Column
Chromatography
Nina Salamah, MSc., Apt
Kromatografi Kolom
Sederhana
Bergerak / aliran karena gaya grafitasi
↓
Pemilihan fase diam + fase gerak
↓
Kepolaran
↓
Pita-pita kromatogram
↓
Terbentuk fraksi-fraksi
↓
Dianalisis dengan KLT / KK↓
Pengisian Kolom
Fase diam
Pasir
• pengisian kolom homogen
• fase diam ukuran sama
• fase diam bentuk homogen
• bebas gelembung udara
Kapas / glass
wool
Tehnis : fase diam + pelarut → bubur
(f. gerak)
Klasifikasi Sistem
Kromatografi
Umum
Tehnik
Spesifik
1. K. Cair  LLC
(LC)
 LSC
 IEC
2. K. Gas
(GC)
Fase diam
Cair pd padatan
Padatan
Resin
Cair pd padatan
GLC
Padatan
 GSC
 Gas terikat Padatan

Keseimba
ngan
Partisi
Absorbsi
Tukar ion
Partisi
Absorbsi
P/A
LIQUID COLUMN CHROMATOGRAPHY
A sample mixture is passed through a column
packed with solid particles which may or may
not be coated with another liquid.
With the proper solvents, packing conditions,
some components in the sample will travel the
column more slowly than others resulting in
the desired separation.
Principles of Separation on a
column
Principles of Separation
Principles of Separation
Principles of Separation
Principles of Separation
Principles of Separation
Principles of Separation
 Gravitasi
 Pressure/tekanan
 Vacum
 pompa
How does reverse phase chromatography
compare
to normal phase chromatography ?
Normal Phase Column
Chromatography …




The stationary phase is POLAR
The more polar component interacts
more strongly with the stationary
phase
The more polar component moves
more slowly.
The non-polar component moves
more rapidly.
Reverse Phase
Chromatography…
Silica is alkylated with long chain hydrocarbon groups, using
18 carbons long. This is usually referred to as C-18 silica.
CH3
CH2
CH3
CH2
17
Si
CH3
Si
O
O
O
Si
O
O
Si
O
O
O
O
Si
O
O
Si
O
Si
O
O
CH3
Si
O
O
Si
O
O
Si
Si
CH3
O
O
O
O
Si
Si
O
SiCH3)3
CH3
O
SiCH3)3
SiCH3)3
17
O
O
O
O
Si
O
O
O
O
Reverse Phase Column
Chromatography….





