Low

Pressure
Column
Chromatography
(LPCC)/LPLC

Taufik Turahman, M.Farm.,Apt

 Column chromatography

 Column chromatography is a separation

technique in which components of mixture is
separated by using a glass column packed
with stationary phase and the liquid mobile
phase flowing continuously through the
column.
 Column chromatography is a widely
used method for the purification or
separation of chemical compound mixture
in lab
 Classification of Chromatography to physical state of mobile phase

chromatography

Mobile phase: liquid Mobile phase: Gas

Liquid chromatography Gas chromatography

eg: GLC
Liquid -solid Liquid-Liquid GSC
chromatography chromatography

eg: column adsorption eg: column partition

TLC,HPTLC HPLC HPCPC
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 Classification based up on physical nature of stationary phase

Stationary phase: solid Stationary phase: liquid

Adsorption chromatography Partition chromatography

eg: column adsorption eg: column partition

Ion exchange Size Paper partition
exclusion HPCPC GC(GLC)
TLC,HPTLC,HPLC

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Principles of column chromatography:

Column adsorption chromatography

Partition chromatography
Ion-exchange chromatography
Size exclusion chromatography or Gel
permeation chromatography:
Affinity chromatography
 Column adsorption chromatography

 The principle underlying the separation of the compounds is

adsorption at the solid-liquid interphase.
 solid stationary phase like silica gel and liquid mobile phase
is used.
 The separation is based on differences in affinities towards
the stationary phase.
The ratio of activities of the solute into two immiscible liquid phases is
known as a partition coefficient.
Partition on a column
 Ion-exchange
chromatography
 Kromatografi pertukaran ion adalah proses dari campuran bermuatan ion yg
dapat memisahkan dengan menggunakan resin penukar ion.
 The principle of separation is by reversible exchange of ions between the
ions present in the solution.
 Eg: a mixed bed cation-anion exchanger remove salts (ex CaCl2) from water
by exchanging them for H2O :Deionization of water

Example:
 CH=CH 2 CH=CH 2

CH=CH 2

Polymer matrix

Induction of
HSO3CL,H2SO4 basic groups
sulfonated
(Quartenary
ammonium
hydroxide/
halide/nitrate

-CH-
CH

Counter ion

counter ion
SO3- H+
CH-N + (CH 3)3 x-
strong acid cation exchange resin
Strong base anion exchange resin
H2O R-SO3-H+ )
( R-SO3
X-=OH-, CL-, NO3-
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 Gel permeation chromatography

 It is also called as gel filtration or size

exclusion chromatography.
 In size exclusion chromatography, the
stationary phase is a porous matrix
 Pemisahan ini didasarkan pada ukuran
molekul mereka (analytes) karena gel
berperilaku seperti saringan moleculae.
 Affinity chromatography

It is based on the Lock & key mechanism prevalent in biological

system
Requirements for column chromatography:

1. Column characteristics & selection

2. Stationary phases

3. Mobile phases

4. Preparation of column

5. Introduction of sample

6. Development of column

7. Detection of components

8. Recovery of components
Vignan Pharmacy College, Vadlamudi, guntur Dist (AP) pin: 522213 21
 Column characteristics & selection:
Table:1: selecting a suitable column
dimension
Good quality neutral
Materials of construction glass,plastic or
nylon
Adsorbent(stationary phase)/adsorbate (mixture) 30:1
weight ratio
Column length to diameter ratio(cm) 10-15:1 or 30-100:1

Multi component system is present Long column is used

Components with similar affinities for adsorbent Long column is used

are present
Components with different affinities for adsorbent Short column is
are present used

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 Stationary phases in column chromatography:

 Stationary phases used in column adsorption chromatography are also

known as adsorbents.

