Q: HPLC technique families: Which set correctly lists the major high-performance liquid chromatography modes commonly used in laboratories?

Introduction / Context: HPLC is not one technique but a platform supporting multiple separation mechanisms. Knowing the main HPLC modes guides analysts in matching chemistry to the problem: polarity differences, charge, or size. Given Data / Assumptions: Partition (liquid/liquid, now typically liquid–stationary bonded phase) separates by differential partitioning. Adsorption (liquid/solid, e.g., normal-phase on silica) separates by surface interactions. Ion-exchange separates by electrostatic interactions; size exclusion (gel permeation) separates by hydrodynamic volume. Concept / Approach: HPLC modes correspond to analyte–stationary phase interaction types. Reversed-phase (a partition variant) dominates for small molecules, ion-exchange suits ionic biomolecules, and size exclusion is preferred for polymers and proteins where minimal interaction is desired. Step-by-Step Solution: Enumerate common modes: partition, adsorption, ion-exchange, and size-exclusion.Confirm each option names a legitimate HPLC mode.Choose the comprehensive option listing all. Verification / Alternative check: Method compendia (e.g., pharmacopeias) and HPLC vendor manuals organize columns and applications by precisely these modes. Why Other Options Are Wrong: Options (a), (b), (c) are subsets; none alone is exhaustive. Common Pitfalls: Equating reversed-phase only with HPLC; while dominant, it is one of several modes. Final Answer: all of the above

Q: Reversed-phase HPLC setup: Which pairing correctly describes the polarity of solvent (mobile phase) and column (stationary phase)?

Introduction / Context: Reversed-phase HPLC (RP-HPLC) is the most widely used HPLC mode for small molecules. It inverts the polarity arrangement relative to classical normal-phase chromatography. Given Data / Assumptions: Stationary phase: non-polar (e.g., C18, C8 alkyl-bonded silica). Mobile phase: relatively polar (e.g., water mixed with methanol or acetonitrile). Analyte retention increases with hydrophobicity. Concept / Approach: In RP-HPLC, non-polar analytes interact more strongly with the hydrophobic stationary phase and elute later, while polar analytes elute earlier in polar aqueous-organic mobile phases. This is the opposite of normal-phase, where a polar stationary phase and non-polar mobile phase are used. Step-by-Step Solution: Identify the hallmark of reversed-phase: non-polar stationary phase.Recognize that mobile phases are polar mixtures (water + organic modifier).Select the polar solvent/non-polar column pairing. Verification / Alternative check: Column manufacturer guides specify C18 columns with water–acetonitrile or water–methanol gradients—clear evidence of polar mobile phase and non-polar stationary phase. Why Other Options Are Wrong: Non-polar solvent/polar column describes normal-phase, not reversed-phase. Non-polar/non-polar is not a standard chromatographic mode. “Any of the above” is incorrect because reversed-phase has a specific polarity arrangement. Common Pitfalls: Confusing reversed-phase with hydrophilic interaction liquid chromatography (HILIC), which uses a polar stationary phase but different retention mechanism. Final Answer: polar solvent/non-polar column

Q: Gradient elution in HPLC: How does the solvent composition behave during a gradient program?

Introduction / Context: Gradient elution is a powerful tool to resolve mixtures with a wide range of polarities. By changing the mobile-phase composition over time, one can elute early the weakly retained species and still bring out strongly retained species within the same run. Given Data / Assumptions: Mobile phase is typically a binary mix (e.g., water–acetonitrile or water–methanol). Stronger solvent fraction is increased with time to decrease retention. Gradients can be linear (continuous) or stepwise. Concept / Approach: Retention factor decreases as the proportion of stronger solvent increases. A programmed change in composition improves peak shape, reduces analysis time, and helps elute late-retaining components. Isocratic elution, in contrast, uses constant composition. Step-by-Step Solution: Define gradient elution: time-dependent solvent composition.Recognize acceptable profiles: linear ramps or discrete steps.Select the option stating the composition changes continuously or stepwise. Verification / Alternative check: HPLC system methods show programmed gradients (%B vs time tables), confirming variable composition during the run. Why Other Options Are Wrong: Constant composition defines isocratic, not gradient. “Both” cannot be true since constant and changing are mutually exclusive. “None” is incorrect because (b) is correct. Common Pitfalls: Ignoring dwell volume and gradient delay when transferring methods between instruments; these impact the effective gradient reaching the column. Final Answer: is changed continuously or in a series of steps

Q: Eluent strength in liquid chromatography: What does this parameter primarily measure when comparing mobile phases?

