Introduction of Theory

Chromatography
 Theory

1. Chromatography Basic
2. Efficiency of Separation
3. Why Bands Spread
CHROMATOGRAPHIC COLUMN
 Chromatography Basics
Typical chromatogram:
Response
Detector

time or volume
 Chromatography Basics
tr2
tr1
Response
Detector

tm
tr1

injection time time or volume

tm = time for mobile phase to travel tr = retention time
length of column (dead time)
tr = adjusted retention time
= tr2/tr1 = relative retention
= tr - tm
 Chromatography Basics
Mobile phase flow rate:
volumetric flow rate (F): ml/min
linear flow rate (v): cm/min (mm/min)

Two ways to describe solute retention

retention time, tr
retention volume, Vr

Vr = Ftr
 Chromatography Basics
Partition coefficient K = Cs/Cm
C = Concentration of analyte
s = stationary phase
m = mobile phase

Vs = volume of stationary phase

Vm = volume of mobile phase
 Chromatography Basics
Capacity factor, k: a measure of retention
higher k = greater solute retention

Vs moless
k = K = moles
Vm m

ts tr - tm
k = t = t
m m
 Theory

Efisiensi Chromatography
1. Harga N (jumlah pelat teori) besar
2. Harga H (Jarak setara pelat teori/HETP) kecil
3. Harga L (panjang kolom) yang pendek
4. Harga t (waktu tambat) yang singkat
5. Harga R (resolusi) yang besar
 Efficiency of Separation
Two factors affect how well two components
are separated
difference in retention time
peak widths
 Efficiency of Separation
Solutes in a column spread into a Gaussian profile:
Gaussian peak shape:

s s
h
w1/2=2.35s
1/2h
w=4s

t0 tr
time
 Efficiency of Separation
(HETP) Height Equivalent of a Theoretical Plate / JSTP (Jarak
Setara Pelat Teori) adl panjang kolom kromatografi (dlm mm)
yang diperlukan sampai terjadinya satu kali keseimbangan
molekul komponen dalam fase gerak dan fase diam
 Efficiency of Separation
Plate theory (HETP):
treats separation in discrete stages, more stages = more plates.
The smaller HETP, the narrower the eluted peak
Theoretical plates (N): a number indicating how
good a column is for a separation
 Efficiency of Separation
N depends upon the length of the column
Independent of the column length is the
Height Equivalent of a Theoretical Plate
2
L Lw
HETP 2
N 16tr
As HETP , resolution increases (N )
 Efficiency of Separation
The resolution (separation) of two solutes:
Dtr DVr
Resolution (R) =
wavg = wavg

Dtr = difference between retention times of two peaks = (tr2 - tr1)

wavg = average of the peak widths at baseline ( 4s)

 Efficiency of Separation
Resolution: higher R, better separation
R=0.50 R=0.75

t0 2s time t0 3s time

R1 R=1.00 R=1.50
is good

t0 4s time t0 6s time
 Efficiency of Separation
2 2 2
tr 16tr 5.55tr
N
s 2
w 2
w1 2
2

N is specific for each solute on a given

column
Increasing retention time increases N
 Efficiency of Separation
Ns relation to Resolution (R):

N 1 k2
R
4 1 kavg
tr2 k2 K2
= relative retention = = =
tr1 k1 K1
 Efficiency of Separation
N required to obtain a certain resolution:
2 1 k 2
2 avg

N 16R
1 k2
N1 N2 N2>N1

t0 time t0 time
 Why Bands Spread
Band broadening

Causes of band broadening:

Longitudinal diffusion (difusi molekul solut)
Resistance to mass transfer (RMT) (tahanan alih massa)
Eddy diffusion (difusi pusaran)
 Why Bands Spread
Longitudinal diffusion: solute [ ] is lower at
the edges of a band; solute diffuses to the edges.

Low High Low

Same Conc.at Equil.
Conc. Conc. Conc.

