Mixed 2 Flashcards
Which of the following statements about the charged groups in IEX stationary phases is correct?
A) They are involved in the exchange process and determine the specificity and strength of analyte binding.
B) They determine whether the stationary phase is an anion exchanger or a cation exchanger.
C) Cation exchangers have positively charged functional groups and possess exchangeable anions.
D) All of the above.
D) All of the above.
True or False: The matrix in IEX stationary phases determines the physical and chemical stability, flow characteristics, and may be responsible for unspecific binding effects.
True
Identify the material that was originally used as a matrix for ion exchange chromatography.
A) Cellulose
B) Dextran
C) Agarose
D) Polystyrene-divinylbenzene copolymers
D) Polystyrene-divinylbenzene copolymers
Which of the following statements about the porosity of the matrix in IEX stationary phases is incorrect?
A) Non-porous matrices are preferable for high-resolution separations to avoid diffusion effects.
B) Micropores increase the binding capacity but cause band broadening.
C) Macroporous particles with pore diameters of about 600-800 nm overcome the disadvantages of micropores.
D) High porosity offers a large surface area covered by charged groups, which is an advantage when separating small molecules.
D) High porosity offers a large surface area covered by charged groups, which is an advantage when separating small molecules.
What is the advantage of continuous bed matrices in IEX stationary phases?
A) They offer higher binding capacity compared to bead-based matrices.
B) They provide more homogeneous mobile phase flow and shorter diffusion distances for the analytes.
C) They are more physically and chemically stable than bead-based matrices.
D) They are less expensive to manufacture compared to bead-based matrices.
B) They provide more homogeneous mobile phase flow and shorter diffusion distances for the analytes.
Which of the following factors does NOT directly affect the chromatographic resolution in IEX?
A) Porosity of the matrix
B) Size and size distribution of the particles
C) Pore size of the matrix
D) Type of mobile phase used
D) Type of mobile phase used
True or False: The physical and chemical stability of the matrix in IEX is important for facilitating high flow rates during cleaning or re-equilibration steps.
True
Which of the following statements about mobile phases in IEX is incorrect?
A) Sodium chloride is a widely used eluent for protein separation because it has no significant effect on protein structure.
B) The competing ion in the mobile phase has the function of eluting sample components through the column.
C) The higher the concentration of the competing ion, the more effective the displacement of analyte ions from the stationary phase.
D) The nature of the competing ion does not affect the elution of solute ions.
D) The nature of the competing ion does not affect the elution of solute ions.
Identify the correct order of preference for exchanging ions at low concentrations and ordinary temperatures.
A) Higher valency > Higher atomic number
B) Higher atomic number > Higher valency
C) Higher valency = Higher atomic number
D) None of the above
A) Higher valency > Higher atomic number
Which of the following statements about the effect of flow rate and temperature in IEX is correct?
A) Higher flow rates cause lower elution volumes, while higher temperatures decrease chromatographic efficiency.
B) Higher flow rates cause higher elution volumes, while higher temperatures increase chromatographic efficiency.
C) Higher flow rates cause lower elution volumes, while higher temperatures increase chromatographic efficiency.
D) Higher flow rates and higher temperatures both cause higher elution volumes.
C) Higher flow rates cause lower elution volumes, while higher temperatures increase chromatographic efficiency.
Which of the following additives can be used in IEX mobile phases for solubilization of proteins?
A) Urea and guanidine hydrochloride
B) Detergents
C) Both A and B
D) None of the above
C) Both A and B
Which of the following is NOT a desired property of buffers used in IEX mobile phases?
A) High buffering capacity at the working pH
B) High solubility
C) High purity
D) High conductivity
D) High conductivity
Possibility of precipitation can occur when ______ buffer and ______ metal ions are mixed in IEX mobile phases.
A) phosphate, divalent
B) acetate, monovalent
C) citrate, trivalent
D) borate, divalent
A) phosphate, divalent
Which type of detector is the detector of choice for the majority of IEX applications?
A) UV-Visible absorbance detector
B) Refractive index detector
C) Electrical conductivity detector
D) Fluorescence detector
C) Electrical conductivity detector
True or False: The output signal from an electrical conductivity detector is inherently linearly related to the ion concentration in the detector cell.
False. The ‘out of balance signal’ is not inherently linearly related to the ion concentration in the cell. Thus, the electronic circuit modifies the response of the detector to provide an output that is linearly related to the ion concentration.
Which detection technique in IEX is suitable for analytes that exhibit a significant absorbance profile in the UV-Vis spectrum?
A) Amperometric detection
B) Photometric detection
C) Mass spectrometry
D) Electrical conductivity detection
B) Photometric detection
Identify the type of ionization technique used in mass spectrometry detection for IEX, which is suitable when the solute ions are anionic.