Introduction / Context: Eluent (elution) strength is a practical descriptor in normal-phase and reversed-phase method development. It quantifies how strongly a mobile phase can compete with analyte–stationary phase interactions to move analytes along the column. Given Data / Assumptions: The term is linked to Snyder’s solvent strength concept and eluotropic series. Stronger eluents reduce retention factors (k) and shorten retention times. Comparisons are made for a specific stationary phase chemistry. Concept / Approach: In adsorption-dominated separations, eluent strength reflects the relative adsorption energy of the solvent on the stationary phase surface. A solvent with higher adsorption energy displaces analytes more effectively, weakening analyte adsorption and enhancing elution. Step-by-Step Solution: Relate eluent strength to competitive adsorption.Identify that “adsorption energy” (not bulk absorption, diffusivity, or mixing) is the correct underlying concept.Choose the option that names solvent adsorption energy. Verification / Alternative check: Empirical plots of log k versus solvent strength parameter show predictable retention changes, validating the concept for a given stationary phase. Why Other Options Are Wrong: Absorption energy (b) refers to bulk uptake; chromatography is about surface adsorption in this context. Diffusivity (c) and mixing index (d) affect kinetics and system plumbing, not thermodynamic elution strength. Common Pitfalls: Applying a single solvent strength scale across different stationary phases; values are phase-dependent. Final Answer: solvent adsorption energy

Q: In HPLC system plumbing, how is dwell volume best defined for gradient methods?

Introduction / Context: Dwell volume (also called gradient delay volume) is critical when transferring gradient HPLC methods between instruments. It explains why the same programmed gradient can produce different chromatograms on two systems. Given Data / Assumptions: Solvents are mixed either at a low-pressure proportioning valve (pre-mix) or at a high-pressure mixer. The mixed solvent travels through tubing, mixer, pump heads, and injector before entering the column. The “distance” is measured as a volume because flow rate converts volume to time. Concept / Approach: Dwell volume is defined as the volume from the mixing point to the head of the column. At a given flow, this volume corresponds to a delay (dwell time) before a programmed change in composition reaches the column inlet. Minimizing or matching dwell volumes is key to method transfer. Step-by-Step Solution: Locate the solvent mixing point in the system diagram.Trace the flow path up to the column inlet; integrate all volumes in between.Recognize that the measured physical quantity is volume; the related time is derived from flow rate. Verification / Alternative check: A practical test injects a tracer under a programmed step change in %B and measures the response at the detector to compute dwell volume and corresponding dwell time. Why Other Options Are Wrong: (a) Refers to column void volume, not dwell volume. (b) Is the dwell time (a consequence), not the definition; the question asks for volume. (d) Incorrect because (c) states the accepted definition. Common Pitfalls: Confusing dwell volume with extra-column volume downstream of the column or with the column dead volume (void volume). Final Answer: the volume of the column between the point at which solvents are mixed and the beginning of the column

Q: Compared with traditional low-pressure liquid chromatography (LPLC), which advantages are typically associated with HPLC systems?

Introduction / Context: HPLC modernized liquid chromatography by adding high-pressure pumping, smaller particles, precise detectors, temperature control, and autosamplers. These upgrades expand application range and improve data quality versus traditional low-pressure setups. Given Data / Assumptions: HPLC uses tightly packed, small-particle columns that increase efficiency. Detectors (UV/Vis, fluorescence, MS) provide high sensitivity. Specialty modes (ion-exchange, ion-pair, SEC) handle ionic species and large biomolecules. Concept / Approach: Higher efficiency yields narrower peaks and better resolution, enhancing sensitivity. Columns are reusable with proper conditioning. Modes like ion-exchange and SEC, along with biocompatible hardware and volatile buffers, improve sample recovery for proteins and polymers. Step-by-Step Solution: Assess each option against HPLC capabilities.Confirm that sensitivity, reusability, and applicability to ionic/large analytes are legitimate benefits.Select the comprehensive choice covering all points. Verification / Alternative check: Vendor application notes demonstrate sub-ppm detection and robust column lifetimes across modes, with validated recoveries in bioanalytical methods. Why Other Options Are Wrong: (a), (b), (c) are each true but incomplete; (d) captures the full advantage set posed in the question. Common Pitfalls: Assuming all HPLC modes suit every macromolecule; shear or adsorption can reduce recovery without appropriate column and mobile-phase selection. Final Answer: All of the above