Type of mesh size in microns

60/120 mesh 120-250 micron
100/200mesh 75-150 micron
70/230 mesh 63-200 micron
230/400 mesh 37-63 micron
70/ 325 mesh 45-200 micron

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 Adsorbents used in column chromatography

Weak activity Medium activity Strong activity

Sucrose Calcium carbonate Activate magnesium

silicate
Starch Calcium phosphate Activated alumina

Inulin Magnesium Activated charcoal

carbonate
Talc Magnesium oxide Activated magnesia

Sodium carbonate Calcium oxide Activated silica

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 Preparation (packing) of the column:

 100–500 g fase diam/ 1 gram ekstrak

 massa adsorbent : extract = 40:1
 Tinggi kolom : diameter = 15 :1
 Metode packing :
slurrypacking
dry packing
Aplikasi sampel
 Sampel dilarutkan dg sedikit
fase gerak awal
 Lalu dg pipet dimasukkan ke
atas kolom
 Katup bawah dibuka supaya
sampel teradsorbsi lalu
ditutup
 Lalu diberi fase gerak
 Di bagian atas diberi lapisan
pasir 5-10 mm atau kertas
saring dengan diameter yg
sama dg kolom
 Sampel tidak dilarutkan dg
fase gerak awal
 Biasanya utk silika gel krn
fase gerak awal nonpolar
 Sampel dilarutkan solven
(EtOAc atau MeOH) lalu
diberi 10 kali berat silika gel
(atau bahan lain mis celite)
 Lalu serbuk kering
dimasukkan ke kolom
 Proses elusi
 Gravitasi
 Tekanan (e.g., flash chromatography / FC)

 Vakum (e.g., VLC),

 Pompa (low/medium)

Sistem gradien vs isokratis

Isocratic elution: (Iso means same or similar)
In this elution technique, the same solvent composition or
solvent of sample polarity is used throughout the process of
separation.e.g.(chloroform only as a solvent or
CHCL3:MeOH=1:1)

Gradient elution:(gradient means gradual)

In this elution tecenique, solvents of gradually increasing
polarity or increasing elution length are used during the
process of separation.
 Menentukan fase gerak

 Jika tahu golongan komponen apa

 protokol hasil publlikasi
 Jika belum tahu  gunakan KLT
 Siapkan larutan ekstrak kasar atau campuran
senyawa pd pelarut organik bertitik didih rendah (kira-
kira 10 mg/mL)
 Larutan 2–5 μL di-KLT dgn beda fase gerak
 Visualisasi di UV lamp atau semprot dg pereaksi yg
sesuai
 Bandingkan hasil KLT, pilih fase gerak yg sesuai
DETECTION & RECOVERY OF COMPONENTS:

Fractions are collected by elution analysis

Each fraction is examined by TLC using suitable experimental

conditions

Those fractions which give same Rf values in TLC are added as a

common fraction

From the column fraction, solvent is evaporated, dried & the

materialis collected in eppendorf container

After spectral analysis (NMR, MS, X-RD etc.)the compound is

identified
45
 Gravitasi

 Pressure/tekanan

 Vacum

 pompa
Flash
Chromatography
Cartridges
 CONCLUSION

 Column chromatography is a conventional tool for separation of

phytohemicals, removal of impurities and purification of drugs.
 Effective separation of constituents from different sources in
preparative scale (milligram to gram) can be achieved by column
chromatography.
 Availability of wide range of stationary phases makes the technique
to be used for different kinds of mechanisms.
 Understanding the basic principles of column chromatography
enables us to find solutions for current research problems.
 Refrens
 Mahfouz m abdel-gawad, maher a el-hashash, mortada m el-sayed..,
Chromatographic isolation of Allium cepa (ssp. Red onion) and its cytotoxic
activity against human liver carcinoma cell lines (HEPG2); International
Journal Of Pharmcy and Pharmaceutical Sciences, vol 6, Page no: 109-110.
 Rachana gohel, bhavna solanki, nilesh gurav.., Isolation and characterization
of shatavarin iv from root of asparagus racemosus willd ; International
Journal Of Pharmcy and Pharmaceutical Sciences, vol 7, page no: 363-365
 End