The stationary phase (column packing)
is now NON-POLAR
Non-polar compounds will move more
slowly because they are attracted to the
column packing.
The more polar component moves more
quickly down the column.
Polar solvents, such as water and
methanol are used in reverse phase
chromatography
Used mainly in columns, such as HPLC
Diagram of Simple Liquid Column Chromatography
DIAGRAM O F S IMPLE LIQ UID C O LUMN C HRO MATO G RAPHY
Solvent (m obile or
moving phase)
A+ B+C
Sam ple
(A+B+C)
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOO OOOO
Column
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOO OOOO
OOOOOO OOOOO
Solid P articles
OOOOOO OOOOO (packing materialOOOOOO OOOO stationary phase)
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOO OOOO
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOO OOOO
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOO OOOO
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOO OOOO
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOO OOOO
Eluant (eluat e)
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOA OOOO
OOOOOO OOOOO
OOOOOO OOOO
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOO OOOO
OOOOOB OOOO
OOOOOO OOOOO
OOOOOO OOOO
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOO OOOO
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOO OOOO
OOOOOC OOOO
OOOOOO OOOOO
OOOOOO OOOO
OOOOOO OOOOO
OOOOOO OOOOO
OOOOOO OOOO
OOOOOO OOOOO
OOOOOO OOOOO
FOUR BASIC LIQUID CHROMATOGRAPHY
The 4 basic liquid chromatography modes are named according
to the mechanism involved:
1. Liquid/Solid Chromatography (adsorption chromatography)
A. Normal Phase LSC
B. Reverse Phase LSC
2. Liquid/Liquid Chromatography (partition chromatography)
A. Normal Phase LLC
B. Reverse Phase LLC
3. Ion Exchange Chromatography
4. Gel Permeation Chromatography (exclusion chromatography)
Types of Chromatography
LIQUID SOLID CHROMATOGRAPHY
Normal phase LS
Reverse phase LS
d- d+
Si - O - H
30 m
Silica Gel
The separation mechanism in LSC is based on the
competition of the components of the mixture sample
for the active sites on an absorbent such as Silica Gel.
LIQUID SOLID CHROMATOGRAPHY
OH
HEXANE
Si - OH
CH 3
OH
CH 3
C-CH
CH 3
CH 3 - C
CH 3
CH 3
3
WATER-SOLUBLE VITAMINS
1.
Niacinamide
2.
Pyridoxine
H 3C
N
N
HO
CH 2OH
CONH 2
Riboflavin
CH 2OH
HOCH
HOCH
HOCH
CH 2
H3C
N
N
CH 2OH
3.
H3C
4. T hiamin
O
NH
N
O
H 3C
N
N
NH 2
CH 2
S
N
CH 2CH 2OH
Cl
CH 3
WATER-SOLUBLE VITAMINS
LIQUID-LIQUID CHROMATOGRAPHY
ODPN (oxydipropionylnitrile)
Normal Phase LLC
Reverse Phase LLC
NCCH3CH2OCH2CH2CN(Normal)
CH3(CH2) 16CH3 (Reverse)
The stationary solid surface is coated with a 2nd liquid (the
Stationary Phase) which is immiscible in the solvent (Mobile) phase.
Partitioning of the sample between 2 phases delays or retains some
components more than others to effect separation.
ION-EXCHANGE CHROMATOGRAPHY
SO3- Na+
Separation in Ion-exchange Chromatography is based on the
competition of different ionic compounds of the sample for the
active sites on the ion-exchange resin (column-packing).
Types of Ion Exchange Resins
Type of
Exchanger
Functional Exchanger Group
Trade Name
Cation
Strong Acid
Sulfonic acid (-SO3-H+)
Dowex 50;
Amberlite IR 120
Weak acid
Carboxyclic acid (-CO2-H+)
Amberlite IRC 50
Anion
Strong base
Quaternary ammonium ion (- Dowex 1;
NR3+OH-)
Amberlite IRA 400
Weak base
Amine group (-NH3+OH-)
Dowex 3;
Amberlite IR 45
Chromatography
Conditions associated with each kind of chromatography
•Ion exchange chromatography
•Organic cation exchange resins involve crosslinked polystyrene containing
either SO3- or COO- functional groups with an associated cation
H H H H
C
C
C
C
H
+
Na SO3
H
-
-
+
SO3 Na
•Organic anion exchange resin involve
•crosslinked polystyrene containing NH3+
•functional groups with an associated anion
H H H H
C
C
H
-
Cl NH3
C
C
H
+
NH3 Cl
-
The affinity of dissolved ions
for the resin varies
with the ion and the
composition of the
solution

nRzSO3–H+ + Mn+
(RzSO3)nM + nH+

nRzCO2–H+ + Mn+
(RzCO2)nM + nH+

nRzNR3+OH-+ An-
(RzNR3)nA + nOH-
MECHANISM OF ION-EXCHANGE
CHROMATOGRAPHY OF AMINO ACIDS
pH2
SO 3
-
Na
+
H3N
+
COOH
Ion-exchange Resin
SO 3
-
H 3N
Na
+
+
COO
-
pH4.5
Chromatography of Amino Acids
Statio nar y P h ase
Mo bile P h ase
H3 N
-
SO 3 Na+
+
COOH
+
Na
SO 3
OH
-
H3 N
+
COOH
Ex ch an ge Resin
-
SO 3 H3N+
COOH
SO 3
p H3 .5
OH
-
H3 N+
+
-
Na
COO
H
+
-
OH = H2 O
+
Na
SO 3
-
H3 N
+
-
COO
H
+
-
OH = H 2 O
-
SO 3Na+
p H4 .5
Some Applications of Ion Exchange
Chromatography

Purifications
a mixed bed cation-anion exchanger
remove salts (ex CaCl2) from water by
exchanging them for H2O :Deionization of
water

Concentrations
The concentration of trace elements in
seawater.