HB = B/v
B = constant, v = flow rate
decrease HB by increasing v
 Why Bands Spread
Resistance to mass transfer (RMT):
Mobile
phase slow equil.
Stationary
phase
bandwidth bandwidth

HC = Cv
C = constant, v = flow rate
decrease HC by decreasing v
 Why Bands Spread
Eddy diffusion :

time

HA = A
A = constant, depends on size of particles
 Why Bands Spread
Van Deemter mengemukakan syatu persamaan
relasi antara JSTP terhadap laju reaksi
Van Deemter Equation:

HETP = HA + HB + Hc
HETP = A + (B/v) + Cv
 Why Bands Spread
van Deemter Plot:

H B Cv
v
Hmin
A

vopt
v
 Why Bands Spread
Asymmetric peak shapes: K depends on [ ]
at high [ ] (solute becomes solvent)

(+) overloaded
Linear ideal
peak shape
Cs
(-) tailed

Cm
 Why Bands Spread
Asymmetric peak shapes Observed
slow chromatogram
slow
(+) deviation:

fast fast
fast

fast fast

(-) deviation:
slow slow
slow
time
 High Performance Liquid
Chromatography

HPLC is characterized by the use of high pressure

to push a mobile phase solution through a column
of stationary phase allowing separation of
complex mixtures with high resolution.
Instrument Schematics
High Pressure Liquid Chromatography (HPLC)
STATIONARY PHASES
ADSORBENT PARTICLE
Columns & Guard Columns
pHidelity Columns
Pinnacle DB Columns
Pinnacle II Columns
Allure Columns
Ultra Columns Viva
Wide Pore Columns
Guard Columns
Prep Columns
Methods Development and Validation Kits Fast LC
Columns by Phase C18 Columns C8 Columns Biphenyl
Columns Cyano Columns Phenyl Columns Silica Columns Other
Phase Columns
Columns by Use
pH Stable Columns
Columns for Acidic Analytes
Columns for Basic Analytes
Columns for Hydrophobic Neutral Analytes
Columns for Hydrophilic Neutral Analytes
Columns for Mixed Analytes
Columns for LC/MS Columns: Unique Phases
Columns: Other Phases
Applications Applications:
Biochemical Applications:
Environmental Applications:
Foods & Flavors Applications:
Forensic Applications:
General Applications:
Nutraceutical Applications:
Pharmaceutical Application
 Chromatography Stationary Phases

Silica Gel Derivatized Silica Gel

Where R = C18H37
hydrocarbon chain
(octadecylsilyl deriv.
silica or C18)

bulk (SiO2)x surface bulk (SiO2)x surface

relatively polar surface relatively nonpolar surface

normal phase reversed phase
Normal vs. Reversed Phase Chromatography
Chromatographic Modes, Sorbent types, Typical elution solvent
 Solvent Extraction (pH effects)
Equilibrium constant for this partitioning is
K (partition coefficient)

[S]2
K=
[S]1
 Solvent Extraction (pH effects)
with organic acids/bases:

Ka
HA H+ + A-
Kb
B + H2O BH+ + OH-
Generally, neutral species are more soluble
in an organic solvent and charged species
are more soluble in aqueous solution
 Solvent Extraction (pH effects)
Partitioning of organic acids between two
phases:
very little here, ions
have poor solubility
organic HA H+ + A-

Ka
aqueous HA H+ + A-
 Solvent Extraction (pH effects)
When the solute (acid/base) can exist in different
forms, D (distribution coefficient) is used instead
of K (partition coefficient)
 Solvent Extraction (pH effects)

total conc. in phase 2

D=
total conc. in phase 1
HA [HA]org
D=
K [HA]aq + [A-]aq
Ka
HA H+ + A-
 Solvent Extraction (pH effects)
Substitute for [A-] in D eq. and rearrange

[H+][A-] Ka [HA]
Ka = [A-] =
[HA] [H+]
[HA]2
D=
Ka [ HA]1
[HA]1
[H ]
 Solvent Extraction (pH effects)
[HA]2 [HA]2
D=
Ka [ HA]1 K
[HA]1 [HA]1 1
a
[H ] [ H ]

Ka [HA]2
D 1 = =K
[ H ] [HA]1
Solvent Extraction (pH effects)

Ka
D 1 = K
[ H ]

