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V. CHROMATOGRPAHY
*Chromatographic methods:
1.
2.
3.
4.

Plane chromatography (PC and TLC)

Gas chromatography (GC)
Liquid chromatography (LC)
Other separation methods: Supercritical fluid
chromatography (SFC) and capillary electrophoresis

*Separation based on rate process:

Field: Electrophoresis, centrifugation, mass spectrometry
Barrier (porosity): Membrane (UF, RO), dialysis, gas diffusion
*Separation based on equilibrium between phases:
Gas-liquid: Gas-liquid chromatography (GLC), distillation
Gas-solid: Gas-solid chromatography (GSC), sublimation
Liquid-liquid: Liquid-liquid chromatography (LLC), extraction
Liquid-solid: Liquid-solid chromatography (LSC), crystallization
or precipitation
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*Phases: Gas, supercritical fluid (gas under pressure at certain
temp.), liquid and solid.
*Chromatography: Tswett (1906)
Chromatography = Chroma + graphein (Greek)
Color
write
First used to describe the research work on the separation of
colored pigments into bands on a column of chalk.
*Definition of chromatography:
"Chromatography is a method in which the components of a
mixture are separated on an adsorbent column in a flowing
system."Tswett (1906)
"A method used primarily for the separation of the components of
a sample, in which the components are distributed between two
phases, one of which is stationary while the other moves. The
stationary phase may be a solid, or a liquid supported on a solid,
or a gel. The stationary phase may be packed in a column, spread
as a layer, or distributed as a film, etc.; in these definitions
'chromatographic bed' is used as a general term to denote any of
the different forms in which the stationary phase may be used.
The mobile phase may be gaseous or liquid."
International Union of Pure and Applied Chemistry (1974)
*Chromatographic methods: Classified based on equilibration
process, which is governed by the type of stationary phase.
1. Adsorption chromatography: Based on adsorption.
Stationary type: Plane: TLC (solid supported on an inert plate)
Column: Solid on which sample components are adsorbed.
Mobile phase: Liquid: LSC or CC; gas: GSC
The components distribute between the two phases through a
combination of adsorption and desorption processes.
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2. Partition chromatography: Based on solubility.
Stationary type:
Plane: PC (a layer of water adsorbed on a sheet of paper)
Column: Liquid supported on an inert solid.
Mobile phase: Liquid: LLC; gas: GLC
3. Bonded phase chromatography:Based on partition.
Stationary phase: Organic species bonded to a solid surface.
Mobile phase: Liquid: BPLC; gas: BPGC; supercritical fluid: SFC
4. Ion-exchange chromatography: Based on ion-exchange.
Stationary phase: ion-exchange resin.
Separation mechanism is based on ion-exchange equilibrium.
5.

Molecular exclusion chromatography (gel permeation

chromatography, gel filtration): Based on pore penetration.
Stationary phase: molecular sieve.
Molecules are separated according to their size by their ability to
penetrate a sieve-like structure.

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*Elution development (elution chromatography):
Most widely used in the GSC, GLC, LLC and LSC.
Small sample is introduced onto the column and eluted with a
mobile phase, which has a lesser affinity for stationary phase
than sample components.
Used for analytical separation due to complete separation.
Simple elution: Eluted with the same solvent.
Stepwise elution: Changing the eluent after predetermined period
of time to increase eluting power.
Gradient elution: Using a gradual change in composition of the
eluting solvent to achieve separation.
May be linear, steadily increasing or decreasing, or logarithmic
and may be a concentration, pH, polarity or ionic strength
gradient.

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*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

(1)
(2)

Where KD: Distribution (partition) coefficient.

nM: No. of molecule (solute) in the mobile phase.
nS: No. of molecule (solute) in the stationary phase.
*Chromatogram:

Response

tR
Start
tM

Retention Time
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V. CHROMATOGRPAHY

*Retention time:

tR = tR' + tM

(3)

Where tR: Solute retention time.

tR': Adjusted retention time.
tM: Column dead time (for an unretained component, e.g.
air (or methane) for GC).
*Retention volume:

VR = VM + KD VS

(4)

WhereVR: Retention volume.

