17 Flashcards
(20 cards)
What is chromatography and what are its two fundamental phases?
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).
How did the term ‘chromatography’ originate and what was its initial application?
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.
What was David Talbot Day’s contribution to the development of chromatography?
Around 1900, Day used column chromatography with clay or limestone to separate crude petroleum into useful fractions, demonstrating industrial-scale separation.
Who introduced column chromatography for plant pigments, and what stationary phase did they use?
Mikhail Tsvet in 1906 used a calcium carbonate–packed column to separate plant pigments into distinct color fractions.
List three forensic applications of chromatography.
Chromatography is used in forensics to analyze blood samples, detect accelerants in arson investigations, and perform postmortem toxicology screens.
Name two applications of chromatography in the food industry.
Chromatography can detect food adulteration (e.g., horsemeat in beef products) and quantify nutritional or spoilage markers to ensure food safety and quality.
Why is chromatography essential in pharmaceutical manufacturing?
It purifies antibodies and other biologics, confirms the purity of vaccine preparations, and ensures that drug products meet strict regulatory standards.
What distinguishes column chromatography from planar (or thin-layer) chromatography?
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.
Define ‘small molecule’ in the context of chromatography and give examples.
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.
Why can separating small molecules be more challenging than separating proteins?
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.
What are the primary types of chromatography used for small molecules?
Common techniques include gas chromatography (GC), liquid chromatography (LC), high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), and capillary electrophoresis (CE).
Apart from chromatography, what are two detection techniques for small molecules?
Nuclear magnetic resonance (NMR) spectroscopy (robust but expensive) and mass spectrometry (MS), which is suited for complex biological samples.
How does UV/Vis spectroscopy serve as a detection method in chromatography?
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.
What principle underlies high-performance liquid chromatography (HPLC)?
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.
In reverse-phase HPLC, what is the nature of the stationary phase and its effect on elution order?
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.
Contrast normal-phase HPLC with reverse-phase HPLC in terms of stationary phase polarity.
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.
What role does the mobile phase composition play in HPLC separation?
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.
How does adjusting the pH of the mobile phase affect HPLC separation?
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.
Describe hydrophilic interaction liquid chromatography (HILIC).
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.
What is the main factor determining elution order in reverse-phase HPLC?
