All Doc Q Flashcards
Polar stationary phase
Column I: Normal Phase Chromatography
Column II: Hydrophilic Interaction Chromatography
Choose:
A. If the phrase applies to column I
B. If the phrase applies to column II
C. If the phrase applies to both columns
D. If the phrase does not apply to columns
Answer: C
Rationale:
Column I: True (NPC uses a polar stationary phase e.g. silica)
Column II: True (HILIC specifically relies on polar SP that interacts with water in the MP)
Nonpolar mobile phase
Column I: Ion-exchange chromatography
Column II: Hydrophilic Interaction Chromatography
Choose:
A. If the phrase applies to column I
B. If the phrase applies to column II
C. If the phrase applies to both columns
D. If the phrase does not apply to columns
Answer: D
Rationale:
Column I: False. IEC commonly uses polar MP, often containing water, to facilitate ionic interactions between the charged molecules and the SP
Column II: False. Similar to column I, HILIC relies on polar MP with a significant amount of water to interact with the polar SP
Measure of chromatographic efficiency
Column I: Rs (Resolution)
Column II: α (alpha factor)
Choose:
A. If the phrase applies to column I
B. If the phrase applies to column II
C. If the phrase applies to both columns
D. If the phrase does not apply to columns
Answer: A
Rationale:
Column I: General parameter used in most chromatography method, including IEC. It can be calculated using retention times and peak widths from the chromatogram
Column II: More commonly used in linear chromatography techniques e.g. RPC and NPC. It might not always be the preferred metric for IEC, which relies on ionic interactions for separation
Measure of chromatographic selectivity
Column I: N
Column II: HETP
Choose:
A. If the phrase applies to column I
B. If the phrase applies to column II
C. If the phrase applies to both columns
D. If the phrase does not apply to columns
Answer: D
Rationale:
Selectivity refers to a column’s ability to distinguish between different components in a mixture based on their interactions with the SP
Columns I and II: metrics used to evaluate column efficiency, not selectivity.
Column I: N reflects how well a column separates bands based on their spreading
Column II: HETP is inversely proportional to N and indicates band broadening within the column
Separation of neutral and cationic molecules
Column I: Cation exchanger
Column II: Anion exchanger
Choose:
A. If the phrase applies to column I
B. If the phrase applies to column II
C. If the phrase applies to both columns
D. If the phrase does not apply to columns
Answer: A
Rationale:
Column I: Cation exchanger has negatively charged FGs on the SP that attract and retain positively charged cations from the sample. Neutral molecules with no charge wouldn’t interact significantly with the cation exchanger and would likely pass through the column unretained.
Column II: Anion exchanger has positively charged FGs that attract and retain negatively charged molecules (anions) from the sample. They wouldn’t specifically interact with neutral molecules
Partitioning as mechanism of separation
Column I: Paper Chromatography
Column II: TLC
Choose:
A. If the phrase applies to column I
B. If the phrase applies to column II
C. If the phrase applies to both columns
D. If the phrase does not apply to columns
Answer: A
Rationale:
Partitioning refers to the distribution of a solute between two immiscible phases based on their relative affinities. In chromatography, this translates to the separation of components in a mixture due to their differing preferences for the mobile and stationary phases.
Column I: The primary separation mechanism in paper chromatography is indeed partitioning. The mobile phase (solvent) and the stationary phase (water held within cellulose fibers) create a system where components partition based on their polarities. More polar components interact more with the water, while less polar components spend more time in the mobile phase, leading to separation.
Column II: While partitioning is also involved in TLC, there can be an additional element of adsorption depending on the specific stationary phase used. Adsorption involves a more direct interaction between the solute molecules and the active sites on the adsorbent (e.g., silica gel).
Mechanism of separation in Size Exclusion Chromatography
Column I: Mechanical Entrapment
Column II: Adsorption to Mechanical Beads
Choose:
A. If the phrase applies to column I
B. If the phrase applies to column II
C. If the phrase applies to both columns
D. If the phrase does not apply to columns
Answer: A
Rationale:
Size Exclusion Chromatography (SEC): SEC separates molecules based on size. Larger molecules are indeed excluded from the pores of the packing material, but the term “mechanical entrapment” needs some refinement.
Column I: Mechanical Entrapment: While not a perfectly accurate term, it can be conceptually helpful to understand the basic principle of SEC. Molecules larger than the pores cannot enter them and are effectively excluded from a significant portion of the stationary phase volume. This “exclusion” can be thought of as a kind of entrapment within the larger channels of the column.
Column II: Adsorption to Mechanical Beads: This is not the primary mechanism in SEC. There might be some weak interactions between the sample and the beads, but they don’t significantly contribute to separation based on size.
Affected by flow rate
Column I: Eddy diffusion
Column II: HETP
Choose:
A. If the phrase applies to column I
B. If the phrase applies to column II
C. If the phrase applies to both columns
D. If the phrase does not apply to columns
Answer: A
Rationale:
Affected by flow rate: Flow rate can influence both phenomena, but to varying degrees.
Column I: Eddy diffusion is directly impacted by flow rate. Higher flow rates lead to greater turbulence and consequently, more significant eddy diffusion, broadening the bands of sample components.
Column II: HETP (Height Equivalent to a Theoretical Plate): a combined measure of various band broadening effects, including eddy diffusion. While flow rate can affect HETP, it’s not the only factor. Other factors like mass transfer and mobile phase diffusion also contribute. The impact of flow rate on HETP can also be complex, with an optimum flow rate for minimizing band broadening.