VM: Mobile phase volume.
VS: Stationary phase volume.
KD: Distribution coefficient.
*Retention time vs. retention volume:
VR = tR Fc
Where

and

VR' = tR' Fc

(5)

Fc: Column flow rate.

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*Resolution: R = (Efficiency) (Capacity factor) (Selectivity)
(6)
R = (1/4) [L/H]1/2 [k/(k+1)][ (-1)/
(7)
R = (1/4) [N]1/2 [k/(k+1)][ (-1)/
Where

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).

*Capacity factor, k: Rate of solute migration.

Ideally, k = 1-10 for both resolution and retention time.
k = (Time in phase S)/(Time in phase M)
k = tR'/tM = (tR - tM)/tM = tR/tM - 1
k = VR'/VM = (VR - VM)/VM = KD (VS/VM)
=>
tR = (1 + k) tM
=>
tR = (1 + k) (L/)
(Since tM = L/)
Where : Average mobile phase velocity.

(8)
(9)
(10)
(11)

*To reduce tR, shorten column length or increase flow rate.

Changes in mobile phase flow rate affect the retention times, but k
remains unchanged, see (11).
k = 0 =>
No resolution.
k = 1 =>
k/(k+1) = 0.5=>
50% increase in R.
k = 9 =>
k/(k+1) = 0.9=>
90% increase in R.
k moves from 0 to 9 => Increase in R moves from 0 to 90%.
Increases in k enhance R, but elongate elution times.
Optimizing k to improve resolution:
1. GC, increasing column temp. or temp. programming.
2. SFC, reducing density.
3. HPLC, changing solvent composition (gradient elution or
solvent programming).
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Example:
tM

t Rb

tRc

a
1
2
ka = (tRa - tM)/tM = 0;
k=0

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

(12)
(13)

*Elution problem:
1a
1b

2a
2b
2c

1.
2.

a and b: Same k, same , but different N.

a > b: Change in N (efficiency) by changing the column.
a > c: Change in (selectivity).
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To increase while maintaining k in the range of 1 to 10:
1. Changing mobile phase composition including change in pH.
2. Changing column temperature.
3. Changing stationary phase composition.
4. Using special chemical effects, e.g., adsorbent impregnated
with silver salt improves separation of olefins.
*Parameters for the peak curve:
Where
h

Wh: Peak width at 1/2 height.

Wb: Peak width at the base.
h: Height of a triangle formed

Wh
h/2
Wb

by drawing tangent to the

curve (peak).

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).
or = Wb/4
(14)
Wb = 4
Wh = [5.54]1/2
(15)

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*Peak shape terms:

1.
2.
3.

Gaussianideal shape.
Tailingsurface adsorption effects or dead volume.
Frontingoverloaded or sample capacity exceeded.

*Peak width vs. retention time:

Wb1/Wb2 = [t1/t2]1/2

At the same peak height

(16)

Peak broadening is due to longitudinal diffusion molecular

diffusion through the column.

TLC plate

Chromatogram

The longer the retention time, the broader the peak.

*Resolution: Resolution is complete at R = 1.5.
A measure of the degree of separation of adjacent peaks.
R = tR/4 = (tR1-tR2)/[0.5(Wb1+Wb2)]
= 2 (tR1 - tR2)/(Wb1 + Wb2)
*Efficiency of separation (theoretical plates, n or N):
n = [tR/]2 = 16 [tR/Wb]2 = 5.54 [tR/Wh]2

(17)
(18)
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*Bandspreading:
Open tube (capillary)

Long

Packed column

Short

Stationary phase coated on the wall

Bandspreading

*Effective theoretical plates, Neff:

Neff = 16 [tR'/Wb]2
=> Neff = 16[tR'/Wb]2[tR/tR]2 = 16 [tR/Wb]2 [tR'/tR]2
Neff = N [k/(1+k)]2
At large k, k+1 ~ k => Neff = N
106

