Chromatography Theory - The Basis of Separation Flashcards
(21 cards)
Solid stationary phase particles should be (3)
- Spherical - coated w adsorbant chemistry
- Small - 5->30 um diameter
- Tightly Packed
what constitiutes a good chromatogram (4)
- peaks should be tall, slender, symmetrical w minimal trailing
- peak should be separated from neighbouring peaks = good resolution
- smooth baseline signal - no interference
- peak should be resolved at least 2 mins from Dead Time (t0)
what is Vm
Column Dead Volume = the volume that flows through the system from injection to detector without interacting with the column
Vm calc
(0.5 x L x (d)2) / 1000
where L = length
d = diameter
what is t0
Dead Time = the time taken for mobile phase (or solvent) to pass through column, specifically for components not retained by stationary phase
(time taken for non interacting compund to pass through column)
t0 calc
t0 = Vm / F
Vm = Column dead vol
F = Flow rate of mobile phase in ml/min
what is tR
Retention time = time between sample injection and an analyte peak reaching a detector at the end of the column
(read off chromatogram, time 0 to top of peak)
what is k’
Retention Factor = the migration rate of an analyte on a column
K’ calc
K’ = tR- t0 / t0
tR = retention time
t0 = dead time
what is ideal retention factor (K’) of analyte between
one and five
what is the selectivity factor (alpha symbol)
the separation of two species on the column
selectivity factor calc
alpha = K’b / K’a
(retention factor of both analytes)
what is resolution factor (Rs)
how well adjacent peaks are separated
Rs calc
Rs = (tR1 - tR2) / 0.5 (tw1 + tw2)
where tR1 = retention time of peak 1
tw1 = width of peak 1
how to obtain high resolution
- control the capacity/retention factor by changing temp (in GC) and by changing composition of mobile phase (in LC)
- manipulate selectivity factor by: changing mobile/stationary ohase composition, changing column temp, using special chemical effects
5 ways to reduce tailing & peak asymmetry
- lower pH (add acidic buffer)
- use a C18 column (reverse phase column)
- reduce sample size (more product = more tailing)
- speed up velocity of mobile phase
- pre clean sample to remove matrix
what is the theoretical plate
the step of adsorption & desorption for an analyte (analyte with high affinity for mobile phase stilladsorbs & desorbs from stationary phase thousands of times)
what does an increased number of theoretical plates lead to
- better separation
- better peak shape
- better resolution
theoretical plates (N) calc
N = 16 (tr / W)^2
where tR = retention time
W = peak width at base line
height equivalent to theoretical plates calc
HETP = No. Theoretical Plates/Length of Column
(calculates distance between adjacent plates)
what does the smaller the HETP mean
the better the column
more theoretical plates