Analytical Separations
Separations of metal ions and amino acid or
halide ions
SIZE EXCLUTION CHROMATOGRAPHY
Gel-Permeation Chromatography is a mechanical sorting of molecules
based on the size of the molecules in solution.
Small molecules are able to permeate more pores and are, therefore,
retained longer than large molecules.
SIZE EXCLUTION
CHROMATOGRAPHY




Molecules that can penetrate the gel particles are
separated based on size and shape. Others pass
straight through the column.
Gel filtration chromatography : mobile phase is
water.
Gel permeation chromatography : mobile phase is
an organic solvent.
Sephadex is popular molecular-sieve material 4 the
separation of proteins.
SOLVENTS
Polar Solvents
Water > Methanol > Acetonitrile > Ethanol >
Oxydipropionitrile
Non-polar Solvents
N-Decane > N-Hexane > N-Pentane >
Cyclohexane
Kekuatan elusi pelarut pada silika dan polaritas pelarut
Schematic of a chromatogram
PARAMETER PEMISAHAN
DALAM KROMATOGRAFI KOLOM
1. KAPASITAS
Kapasitas menggambarkan kemampuan fase diam dalam
menahan analit. Jika waktu tambat lama, berarti
kapasitasnya besar.
2. SELEKTIVITAS
Selektivitas menggambarkan kemampuan fase diam
untuk dapat memisahkan suatu campuran senyawa.
Semakin besar nilai , campuran senyawa semakin
terpisah.
3. RESOLUSI
Resolusi menggambarkan kemampuan kolom dalam
memisahkan campuran senyawa
4. JUMLAH PLATE TEORITIS
Jml pelat teoritis N, dalam kolom dapat diketahui dari
hasil kromatogram.
5. TINGGI PLATE TEORITIS = H
HETP = High Equivalent to A Teoritical Plate
Adl : ukuran yang menunjukkan ruang yg ditempati oleh
setiap pelat teoritis.panjang kolom
t -t
K'  R M
t
M
K'
α 2
K'
1
(t2 - t1)
R 
0.5(w1 + w2)
N
HETP
 4 tR  2


 W 
= L/N
L = PANJANG KOLOM
t  t -t
t
r
Adjusted Retention Time
r
m
tr = retention time
tm = min. time for unretained mobile phase to travel through column
In GC tm is the time CH4 takes to travel through the column
t

t
so   1
Relative Retention
tr' 2  tr' 1
'
r2
'
r1
Capacity Factor
tr - tm
k'
tm
Resolution
Nilai R yg baik > 1,5. Jika R = 1
masih tjd tumpang tindih di
antara kedua puncak  2%
Untuk memperbaiki R :
1. memperbesar  tR = t2 – t1
kolom diperpanjang
jumlah fase diam diperbesar
manipulasi faktor pemisahan
pengoptimalan suhu
pilih f.d dan f.g yang cocok
2. Memperkecil lebar puncak, W
pilih ukuran fase diam kecil (halus) dan pengisian
dalam kolom diperbaiki (seragam dan kompak.
Kecepatan alir fase gerak optimum
Kurangi dead space dalam kolom
Kurangi jumlah sampel
Diameter kolom diperkecil.
Resolution
tr Vt 0.589tr
Resolution


wav wav
w1/ 2 av
w = peak width at the baseline between tangents drawn to the
steepest parts of the peak
w1/2 = measured at ½ the peak height
A peak with a retention time of 407 s has a width at the base of 13 s.
A neighboring peak is eluted at 424 s with a width of 16 s. Find the
resolution for these two components.
tr
424- 407
Resolution 

 1.1
wav 1 / 2(13 + 16)
Chromatography
Chromatographic column theory of packed columns
•The effect of column efficiency and column selectivity on resolution
Poor resolution because of poor
column efficiency
Good resolution because of good
column efficiency, although
column selectivity is not
great
Good resolution because of good
column selectivity, although
column efficiency is poor
Poor resolution because of poor
column selectivity, although
column efficiency is good
Theories of
Elution Chromatography
some zone broadening
zone separation
Plate height: constant of proportionality between the variance (s2)
of the band and the distance traveled (x)
Smaller plate height = narrow peaks = better separations
H
s2
Plate height (H)
x
16t r2  t r2 
N  2   2  Number of plates (N)
w
s 
A solute with a retention time of 407 s has a width at the base of 13
s on a column 12.2 m long. Find the plate height and number of
plates.
L L = column length
H
N
16t r2
N 2
w
16t r2 16  4072
4
N 2 

1
.
57

10
w
132
L
12.2m
H 
 0.78m m
4
N 1.57 10
N
Resolution
4
'
  - 1  k2 


' 

1
+
k


av 
N is the number of theoretical plates
 is the relative retention of two peaks
k’2 is the capacity factor for the more retained component
k’av is the average capacity factor for both components
Remember that variance is additive but standard deviation is not
Applications of
Chromatography
Qualitative Analysis
 Quantitative Analysis

Analyses Based on Peak Height
 Analyses Based on Peak Areas
 Calibration and Standards
 The Internal Standard Method
 The Area Normalization Method

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