(19)
(20)

Practical useful efficiency

Neff

0
0

20
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V. CHROMATOGRPAHY
*HETP (height equivalent to a theoretical plate):
(21)
H = 2/L
H = L/n
or
H = L/N
(22)
=> n = L/H = L/(2/L) = L2/2 = L2/[Wb/4]2 = 16 [L/Wb]2 (23)
Substitute L, Wb in length with tR, Wb in time
=> N = 16 [L/Wb]2 = 16 [tR/Wb]2
(17) (23)
Where H is constant for a given system.

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. Uneven flow velocity across the column. (packed column).
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*Resolution vs. efficiency:
R = (Efficiency) (Capacity factor) (Selectivity)
Diffusion
Interaction
R = (1/4) [L/H]1/2 (k/k+1) (-1/)
=>
R = (1/4) [N]1/2 (k/k+1) (-1/)
=>
N = 16 R2 [k+1/k]2 [/]2
=>
R=(1/4)[0.5(N1+N2)]1/2[(tR1'-tR2')/0.5(tR1'+tR2')]
From (10)(21) => tR = (NH/) (k+1)
=>
tR = 16 R2(H/) [(k+1)3/k2] [/]2
=>
N1 : N2 = R12 : R22 or tR1 : tR2 = R12 : R22 or
H1 : H2 = R22 : R12

(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,
(29)
= Fc/Ac = Fc/r2Ee
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:
(30)
H = A + B/ + C = A + B/ + (Cs + Cm)
(31)
H = HE + HL + HS + HM
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.
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*Eddy diffusion (radial diffusion)A term: Uneven flow path.
HE = A = 2 dp
(32)
Where : Packing factor, 1-8.
20-40 mesh: = 1, 200-400 mesh: = 8.
dp: Average packing particle diameter.
Independent of linear velocity (), diffusion coefficient (D).
A term is 0 for open tubular GC columns due to single path flow.
*Longitudinal diffusionB/ term: Bandspreading process.
HL = B/ = 2 DM/
(33)
Where : Linear velocity of mobile phase.
Obstruction factor, packed: 0.6-0.8, open: 1.

DM: Diffusion coefficient in mobile phase.

Important at low mobile-phase velocity.
Important in GC than LC, since diffusion coefficient (diffusivity)
of solutes in gases are at least 104 greater than in liquids.
Diffusion
Gas
Supercritical
Liquid
Solid
Diffusion as (MW):
MW
10
100
1,000
10,000
100,000
1,000,000

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

Drops by ca. 1/2 for one log of MW.

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*Stationary phase mass transfer (open tube)Cs term:
HS = Cs = [2k/3(1+k)2][df2 /DS]
Where df: Film thickness.
DS: Diffusion coefficient in stationary phase.
Unimportant in SEC due to no stationary phase.
*Mobile phase mass transfer (open tube)Cm term:
HM = Cm = [(1+6k+11k2)/96(1+k)2][dc2/DM]
Where dc: Column diameter.

(34)

(35)

*HETP for open tube capillary column:

HETP = 2 DM/ + [2k/3(1+k)2][df2/DS] +
[(1+6k+11k2)/96(1+k)2][dc2/DM]
(36)
No A term, and = 1.
*HETP for packed GC column:
HETP = 2 dp + 2 DM/ + [2k/3(1+k)2][df2 /DS] +
(37)
[dp2/DM]
*HETP for packed LSC column:
HETP = 2 dp + 2 DM/ +
[(a+bk+ck2)/24(1+k)2][dp2/DM]
(38)
No CS (DS) due to adsorption rather than partition in stationary
phase.

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*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. The column resolution;
b. The average number of plates in the column;
c. The plate height;
d. The length of column required achieving a resolution of 1.5;
e. The time required to elute substance B on the longer column;
f. The plate height required for a resolution of 1.5 on the original
30-cm column and in the original time.
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.