Can be used to identify compounds
Column I: Retention time
Column II: Capacity Factor
Choose:
A. If the phrase applies to column I
B. If the phrase applies to column II
C. If the phrase applies to both columns
D. If the phrase does not apply to columns
Answer: C
Rationale:
Identification of unknown compounds in a mixture often relies on comparing their properties with known standards. In chromatography, retention time and capacity factor can both be used for this purpose.
Column I: Retention time: Time it takes for a particular compound to elute from the column. By comparing the retention time of an unknown compound to the retention times of known standards under the same chromatographic conditions, tentative identification can be achieved.
Column II: Capacity factor (k’): a parameter calculated from the retention time of the unknown compound and the void volume of the column. It provides a normalized value for the retention time, making it less sensitive to changes in flow rate compared to raw retention time. By comparing the capacity factor of an unknown compound to those of known standards, identification can be aided.
Affected by particles size of stationary phase
Column I: Eddy diffusion
Column II: Resistance to mass transfer
Choose:
A. If the phrase applies to column I
B. If the phrase applies to column II
C. If the phrase applies to both columns
D. If the phrase does not apply to columns
Answer: B
Rationale:
Particles size of stationary phase: Smaller particles generally lead to a higher surface area and potentially more efficient columns.
Column I: Eddy diffusion: While smaller particles can create a more complex flow path and potentially increase eddy diffusion, the impact on resistance to mass transfer might be more significant.
Column II: Resistance to mass transfer: refers to the difficulty molecules encounter when moving between the mobile and stationary phases. The size of the stationary phase particles can influence the thickness of the stagnant mobile phase layer around the particles. Smaller particles generally create a thinner layer, reducing the distance molecules need to diffuse and potentially lowering resistance to mass transfer.
Affects column efficiency
Column I: Flow rate
Column II: Particle shape of stationary phase
Choose:
A. If the phrase applies to column I
B. If the phrase applies to column II
C. If the phrase applies to both columns
D. If the phrase does not apply to columns
C. If the phrase applies to both columns
Rationale:
Flow Rate: Higher flow rates can cause band broadening and decrease column efficiency
Particle shape: Irregularly shaped particles can also lead to band broadening and reduced efficiency
Separation of proteins
Column I: RPLC
Column II: SEC
Choose:
A. If the phrase applies to column I
B. If the phrase applies to column II
C. If the phrase applies to both columns
D. If the phrase does not apply to columns
Answer: C
Rationale:
Proteins: These are complex biomolecules with a variety of sizes and functional groups.
Column I: RPLC (Reversed-Phase Liquid Chromatography): RPLC is a versatile technique widely used for separating proteins. It relies on the interaction between the hydrophobic regions of proteins and the hydrophobic stationary phase. Proteins with more hydrophobic regions bind more strongly to the stationary phase and elute later.
Column II: SEC (Size-Exclusion Chromatography): SEC is another valuable tool for protein separation. It separates molecules based on their size. Larger proteins are excluded from the pores of the stationary phase and elute earlier, while smaller proteins can enter the pores and elute later.
What does the retention time (tR) represent in an HPLC system?
A. The distance between two peaks
B. The time an analyte spends in the stationary phase
C. The distance of the peak maxima from the injection point
D. The flow rate of the mobile phase
C. The distance of the peak maxima from the injection point.
It is expressed in time units
Water is the strongest eluent
Column I: HILIC
Column II: RPLC
Choose:
A. If the phrase applies to column I
B. If the phrase applies to column II
C. If the phrase applies to both columns
D. If the phrase does not apply to columns
Answer: A
Rationale:
Column I: In HILIC, the stationary phase is typically polar, and the mobile phase is relatively non-polar. Water, being highly polar, is used as a strong eluent in HILIC. When water content in the mobile phase is increased, it competes with polar analytes for interactions with the stationary phase, causing the analytes to elute faster. Thus, water is considered the strongest eluent in HILIC because it can disrupt the interactions between the polar stationary phase and the analytes effectively.
Column II: In RPLC, the stationary phase is non-polar, and the mobile phase is polar. Typically, water is used as the weaker component of the mobile phase. Organic solvents like acetonitrile or methanol are added to increase the eluting strength. Therefore, in RPLC, water is not the strongest eluent. Instead, organic solvents are stronger eluents because they reduce the retention of non-polar analytes by decreasing their interactions with the non-polar stationary phase.
This chromatographic parameter is a measure of efficiency.
Plate height
A high value of _____ _____ _____ _____ means high efficiency
number of theoretical plates
_____ is both a measure of selectivity and efficiency
Resolution
To measure the void time a _____ _____ _____ is used.
void volume marker
A higher value of _____ _____ means higher HETP
peak width
Another term for retention factor is _____ _____
capacity factor
_____ is ability of chromatographic system to discriminate different analytes
selectivity
The plot of the detector response vs. time is called a _____
chromatogram
_____ _____ is the distance of the peak maxima from the injection point expressed in time units.
Retention time
The difference between the retention volume and the void volume is called _____ _____ _____
reduced retention time
Polarity: The longer the carbon backbone, the more _____ is the molecule
nonpolar
100% silanol coverage is not possible because C18 exhibits this structural effect
A. Electron donating inductive effects
B. Resonance effects
C. Steric effects
C. steric effects
T/F: The main purpose of endcapping is to cover the residual silanol groups.
True. Silica gel has reactive silanol groups (OH) on its surface. Endcapping replaces these with non-polar groups, reducing unwanted interactions and improving chromatographic performance.
T/F: Generally, anion exchange resins are negatively charged.
False