Question 1

Detector characteristics in HPLC Which statement about a refractive index (RI) detector in HPLC is correct under routine analytical conditions?

Accepted Answer

Introduction / Context: Detector choice in HPLC determines sensitivity, selectivity, and compatibility with elution modes. Refractive index (RI) detectors are universal but have specific limitations compared with UV, fluorescence, or MS detectors. Given Data / Assumptions: RI detectors measure differences in refractive index between sample and reference streams. RI signal drifts with temperature and composition changes. Gradient elution changes mobile phase composition during a run. Concept / Approach: Because RI signal is highly sensitive to mobile-phase composition, gradient elution causes large baseline shifts that obscure peaks. Therefore, RI is typically restricted to isocratic runs. UV detectors, in contrast, handle gradients well and are usually more sensitive for chromophoric analytes. Step-by-Step Solution: Identify the operational limitation: composition sensitivity.Relate gradients to composition changes → unstable baseline.Select the option stating isocratic-only compatibility. Verification / Alternative check: Method development guides advise using RI for sugars, polymers, and other non-UV-absorbing solutes in isocratic mode with precise temperature control. Why Other Options Are Wrong: Option a: RI is generally less sensitive than UV/fluorescence.Option c: RI is broadly responsive (near-universal) rather than highly selective; its limitation is sensitivity, not lack of response.Option d: incorrect because option b is true. Common Pitfalls: Attempting gradient methods with RI; neglecting temperature stabilization leading to drift and noise. Final Answer: It can only be used for isocratic elutions (not gradients).

Question 2

Mobile phase selection in HPLC Which statement best reflects what can be used as a mobile phase in high-performance liquid chromatography (HPLC)?

Accepted Answer

Introduction / Context: In HPLC, the mobile phase is a liquid that transports analytes through the stationary phase. Proper solvent selection is critical for solubility, chromatographic retention, peak shape, detector compatibility, and safety. Given Data / Assumptions: Common mobile phases include water, methanol, acetonitrile, buffers, and their mixtures. Analytes must dissolve to be injected without precipitation in the system. Solvent must be compatible with the stationary phase and detector (e.g., UV transparency for UV detection). Concept / Approach: The correct principle is that the mobile phase must be a liquid (or mixture) that dissolves the analyte and works with the column and detection method. Insoluble or poorly soluble systems will cause blockages, poor recovery, and unreliable chromatography. Step-by-Step Solution: Require adequate analyte solubility in the chosen solvent system.Ensure compatibility with bonded phase (e.g., avoid solvents that strip ligands) and equipment seals.Consider detector needs (e.g., low UV cutoff for UV detection).Select the option stating these conditions. Verification / Alternative check: Method validation often includes solubility screens and UV cutoff checks; successful runs confirm the practical solvent compatibility requirement. Why Other Options Are Wrong: Limited or zero solubility (options b and c) are incompatible with HPLC operation.“Any of the above” (option d) incorrectly implies insoluble systems are acceptable. Common Pitfalls: Choosing mobile phases with high UV absorbance for UV detection; ignoring buffer volatility for MS; exceeding pressure limits with viscous solvents. Final Answer: Any liquid (or liquid mixture) in which the analyte is sufficiently soluble and which is compatible with the column and detector.

Question 3

Chromatography terminology — void volume In column liquid chromatography, the term “void volume” most commonly refers to which volume?

