flashcard 9

Question 1

What is chromatography and what are its two fundamental phases?

Answer 1

Chromatography is a technique for separating mixtures of gases, liquids, or dissolved substances. It relies on a stationary phase (which remains fixed) and a mobile phase (which moves over or through the stationary phase).

Question 2

How did the term ‘chromatography’ originate and what was its initial application?

Answer 2

The term appeared in the early 1900s, derived from earlier mid-19th-century work referred to as ‘colour writing,’ used to separate plant pigments like chlorophyll and carotenoids.

Question 3

What was David Talbot Day’s contribution to the development of chromatography?

Answer 3

Around 1900, Day used column chromatography with clay or limestone to separate crude petroleum into useful fractions, demonstrating industrial-scale separation.

Question 4

Who introduced column chromatography for plant pigments, and what stationary phase did they use?

Answer 4

Mikhail Tsvet in 1906 used a calcium carbonate–packed column to separate plant pigments into distinct color fractions.

Question 5

List three forensic applications of chromatography.

Answer 5

Chromatography is used in forensics to analyze blood samples, detect accelerants in arson investigations, and perform postmortem toxicology screens.

Question 6

Name two applications of chromatography in the food industry.

Answer 6

Chromatography can detect food adulteration (e.g., horsemeat in beef products) and quantify nutritional or spoilage markers to ensure food safety and quality.

Question 7

Why is chromatography essential in pharmaceutical manufacturing?

Answer 7

It purifies antibodies and other biologics, confirms the purity of vaccine preparations, and ensures that drug products meet strict regulatory standards.

Question 8

What distinguishes column chromatography from planar (or thin-layer) chromatography?

Answer 8

In column chromatography, the stationary phase is packed into a tube and the mobile phase flows through under gravity or pressure. In planar chromatography, the stationary phase is coated onto a flat surface (e.g., a TLC plate) and the mobile phase travels up by capillary action.

Question 9

Define ‘small molecule’ in the context of chromatography and give examples.

Answer 9

A small molecule is any compound with a molecular weight below about 1,000 Da. Examples include amino acids, lipids, sugars, fatty acids, and alkaloids.

Question 10

Why can separating small molecules be more challenging than separating proteins?

Answer 10

Because small molecules have similar, low molecular weights (10–1,000 Da), they often have subtle differences in polarity or size, requiring more sophisticated chromatographic techniques to resolve them.

Question 11

What are the primary types of chromatography used for small molecules?

Answer 11

Common techniques include gas chromatography (GC), liquid chromatography (LC), high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), and capillary electrophoresis (CE).

Question 12

Apart from chromatography, what are two detection techniques for small molecules?

Answer 12

Nuclear magnetic resonance (NMR) spectroscopy (robust but expensive) and mass spectrometry (MS), which is suited for complex biological samples.

Question 13

How does UV/Vis spectroscopy serve as a detection method in chromatography?

Answer 13

UV/Vis detectors measure absorbance at specific wavelengths, identifying compounds based on their characteristic absorbance peaks; a diode array detector (DAD) can scan a full spectrum simultaneously.

Question 14

What principle underlies high-performance liquid chromatography (HPLC)?

Answer 14

HPLC uses a pressurized liquid mobile phase pumped through a column packed with a stationary phase, separating analytes based on their relative affinities for each phase.

Question 15

In reverse-phase HPLC, what is the nature of the stationary phase and its effect on elution order?

Answer 15

The stationary phase is hydrophobic (e.g., C8 or C18 chains bonded to silica). Hydrophilic analytes elute first, and more hydrophobic analytes elute later, because they interact more strongly with the nonpolar surface.

Question 16

Contrast normal-phase HPLC with reverse-phase HPLC in terms of stationary phase polarity.

Answer 16

Normal-phase HPLC uses a polar stationary phase (e.g., silica with NH₂ groups), so hydrophobic analytes elute first and polar analytes elute last. Reverse-phase uses a nonpolar stationary phase, causing polar analytes to elute first.

Question 17

What role does the mobile phase composition play in HPLC separation?

Answer 17

The mobile phase polarity (e.g., percentage of acetonitrile vs. water) influences analyte retention: increasing the organic fraction typically reduces retention times of hydrophobic compounds.

Question 18

How does adjusting the pH of the mobile phase affect HPLC separation?

Answer 18

Modifying pH can change the ionization state of acidic or basic analytes, altering their affinity for the stationary phase and thus changing retention order or peak resolution.

Question 19

Describe hydrophilic interaction liquid chromatography (HILIC).

Answer 19

HILIC uses a polar stationary phase and a mostly organic mobile phase, retaining polar analytes via a water-enriched layer on the stationary phase surface; hydrophobic molecules elute quickly.

Question 20

What is the main factor determining elution order in reverse-phase HPLC?

Answer 20

The polarity of the stationary phase is the primary determinant; analytes partition between a nonpolar (hydrophobic) stationary phase and a polar mobile phase.

Question 21

Why might one choose a longer HPLC column despite increased run time?

Answer 21

Longer columns provide more surface area for analyte-stationary phase interactions, resulting in better separation (higher resolution) of closely eluting compounds.

Question 22

How does column internal diameter affect HPLC performance?

Answer 22

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.