(21)
(28)
=>
=>

H = L/N = 30/3.44 x 103 = 8.71 10-3 cm

N1 : N2 = R12 : R22 => 3445 : N2 = (1.06)2 : (1.5)2
N2 = 6.9 x 103
L2 = (6.9 x 103) (8.71 10-3 cm) = 60.1 cm

e.

(28) tR1 : tR2 = R12 : R22 => 17.63 : tR2 = (1.06)2 : (1.5)2
=> tR2 = 35.3 min

f.

(28) H1 : H2 = R22 : R12 => H2 = (8.71 10-3)(1.06)2/(1.5)2

=> H2 = 4.3 10-3 cm
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Concentration (C)

V. CHROMATOGRPAHY
*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)

Zero moment M0: c(t) dt

First moment M1: (1/A) c(t) dt

Area

(39)

Retention time (tR) (40)

Second moment M2: (1/A) c(t - tR)2 dt

Variance (2)

Third moment M3: [1/A(t2)] c(t - tR)3 dt

Skew

(41)

(42)

Skew = 0, if it is Gaussian distribution.

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*Molecular basis for separation:
Molecule i interacting with phase j
Eij = Ed + Eo + Ei + Eab

(43)

WhereEij: Total interaction energy.

Ed: London dispersion force; small number.
Ed = - (3/2) [Ii Ij/(Ii+Ij)] (ij/r6) ~ 1 kcal/mol

(44)

Eo: Orientation dipole interaction; moderately large no.

Eo = - (2/3) (i2j 2/kTr6) = 1-10 kcal/mol

(45)

Ei: Induced dipole interaction; moderately large no.

Ei = - (i2j/r6) = 1-10 kcal/mol
Ed + Eo + Ei: Van der Waals forces.

(46)

Eab: All acid-base interaction including H-bonding and

electrostatic forces. Hard to quantitate. Large no. when
present.
Eab ~ EAEB + CACB = 1-50 kcal/mol

(47)

Where i or j: Respective dipoles of molecule i or j.

r: Distance between centers.
T: Temperature (K).
Ii or Ij: Ionization potentials.
i or j: Polarizability.
EA or EB: Hard effects, electrostatic charge-transfer.
CA or CB: Soft covalent effects, electron sharing Lewis
acid-base.
Useful for predicting relative behavior but hard to calculate or
estimate on absolute terms due to nonideal behavior and
difficulty of selecting an accurate mode.
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*Kovats retention index (RI):
For a normal alkane standard, retention index is equal to 100 times
the number of carbons in the compound regardless of column
packing, temperature or other gas chromatographic conditions.

Log t R' (sec)

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

Heptane
Hexane

5
6
7
8
9
Number of n-alkane carbon atoms

Kovats retention indices and McReynolds numbers are commonly

used to define overall polarity of stationary phase.
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*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).
(50)
Response factor (RF) = (AIS per g/(ASPL per g)
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)
(52)
=> RC = (C in A - C in B)/ g C fortified per g sample
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*External standard method: Calibration curve method.
Using the same component, a curve was established by plotting
concentration vs. peak areas or heights.
The curve should consist of 6 pts, and pass through the origin.
Frequent restandardization is necessary for highest accuracy,
especially for the slope correction.
*Internal standard method:
Highest precision for quantification of chromatography due to
difficulty of accurate injection.
Resolution of IS from other components should be > 1.5.
IS is added prior to sample preparation. If there is a loss of sample
during extraction or other sample preparation procedure, the
losses of sample compound and IS are assumed to be the
same.
*Double internal standard method:
First IS is added prior to sample preparation mainly for relative
recovery of sample, especially in the low recovery of volatile
compounds.
Second IS is added after sample prepared and prior to
chromatographic analysis mainly for quantitation.
*Combination of internal and external standard method:
Internal standard is added prior to sample preparation for the
determination of relative recovery of analytes.
Component quantification of sample in the chromatograms was
mainly based on each external standard due to the differences
in response factors for multiple components.

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73