Accepted Answer

Introduction / Context: Understanding void volume (often denoted Vm or V0) is important for calculating retention factors, estimating dead time, and interpreting unretained marker peaks in HPLC and other column methods. Given Data / Assumptions: Packed columns contain solid particles (stationary phase) that displace volume. Mobile phase occupies the interstitial (interparticle) spaces that allow flow. Unretained compounds elute at roughly the void time corresponding to this volume. Concept / Approach: Void volume is the volume of mobile phase inside the packed bed that flows through the interparticle channels (and, for some modes like size-exclusion, corresponds to the volume outside porous beads). It is central to retention factor calculations: k = (tR − t0) / t0 where t0 is the void time. Step-by-Step Solution: Recognize that packing occupies part of the column volume.Define void volume as the volume available to the flowing liquid phase within the bed.Select the option that explicitly describes this interparticle eluent volume. Verification / Alternative check: Inject an unretained tracer (e.g., uracil in RP-HPLC) and compute V0 from t0 and flow rate; this matches the interparticle mobile-phase volume. Why Other Options Are Wrong: Option b includes reservoirs and tubing, which are outside the packed bed definition.Option c is a time, not a volume.Option d refers only to extra-column volume ahead of the column, not the bed's void volume. Common Pitfalls: Confusing extra-column volumes (mixers, tubing) with true column void volume; misusing t0 from poorly chosen unretained markers. Final Answer: The total volume of eluent (mobile phase) occupying the interparticle space in the packed column, the remainder being occupied by the packing material.

Question 4

In analytical chemistry, HPLC stands for what full form used to describe modern high-resolution liquid chromatography?

Accepted Answer

Introduction / Context: High-performance liquid chromatography (HPLC) is a cornerstone separation technique in pharmaceutical analysis, biochemistry, food testing, and environmental monitoring. The question checks whether you know the correct expansion of the acronym and, by extension, that HPLC denotes an instrumented, high-pressure, high-resolution form of liquid chromatography. Given Data / Assumptions: HPLC uses high-pressure pumps, a packed column, a controlled mobile phase, and sensitive detectors. Historically, some sources used “high-pressure” early on; the standardized modern term is “high-performance.” The context is routine analytical chemistry terminology. Concept / Approach: The accepted, formal name emphasizes performance—resolution, speed, sensitivity—not merely the pressure used. While high pressure is indeed involved (typically 100–400 bar for conventional HPLC and higher for UHPLC), the field recognized that performance captures the broader advantage: small particle columns, precise gradients, and superior reproducibility. Step-by-Step Solution: Identify the most widely accepted expansion used in academic texts and instrument manuals.Acknowledge the historical “high-pressure” phrasing but prioritize the standardized term.Select the option that reflects current consensus usage in laboratories and textbooks. Verification / Alternative check: Check any modern chromatography textbook or chromatography system software splash screen; “High Performance Liquid Chromatography” appears universally. UHPLC (ultra-high-performance) further underlines the performance-centric nomenclature. Why Other Options Are Wrong: High Pressure Liquid Chromatography: historic/colloquial, but not the preferred standard term. Both (a) and (b): implies equivalence today; current best practice prefers “performance.” Highly Placed Liquid Chromatography: not a recognized term. Common Pitfalls: Believing pressure alone defines HPLC; in reality, column technology, particle size, and detector performance are equally important. Final Answer: High Performance Liquid Chromatography

Question 5

In chromatography theory, what does an eluotropic series describe when selecting solvents for a given adsorbent or stationary phase?

