchanged.
    Answer: Decreasing mobile-phase polarity decreased $R_f$ because the pigment is less soluble in the mobile phase This question assesses how solvent polarity affects Rf in paper chromatography. Paper chromatography uses polar cellulose, where less polar solvents reduce compound solubility in the mobile phase, increasing retention and lowering Rf. Switching to a less polar solvent decreases chlorophyll b's Rf from 0.3 to 0.15, as it partitions more into the stationary phase. This occurs because the pigment is less solvated by the nonpolar mobile phase. Choice B errs by claiming decreased polarity increases Rf, ignoring reduced solvation's effect on retention. For similar experiments, note Rf changes with mobile phase polarity. A transferable strategy is to predict Rf decreases when mobile-stationary polarity mismatch increases for the analyte.
19. Two columns are evaluated for separating a drug and its metabolite using HPLC at the same flow rate and mobile phase composition. Column 1 produces narrow peaks with baseline separation; Column 2 produces broader, partially overlapping peaks. Which statement best describes Column 1 relative to Column 2? Assume equal injection volume and detector settings.
    Answer: Column 1 has higher separation efficiency (greater number of theoretical plates) This question assesses column efficiency in HPLC through peak width and resolution. HPLC efficiency is quantified by theoretical plates, where narrower peaks indicate higher efficiency due to less band broadening and better mass transfer. Column 1 shows narrow, baseline-separated peaks compared to Column 2's broader, overlapping ones under identical conditions. Thus, Column 1 has higher efficiency, enabling better separation. Choice D errs by claiming broader peaks mean more efficiency, confusing broadening with equilibration; actually, broadening reduces resolution. In similar evaluations, compare peak widths at half-height for efficiency. A transferable strategy is to remember that efficiency improves resolution for closely eluting compounds without changing selectivity.
20. A crude reaction mixture is purified by silica column chromatography. The product contains a carboxylic acid; the major impurity is a nonpolar alkane. The mobile phase is 90:10 hexanes:ethyl acetate. Which observation is most consistent with this separation? Assume the acid is not ionized and can hydrogen bond strongly to silica.
    Answer: The alkane elutes in early fractions; the carboxylic acid elutes later (or may require more polar solvent) This question evaluates elution order in normal-phase silica chromatography for compounds of differing polarity. Silica retains polar compounds like carboxylic acids via hydrogen bonding, while nonpolar alkanes elute quickly in nonpolar solvents. With 90:10 hexanes:ethyl acetate, the alkane impurity elutes early, but the acid requires more polar conditions. This follows because the acid's strong silica interactions delay elution. Choice B is incorrect as it claims the acid elutes first due to mobile phase attraction, but polar compounds are retained more on polar stationary phases. In similar purifications, rank compounds by polarity and H-bonding. A useful check is to expect nonpolar first in normal-phase with nonpolar mobile phases.

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