K K[ H ]
D=
Ka [ H ] Ka
1
[ H ]
 Solvent Extraction (pH effects)
pH effect on D for organic acids
[H+]=Ka
[H+]>>Ka
pH=pKa
K

mainly mainly
D HA A-

[H+]<<Ka
[HA]org
D=
[HA]aq + [A-]aq pH
 Solvent Extraction (pH effects)
Example problem: Want to separate two organic
acids using a scheme based on pH.
Acid 1 (pKa = 4), Acid 2 (pKa = 8)

K1 Acid 2 stays in
K2 organic phase,
acid 1 is extracted
D into aqueous phase

4 pH 8
 Solvent Extraction (pH effects)
Analogous treatment for organic bases (proton
acceptors, not KOH)

[H+]=Ka
pH=pKa [H+]<<Ka
K
K Ka mainly mainly
D= D BH+ B
[H+] + Ka
[H+]>>Ka

pH
 HPLC System
9060 Polychrom Computer
(Diode Array) Detector Workstation

9050 Variable
9010 Solvent UV/Vis Detector
HPLC Solvent Delivery System
Reservoirs

Rheodyne
Injector

Keep an eye on
HPLC these 4 screens!
Column
Solvent Delivery System
 %A %B %C Flow Rate Pressure to column
{H2O} {MeOH} (mL/min) (atmos.)
load

Ready
inject

Rheodyne
Injector
Varian 9010 Solvent Delivery System to injector

through pump

Column
through C
pulse
dampener
A B

from solvent to
Ternary Pump reservoir detector
Variable UV/Vis Detector
ABS AUFS l RunTime EndTime
0.001 2.000 238 0.00 min 10.0 min

Ready
 Ready
UV Spectrum
UV Spectrum {shows full UV abs.}
UVmax
UVmax

Chromatogram

ABS.
Reset
Wavelength

Rt Rt

ABS.
Time

Chromatogram
{shows peaks, Rt}

Varian 9060
Polychrom Detector
 Approximation
HPLC Chromatograms of peak area by
triangulation
Absorbance

Peak A Peak B

height

0 1 2 3 4 5 6 7
Time (minutes) base

Rt = 3.0 min. Rt = 5.2 min. base x height

faster moving slower moving Area =
2
less retained more retained
 Reduce Particle Size to Improve Efficiency and
Resolution- Small Molecule Separations
High Speed Separation of Analgesics on Columns
with Different Particle Sizes
mAU 1
175 4
150 2 6
125
3 5
100 7 1.8 um 1 4-Acetamidophenol
75
50 2 Caffeine
25
0
3 2-Acetamidophenol
0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 min
mAU
4 Acetanilide
175 5 Acetylsalicylic Acid
150 1 4
2 6 Phenacetin
125
6
100
5 7 7 Salicylic Acid
75 3 3.5 um
50
25
0 LC Conditions
0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 min
Column: SB-C18, 4.6 x 30 mm
mAU Detector: 254 nm
175
Injector: 1 ul,
1
150

4 Mobile Phase: 1% Formic Acid

2 6
125

100
Acetonitrile
75
3 5 7 5 um
50
(82:18)
25
Flow: 2.0 ml/min
0

0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 min

 Chromatographic Comparisons on Columns
with Different Particle Sizes
Separation of Analgesics
Particle Size
Chromatographic Parameter 5 um 3.5 um 1.8 um
Peak width, L Acetylsalicylic acid 0.056 0.043 0.028
Peak width, L Salicylic acid 0.077 0.059 0.049
Resolution Acetylsalicylic acid/ Phenacetin 4.7 6.0 8.5
Resolution Phenacetin/Salicylic acid 1.61 2.07 2.25
Efficiency (plates) Acetylsalicylic acid 1980 3170 6474
Efficiency (plates) Salicylic acid 1917 3430 6049
Peak Capacity (kmax=10, Rs =1,
27 35 48
n=1+(N/4)ln(1+kmax)
The Rapid Resolution HT column with 1.8 um particles provides the best res
measured by any parameter.
Short Columns Packed with Smaller Particles Provide
Very High Efficiency and Very Short Analysis Times
Columns: ZORBAX SB-C18, 4.6 x 50 mm Mobile Phase: 25% Water: 75% MeOH Flow Rate: 1.5 mL/min
Temperature: RT
Detection: UV 254 nm Sample: QC: 1. Uracil 2. Phenol 3. 4-Cl-Nitrobenzene 4. Toluene

