Theory Chromatography

Theory Chromatography

1. Chromatography Basic
2. Efficiency of Separation
3. Why Bands Spread
4. Effect of pH eluent in chromatography
CHROMATOGRAPHIC COLUMN
 Chromatography Basics
 Typical chromatogram:
Response
Detector

time or volume
 Chromatography Basics
tr2
tr1
Response
Detector

tm
tr1

injection time time or volume

tm = time for mobile phase to travel tr = retention time
length of column (dead time)
tr = adjusted retention time
 = tr2/tr1 = 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
 Efficiency of Separation
 Solutes in a column spread into a Gaussian profile:
 Gaussian peak shape:

s s
w1/2=2.35s
h

1/2h
w=4s

t0 tr
time
 Theory

Efisiensi Chromatography
1. Harga R (resolusi) yang besar >1,5
2. Harga N (jumlah pelat teori) besar
3. Harga H (Jarak setara pelat teori/HETP) kecil
4. Harga L (panjang kolom) yang pendek
5. Harga t (waktu tambat) yang singkat
 Efficiency of Separation
 The resolution (separation) of two solutes:
 Resolusi adalah daya pisah antar dua puncak
Dtr DVr
 Resolution (R) = wavg = wavg

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

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

 Efficiency of Separation
 Two factors affect how well two components
are separated
 difference in retention time
 peak widths
 Efficiency of Separation
 Resolution: higher R, better separation
R=0.50 R=0.75

t0 2s time t0 3s time

R=1.00 R=1.50 R1 ,5

is good

t0 4s time t0 6s time
 N (theoretical plates)
 The plate model supposes that
the chromatographiccolumn is contains a
large number of separate layers, called
theoretical plates. Separate equilibrations
of the sample between the stationary and
mobile phase occur in these "plates".
 Efficiency of Separation
2 2 2
tr 16 t r 5 . 55 t r
N   
 2
w 2
w1 2
2

 N is specific for each solute on a given

column
 Increasing retention time increases N
 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 )
 soal
 KROMATOGRAM 1
Kondisi analisis  metanol : air (1:1)
Analit A  tr =3,2 w=3,0
Analit B  tr= 4,4 w=3,5
 KROMATOGRAM 2

Kondisi analisis  metanol : air (2:1)

Analit A  tr =3,0 w=1,5
Analit B  tr= 5,4 w=1,2
Tentukan kromatogram mana yang lebih efisiensi, bila
panjang kolom 250mm ?
 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
 https://www.youtube.com/watch?v=fRJ4dJ6-66U
 Eddy diffusion
https://www.youtube.com/watch?v=p2KvzK81s2g&list=PLmRp
OX9aPBrL3ha0tZ15HyDQTlo27W59V
 Longitudinal diffusion
https://www.youtube.com/watch?v=wG5nDzKuGDU&index=3
&list=PLmRpOX9aPBrL3ha0tZ15HyDQTlo27W59V
 Mass transfer
https://www.youtube.com/watch?v=vCt0C5OvsxM&index=7&l
ist=PLmRpOX9aPBrL3ha0tZ15HyDQTlo27W59V
 Why Bands Spread
 Van Deemter mengemukakan suatu persamaan
relasi antara JSTP terhadap laju reaksi dan faktor
penyebab pelebaran pita
 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

OOO OOO
| | | | | |
O
S
iO
S
i
 O
S
i
 O
H
 O
S
iO
S
i
 O
S
i
 O
R
 Where R = C18H37
| | | | | |
OOO OOO hydrocarbon chain
| | | | | | (octadecylsilyl deriv.
O
S
iO
S
i
 O
S
i
 O
H
 O
S
iO
S
i
 O
S
i
 R silica or “C18”)
O

| | | | | |
OOO OOO

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

relatively polar surface relatively nonpolar surface

“normal phase” “reversed phase”
 Normal vs. Reversed Phase Chromatography

Normal Phase Reversed Phase

Stationary phase Polar (silica gel) Non-polar (C18)
Non-polar Polar
Mobile phase
(organic solvents) (aqueous/organic)
Sample movement Non-polar fastest Polar fastest
Different polarities Different
Separation based on
(functionality) hydrocarbon content
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  
a
[HA]1  1   
[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 ]  K a
 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
[H+]<<Ka
K pH=pKa
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
 HPLC Chromatograms Approximation
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.

faster moving slower moving base x height
Area =
less retained more retained 2
 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
mAU
0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 min
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
Injector: 1 ul,
175

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, n=1+
27 35 48
(N/4)ln(1+kmax)

Rapid Resolution HT column with 1.8 um particles provides the best res
sured 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 mm 4.6 x 50 mm, 1.8 mm

2

Plates (N)
2
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 analy

 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
1 1
5 Eclipse XDB-C18, 5 HT
3.5 m Eclipse XDB-
N7 =
7
6,838 C18,1.8 m
2 Rs = 1.2 2 N7 = 11,985
7
6 4,5 6
4 4 Rs4,5 = 1.6
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 mm

N=21848 29.65
4N=22680

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

0 1 5 10 15 20 25 30 min
93%
.

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

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

0 5 10 15 20 25 30 min

ns: ZORBAX SB-C18 Mobile Phase: 50% 20 mM NaH 2PO4, pH 2.8: 50% ACN Flow Rate: 1 mL/min Temperature: R
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 mm 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
5 min

B. 0.75 mL/min

0 0.2 0.4 0.6 0.8 1

1.2 min

• 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…