A) Positive-mode electrospray ionization (ESI)
B) Negative-mode electrospray ionization (ESI)
C) Atmospheric pressure chemical ionization
D) Electron impact ionization
B) Negative-mode electrospray ionization (ESI)
In the chromatograms shown in Figure 10, which type of IEX is used for the separation of inorganic anions (a) and inorganic cations (b)?
A) Anion exchange chromatography (a), Cation exchange chromatography (b)
B) Cation exchange chromatography (a), Anion exchange chromatography (b)
C) Both are anion exchange chromatography
D) Both are cation exchange chromatography
A) Anion exchange chromatography (a), Cation exchange chromatography (b)
In IEX, ______ have no or only weak UV chromophores, making ______ the detector of choice for most applications.
A) anions, conductivity
B) cations, conductivity
C) both anions and cations, conductivity
D) both anions and cations, UV-Vis absorbance
C) both anions and cations, conductivity
True or False: The mobile phase in IEX typically consists of an aqueous solution of a suitable salt or mixtures of salts, with the addition of a small percentage of an organic solvent.
True
What does HILIC stand for?
Hydrophilic Interaction Chromatography
HILIC can be described as a variation of ___________ performed using a polar stationary phase.
Reversed Phase Liquid Chromatography (RPLC)
In HILIC, the mobile phase typically contains _____ of aqueous solvent/buffer.
a) >60%
b) <60%
c) Exactly 60%
d) None of the above
b) <60%
What are the three mechanisms of retention in HILIC?
1) Hydrophilic partitioning,
2) Hydrogen bonding,
3) Electrostatic interactions
In the chromatogram shown in Figure 4, which peak corresponds to epinephrine in HILIC?
a) Peak 1
b) Peak 2
c) Both peaks 1 and 2
d) Neither peak 1 nor 2
a) Peak 1
Name the different types of stationary phases available for HILIC separation.
Silica, Amino, Cyano, Diol, Zwitterionic, Polymer-based
True or False: An increase in the percentage of organic solvent in the mobile phase leads to a decrease in retention in HILIC.
False
Which of the following organic solvents has the strongest elution strength in HILIC?
a) Acetonitrile
b) Methanol
c) Isopropanol
d) Tetrahydrofuran
d) Tetrahydrofuran
The recommended buffers for HILIC are ___________ of acetic and formic acids.
ammonium salts
In Figure 8, the retention of salicylic acid (negatively charged) on Hypersil GOLD HILIC (positively charged) decreases as the buffer concentration _____.
a) increases
b) decreases
c) remains constant
d) None of the above
a) increases
True or False: Increased salt concentrations result in decreased retention of positively charged solutes on stationary phases with the same charge.
True
Which of the following statements is NOT a recommendation for mobile phase selection in HILIC applications?
a) Use acetonitrile or other polar, water-miscible organic modifiers.
b) Have a high organic content, between 60 to 97%.
c) Use buffer salts such as ammonium acetate and ammonium formate.
d) Run gradients from 100% organic to 100% aqueous.
d) Run gradients from 100% organic to 100% aqueous.
In HILIC, the resulting elution order of solutes is _____ that observed in a reversed phase separation.
a) the same as
b) roughly the opposite of
c) unrelated to
d) none of the above
b) roughly the opposite of
What is the mechanism behind the formation of an aqueous layer on the surface of the polar stationary phase in HILIC?
Polar analytes preferentially partition into this aqueous-rich layer.
In HILIC, a minimum of _____ water is necessary to ensure sufficient hydration of the stationary phase.
3%
True or False: As the buffer pH increases, the retention of acetylsalicylic acid (pKa = 3.5) in HILIC also increases.
True
In Figure 10.2, the retention of cytosine and cytidine on Hypersil GOLD HILIC increases with increasing ammonium acetate concentration due to _____.
a) increased electrostatic interactions
b) increased hydrophobic interactions
c) enhanced hydrogen-bonding interactions
d) none of the above
c) enhanced hydrogen-bonding interactions
Which of the following statements is correct regarding the effect of buffer pH in HILIC?
a) Charged compounds are less hydrophilic and therefore less retained.
b) Charged compounds are more hydrophilic and therefore more retained.
c) Buffer pH has no effect on the retention of charged compounds.
d) The effect of buffer pH depends on the type of stationary phase used.
b) Charged compounds are more hydrophilic and therefore more retained.
What is the name of the chromatographic technique in which the mobile phase used is more polar than the stationary phase?
Reversed Phase Liquid Chromatography (RPLC)
The most widely used stationary phase in RPLC is based on _____ bonded to the surface of a silica support particle.
Octadecylsilane (ODS)
Which of the following statements about polar end-capped, polar embedded phases is NOT true?
a) They provide an additional avenue for potential modification of the chromatographic selectivity.
b) They offer an enhancement of retention of polar analytes.
c) They cannot be used with high aqueous mobile phases.
d) They can be used with 100% aqueous mobile phases without loss of analyte retention.
c) They cannot be used with high aqueous mobile phases.