Question 23

What parameters can be adjusted in GC to optimize separation?

Answer 23

One can vary column length, internal diameter, stationary phase film thickness, carrier gas flow rate, and temperature programming to balance resolution and analysis time.

Question 24

How does temperature programming in GC improve separation of analytes with different boiling points?

Answer 24

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.

Question 25

Explain why GC-MS is considered a gold standard for trace analysis.

Answer 25

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.

Question 26

What defines a supercritical fluid, and how is it used in supercritical fluid chromatography (SFC)?

Answer 26

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.

Question 27

Give one advantage of using supercritical CO₂ for decaffeinating coffee beans.

Answer 27

Supercritical CO₂ penetrates intact coffee beans without grinding, selectively dissolving caffeine at moderate temperatures and leaving flavor compounds largely unaffected.

Question 28

What is the function of a DAD (diode array detector) in HPLC?

Answer 28

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.

Question 29

Why is mass spectrometry (MS) more sensitive than UV/Vis detection for small molecules?

Answer 29

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.

Question 30

What are the three quadrupoles in a triple quadrupole mass spectrometer used for?

Answer 30

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).

Question 31

How does time-of-flight (TOF) MS separate ions?

Answer 31

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.

Question 32

Describe the principle of an Orbitrap mass analyzer.

Answer 32

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.

Question 33

What is the primary challenge in achieving good separation of multiple small molecules simultaneously?

Answer 33

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.

Question 34

In HPLC method selection, why is the sample matrix a critical consideration?

Answer 34

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.

Question 35

How does the film thickness of a GC column’s stationary phase affect sensitivity and resolution?

Answer 35

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.

Question 36

Why is carrier gas flow rate in GC a trade-off between analysis time and resolution?

Answer 36

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.

Question 37

What feature makes SFC particularly useful for polar analytes that are difficult to separate by HPLC or GC?

Answer 37

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.

Question 38

How does the Na⁺/H⁺ exchanger in the kidney’s proximal tubule relate to ion-exchange chromatography?

Answer 38

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.

Question 39

What is the significance of retention time in chromatography?

Answer 39

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.

Question 40

Explain why a DAD is more versatile than a single-wavelength UV detector in HPLC.

Answer 40

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.

Question 41

What determines whether a GC detector like an FID or an ECD is chosen?

Answer 41

The detector choice depends on analyte properties: FID is universal for organic compounds (responds to carbon content), while ECD is highly sensitive to electronegative species (e.g., halogenated compounds).

Question 42

Why are plasma-based detection methods (e.g., ICP-MS) less common in small-molecule GC/LC?

Answer 42

Plasma-based detectors (e.g., ICP-MS) target elemental analysis rather than molecular structure, making them more suitable for inorganic or metal analysis rather than organic small-molecule profiling.

Question 43

How does isoelectric point (pI) play a role in ion-exchange chromatography?

Answer 43

At a given pH, analytes with net charges opposite to the stationary phase’s charge will bind; adjusting pH to the analyte’s pI reduces its net charge and elutes it from the column, enabling selective separation.

Question 44

In the context of small-molecule chromatography, what is the benefit of using capillary electrophoresis (CE)?

Answer 44

CE separates analytes based on electrophoretic mobility (charge-to-size ratio) in an electric field, offering high efficiency and rapid separation for charged small molecules without a packed stationary phase.

Question 45

Describe one scenario where mass spectrometric detection reveals compounds that UV/Vis cannot.

Answer 45

Non-chromophoric analytes—molecules lacking strong UV-absorbing groups—remain invisible to UV/Vis but can be ionized and detected by MS, enabling identification of otherwise undetectable species in complex mixtures.

Question 46

How does a detector’s dynamic range influence the choice of chromatography-detection combination?

Answer 46

A detector with a wide dynamic range (e.g., MS with linear response over several orders of magnitude) can quantify both trace and abundant analytes in the same run, whereas narrow-range detectors may saturate or miss low-level components.

Question 47

What is 'matrix suppression' in LC-MS, and how can chromatographic choices mitigate it?

Answer 47

Matrix suppression occurs when co-eluting matrix components reduce ionization efficiency of analytes in the mass spectrometer. Optimizing chromatographic separation (e.g., using longer or different-phase columns) reduces co-elution and minimizes suppression.

Question 48

Why is GC not suitable for analyzing nonvolatile or thermally labile small molecules without derivatization?

Answer 48

Nonvolatile or thermally unstable compounds decompose or fail to vaporize in GC injectors. Derivatization converts them into more volatile, thermally stable derivatives, enabling GC analysis but adding complexity.

Question 49

What considerations determine whether to use GC-MS versus LC-MS for a given small-molecule analysis?

Answer 49

GC-MS is ideal for volatile, thermally stable analytes that can be vaporized; LC-MS is chosen for nonvolatile, polar, or thermally labile compounds that cannot readily undergo GC analysis.

Question 50

Summarize the key factors in selecting an optimal chromatography method for small molecules.

Answer 50

Choose stationary/mobile phases to exploit analyte polarity or charge; balance resolution against run time and sample throughput; account for matrix complexity and potential interferences; and match detection method (UV/Vis, DAD, or MS) to analyte properties and required sensitivity.