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)
Describe GC columns:
Very fine capillary tubing wrapped on fine metal ring
Relatively simple and cheap to make from glass, stainless steel, polyethylene or Teflon
May be straight, U-shaped or coiled (100 m long)
A capillary column usually has an internal diameter of 0.1-0.3mm and is 50-100m (give solid phase characteristics)
Packing material added to fill tube
Describe GC detectors:
GC is often used in combination with mass spectrometry (normally need a combination of different techniques):
- Detector is used to monitor a particular mass - charge ratio (m/e)
- And also to give a complete mass spectrum
- 20 minutes (slow) but has a very fine resolution
Mass spec. isn’t perfect, NMR would also be needed
1 run can distinguish many different drugs
Describe liquid chromatography:
This technique combines TLC and GC techniques
Automatic recording apparatus used in the chromatographic analysis of mixtures of amino acids:
- Change flow rates to change pressure
- Different pH changes ionisation (affects interaction with other molecules)
- Change liquid/solvent during run (increase ability to resolve/separate compounds)
LC uses a stationary phase column and a liquid mobile phase organic/aqueous
Uses pressure to force mobile phase through column
Describe flash chromatography:
(Low/medium pressure liquid chromatography)
Isolate compound of interest
Low resolution (2-3 compounds distinguished)
Speed
Volume (capacity)
Describe high performance liquid chromatography (HPLC):
Formerly high pressure LC
Describe column selection for >2000 bp HPLC:
Organic soluble: - Gel permeation Aqueous soluble: - Gel filtration - Ion exchange - Reversed phase - Hydrophobic interaction - Affinity bioaffinity
Describe column selection for
Organic soluble: - Methanol/water soluble (normal phase, reverse phase) - Hexane soluble - THF soluble (gel permeation) Aqueous soluble: - Non-ionic (reverse phase) - Ionic (reverse phase, ion exchange) - Peptides/proteins (reverse phase)
Describe LC stationary phases:
Normal phase - plain silica
Reverse phase - basic column, reverse order of elution (stick on lipophilic element, less polar)
Reverse phase, more retentive than C8, excellent for ion-pairing
Normal- or reverse-phase, selectivity for polar compounds
Reverse-phase, good for aromatic compounds
Ion exchange, separate bases (by using acids)
Ion exchange, separate acids (by using bases)
Describe HPLC reverse phase:
- Analytes retained on the less polar stationary phase until eluted with a sufficiently polar mobile phase
- Simple to operagte
- High efficiency (good resolution)
- Column stability (last for a long term)
- Ability to analyse a broad spectrum of both closely related and widely different compounds
Good pharmacology-wise
Describe reverse phase in drug metabolism studies:
Drugs need to be liphilic to get in, but excretion is water based, thus most metabolite are water soluble
Most metabolic pathways make the drug more hydrophilic
For normal phase, metabolites may come off after parent - difficult to establish assays for unknown metabolites - may have very long run times
For reverse phase, the metabolites will elute before the parent compound, so when that elutes, you know the metabolites have already eluted (with exceptions e.g. acetylation)
Describe column selection for HPLC:
Packing can be spherical or irregular:
Irregular is cheaper and has a larger surface area
Spherical have lower back pressure, better resolution and reproducibility (liquid flow is much smoother, decrease pressure, increase flow rate, faster chromatography)
Size:
Larger is faster, with less back pressure but poorer resolution
Smaller has greater resolution
Usually 5um
More particles=more interactions (and strength)=increased ability to separate compounds
Describe carbon loading in HPLC:
Carbon load indicates the amount of functional bonded phase attached to the base silica:
Lower loading=less lipophilic=shorter retention times (=less reolution)
Describe reverse phase HPLC flexibility in vitamin studies:
Different vitamins are fat/water soluble
Uses different solvents with the same column to separate (fat soluble vitamins have lipophilic solvents and water soluble vitamins have ion-pairing reagents)
It is very cheap to change solvents
Describe HPLC detection:
Many types of detection systems are used:
Spectrophotometric (modern systems have diode array detectors that can only monitor 256 wavelengths simultaneously and can be tweaked so they only see the compounds they want to see)
Fluorescence
Radioactivity
Electrochemical (but so sensitive)
Mass spec.
NMR (extremely good resolution, slower but unambiguous)
DO need to know excitation and emission wavelengths (combine UV, fluorescence and mass spec. in series)
Describe HPLC set-up:
Pump
Vacuum
Autosampler (automate, high throughput applications)
Also have series well plate handler which can load 1000s of samples at once
Only a few ug on column (may have to do 30-40 runs to collect fraction)
List some applications of HPLC:
Diode aray
Screening for drug metabolites
Screening for differences in metabolisms between species
Frequency and administration of drugs
Detecting a drug in plasma, urine and metabolites
Describe HPLC in theory vs. practise:
A lot of theory is on the subject, but most reverse-phase HPLC conditions are derived by trial and error
