18 Flashcards
(21 cards)
What is the main factor determining elution order in reverse-phase HPLC?
The polarity of the stationary phase is the primary determinant; analytes partition between a nonpolar (hydrophobic) stationary phase and a polar mobile phase.
Why might one choose a longer HPLC column despite increased run time?
Longer columns provide more surface area for analyte-stationary phase interactions, resulting in better separation (higher resolution) of closely eluting compounds.
How does column internal diameter affect HPLC performance?
Columns with smaller internal diameters give higher resolution due to a larger surface-to-volume ratio, but they require higher pressure and have lower sample capacity.
What parameters can be adjusted in GC to optimize separation?
One can vary column length, internal diameter, stationary phase film thickness, carrier gas flow rate, and temperature programming to balance resolution and analysis time.
How does temperature programming in GC improve separation of analytes with different boiling points?
Starting at a lower temperature allows volatile compounds to separate. Gradually increasing oven temperature helps elute less volatile compounds later, improving overall resolution for a wide boiling-point range.
Explain why GC-MS is considered a gold standard for trace analysis.
GC-MS combines the high separation efficiency of gas chromatography with the specificity and sensitivity of mass spectrometry, enabling accurate identification and quantification of low-level compounds.
What defines a supercritical fluid, and how is it used in supercritical fluid chromatography (SFC)?
A supercritical fluid exists above its critical temperature and pressure, combining gas-like diffusivity with liquid-like solvating power. In SFC, supercritical CO₂ serves as a tunable mobile phase for efficient separation.
Give one advantage of using supercritical CO₂ for decaffeinating coffee beans.
Supercritical CO₂ penetrates intact coffee beans without grinding, selectively dissolving caffeine at moderate temperatures and leaving flavor compounds largely unaffected.
What is the function of a DAD (diode array detector) in HPLC?
A DAD collects absorbance data across multiple wavelengths simultaneously, eliminating the need to preselect a single detection wavelength and capturing full UV/Vis spectra of eluting compounds.
Why is mass spectrometry (MS) more sensitive than UV/Vis detection for small molecules?
MS measures mass-to-charge ratios of ionized molecules, providing both structural information and high sensitivity, whereas UV/Vis relies solely on chromophores and can miss non-UV-active analytes.
What are the three quadrupoles in a triple quadrupole mass spectrometer used for?
Q1 filters ions by m/z, Q2 serves as a collision cell fragmenting selected ions, and Q3 filters fragment ions before they reach the detector for highly specific quantification (MS/MS).
How does time-of-flight (TOF) MS separate ions?
In TOF MS, ions are accelerated into a field-free flight tube; lighter ions travel faster and reach the detector before heavier ions. The measured flight times are used to calculate m/z values.
Describe the principle of an Orbitrap mass analyzer.
Ions enter an electrostatic field in the Orbitrap, orbiting around a central spindle. The frequency of their oscillations is inversely related to m/z, producing high-resolution mass spectra based on induced image currents.
What is the primary challenge in achieving good separation of multiple small molecules simultaneously?
The challenge lies in resolving analytes with very similar physicochemical properties (e.g., polarity or size), which requires careful selection of stationary/mobile phases, column parameters, and detection methods.
In HPLC method selection, why is the sample matrix a critical consideration?
Complex matrices (e.g., biological fluids) may contain interfering substances that co-elute or bind to the stationary phase, necessitating additional sample preparation or a longer column to achieve adequate separation.
How does the film thickness of a GC column’s stationary phase affect sensitivity and resolution?
Thicker films increase the column’s capacity for analytes, improving sensitivity for trace compounds, but may also lengthen retention times and reduce resolution for highly volatile analytes.
Why is carrier gas flow rate in GC a trade-off between analysis time and resolution?
A faster flow rate shortens analysis time but reduces interaction between analytes and the stationary phase (lower resolution). A slower flow improves resolution but lengthens run times.
What feature makes SFC particularly useful for polar analytes that are difficult to separate by HPLC or GC?
Adjusting the density of the supercritical fluid (by controlling pressure and temperature) allows tuning of mobile-phase polarity, enabling efficient extraction and separation of polar compounds.
How does the Na⁺/H⁺ exchanger in the kidney’s proximal tubule relate to ion-exchange chromatography?
Both rely on reversible binding of charged species: in ion-exchange chromatography, charged analytes bind to oppositely charged functional groups on the stationary phase, analogous to how Na⁺ and H⁺ exchange across the tubular membrane.
What is the significance of retention time in chromatography?
Retention time is the time it takes for a compound to elute from the column after injection; it reflects each analyte’s affinity for the stationary phase relative to the mobile phase and is used for identification and quantification.
Explain why a DAD is more versatile than a single-wavelength UV detector in HPLC.
A DAD records absorbance across a broad wavelength range, capturing full spectral data for each peak, which aids in compound identification and detection of co-eluting impurities—capabilities beyond single-wavelength detectors.
