High Performance Liquid

Chromatography (HPLC)

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 Tipe Kromatografi

Chromatography
Planar Column
Liquid Gas Supercritical Fluid
Partition Gas - Liquid

Bonded Phase Gas – Bonded Phase

Adsorption Gas - Solid

Ion Exchange

Size Exclusion

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 Komponen Dasar pada HPLC:
1. Pompa. Mobil phase pressures up to 6000 psi are necessary to
achieve reasonable column elution times (~ minutes). Typical flow
rates are 0.1 to 10 mL/minute.
2. Sistem Injeksi. Used to introduce small samples (0.1 to 500 µL)
into the carrier stream under high pressure.
3. Reservoirs (Solvents). Multiple solvents are necessary for
performing gradient elution's (i.e. changing the polarity of the mobil
phase during a run).
4. Kolom Kromatografi Typically 10-30 cm in length containing a
packing of 5-10 µm diameter. Many types of columns are available,
depending on the type of liquid chromatography desired.
5. Detektor. Many types are available including UV, IR, refractive
index, fluorescence, conductivity, mass spectrometry, and
electrochemical. Diode array detectors are used when wavelength
scans are desired.

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 Pompa
Desirable Features:
• Must generate pressures
up to 6,000 psi
– To allow for separation in
reasonable time frames
• Flow-rates range from 0.1
to 10 mL/minute
• Limited pulsing in the
system
– Many HPLC systems have
a dual pump system to
minimize pulsing
• Flow control
• Corrosion resistance
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 Sistem Injeksi
Used to
introduce small
samples (0.001
to 0.5 mL) into
the carrier
stream under
high pressure

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 Detektor
• Tipe
– General – respond to mobil phase bulk
properties which vary in the presence of
solutes (e.g. refractive index)
– Specific – respond to some propert of the
solute (not possessed by the mobil phase
(e.g. UV)
– “Hyphenated” detector – LC-MS

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DETEKTOR
• Detektor umum didasarkan pada perubahan indeks bias pelarut,
sehingga kurang sensitiv (limit deteksi <<)
- tidak sesuai untuk elusi bergradien
- dapat mendeteksi solut 10-6 gram
- digunakan untuk krom. Preparatif (isolasi bahan alam)
- dapat mendeteksi semua solut yang keluar dari kolom karena
berdasarkan perubahan indeks bias pelarut

• Detektor selektif/khusus didasarkan pada struktur molekul

solut/analit
- sesuai untuk elusi bergradien
- dapat mendeteksi solut 10-9 gram
- untuk analisis rutin, dengan solut yang runut
- hanya tepat untuk mendeteksi solut yang memiliki sifat optis/dapat
menyerap rentang panjang gelombang UV
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 Absorbance Detectors
• The UV/Vis source usually comes
from a monochromator so the
wavelength can be selected, or
scanned.
• Absorbance increases as eluate
passes through the cell.
• If wavelength scanning is desired, the
flow is stopped long enough for the
scan to take place.
• It’s possible to have the same setup
using IR light, although not as
common since many useful solvents
are not IR transparent.

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 Diode
Array
Detector

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 Photo Diode Array
Detektor diode array merupakan spektrometer
yang terdiri atas satu tatanan yang teratur (array)
dari foto dioda aktif dalam jumlah yang sangat
banyak (330 buah). Dan tiap-tiap foto dioda aktif
dalam tatanan tersebut memberikan respon yang
spesifik terhadap radiasi dengan panjang
gelombang tertentu. Dengan demikian radiasi
polikromatis akan diterima dengan cepat dan
serempak oleh foto dioda yang ada dalam tatanan
tersebut, sehingga scanningnya menjadi sangat
cepat.
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HPLC Detectors

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 Kolom HPLC
• Must operate in high pressure
– Usually constructed of metals
• Typical dimensions
– 10-30 cm long
– 1-3 cm ID
• Contains packing material
which holds the stationary
phase
– Many types exist
– Typical packing materials are 5-
10 µm in diameter
• Guard column used to extend
life of main column

