CHROMATOGRAPHY

Chromatography is the most powerful

tool for separating & measuring the
components of a complex mixture.
Quantitative & qualitative analysis
Chromatography is a physical method of separation in which the
components to be separated are distributed between two phases, one of
which is stationary while the other moves in a definite direction (IUPAC,
1993)

PRINCIPLE
Types of Chromatography : Based on interaction of the solute v.s.
the stationary phase
Adsoption Partition Chromatography
Ion. Exchange Molecular Exclusion Chromatography
Affinity Chromatography

PRINCIPLE
Types of Chromatography
.

the surface hydroxyl groups interact with the analytes, the

more the interaction the more the analytes are retained and
therefore the longer they take to pass through the column

PRINCIPLE
Types of Chromatography
Adsorption Chromatography

. surface hydroxyl groups interact with the analytes,

the
the more the interaction the more the analytes are
retained and therefore the longer they take to pass
through the column
The mobile phase (solvent) and analyte molecules
compete for the adsorbent sites of the stationary phase.
The more polar the molecules the longer they are
retained.
Strong solvents result in short retention times as the
solvent molecules successfully compete for the
stationary phase active sites which means there are
fewer available sites to retain the analyte molecules

Si OH
O
Si OH
O

PRINCIPLE

PRINCIPLE
Types of Chromatography
Partition Chromatography

.a solid support coated with a high boiling liquid which

Solid
support

acts as the stationary phase

Separation occurs because of the differences in solubility
for the analytes in the stationary and mobile phases
The partition coefficient is defined as:

K =

Concn. in stationary phase

Concn. in mobile phase

Stationary phase

Analyte

PRINCIPLE
Types of Chromatography
There are two types of partition chromatography normal phase and
reversed
phase, they are defined by the relative polarities of the
.
mobile and stationary phases

Stationary phase

Mobile phase

Normal
phase

High polarity

Low polarity

(hydrophilic)

(hydrophobic)

Reversed
phase

Low polarity

High polarity

(hydrophobic)

(hydrophilic)

NORMAL PHASE
Stationary Phase

Silica gel: -Si-OH

Cyano type: -Si-CH2CH2CH2CN
Amino type: -Si-CH2CH2CH2NH2
Diol type: -Si-CH2CH2CH2OCH(OH)-CH2OH

REVERSE PHASE
C18 (ODS) type
C8 (octyl) type
C4 (butyl) type

Si

-O-Si

Phenyl type
TMS type

CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2

CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3

C18 (ODS)

THEORY OF CHROMATOGRAPHY

Chromatography is a physical method of separation in which the

components to be separated are distributed between two phases, one of
which is stationary while the other moves in a definite direction (IUPAC,
1993)

PRINCIPLE
Solvent Extraction :
transfer of a solute from phase 1 phase 2

S (in phase1) S (in phase 2)

Partition coefficient
K = Co/Cw
Co is concentration in the organic
phase (solvent)
Cw is the concentration in the
aqueous phase (water)

s2
K
s1

PRINCIPLE
K = Co/Cw
Co is concentration in the organic
phase (solvent)
Cw is the concentration in the
aqueous phase (water)

s2
K
s1

PRINCIPLE

0.83 mg

0.69
0.83 mg

0.69 mg
0.58

1.0 mg

How would you make this broad peak more narrow?

PRINCIPLE

PRINCIPLE

PRINCIPLE

PRINCIPLE
Efficiency is a factor that is typically used to
describe peak width.
0.83 mg

High Efficiency - narrow peaks

The term that is generally used to describe
column efficiency is number of theoretical plates or N

1.0 mg

N = L/H

H (heigh equivalent to Theoritical Plate)

Where: L =column length

H = plate height (both in the same units)

PRINCIPLE
RETENTION : CAPACITY FACTOR or RETENTION FACTOR
Higher values of k mean the analyte will stay in the column longer. The longer it stays,
the more time there is for the peak will widen.
0.83 mg

1.0 mg

k = (tr to)/ to
Where tr = the retention time of the
compound, and to = the dead time

H (heigh equivalent to Theoritical Plate)

PRINCIPLE
kx = ( tR - tm) / tm
Rf = x / = 1/(1+k)
Rf = tm /( tm+tR(x) = 1/(1+k)
k = (1 Rf) / Rf (13)

tR(x)
tm

Rf = 0 k ~ (sampel tdk migrasi)

Rf = 0,5 k = 1
t (menit)

Rf = 1 k = 0 (sampel terbawa pelarut)