Accepted Answer

Introduction / Context: An eluotropic series is a practical guide used by chromatographers to choose mobile phases. It orders solvents by their eluting power on a particular stationary phase, which is crucial for method development and troubleshooting. Given Data / Assumptions: The stationary phase (e.g., silica) is fixed. Solvents can be ordered from weak (low elution strength) to strong (high elution strength). The series is adsorbent-dependent (ranking changes with the stationary phase chemistry). Concept / Approach: Eluting power reflects how effectively a solvent competes with analyte–adsorbent interactions. A stronger solvent displaces analytes more readily, reducing retention and speeding elution. The eluotropic series captures this in a ranked list (e.g., for silica, hexane < toluene < dichloromethane < ethyl acetate < acetone < acetonitrile < methanol < water). Step-by-Step Solution: Identify what is being ranked: solvents, not the column material.Define the criterion: relative ability to displace solutes from the stationary phase.Select the option that explicitly states this ranking role. Verification / Alternative check: Method development guides show solvent switches along an eluotropic series to tune retention times and resolution. Empirical retention changes confirm the rankings. Why Other Options Are Wrong: Ranking column packing (b) confuses stationary phase selection with solvent choice. “Measure of solvent adsorption energy” (c) is not the standard definition; the series is empirical and contextual. None of the above (d) is incorrect because (a) defines it correctly. Common Pitfalls: Assuming one universal series; rankings depend on the stationary phase and sometimes on temperature or additive content. Final Answer: ranks solvents by their relative abilities to displace solutes from a given absorbent

Question 6

HPLC technique families: Which set correctly lists the major high-performance liquid chromatography modes commonly used in laboratories?

Accepted Answer

Introduction / Context: HPLC is not one technique but a platform supporting multiple separation mechanisms. Knowing the main HPLC modes guides analysts in matching chemistry to the problem: polarity differences, charge, or size. Given Data / Assumptions: Partition (liquid/liquid, now typically liquid–stationary bonded phase) separates by differential partitioning. Adsorption (liquid/solid, e.g., normal-phase on silica) separates by surface interactions. Ion-exchange separates by electrostatic interactions; size exclusion (gel permeation) separates by hydrodynamic volume. Concept / Approach: HPLC modes correspond to analyte–stationary phase interaction types. Reversed-phase (a partition variant) dominates for small molecules, ion-exchange suits ionic biomolecules, and size exclusion is preferred for polymers and proteins where minimal interaction is desired. Step-by-Step Solution: Enumerate common modes: partition, adsorption, ion-exchange, and size-exclusion.Confirm each option names a legitimate HPLC mode.Choose the comprehensive option listing all. Verification / Alternative check: Method compendia (e.g., pharmacopeias) and HPLC vendor manuals organize columns and applications by precisely these modes. Why Other Options Are Wrong: Options (a), (b), (c) are subsets; none alone is exhaustive. Common Pitfalls: Equating reversed-phase only with HPLC; while dominant, it is one of several modes. Final Answer: all of the above

Question 7

Reversed-phase HPLC setup: Which pairing correctly describes the polarity of solvent (mobile phase) and column (stationary phase)?

Accepted Answer

Introduction / Context: Reversed-phase HPLC (RP-HPLC) is the most widely used HPLC mode for small molecules. It inverts the polarity arrangement relative to classical normal-phase chromatography. Given Data / Assumptions: Stationary phase: non-polar (e.g., C18, C8 alkyl-bonded silica). Mobile phase: relatively polar (e.g., water mixed with methanol or acetonitrile). Analyte retention increases with hydrophobicity. Concept / Approach: In RP-HPLC, non-polar analytes interact more strongly with the hydrophobic stationary phase and elute later, while polar analytes elute earlier in polar aqueous-organic mobile phases. This is the opposite of normal-phase, where a polar stationary phase and non-polar mobile phase are used. Step-by-Step Solution: Identify the hallmark of reversed-phase: non-polar stationary phase.Recognize that mobile phases are polar mixtures (water + organic modifier).Select the polar solvent/non-polar column pairing. Verification / Alternative check: Column manufacturer guides specify C18 columns with water–acetonitrile or water–methanol gradients—clear evidence of polar mobile phase and non-polar stationary phase. Why Other Options Are Wrong: Non-polar solvent/polar column describes normal-phase, not reversed-phase. Non-polar/non-polar is not a standard chromatographic mode. “Any of the above” is incorrect because reversed-phase has a specific polarity arrangement. Common Pitfalls: Confusing reversed-phase with hydrophilic interaction liquid chromatography (HILIC), which uses a polar stationary phase but different retention mechanism. Final Answer: polar solvent/non-polar column

Question 8

Gradient elution in HPLC: How does the solvent composition behave during a gradient program?

