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V. CHROMATOGRPAHY
*Chromatographic methods:
1.
2.
3.
4.
*Chromatographic requirements:
1)Reproducible chromatograms: Stable mobile phase flow rate
and pressure, achieved using accurate control system.
2) Stable stationary phase, with no column bleed (GC) or
dissolution (HPLC), achieved using bonded stationary phases
or operating the column at temperatures well below the
operating limits.
3) Obtaining sharp chromatographic peaks with minimal
tailing (sharp narrow peaks produce the greatest
concentrations of solute in the mobile phase).
4) Using a mobile phase that can be efficiently removed from the
eluent in an interface, has minimal spectral interference with
the spectra of the eluted components, is thermal stable and
does not react with the components at the elevated
temperatures of an interface.
*Partition: Competition between two phases for solute molecules.
At equilibrium: nM <=> nS
=>
KD = nS/nM
Where KD: Distribution (partition) coefficient.
nM: No. of molecule (solute) in the mobile phase.
nS: No. of molecule (solute) in the stationary phase.
Response
*Chromatogram:
tR
Start
tM
Retention Time
(1)
(2)
*Retention time:
t R = t R' + t M
(3)
VR = VM + KD VS
(4)
VR = tR Fc
Where
and
VR' = tR' Fc
(5)
k: Capacity factor.
= k2/k1: Relative retention, selectivity.
L/H = N: Efficiency, theoretical plates.
L: Length of column.
H: HETP (height equivalent to a theoretical plate).
(8)
(9)
(10)
(11)
Example:
tM
tRb
a
1
2
ka = (tRa - tM)/tM = 0;
k=
0
tRc
b
3
min
kb = 1; kc = 2 => b,c = 2/1 = 2
k=5
Time-consuming,
k = 105 wasting solvent or gas,
losing sensitivity.
*Relative retention, selectivity, : For k2 > k1, > 1
= tR2'/tR1' = (tR2 - tM)/(tR1 - tM) = k2/k1
= VR2'/VR1' = (VR2 - VM)/(VR1 - VM) = k2/k1
*Elution problem:
1a
1b
2a
2b
2c
1.
2.
(12)
(13)
Wh
h/2
Wb
The width of each peak (Wb) is a measure of the statistical
distribution of the retention time.
, standard deviation of the curve = One-half the width at the
half-height (HWHM, half width at half maximum).
Wb = 4
or = Wb/4
(14)
Wh = [5.54]1/2
(15)
1.
2.
3.
Gaussianideal shape.
Tailingsurface adsorption effects or dead volume.
Frontingoverloaded or sample capacity exceeded.
(16)
Chromatogram
*Bandspreading:
Packed column
Long
Short
(20)
Nef
(19)
20
(17) (23)
The lower the HETP, the more efficient the column will be.
For high efficiency, high N is required. To avoid long
column, HETP is as short as possible. Increase in N is
effective by reducing H rather than lengthening the
column.
Decrease in particle size of packing improves H, but for liquid
mobile phase, where B/is negligible, reduction in solvent
viscosity increases diffusion coefficient in mobile phase, thus
improves H.
*Factors affecting bandspreading:
1. Resistance to mass transfer in stationary and mobile phases.
2. Diffusion through the column (longitudinal diffusion).
3.
(6)
(7)
(24)
(25)
(26)
(27)
(28)
*Example: R = 1, k = 3, = 1.3
n = 16 [1]2 [(1+3)/3]2 [1.3/(1.3-1)]2 = 534
For typical = 1.01,
n = 16 [1]2 [(1+3)/3]2 [1.01/(1.01-1)]2 = 290161
*Linear velocity,
= Fc/Ac = Fc/r2Ee
(29)
Where Ac: Cross section area of column.
Ee: Interparticle porosity (usually ca. 0.5).
The larger the column, the lower the linear velocity will be.
*Van Deemter equation:
H = A + B/+ C= A + B/ + (Cs + Cm)
(30)
H = HE + HL + HS + HM
(31)
Where A, B and C are approx. constant for a given system.
: The linear velocity of the carrier fluid in cm/sec.
HE: Radial diffusion (Eddy diffusion).
HL: Longitudinal diffusion.
HS: Stationary phase mass transfer.
HM: Mobile phase mass transfer.
Coefficient (cm2/s)
10-1
10-3
10-5
?
D ( 10-5)
2.2
0.7
0.25
0.11
0.05
0.025
Mol. diameter ()
2.9
6.2
13.2
28.5
62.0
132
(34)
(35)
(36)
*Example:
Substances A and B were found to have retention times of 16.40
and 17.63 min, respectively, on a 30.0-cm column. An
unretained species passed through the column in 1.30 min.
The peak widths (at base) for A and B were 1.11 and 1.21
min, respectively.
Calculate:
a.
b.
c.
d.
e.
f.
Solution:
a. (17) R = 2(17.63-16.40)/(1.11+1.21) = 1.06
b. (18) NA = 16[16.40/1.11]2 = 3493
(18) NB = 16[17.63/1.21]2 = 3397
NAV = (3493 + 3397)/2 = 3445 = 3.45 103
c.
d.
e.
f.
Concentration (C)
*Statistical moments:
Bandspreading in elution chromatography (separation processes)
can be described by a Gaussian distribution for random
processes (e.g., diffusion).
Gaussian peak
Time (t)
Area
Variance (2)
(39)
Skew
(41)
(42)
(43)
(44)
(45)
(46)
(47)
RI = 100z + 100 [(log tx' - log tz')/(log tz+1' - log tz')] (48)
Where RI: Retention index.
tx': Adjusted retention time for substance x.
tz' and tz+1': Corresponding adjusted retention times for
normal hydrocarbons with z and z+1 carbon atoms,
respectively.
Example: RI for heptane: 700.
Reference compounds: n-Alkane, such as pentane and hexane.
Index differences (I) between columns can yield supporting data
related to molecular structure.
tM can be checked out using butane from a lighter.
Within a homologous series, a plot of log tR' vs. C# is linear.
Nonane
Octane
Pentane
Butane
4
Heptane
Hexane
5
6
7
8
9
Number of n-alkane carbon atoms
*Quantitation in chromatography:
Precision (): The degree of agreement between replicate
measurement of the same quantity and does not necessarily
imply accuracy.
Accuracy (): The degree of agreement between the measured
value and the true value (accepted).
Peak areas are independent of broadening and are more
satisfactory parameter than peak heights.
Peak area, from integration of detector signal during elution of a
component, is proportional to component concentration.
*Quantitation of a sample peak:
Sample content (g/g) = (ASPL/AIS)(WIS/WSPL)(RF/RC) (49)
Where ASPL: Integrated area of a sample (Vsec).
AIS: Integrated area of internal standard (Vsec).
WIS: Total amount of internal standard added (g).
WSPL: Total sample weight (g).
RC: Relative recovery (dimensionless).
RF: Response factor (dimensionless).
Response factor (RF) = (AIS per g/(ASPL per g) (50)
For more accuracy: Response factor (RF) = (SIS/SSPL) (51)
Where SIS: Slope of IS regression line of 6 points.
SSPL: Slope of SPL regression line of 6 points.
*Recovery test: 6 different concentrations.
1. Absolute recovery: 10 or less to 70%.
2. Relative recovery: 90 to 110%, for volatiles, 70 to 90%.
Sample A: IS + component C
Sample B: IS only.
C in A (g/g) = (AC/AIS)(WIS/WA)(RF)
C in B (g/g) = (AC/AIS)(WIS/WB)(RF)
=> RC = (C in A - C in B)/ g C fortified per g sample
(52)