Pengaruh polaritas fase gerak terhadap retensi

Utk suatu analit :
Log k2/k1 = (P1 P2)

kB < kA, tRB < tRA

Fase Normal

Fase diam: polar

Fase gerak: kurang polar

Fase Balik

Fase diam:kurang polar

Fase gerak: polar

kA > kB, tRA > tRB

Utk suatu analit :
Log k2/k1 = (P2 P1)

PRINCIPLE
RETENTION : SELECTIVITY
the selectivity factor and is an indication of how well the compounds will separate.
Higher means larger difference in retention time and more separation
0.83 mg

1.0 mg

H (heigh equivalent to Theoritical Plate)

= kB/kA

PRINCIPLE
Retention (the time it takes for the analytes to elute, related to k)
Selectivity (how different the analytes are from each other and related to )
Efficiency (how good the column is, related to N)

RESOLUTION

PRINCIPLE

RESOLUTION
Rs = tRB tRA / (WB+WA)
= 2 (tRB tRA ) /(WB+WA)
A and B are the two peaks
tR = retention time and
w = the peak width at the base
of each
peak

tRA
tRB
tm

A
Wa

Wb

PRINCIPLE

PRINCIPLE
N = L/H
Where: L =column length
H = plate height (both in the same units)

The value of H depends primarily on four factors:

1) the velocity of the mobile phase,
2) eddy diffusion or multipath diffusion,
3) the diffusion of the compound in the mobile phase
4) the transfer of the compound between the stationary phase and the
mobile phase.

PRINCIPLE
H = A + B/u + (Cs + Cm) u
u = the average linear mobile phase velocity
A is a term expressing multipath diffusion
B/u is the term for longitudinal diffusion
Cs is the mass transfer term in the stationary phase
Cm is the mass transfer term in the mobile phase

PRINCIPLE
Flow
Direction

Multipath

The amount of spreading is affected by the nature of the

column material and how well the column is packed. This
factor is generally proportional to the particle size of the
packing material.

2
Pathways of two molecules during elution. Distance traveled by
molecule 1 is longer than that traveled by molecule 2, thus molecule 1
will take longer to elute.

PRINCIPLE

Longitudinal Diffusion

At low velocities longitudinal diffusion has a negative effect on resolution, but this
effect is negligible at higher velocities. This term is very important in gas
chromatography as diffusion coefficients in gasses are orders of magnitude higher
than in liquids. In liquid chromatography, this term is typically close to zero
relative to the other terms.

Molecules diffuse from areas of high

concentration to areas of low concentration.

Flow
Over time.

Flow

PRINCIPLE
Longitudinal diffusion :
the faster the flow
the less a band spends in column.
the less time for diffusion.
broadening 1

PRINCIPLE

mass transfer

The faster the mobile phase moves, the less time there is
for equilibrium between the phases and the mass transfer
effect on peak broadening is directly related to mobile
phase velocity.

m.p.
s.p.

PRINCIPLE

CHROMATOGRAM OF GC AND HPLC

tR- retention time - determines sample identity
to - elution time of unretained peak

QUALITATIVE

tR
QUANTITATIVE

tR
mAU

Area is proportional
to the quantity of
analyte.

to
Time (minutes)

QUALITATIVE ANALYSIS
Melamine Determination in several Milk

a : Melamin Standard 0,5 ppm

b : Strawbery Milk
c : strawberry Milk spike 1 ppm
d : strawberry Milk spike 2 ppm

Look at the retention time of standard and

compare it with the retention time of
sample

QUALITATIVE ANALYSIS
How if my sample contain a lot of peaks? Which one should I take?

Look at the retention time of standard and

compare it with the retention time of
sample

QUANTITATIVE ANALYSIS
Using AUC (Area Under Curve) make calibration curve

Calibration Curve
Type of calibration curve :
1. External Standard Calibration Curve
2. Internal Standard Calibration Curve
3. Standard Addition Calibration Curve

One point method

QUANTITATIVE ANALYSIS

AUC

External Standar Calibration Curve

Y = mx + h
AUC = slope x concentration + h
concentration

1. Inject several concentration (minimum

5) into HPLC
2. Record the AUC of each concentration
3. Plot graph (in excel) where the axis is
concentration and the y is AUC
4. Find the regression linear equation from
the graph
5. Inject the sample into HPLC and record
the AUC in defined Retention time
6. Input the AUC into the equation and
calculate the concentration

QUANTITATIVE ANALYSIS
Using AUC (Area Under Curve)
4500
4000
3500
3000
2500
2000
1500

1000

y = 69.838x - 46.223
R = 0.9979

500
0
0

10

20

y = 69.838x - 46.223

30

40

50

60

2200 = 69.838x 46.223

C (ppm)
5
15
25
35
45
55
Sample
Sample

AUC
334.8
1010.3
1670.5
2389.5
3006.4
3882
2200
5000

X = ..