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 Jenis-jenis Kromatografi Cair
1. Liquid-Liquid or Partition Chromatography
– The stationary phase is a liquid adsorbed on a solid
2. Liquid-Bonded Phase Chromatography
– The stationary phase is an organic species bonded to a
solid surface
3. Liquid-Solid or Adsorption Chromatography
– The stationary phase is a solid
4. Ion-Exchange Chromatography
– The stationary phase is an ion-exchange resin
5. Size Exclusion or Gel Permeation Chromatography
– The stationary phase is a liquid in the intersticies of a 15
polymeric solid
Types
of LC

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 Separation Principles in HPLC
General Rule of Thumb:
Polarity of analytes ≈ polarity of stationary
phase ≠ polarity of mobil phase

To achieve good separation, the analytes

should interact with the stationary phase, but
not too strongly or the retention time will
become very long
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 1. Liquid-Liquid or Partition
Chromatography
Characteristics
• A liquid stationary phase is retained on
the surface of the packing.
• Separation is based on differences in the
polarity of the solutes.
• One phase will be polar and the other
non-polar.

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Kromatografi cair-cair mengikuti prinsip
ekstraksi cair-cair
• Diperlukan fasa diam yang tidak larut dalam fasa gerak
(kenyataannya tdk ada yang benar2 tidak larut, karena
dilewati berliter-liter fasa gerak dengan tekanan tinggi

• Fasa diam disalutkan secara fisis, sehingga mudah

tererosi. Contoh Fs: Corbowax, Oksidipropionitril

• Biasanya diatasi dengan menjenuhkan fasa gerak

dengan fasa diam

• Fs Tidak tahan terhadap perubahan suhu karena akan

mengubah kelarutannya
• K = Cs/Cm
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 Normal and Reversed Phased (Partition)
Chromatography
A. Characteristics of Normal Phase Chromatography
• Highly polar stationary phase
– Silica or alumina oxides
• Relatively non-polar solvent
– e.g. hexane or i-propylether
• Least polar solutes elute first
• Increasing mobil phase polarity decreases elution
times – short time (i.e. polar compounds remain in the
mobil phase longer)

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B. Characteristics of Reversed Phase Chromatography

• Non-polar stationary phase

–e.g. a hydrocarbon
• Relatively polar mobil phase
–e.g. water, methanol or acetonitrile
• More polar solutes elute first
• Penurunan kepolaran fasa gerak akan
meningkatkan waktu elusi

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Reversed order
of elution

Increasing Mobil
phase Polarity,
Decreases
Elution Time

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 Typical Applications of Partition
Chromatography
Field of Application Separation
Pharmaceuticals Antibiotics, Sedatives, Steroids, Analgesics

Biochemical Amino acids, Proteins, Carbohydrates, Lipids

Food Products Artificial Sweeteners, Antioxidants,

Preservatives
Industrical Chemicals Condensed Aromatics, Surfactants,
Propellants, Dyes
Forensic Chemistry Drugs, Poisons, Blood Alcohol, narcotics

Clinical Medicine Bile Acids, Drug Metabolites, Urine Extracts,

Estrogens
Pollutants Pesticides, Herbicides, Phenols, PCBs

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Partition Chromatography –
Example Chromatograms

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 2. Adsorption or Liquid-Solid
Chromatography
• The classic form of liquid chromatography (introduced in
the 1900’s) à Michael Tswett dengan pemisahan klorofil
• Two common stationary phases
– Silica (most common)
– Alumina
• Highly polar stationary phase, less polar mobile phase
• Separation occurs based on stationare phase strength
for retaining the solute
– Mobil phase discussion given later
• Use overlaps with normal phase (partition)
chromatography
• Suitable for: non-polar compounds of low molecular
weight
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 Mekanisme Kromatografi Cair Padat
• Molekul solut akan terikat pada gugus aktif fasa diam, dengan
persamaan adsorpsi:
Xm + nSads == Xads + nSm
K=
• Fasa Diam dalam KCP: Silika (SiO2)x dan alumina ((Al2O3)x
• Contoh elusi dalam KCP sesuai dengan kepolaran solut/analit:
Sebuah sampel dengan komposisi: benzena+asetofenon+benzil
alkohol. Maka yang akan terelusi lebih dulu dalam Fs silika:
Benzena-asetofenon-benzil alkohol. MENGAPA ??
• Silika dan alumina mempunyai gugus hidroksil, sehingga sifat
adsorpsinya mengikuti reaksi asam-basa lewis
• Contoh memisahakan isomer hidrokuinon