Accepted Answer

Introduction / Context: Gradient elution is a powerful tool to resolve mixtures with a wide range of polarities. By changing the mobile-phase composition over time, one can elute early the weakly retained species and still bring out strongly retained species within the same run. Given Data / Assumptions: Mobile phase is typically a binary mix (e.g., water–acetonitrile or water–methanol). Stronger solvent fraction is increased with time to decrease retention. Gradients can be linear (continuous) or stepwise. Concept / Approach: Retention factor decreases as the proportion of stronger solvent increases. A programmed change in composition improves peak shape, reduces analysis time, and helps elute late-retaining components. Isocratic elution, in contrast, uses constant composition. Step-by-Step Solution: Define gradient elution: time-dependent solvent composition.Recognize acceptable profiles: linear ramps or discrete steps.Select the option stating the composition changes continuously or stepwise. Verification / Alternative check: HPLC system methods show programmed gradients (%B vs time tables), confirming variable composition during the run. Why Other Options Are Wrong: Constant composition defines isocratic, not gradient. “Both” cannot be true since constant and changing are mutually exclusive. “None” is incorrect because (b) is correct. Common Pitfalls: Ignoring dwell volume and gradient delay when transferring methods between instruments; these impact the effective gradient reaching the column. Final Answer: is changed continuously or in a series of steps

Question 9

Eluent strength in liquid chromatography: What does this parameter primarily measure when comparing mobile phases?

Accepted Answer

Introduction / Context: Eluent (elution) strength is a practical descriptor in normal-phase and reversed-phase method development. It quantifies how strongly a mobile phase can compete with analyte–stationary phase interactions to move analytes along the column. Given Data / Assumptions: The term is linked to Snyder’s solvent strength concept and eluotropic series. Stronger eluents reduce retention factors (k) and shorten retention times. Comparisons are made for a specific stationary phase chemistry. Concept / Approach: In adsorption-dominated separations, eluent strength reflects the relative adsorption energy of the solvent on the stationary phase surface. A solvent with higher adsorption energy displaces analytes more effectively, weakening analyte adsorption and enhancing elution. Step-by-Step Solution: Relate eluent strength to competitive adsorption.Identify that “adsorption energy” (not bulk absorption, diffusivity, or mixing) is the correct underlying concept.Choose the option that names solvent adsorption energy. Verification / Alternative check: Empirical plots of log k versus solvent strength parameter show predictable retention changes, validating the concept for a given stationary phase. Why Other Options Are Wrong: Absorption energy (b) refers to bulk uptake; chromatography is about surface adsorption in this context. Diffusivity (c) and mixing index (d) affect kinetics and system plumbing, not thermodynamic elution strength. Common Pitfalls: Applying a single solvent strength scale across different stationary phases; values are phase-dependent. Final Answer: solvent adsorption energy

Question 10

In HPLC system plumbing, how is dwell volume best defined for gradient methods?

Accepted Answer

Introduction / Context: Dwell volume (also called gradient delay volume) is critical when transferring gradient HPLC methods between instruments. It explains why the same programmed gradient can produce different chromatograms on two systems. Given Data / Assumptions: Solvents are mixed either at a low-pressure proportioning valve (pre-mix) or at a high-pressure mixer. The mixed solvent travels through tubing, mixer, pump heads, and injector before entering the column. The “distance” is measured as a volume because flow rate converts volume to time. Concept / Approach: Dwell volume is defined as the volume from the mixing point to the head of the column. At a given flow, this volume corresponds to a delay (dwell time) before a programmed change in composition reaches the column inlet. Minimizing or matching dwell volumes is key to method transfer. Step-by-Step Solution: Locate the solvent mixing point in the system diagram.Trace the flow path up to the column inlet; integrate all volumes in between.Recognize that the measured physical quantity is volume; the related time is derived from flow rate. Verification / Alternative check: A practical test injects a tracer under a programmed step change in %B and measures the response at the detector to compute dwell volume and corresponding dwell time. Why Other Options Are Wrong: (a) Refers to column void volume, not dwell volume. (b) Is the dwell time (a consequence), not the definition; the question asks for volume. (d) Incorrect because (c) states the accepted definition. Common Pitfalls: Confusing dwell volume with extra-column volume downstream of the column or with the column dead volume (void volume). Final Answer: the volume of the column between the point at which solvents are mixed and the beginning of the column

Question 11

Compared with traditional low-pressure liquid chromatography (LPLC), which advantages are typically associated with HPLC systems?

