HPLC/GC Flashcards
Define chromatography:
A simple technique for separating based on colour (visualisation), because different compounds have different solubilities and chemical properties
Describe the reasons chromatography is used in pharmacology and toxicology:
To follow chemical reactions in drug synthesis
Identification of drugs in biological fluids (drug screening)
Quantification of drug concentrations in plasma, urine etc.
Identify drug metabolites
- If metabolite has pharmacological activity
- Toxicology if metabolite is toxic
- Identify appropriate species for drug development models
Describe thin layer chromatography:
Old technique Rapid development after 1950s TLC allows relatively efficient separation in a very short time Simple Cheap
Describe how TLC works:
An efficient way to separate compounds (thicker=more solvent=slower)
TLC uses readily available sorbents, solvents, and detection reagents:
Used an analytical (qualitative and semi-quantitative) or preparative application (up to 1g)
Similar compounds can be resolved, although speed must be sacrificed for resolution (still quite fast)
Describe the equipment needed for TLC:
TLC plates can be prepared or purchased Often use 20x20cm cut Sorbent usually 0.2-4mm thick Require a development tank UV box or spray (visualisation) Good for semi-analysis
Describe the TLC solid phase:
Silica;
Particle size affects development - larger particles give faster separation at the expense of sharpness
- often contains a fluorescent indicator - blocks fluorescence (don’t need to know about tissue)
Alumina:
- high adsorptive capacity and useful separation of not too polar substances differing in steric arrangement
- presence of C=C bonds increase adsorption onto alumina more than silica
- separation of aromatic hydrocarbons with differnt numbers of carbon atoms and different steric properties - alumina>silica
Describe TLC eluents:
Elute off solid phase
Need to take into account the solubility of the chromatohraphed substances in the mobile phase and the polarity
For polar substances, solvents of non-polar character (saturated or halogenated hydrocarbons) are weak eluents compared with polar solvents (decreases solubility a little bit)
Lipids need non-polar
Don’t want it to be too soluble
List the three factors TLC elution power is influenced by:
Interaction between solvent molecules and chromatophraphed compound
Interaction between adsorbed molecules of the mobile phase and the molecules of the sample in the adsorbed phase (interaction with solid phase)
Interaction between the adsorbed molecules of the mobile phase and the adsorbent
Describe the best resolution of TLC:
Intersection between mobile phase (solvent), analyte (solute) and solid phase
Tweak the phases (harder to tweak the solid phase so tweak the mobile phase)
Compounds that move further from the original spot are more soluble, and have fewer or weaker interactions with the solid phase and greater interactions with the mobile phase
Describe detection in TLC:
Fluorescence: - assumption of quenching fluorescence Chemical detection: - e.g. sulphuric acid (spot the sugar carbons) Radioactivity: - liquid scintillation counter - use radioactive counter (extremely expensive) - use radiography (crossover technique) Advantage - slow Disadvantage - quantification very slow
Describe TLC in drug screening:
Commonly used for the separation and identification of illicitly manufactured drugs
- Rapid (analysis
List the visualisation reagents for amphetamine-type substances:
UV 254nm: Universal method Ninhydrin reagent Acidified potassium iodoplatinate reagent Fast black K Marquis reagent Fluorescent reagent (Fluram) Simon reagent Dragendorff reagent
Describe Ninhydrin reagent:
Many primary and secondary amines attached to an aliphatic carbon atom, such as amphetamine and methamphetamine, result in violet or pink spots
Describe acidified potassium iodoplatinate reagent:
Sensitive general reagent
Most primary and secondary amines give light blue spots
Describe fast black K:
Primary and secondary amines give spots varying in colour from violet (primary amines) to organe or orange-red (secondary amines)
Describe marquis reagent:
Distinction between unsubstituted and ring-substituted ATS
Describe fluorescamine reagent:
Sensitive reagent for primary amines
Recommended for the detection of low concentrations of primary amines
Describe Simon reagent:
General reagent for secondary amines (ephedrine and pseudoephedrine do not react)
Describe dragendorff reagent:
General reagent for alkaloids and nitrogeneous bases
Describe and define the retardation factor of TLC:
After visualisation, mark spots and calculate Rf values:
Rf=migration distance from origin to centre of analyte zone/development distance from origin to solvent front
Describes how much the progress had been retarded
Describe TLC quantification:
TLC is good for certain compounds under certain conditions, but concentration is measured largely by eye
Quantification is:
Difficult
Not very sensitive
Hard to automate
TLC is not accepted as a single technique for drug identification (need a second technique)
Describe gas chromatography:
Uses a gas as a mobile phase and stationary phase is liquid fixed onto an inert support or surface-active adsorbent
Advantages include:
Low viscosity and much higher diffusion rates of gas compared with liquid mobile phase
Allows fast separation and therefore suitable for routine analysis
BUT requires a very expensive GC machine
Describe GC equipment:
Usually quite simple Gas carrier source Device for the introduction of sample Column Detector Recorder
Describe the GC gas carrier:
Gas must be inert with respect to packing material and sample:
- Oxygen-free nitrogen (low cost, safe)
- Hydrogen (low cost, explosive)
- Helium (advantages of N2 and H2 expensive)
- Argon (cheap, hard to obtain)
