chapter 26 introduction to chromatographic separations 1general description of chromatography
DESCRIPTION
Chapter 26 Introduction to Chromatographic Separations 1General Description of Chromatography. 1.1 General description - Sample dissolved in and transported by a mobile phase Some components in sample interact more strongly with stationary phase and are more strongly retained - PowerPoint PPT PresentationTRANSCRIPT
1.1 General description- Sample dissolved in and transported by a mobile phase- Some components in sample interact more strongly with stationary phase and
are more strongly retained- Sample separated into zones or bands
1.2 Classification(based one the types of mobile and stationary)
- Gas chromatography (GC)- Liquid chromatography (LC)- Supercritical fluid chromatography (SFC)
Classification based on the types of mobile and stationary phases
1.3 Elution chromatography:Flushing of sample through column by continual mobile phase addition
- only eluent (portion of sample in
mobile phase) moves down
- migration rate fraction of time spent in mobile phase
Fig. 26-1 (p.764) (a) The separation of a mixture of components A and B by column elution chromatography, (b) the output of the signal detector
Fig. 26-2 (p.765) Concentration profiles of solute bands A and B at two different times in their migration down the column.
- Chromatogram (concentration versus elution time)
- More strongly retained species elutes last (elution order)
- Analyte is diluted during elution (dispersion)
- Zone broadening proportional to elution time
- Adjust migration rates for A and B (increase band separation)- Adjust zone broadening (decrease band spread)
Fig. 26-3 (p.765) Two-component chromatogram illustrating two methods for improving overlapping peaks.
2.1 Distribution constant
Analyte A in equilibrium with two phases
constanton distributi mobile
stationary
stationarymobile
c
cK
AA
tM time for unretained species (dead time),same rate as mobile phase moleculesaverage migration rate
tR retention time for retained speciesaverage migration rate
Ideally: tR independent of volume injected, produces a Gaussian peak
Fig. 26-4 (p.767) A typical chromatography for a two component mixture.
lengthcolumn :L Rt
Lv
Mt
Lu
2.2 Retention time
2.3 Relationship between tR and K
A
MSA
MS
MS
MMS
SSMM
M
A
ku
VVK
estimeatedVV
VV
VcV
VcVc
Vtotal
u
fractionuv
1
1
kfactor retention :/
packingcolumn from :/
)/(K1
1u
/c1
1u
cu
A of mols
phase mobilein A of moles
phase mobilein spendsA timeof
A
A
S
M
2.4 retention factor k
kA is 1.0, separation is poor
kA is >20-30, separation is slow
kA is between 1-10, separation is optimum
M
MRA
AMR
t
ttk
kt
L
t
L
1
1
2.5 Relative migration rate: Selectivity factor ()
Selectivity factor
Larger = better separation
timesretention )(
)(t
factorsretention
constantson distributi
R
MAR
MB
A
B
A
B
tt
t
k
k
K
Ka
3.1 Rate theory of chromatography
- Individual molecule undergoes “random walk”, and many thousands of adsorption/desorption processes.
- Some travel rapidly while other lag add up to give Gaussian peak (like random errors)
- Breadth of band increases down column because of more time
- Zone broadening is affecting separation efficiency – high efficiency requires less broadening
3.2 Column efficiency
N: number of plates
L: length of column
H: height of 1 theoretical plate
Plates are only theoretical –
column efficiency increase with N
H
LN
Fig. 26-6 (p.770) Definition of H. Efficient column has small plate height
– less zone broadening
Experimentally, H and N can be approximated from the width of the base of chromatographic peak.
Fig. 26-7 (p.770) Determination of N.
2)(16W
tN R
3.3 Kinetic variables affecting column efficiency (H)
3.3.1 Mobile phase velocity
- Higher mobile phase velocity, less time on column, less zone broadening
- However, plate height H also changes with flow rate
Fig. 26-7 (p.770) Effect of mobile-phase flow on plate height for GC.
3.3.2 van Deemter Equation
A: multipath term
- Molecules move through different paths
- Larger difference in path length for larger particles
- At low flow rates, diffusion allows particles to switch between paths quickly and reduces variation in transit time
Fig. 26-9 (p.773) Typical pathways of two molecules during elution.
uCuCu
BAH Ms
B/: Longitudinal diffusion term- Diffusion from central zone to front and tail- Proportional to analyte diffusion coefficient- Inversely proportional to flow rate - high flow, less time for diffusion
C:Mass transfer coefficients (CS and CM)
- CS is rate for adsorption onto stationary phase
- CM is rate for analyte to desorb from stationary phase
- Effect proportional to flow rate – at high flow rates less time to approach equilibrium
Fig. 26-10 (p.774) van Deemter plot.
Column resolution
Fig. 26-12 (p.776) Separation at three resolution values
BA
ARBR
BAs WW
tt
WW
ZR
])()[(22
- u (linear flow rate): low flow rate favors increased resolution (van Deemter plot)
-H (plate height) (or N number of plates): use smaller particles, lengthen column, reduce viscosity of mobile phase (diffusion)
- (selectivity factor): vary temperature, composition of column/mobile phase
- kA (retention factor): vary temperature, composition of column/mobile phase
Effect of k and on R
When A and B have similar retention
41
][
4)(
)()(
]))(
(16[
)()(
2
N
k
kkR
t
ttk
N
t
ttR
W
tN
W
ttR
WWW
B
ABs
M
MR
B
ARBRs
BR
ARBRs
BA
R
222222
A
)1
()1
(16)1
()1
(16
2
kk )
1)(
1(
4)
1)(
1(
4
][
k
k
a
aR
k
k
a
aRN
k
k
k
a
aN
k
k
a
aNR
kka
sB
Bs
B
B
Bs
A
B
Effect of R on t
2
32
2 )1()
1(
16
)1()1()(
]1
1 ,
)([
)(
B
Bs
BBBR
BB
B
M
MBRB
B
BR
k
k
u
HR
u
kNH
u
kLt
kuv
uLuL
v
L
t
ttk
v
Lt
Fig. 26-15 (p.780) The general elution problem in chromatography
General elution problem: for multiple components, conditions rarely optimum for all components.
1. Change liquid mobile phase composition – gradient elution or solvent programming
2. Change temperature for gas chromatography – temperature programming
Section 26E
concepts and formulas in
Table 26-4 and 26-5 (p781, Reading assignment)