Accepted Answer

Introduction / Context: HPLC modernized liquid chromatography by adding high-pressure pumping, smaller particles, precise detectors, temperature control, and autosamplers. These upgrades expand application range and improve data quality versus traditional low-pressure setups. Given Data / Assumptions: HPLC uses tightly packed, small-particle columns that increase efficiency. Detectors (UV/Vis, fluorescence, MS) provide high sensitivity. Specialty modes (ion-exchange, ion-pair, SEC) handle ionic species and large biomolecules. Concept / Approach: Higher efficiency yields narrower peaks and better resolution, enhancing sensitivity. Columns are reusable with proper conditioning. Modes like ion-exchange and SEC, along with biocompatible hardware and volatile buffers, improve sample recovery for proteins and polymers. Step-by-Step Solution: Assess each option against HPLC capabilities.Confirm that sensitivity, reusability, and applicability to ionic/large analytes are legitimate benefits.Select the comprehensive choice covering all points. Verification / Alternative check: Vendor application notes demonstrate sub-ppm detection and robust column lifetimes across modes, with validated recoveries in bioanalytical methods. Why Other Options Are Wrong: (a), (b), (c) are each true but incomplete; (d) captures the full advantage set posed in the question. Common Pitfalls: Assuming all HPLC modes suit every macromolecule; shear or adsorption can reduce recovery without appropriate column and mobile-phase selection. Final Answer: All of the above

Question 12

Column efficiency metric: The number of theoretical plates (N) relates to peak width in what way for a given retention time measurement?

Accepted Answer

Introduction / Context: Theoretical plates offer a convenient way to quantify column efficiency. Narrower peaks indicate higher efficiency. In HPLC, N is commonly calculated using peak width at base or at half-height. Given Data / Assumptions: Formulas: N = 16*(tR/Wb)^2 or N = 5.54*(tR/W1/2)^2. tR is retention time; Wb is peak width at the base; W1/2 is width at half-height. Peak shape is assumed approximately Gaussian for these relationships. Concept / Approach: Because width appears in the denominator and is squared, N increases as peak width decreases. This captures the intuitive idea that sharper peaks reflect better separation efficiency. Step-by-Step Solution: Start from N = k*(tR/W)^2 where k is 16 or 5.54 depending on definition.Recognize the inverse-square dependence on W.Conclude that N is inversely proportional to the square of peak width. Verification / Alternative check: Software in chromatographic data systems reports higher N values for the same tR when the measured W is smaller, confirming the mathematical relationship. Why Other Options Are Wrong: Direct proportionality to W^2 contradicts the formula. Cube-root dependence is not part of plate number equations. Options (b) and (d) repeat the same incorrect trend. Common Pitfalls: Mixing up base width and half-height width; use the correct constant and width definition consistently. Final Answer: inversely related to the square of the peak width

Question 13

Isocratic elution in HPLC: During an isocratic method, how does the solvent composition behave over the course of the run?

Accepted Answer

Introduction / Context: Chromatographic methods are broadly categorized as isocratic or gradient. Understanding the distinction is essential for method development, robustness studies, and system suitability testing. Given Data / Assumptions: Isocratic elution: fixed % of solvent components throughout the run. Gradient elution: changing composition with time (continuous or stepwise). Detector and column conditions remain comparable between runs. Concept / Approach: In an isocratic method, the mobile phase composition is constant. This yields steady-state retention factors and often simpler validation. It suits samples with a narrow range of polarities; complex mixtures may require gradients for acceptable analysis time and peak shape. Step-by-Step Solution: Define isocratic: constant composition (%B and %A do not change with time).Contrast with gradient: composition changes over time to elute late-retaining analytes.Select the option stating that composition remains constant. Verification / Alternative check: Instrument method tables for isocratic runs list single fixed percentages for mobile-phase components rather than time-programmed changes. Why Other Options Are Wrong: Changing continuously or stepwise are definitions of gradient elution. “None of these” is incorrect because (a) is correct. Common Pitfalls: Forgetting that temperature shifts can mimic gradient-like effects by altering solvent strength; nonetheless, composition is still constant by definition. Final Answer: remains constant

Question 14

Analytical chemistry — In normal-phase HPLC (high-performance liquid chromatography), which pairing best describes the mobile phase and the stationary phase (column) used?