Name the two types of organic modifiers that can influence the π-π interactions between the solute and the aromatic moiety of the stationary phase in a different way.
Acetonitrile and Methanol
In a reversed phase chromatogram, which analyte elutes first?
a) Hydrophilic analyte
b) Hydrophobic analyte
c) Both hydrophilic and hydrophobic analytes elute simultaneously
d) The elution order cannot be determined based on the given information
a) Hydrophilic analyte
What is the weakest solvent in reversed phase HPLC?
Water
True or False: In HILIC, as progressively more organic modifier is added to the mobile phase, the analyte retention time will continue to increase.
False
Which of the following organic solvents has the highest elution strength (ε0 value) for RPLC separations on a C18 stationary phase?
a) Methanol
b) Acetonitrile
c) Tetrahydrofuran
d) Isopropanol
c) Tetrahydrofuran
The chosen organic solvent for RPLC must be _____ with water.
miscible
Which of the following statements about the viscosity of mobile phase mixtures in RPLC is NOT true?
a) The viscosity of mobile phase mixtures is markedly higher than the pure solvents.
b) A low viscosity mobile phase is favored to reduce dispersion and keep system backpressure low.
c) The use of a low viscosity solvent allows for faster chromatography due to increased rate of mass transfer.
d) The viscosity of mobile phase mixtures decreases as the percentage of organic solvent increases.
d) The viscosity of mobile phase mixtures decreases as the percentage of organic solvent increases.
What is the purpose of using a nomogram in RPLC?
To find equivalent eluotropic strengths between mobile phases that use different organic modifiers.
True or False: Isoeluotropic mobile phases produce separations in approximately the same time frame but may show altered selectivity.
True
Which of the following statements about the effect of changing the organic modifier on selectivity in RPLC is correct?
a) Changing the organic modifier has no effect on selectivity.
b) Changing the organic modifier may improve resolution.
c) Changing the organic modifier always results in better resolution.
d) Changing the organic modifier has no effect on resolution but may change retention times.
b) Changing the organic modifier may improve resolution.
Name the types of stationary phases that are available for RPLC separations.
Silica, Amino, Cyano, Diol, Zwitterionic, Polymer-based
Fluorinated phases in RPLC have shown some _____ and _____ selectivity, particularly for aromatic molecules.
size and shape
In RPLC, the elution order of analytes is based on their _____.
a) Polarity
b) Hydrophobicity or hydrophilicity
c) Molecular weight
d) Charge
b) Hydrophobicity or hydrophilicity
True or False: In RPLC, the organic modifier is added to the mobile phase because it is less polar than water, and therefore, it reduces the retention time of hydrophobic analytes.
True
What is the purpose of using a logarithmic scale for the capacity factor (k) in Figure 8.2?
a) To better visualize the exponential decrease in retention volume with increasing organic solvent composition.
b) To better visualize the linear decrease in retention volume with increasing organic solvent composition.
c) To better visualize the exponential increase in retention volume with increasing organic solvent composition.
d) To better visualize the linear increase in retention volume with increasing organic solvent composition.
a) To better visualize the exponential decrease in retention volume with increasing organic solvent composition.
Which is NOT a property of an ideal solvent for RPLC?
a) Water miscibility
b) Low viscosity
c) Low UV detection
d) High boiling point
d) High boiling point
What are the main parameters used to control HPLC retention and optimize separations of most pharmaceutical compounds?
Mobile-phase pH and composition
True or False: Buffers are not recommended to control the pH stability of the mobile phase in reversed-phase HPLC.
False
What should the specified pH in analytical methods for pharmaceutical analysis represent?
a) The pH of the aqueous solvent
b) The pH of the organic modifier
c) The pH of the mobile phase
d) The pH of the sample solution
a) The pH of the aqueous solvent
In RPLC, the distribution of an analyte between the mobile phase and the surface of the packing material is referred to as the _____ equilibrium.
primary
What is the term used to describe the equilibrium where an analyte can be present in the mobile or stationary phase in two or more different forms?
secondary
For a basic analyte in RPLC, at what pH will the analyte be predominantly in its neutral (un-ionized) form?
a) pH < pKa
b) pH = pKa
c) pH > pKa
d) The pH has no effect on the ionization of the analyte
a) pH < pKa
True or False: For a basic analyte in RPLC, increased retention is observed when the mobile phase pH is lower than the pKa of the analyte.
True
For an acidic analyte in RPLC, at what pH will the analyte be predominantly in its neutral (un-ionized) form?
pH < pKa
In Figure 4.2, which curve represents the retention behavior of a basic analyte?
a) Curve A
b) Curve B
c) Curve C
d) Both Curve A and Curve B
b) Curve B
According to Figure 5.2, the theoretical retention versus pH profile for a zwitterionic component shows a _____ shape.
