forensic toxicology - analytical techniques - HPLC

1. What are the two phases involved in any chromatographic technique? Explain how HPLC uses these two phases. 2. For what types of sample is HPLC analysis most suited? 3. Explain the difference between reversed-phase and normal-phase chromatography. 4. What is solid phase extraction, and how is it related to HPLC? 5. What chromatographic parameter do we commonly use in analysis of HPLC data?

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Last updated: August 13, 2023

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what are chromatographic techniques?	what are the two phases in chromatographic techniques?	what do they involve?	how does this separate the components?
those that involve the separation of mixtures	the mobile phase	the mixture being dissolved in fluid	different components of the mixture will separate based on partitioning between the stationary and mobile phases
	the stationary phase	the mixture being passed through a solid material
what is HPLC?	what does it entail?		how does this separate the components?
high performance liquid chromatography	it takes a pressurised liquid (the mobile phase) and passes it through a column that is filled with an absorbent material (the stationary phase)		the components will separate based on the interaction with the sorbent material
what is the most common sample preparation technique used for HPLC?	what is SPE?		how does this help HPLC?
solid phase extraction (SPE)	a mini form of chromatography used to purify or concentrate the samples before they can be analysed (it also has a mobile & stationary phase)		it provides a more pure sample for HPLC to analyse
what are the 4 basic parameters of a HPLC system?		what do each of these need to do?
a pump		move the mobile phase under pressure
		pump the solvent
		split the appropriate gradient (produce the right ratio of solvents used)
		mix the solvents together effectively
an injector		introduce the sample
		draw the sample from a vial into a needle (load position)
		inject the sample into the flow path of the mobile phase (inject position)
a column		contains the stationary phase
		have an internal diameter (ID) to determine sensitivity & analyte loading
		have stationary phase beads to generate pressure
		be porous to increase surface area for separation
a detector		convert the data into useful information
what is the mobile phase typically made of in HPLC?
water (polar)
either acetonitrile or methanol (non-polar-ish)
a buffer to aid separation (e.g. formic acid)
what is reversed-phase HPLC?	how does this separate the components?		what is the most common reversed-phase HPLC?
a type of HPLC that has a non-polar stationary phase & a moderately-polar mobile phase	hydrophobic molecules will be retained by the stationary phase & eluted when the mobile phase gradient changes from polar to non-polar		C18 (silica with 18-carbon chains attached to the surface)
what is normal-phase HPLC?		how does this separate the components?
a type of HPLC that has a polar stationary phase & a non-polar mobile phase		hydrophilic molecules will be retained by the stationary phase & eluted when the mobile phase gradient changes from non-polar to polar
what are the most common types of detectors for HPLC?	what kind of data do they generate?	how do we analyse these to make this data quantitative?	what is this type of HPLC used for?
diode array detectors (DAD), a type of UV/vis detector	chromatograms	using peak area & internal standards	diagnosis (clinical biochemistry)
			pharmaceutical analysis
			manufacturing quality control
mass spectrometer	-	-	-