Lab Sessions Flashcards
(31 cards)
TLC
sp
mp
to separate what
How to identify analytes
How to achieve better separation
sp silica is polar
mp different ratios of petroleum ether and acetone is non polar and adjustments made to how non polar it is
can separate spinach pigments
Retardation factor: used to compare analytes to known values
Achieve separation by adjusting polarity of mp
Formula for retardation factor
Distance traveled by pigment /
Distance traveled by solvent (solvent is the range of mp)
How to achieve better separation in TLC
Use a range of mp
eg petroleum ether 60:40 acetone produces the best separation due to the chlorophyll being a relatively polar compound
The range of mp are all relatively less polar than silica
Mode of separation in TLC
Adsorption
Polarity of silica
Polar
GC
mp
sp
mode of separation
sample
Oven
Internal standard
mp gas eg helium nitrogen inert gases
sp column packed or capillary
mode = partition
Oven: temperature programming or isothermally
Sample: must be volatile
Calibration curve produced by preparing standards of varying concentrations of ethanol and adding a constant amount of propanol to each
Peak area of ethanol divided by propanol in the chromatogram was graphed against concentration
Explain partition
Partitioning between mp and sp
Which has a shorter retention time ethanol or propanol
ethanol has less carbons than propanol therefore has a shorter retention time
What is a use for GC
Can be used to determine the % alcohol in a product using an internal standard method
HPLC
mp
sp
pump and injector
If sample is like the mp
Detector
mp liquid is polar eg methanol water ratio
sp liquid coated in a column eg C18 is non polar because it is a long chain carbon
Injecting 10ul
Depending on polarity of the sample it will have more of an affinity with the mp or sp
Sample will elute faster and will have a shorter retention time
Photo diode array
mp is polar and sp in non polar
Reverse phase chromatography
why is C18 is non polar
because it is a long chain carbon
How to avoid long retention times in HPLC
Make the mp more like the sample
Area under a chromatogram?
If there’s two peaks
Peak area relates to concentration
Two peaks there is two components in the sample
Photo diode array detector
How to identify caffeine
Light of all wavelengths are passed through the sample and only some will be absorbed depending on what chloroform’s are present in the sample
Caffeine absorbs at 273nm :
If the sample absorbs at 273 there will be a peak at this wavelength
Alternative detectors
Photo diode array
Mass spec detector (no debate as to what the sample is, gives unique fingerprints)
If a substance absorbs UV light
If it has certain chloroforms ( functional groups) present cause the substance to absorb UV light at a wavelength
Keeping the mobile phase constant in HPLC is called
Isocratic elution.
Changing the mobile phase to achieve better separation in HPLC
Gradient elution
Sharpness of peak
How well separated the peaks are
Sharpness of peak = efficiently n, the number of plates
Separation = resolution
Adequately resolved peaks
Can see where one peak end and one peak brings
Resolution value greater than 1.5
How to improve poor resolution
Adjust parameters
Parameters
Flow rate
Type of column
Type of mobile phase
T = 10%
T% = .1
Concentration must be in
moles per litre
