HPLC Flashcards
HPLC is a type of
Analytical chrom
Can use chromatography based on chemical and physical properties, including….
Size exclusion
Reverse phase chrom
Ion exchange chrom
Can be used to analyse classes of compounds, including… (3)
Salts.
Ions and proteins.
Thermally unstable compounds, eg enzymes.
Separates complex mixtures using…
Columns that can withstand high pressure and are resistant to a range of chemicals. Eg steel columns.
The columns are packed with
Small particles.
The MP is pumped through at a ….
Reasonable speed, thus improving efficiancy.
Solvents should be degassed to prevent…
Bubbles from forming. These can interfere with valve operation and also cause peaks on the detector.
Can be used for water samples to…
Identify presence of small organic contaminants.
HPLC columns have an internal diameter of
3 mm to 10 mm
Columns contain spheres of…
High purity silica, packed into a dense matrix.
Van D eq: a reduced plate height, values of A and C, leads to a…
Better separation efficiancy.
Van D eq: the B value is…
Small in HPLC
Sample loops adds a known volume of sample into the column through an….
Injector port, this is isolated from the pump and column, until moved to be connected to the column.
When the MP is pumped, the sample is…
Loaded into the column.
Silica is a good packing material because…
It has excellent absorbent properties.
The column packing separates components bun
Chemical or physical properties.
HPLC is better at separation and analysis of
Non volatile compounds.
Temperature is critical because (2)
High temps may affect stability of analytes.
Retention times are temp dependant.
Not all detectors respond to all…
Compounds.
The signal produced is directly proportional to…
The amount of substance present - Quantitive measurement.
Difference types of detectors: (5)
Photodiode array. Mass spec. Fluorescence. Uv vis. Universal.
How does a uv vis detector work?
Light of particular wavelength is shined through MP flow, containing eluted compounds. The amount of light absorbed is detected. More light absorbed = more solute present.
A disadvantage to uv vis detector:
The compound must contain something that will absorb light at the wavelengths used.
For uv vis, need to know which wavelength…
The solute will absorb.
Photodiode array: uses polychromatic light passed through eluant….
Diff wavelengths passing through sample are absorbed to diff extents.
Adv of photodiode array over uv vis:
Wavelengths are recorded simultaneously. Can analyse species that absorb at diff wavelengths.m
Dis adv of photodiode array:
Low sensitivity. Not all compounds absorb light.
A universal detector detects…
Changes in refractive index. It is less sensitive than uv vis and fluorescence detectors.
Mass spec: produces a mass spectrum for every eluted compound, this is compared with…
A spectra library and the compound can be identified.
If HPLC doesn’t have a MS, retention time of analytes can be….
Compared with known retention times for compounds.