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 Kemasan Kolom KCP

• Kemasan dalam KCP:

Bahan pelikel atau bahan berpori

• Kapasitas kolom berbanding lurus dengan jumlah fase

diam yang dipakai = luas penyerapan

• FM dalam KCP harus menyesuaikan kepolaran dengan

Fs, utamanya jika solut adalah polar -- supaya waktu
retensi tidak terlalu lama. Untuk mengatasi sering
ditambahkan modifier seperti air, alkohol dan asetonitril
pada FM terutama untuk FM yang non polar
• Model elusi solut dapat juga dengan elusi bergradien

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 Adsorption Chromatography -
Compounds That Can be Separated
• Olefins
• Aromatic hydrocarbons
• Halides, sulfides
• Ethers
• Nitro- compounds
• Esters, aldehydes, ketones
• Alcohols, amines
• Sulfones
• Sulfoxides
• Amides
• Carboxylic acids

listed in order of retention time; i.e. from least to greatest interaction

with the stationary phase 29
Adsorption Chromatography –
Example Chromatograms

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 3. Ion Exchange Chromatography
• Separation is based upon
ion-exchange equilibria
between the ions in solution
and the ions of like charge
on the surface of an
insoluble packing.
• Discussed separately later
(di bahas pada materi
berikutnya)

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4. Bonded Phase Chromatography
• Kromatografi Fase Terikat (FKT) memperbaiki
kinerja kromatografi cair-cair karena Fs yang
mudah tererosi
• Fs diikatkan secara kimiawi pada padatan
pendukung
• K = Cs/Cm
• Jenis Fasa Diam:
- Bahan pelikel
- Bahan berpori/mikropori

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 Stationary Phase
Microporous Silica Particles
Aggregate of Particles Sponge-like Structure

H-Bonded Vicinal
Isolated Silanol Groups
Silanol
Groups

• Particles are permeable

to solvent
• ~100 m2/g of particles
• Only use for pH < 8.0
• Use polystyrene
particles for pH 8-12
Geminal Silanol33
Groups
 Bonded Stationary Phase
CH3 CH3

Si OH + Cl Si R Si O Si R
Toluene, 110°C

CH3 CH3
Surface Silanol Surface Bonded
Group Functional Group
• Covalent attachment of the
stationary phase yields a
thermally and hydrolytically
stable bonded phase

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 Common Functional Groups for
Bonded Stationary Phase

Polar Phases (R =) Non-Polar Phases (R =)

(CH2)3NH2 Amino (CH2)17CH3 Octadecyl

(CH2)3CN Cyano (CH2)7CH3 Octyl

(CH3)3OCH2CH(OH)CH2OH Diol (CH2)3C6H5 Phenyl

CH3

Si O Si R

CH3 35
 Kemasan Kolom Kromatografi Fasa Terikat ada 2:
Normal dan Terbalik
• Fasa Terikat Normal
Fasa diam yang terikat pada penyangga lebih polar daripada fasa gerak
seperti pada Kromatografi cair padat.
Contoh Fs: gugus {(Si-(CH2)nCN)} alkil nitril dan {(Si-(CH2)nNH2)} alkil amin
Contoh FM: heksana, tetra hidro furan, metilena klorida

• Fasa Terikat Terbalik

Fasa diam lebih non polar daripada fasa gerak (fasa gerak lebih polar
daripada fasa diam)
Contoh Fs: oktadekil, C18
Contoh FM: metanol, air, asetonitril

KAIDAH UMUM SIFAT ELUSI: urutan elusi solut dalam fase terbalik
berlawanan dengan elusi dalam fasa normal
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 Fasa Gerak pada LC
• Many Types are available
– In contrast to GC
• The mobil phase interacts
(electrostatically) with sample components
– In contrast to GC
• Residence Time = f(solvent type)
• Polarity is important for solute, mobil
phase and stationary phase
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Gradient Elution

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