U-shaped (or plateau-shaped)
True or False: Separations of basic compounds in RPLC are traditionally carried out under ion suppression conditions, where the pH of the mobile phase is raised to produce the neutral molecule.
False
At what pH are the surface silanol groups approximately 50% ionized and 50% non-ionized?
a) pH 1.5
b) pH 3.5
c) pH 5.8
d) pH 7.0
b) pH 3.5
In Figure 9, what is the reason for the improvement in basic analyte peak shape at low pH?
Reduced surface silanol interaction
The unwanted secondary silanol retention in RPLC can be reduced or eliminated by the addition of a small (sterically) highly surface active _____.
base
Which of the following is NOT an example of a sacrificial base used in RPLC?
a) Triethylamine (TEA)
b) Piperazine
c) N,N,N’,N’-tetramethylethylenediamine (TMEDA)
d) Trifluoroacetic acid (TFA)
d) Trifluoroacetic acid (TFA)
True or False: At high mobile phase pH, the analyte is non-ionized, and the interaction with the silica silanol groups is reduced, leading to improved peak shape.
True
In Figure 13, what is the reason for the reduction in retention times as the temperature increases?
Reduced mobile phase viscosity
Which of the following is NOT a disadvantage of working at higher temperatures in RPLC?
a) Increased risk of solvent or sample decomposition
b) Increased risk of bubbles in the detector due to increased solvent vapor pressure
c) Decreased silica solubility
d) Poor reproducibility if thermostating is inadequate
c) Decreased silica solubility
True or False: Both pH and pKa are temperature-independent, which means that chromatographic parameters are not affected by temperature changes in RPLC.
False
An increase in temperature in RPLC reduces the effect of the _____ term of the van Deemter equation, due to the improvement of mass transfer kinetics.
C term
True or False: Column performance in RPLC may increase due to reduced solvent viscosity, system backpressure, and the ability to utilize faster flow rates at higher temperatures.
True
What is the main application of RPLC for amphetamines?
a) Analysis in water samples
b) Separation of standards
c) Synthesis of new compounds
d) Storage of samples
b) Separation of standards
RPLC can be used for the separation of a drug and its __________.
metabolites
What components are analyzed in Betamethasone syrup using RPLC?
Betamethasone
RPLC can be used to separate Cytochrome c ____ _____ in a reproducibility study.
tryptic digest
Which column type is compared in Figure 10 for having an embedded polar group?
a) Symmetry C18
b) SymmetryShield RP18
c) Both a and b
d) None of the above
c) Both a and b
What phase is used in RPLC for the separation of aldehydes and isomers?
Pentafluorophenyl Phase (PFPP)
What is a common use of RPLC in pharmaceutical analysis?
a) Coloration of tablets
b) Analysis of active ingredients
c) Design of drug packaging
d) Marketing of pharmaceuticals
b) Analysis of active ingredients
What does the separation of morphine and its metabolites help in analyzing?
Drug metabolism
In the analysis of Augmentin, the two main components analyzed are amoxicillin and __________ acid.
clavulanic
What is a benefit of using columns with embedded polar groups?
a) Increased separation efficiency
b) Reduced cost
c) Faster analysis
d) Enhanced column durability
a) Increased separation efficiency
The separation of small peptides is performed using a __________ column.
C8
What is the main advantage of using the PFPP phase in RPLC?
a) Lower cost
b) Better separation of polar compounds
c) Improved resolution of aldehydes and isomers
d) Faster analysis time
c) Improved resolution of aldehydes and isomers
What is the main goal of preparative HPLC?
a) Quantitative analysis
b) Qualitative analysis
c) Isolation and purification
d) Chromatogram visualization
c) Isolation and purification
Identify one of the effects of injecting more than a certain amount of sample onto the column in preparative HPLC.
The peak becomes unsymmetrical and shows strong tailing.
In preparative HPLC, concentration overloading occurs when the concentration of the sample is increased but the __________ remains the same.
sample volume injected
What is an advantage of scaling up the analytical system for preparative HPLC?
a) Lower cost
b) High solvent volumes
c) Sharp and symmetrical peaks
d) Reduced resolution
c) Sharp and symmetrical peaks
What particle size is recommended for very complex samples with poor resolutions in preparative HPLC?
5-μm particles
Smaller particle sizes allow greater efficiency and permit the use of __________ columns to increase separation speed.
shorter
Which column dimension is used for larger scale preparative HPLC?
a) 4.6-mm i.d.
b) 7.8-mm i.d.
c) 21.2-mm i.d.
d) 30-mm i.d.
c) 21.2-mm i.d.