RT
15.932
15.608
15.588
15.484
15.257
15.187
15.129
20.000

QUANTITATIVE ANALYSIS

RATIO

Internal Standar Calibration Curve

substance different from the analyte added in a constant

amount to all samples, blanks, and standards
Internal standards are used when the detector response
varies slightly from run to run because of hard to control
parameters. (e.g. Flow rate in a chromatograph)
Even if absolute response varies, as long as the relative
response of analyte and standard is the same, we can find
the analyte concentration.
Plotting the ratio of analyte to internal-standard
Internal standard must not produce signals that interfere
Y = mx + h
with analyte Usually they are separated by wavelength or
AUC = slope x concentration + h time.
concentration

QUANTITATIVE ANALYSIS
Internal Standar Calibration Curve

RATIO

Plotting the ratio of analyte to internalstandard.

Internal standard must not produce signals
that interfere with analyte Usually they are
separated by wavelength or time.
A mixture is prepared with a known amount
of analyte and standard. The detector
usually has a different response for each
species
Y = mx + h
AUC = slope x concentration + h

concentration
Area of
analyte signal
area of standard signal
F

Concentration of analyte
Concentration of standard

QUANTITATIVE ANALYSIS
Internal Standar Calibration Curve

QUANTITATIVE ANALYSIS
Internal Standar Calibration Curve

Example: Pb by ICP Emission

Each Pb solution contains 100
ppm Cu.

Signal
[Pb]
(ppm)

Pb

Cu

20
40
60
80
100

112
243
326
355
558

1347
1527
1383
1135
1440

Pb/Cu
0.083
0.159
0.236
0.313
0.388

No Internal Standard Correction

600

Pb Emission Signal

500

400

300

200

100

0
0

20

40

60

[Pb] (ppm)

80

100

120

Internal Standard Correction

0.450
0.400

Pb Emission Signal

0.350
0.300
0.250
0.200
0.150
0.100
0.050
0.000
0

20

40

60

[Pb] (ppm)

80

100

120

Results for an unknown sample after adding

100 ppm Cu
Run

Pb

Signal
Cu

1
2
3
4
5

346
297
328
331
324

1426
1229
1366
1371
1356

mean

S/N

325
17.8
18.2

1350
72.7
18.6

Pb/Cu
0.243
0.242
0.240
0.241
0.239
0.241
0.00144
167

QUANTITATIVE ANALYSIS
Standard Addition Calibration Curve

spike the sample by adding known amounts of standard solution

to the sample
Assumes that matrix is nearly identical after standard addition
Useful when the analyte is present in a complicated matrix and
no ideal blank is available.
The x-intercept of the standard addition plot corresponds to the
amount of analyte that must have been present in the sample
(after accounting for dilution).

QUANTITATIVE ANALYSIS
Standard Addition Calibration Curve

QUANTITATIVE ANALYSIS
Standard Addition Calibration Curve

1.2

=
+

Signal from standard

Signal from unknown

Signal (V)

0.8

0.6

0.4

= +

0.2

0
-10

-5

0
-0.2

10

15

20

25

Example: Pb in Blood by GFAAS

Concentration
(ppb)

0.50
1.50
2.50
3.50
4.50
5.50

Signal
(AUC)

3.76
9.16
15.03
20.42
25.33
31.87

Results of linear regression:

S = mC + b
m = 5.56 mAbs/ppb

b = 0.93 mAbs

35

30

y = 5.56x + 0.93

mAbs

25

20

15

10

0
0

Pb Concentration (ppb)

A sample containing an unknown amount of

sample gives a AUC of 27.5. Calculate the
sample concentration.
S = mC + b

C = (S - b) / m
C = (27.5 0.92) / 5.56

C = 4.78 ppb

Example: Fe in Drinking Water

Sample
Volume
(mL)
10
10
10
10
10

Standard
Volume
(mL)
Signal (V)
0
5
10
15
20

0.215
0.424
0.685
0.826
0.967

The concentration of
the Fe standard
solution is 11.1 ppm
All solutions are
diluted to a final
volume of 50 mL

1.2

Signal (V)

0.8

0.6

-6.08 mL

0.4

0.2

0
-10

-5

10

-0.2

Volume of standard added (mL)

15

20

25

[Fe] = ?
x-intercept = -6.08 mL
Therefore, 10 mL of sample diluted to 50 mL would give a
signal equivalent to 6.08 mL of standard diluted to 50 mL.

Vsam x [Fe]sam = Vstd x [Fe]std

10.0 mL x [Fe] = 6.08 mL x 11.1 ppm

[Fe] = 6.75 ppm