Accepted Answer

Introduction / Context: Normal-phase HPLC is a classic chromatographic mode that separates analytes primarily by polarity using a polar stationary phase and a relatively non-polar mobile phase. Knowing how phases are paired is essential for choosing solvents, predicting elution order, and troubleshooting retention problems. Given Data / Assumptions: The question asks about normal-phase HPLC configuration. Stationary phase: typically polar (e.g., bare silica with surface silanols). Mobile phase: typically non-polar or weakly polar organic solvents (e.g., hexane with small percentages of isopropanol or ethyl acetate modifiers). Concept / Approach: In normal-phase mode, polar analytes interact strongly with the polar stationary phase and elute later. Less polar analytes spend more time in the non-polar mobile phase and elute earlier. This is the inverse of reversed-phase HPLC, where a non-polar stationary phase is paired with a polar (water-rich) mobile phase. Step-by-Step Solution: Identify normal-phase hallmark: polar stationary phase (silica, alumina).Match the mobile phase: non-polar solvent (e.g., hexane) sometimes with polar modifiers to tune retention.Predict retention: more polar analytes retained longer; less polar elute sooner.Select the configuration that states non-polar solvent / polar column. Verification / Alternative check: Method development guides differentiate normal-phase (polar stationary, non-polar mobile) from reversed-phase (non-polar stationary, polar mobile). Typical solvent systems for normal phase confirm the non-polar mobile phase choice. Why Other Options Are Wrong: Polar solvent / non-polar column describes reversed-phase, not normal-phase. Non-polar solvent / non-polar column does not create useful polarity-based separation. Any of the above is incorrect because normal-phase has a specific pairing. Polar solvent / polar column would lead to very strong retention and is not the standard definition. Common Pitfalls: Confusing normal-phase with reversed-phase; assuming “normal” means water-based. In chromatography, “normal” historically refers to the early silica-based method, not to aqueous systems. Final Answer: non-polar solvent / polar column

Question 15

Chromatography materials — For silica gel used as an HPLC/flash adsorbent, which pore diameters are most commonly selected for general separations?

Accepted Answer

Introduction / Context: Pore size is a critical property of silica gel that affects surface area, mass transfer, and suitability for separating molecules of different sizes. In preparative chromatography and many analytical applications, standard pore sizes are used for robustness and broad applicability. Given Data / Assumptions: Spherical/irregular silica gels are available in multiple pore sizes (measured in Å). General organic separations (small molecules) prefer mid-range pores for a good balance of surface area and flow. Very small or very large pores are specialty options. Concept / Approach: Silica with ~60 Å or ~100 Å pores is widely adopted for small-molecule separations because it offers high surface area without excessively slow diffusion. Larger pores (e.g., 300 Å) are used for peptides/proteins due to size-exclusion considerations, not typical small-molecule work. Step-by-Step Solution: Identify the mainstream “general purpose” pore sizes.Recall common catalog offerings: 60 Å and 100 Å dominate for small molecules.Eliminate extremes (10–50 Å too small; ≥150 Å for macromolecules).Select 60 and 100 Å as the most popular choices. Verification / Alternative check: Manufacturer datasheets and methods frequently recommend 60 Å or 100 Å silica for routine reversed-phase bonding (after derivatization) or normal-phase adsorption columns. Why Other Options Are Wrong: 10 and 50 Å: too small for many applications; less common for general work. 100 and 150 Å: 150 Å skews toward larger molecules; not the most common pair. 150 and 200 Å: typically for large biomolecules. 20 and 30 Å: uncommon for standard workflows. Common Pitfalls: Assuming larger pores are always better; they reduce surface area and change retention, which can harm resolution for small molecules. Final Answer: 60 and 100 Å

HPLC Questions