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Klasifikasi HPLC

1. Partisi (liquid-liquid chromatography)


2. Adsorpsi (liquid-solid
chromatography)
3. Pertukaran ion (ion chromatography)
4. Size exclusion chromatography
5. Affinity chromatography
6. Chiral chromatography

Separation
mechanism

Type of
stationary
phase

Partisi (liquid-liquid chromatography)


Jenis HPLC yang paling banyak digunakan
Fasa diam (stationary phase) berupa cairan
yang bercampur dengan cairan fasa gerak
(mobile phase)
Liquid-liquid
phase

Fasa diam
(adsorpsi fisika)

Sistem partisi

Liquid-bonded
phase

Fasa diam
(ikatan kimia)

Fasa
diam
(polar)

Triethylene
glycol
Water

Normal phase
Fasa
gerak
(non
polar)

Hexane
Propyl ether

Fasa diam
(non polar)

Hydrocarbon

Fasa gerak
(polar)

Water
Methanol
Acetonitrile
THF

Reversed
phase

Typical application of High


Performance Partition
chromatography
Field

Typical mixture separated

Pharmaceutical

Antibiotics, steroids, analgesics

Biochemicals

Amino acids, proteins, carbohydrates, lipids

Food products

Artificial sweeteners, antioxidants, aflatoxins,


additives

Industrial chemicals

Condensed aromatics, surfactants,


propellants, dyes

Pollutants

Pesticides, herbicides, phenols, PCBs

Forensic chemistry

Drugs, poisons, blood alcohol, narcotics

Clinical medicine

Drugs metabolits, urine extracts, estrogens,


75%
aplikasi pemisahan HPLC
bile acids

Lebih dari
menggunakan reversed phase, bonded phase
(octyl or octyldecyl-siloxane) packings dan fasa
geraknya antara lain metanol, acetonitrile, atau
THF

Adsorpsi (liquid-solid)
chromatography
Fasa diam berupa permukaan padatan polar
(silika dan alumina)
Silika lebih dipilih untuk kebanyakan aplikasi
karena kapasitas sampel yang lebih besar
Penggunaan kedua fasa diam ini membuat
waktu retensi lebih lama jika polaritas analit
meningkat
Fasa gerak adalah pelarut organik atau
campuran pelarut organik
komposisi
berpengaruh pada koefisien distribusi

Application of High performance


adsorption chromatography
Pemisahan senyawa nonpolar
Water-insoluble organic compounds (Mr <
5000)
Resolve isomeric mixture
(e.g meta and para substituted benzenes
derivatives)

Affinity chromatography
(Kromatografi Afinitas)
Involves covalently bonding a
reagent, called an affinity ligand, to a
solid support
Typical affinity ligands are antibodies,
enzyme inhibitors, or other
molecules that reversibly and
selectively bind to analyte molecules
in the sample
The retained analytes can be eluted
by changing the mobile phase

Mobile phase
Two distinct roles:
First it must support the strong binding of
the analyte molecules to the ligand
Second, once the undesired species are
removed, the mobile phase must weaken or
eliminate the analyte-ligand interaction so
that the analyte can be eluted
(often changes in pH or ionic strength are
used to change the elution condition during
the two stages of the process)

Stationary phase
A solid such as agarose or a porous
glass bead to which the affinity
ligand is immobilized

Pertukaran ion (ion chromatography)

Lanjutan