4.6 x 50 mm, 3.5 m 4.6 x 50 mm, 1.8 m

2
2
Plates (N) Plates (N)
3
4 1. 3476 3
1. 6560
2. 4585 4
2. 8958
1 3. 5673 1
3. 11508
4. 6180 4. 12266

0 0.5 1 min
0 1 min

Analysis time of < 1 minute for truly high throughput analys

 Particle Size Comparison - Smallest Particle
Size for Highest Resolution and Efficiency
Conditions: Column: Eclipse XDB-C18, 4.6 x 50 mm Mobile Phase: 77% Methanol, 23%
20mM Phosphate Buffer, pH 7
Flow Rate: 1 mL/min, Temperature: Ambient Injection Volume: 1.0 L Inj., Detection: UV
254nm Sample: 1) Uracil, 2) Naproxen, 3) Mefanamic Acid, 4) Butyl Paraben, 5) Propranolol, 6)
Dipropyl Phthalate, 7) Naphthalene.
Rapid Resolution Rapid Resolution HT
1 1
5 Eclipse XDB-C18, 3.5 5 Eclipse XDB-C18,1.8
m m
N7 =7 6,838 N7 = 11,985
2 Rs4,5 = 1.2 2 Rs4,5 = 1.6
7
6 6
4 4
3
3

0 0.5 1 1.5 2 2.5 min 0 0.5 1 1.5 2 2.5 min

Resolution improves and efficiency nearly doubles using the 1.8 um

particles of the Rapid Resolution HT column.
 Combine Short Columns and Small Particle
Sizes for High Speed Analyses
1
2

Rs(1,2) = 4.8 3 4.6 x 250 mm, 5 m

29.65
N=21848 4 N=22680

0 1 5 10 15 20 25 30 min
2
3
Rs(1,2) = 3.5 4.6 x 100 mm, 3.5 m
12.71
4 N=11691

. 0 1 5 10 15 20 25 30 min
2
3 N=6568 4.6 x 30 mm, 1.8 m 93% Shorter
Rs(1,2) = 2.9
4.15 1 mL/min Analysis
4
N=6104 Time
0 5 10 15 20 25 30 min

12 3
N=6463
Rs(1,2) = 2.9 4.6 x 30 mm, 1.8 m
4
2.09 2 mL/min
N=6460 0.5 1 1.5 2 2.5

0 5 10 15 20 25 30 min

Columns: ZORBAX SB-C18 Mobile Phase: 50% 20 mM NaH2PO4, pH 2.8: 50% ACN Flow Rate: 1 mL/min Temperature: RT
Detection: UV 230 nm Sample: 1. Estradiol 2. Ethinylestradiol 3. Dienestrol 4. Norethindrone
 High Throughput Gradient Analysis on Rapid Resolution
HT with Low and High Flow Rates
Columns: ZORBAX Rapid Resolution HT SB-C18, 2.1 x 30 mm, 1.8 m Mobile Phase: A: 20 mM Na2HPO4 pH 2.8, B:
ACN Gradient: A: 30 70%B in 5 minutes, hold for 1min. B: 30 80% B in 1.1 min, hold for 0.3 min, Flow Rate:
see below, Temperature: Ambient Autosampler: 1100, Bypass Mode Detection: UV 230 nm Sample: 1. Estriol 2.
Estradiol 3. Ethynylestradiol 4. Dienestrol 5. Norethindrone

A. 0.25 mL/min

0 1 2 3 4 min
5
B. 0.75 mL/min

0 0.2 0.4 0.6 0.8 1 min

1.2

High flow rates reduce analysis time and increase throughput in gradient
separations as well as isocratic separations.
Any Questions,
comments or
suggestions ?
 Thanks for your Help!

Wassalam