Analisis Farmasi I

Kromatografi Cairan Kinerja Tinggi

High Performance Liquid Chromatography
Oleh :
Datin An Nisa Sukmawati, M.Sc
High Performance Liquid Chromatography
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HPLC is a form of liquid chromatography to separate
compounds that are dissolved in solution.

HPLC instruments consist of reservoir of mobile

phase, a pump, an injector, a separation column, and a
detector.
Compounds are separated by injecting a plug of the
sample mixture onto the column.

The different components in the mixture pass through

the column at different rates due to differences in their
partitioning behavior between the mobile liquid phase
and the stationary phase
Why ???
The reasons of developing HPLC :

1 Non Volatile Compound

Most (85%) of existing compounds
are not volatile enough or stable
enough to be analyzed by GC
2 The amounts solvents and samples
The traditional approach using open
column liquid chromatography requires
very large amounts solvents and
samples.

3 The flow of mobile phase

The flow of mobile phase is very slow
due to gravity dependent, so the
separation takes place in the order of
hours.
4 Cost and Time scale
The collected fractions of separated samples
should also be analyzed separately,
therefore it would increase the cost and time
scale of analysis
Separation Principles in HPLC

General Rule of Thumb:

Polarity of analytes ≈ polarity of stationary phase ≠ polarity of
mobile 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
 Kromatografi Cair Kinerja Tinggi (KCKT)
High Performance Liquid Chromatography (HPLC)

1 Solvent = pelarut = fase gerak = elusi

Terdiri dari campuran pelarut yang dapat bercampur
Misalnya : larutan bufer dengan metanol, H2O dengan
acetonitril
Ada dua sistem pada fase gerak :
1. Fase Normal = fase gerak non polar, fase diamnya polar
( jarang digunakan)
2. Fase terbalik = fase gerak polar, fase diamnya non polar
 Kromatografi Cair Kinerja Tinggi (KCKT)
High Performance Liquid Chromatography (HPLC)

2
Solvent Delivery Sistem = Sistem Elusi dari Fase Gerak
1. Isokratis = komposisi fase gerak tetap selama elusi
2. Gradien = komposisi fase gerak berubah-ubah selama
elusi
 Pump System
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 sys
tem
– Many HPLC systems hav
e a dual pump system to
minimize pulsing
• Flow control and reprodu
cibility < 0.5%
• Corrosion resistance
 Kromatografi Cair Kinerja Tinggi (KCKT)
High Performance Liquid Chromatography (HPLC)

Sampel = 1 mikro liter, 5 mikro liter, 10 mikro liter

4

Injector
Syringe
 Kromatografi Cair Kinerja Tinggi (KCKT)
High Performance Liquid Chromatography (HPLC)
 Kromatografi Cair Kinerja Tinggi (KCKT)
High Performance Liquid Chromatography (HPLC)
Must operate in high pressure
5 ◦ Usually constructed of metals
Column

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

 Kromatografi Cair Kinerja Tinggi (KCKT)
High Performance Liquid Chromatography (HPLC)
5 Column = fase diam
Berupa silika yang dimodifikasi
Column

E.g = Oktadesil silika (ODS) C18 = mampu

memisahkan senyawa-senyawa dengan kepolaran
yang rendah, sedang, maupun tinggi
Sifat :
Non polar
Contoh lain :
Aminopropil C3H6NH2, Asam sulfonat –(CH2)nSO3H
 Kromatografi Cair Kinerja Tinggi (KCKT)
High Performance Liquid Chromatography (HPLC)
 Kromatografi Cair Kinerja Tinggi (KCKT)
High Performance Liquid Chromatography (HPLC)

Detektor harus mempunyai

Detecto

karakteristik sebagai berikut:

1. Mempunyai respon terhadap
solut yang cepat
2. sensitifitas tinggi
3. stabil
r
 Kromatografi Cair Kinerja Tinggi (KCKT)
High Performance Liquid Chromatography (HPLC)

6 Detector
Jenis-jenis detektor
1. Detektor UV-Vis = berguna untuk analisis di bidang farmasi. Berdasarkan
adanya penyerapan radiasi UV dan Visible, panjang gelombang 200-800 nm
2. Photodiode-Array (PDA) = paling banyak dipakai, lebih sensitif dari pada
UV-Vis, panjang gelombang 200-800 nm
3. Fluoresensi = lebih spesifik, hanya senyawa-senyawa yg berfluoresensi.
Panjang gelombang 10-100 nm
4. Indeks bias = universal, tetapi dipengaruhi suhu mudah tidak stabil.
Khusus untuk senyawa yg tidak memiliki gugus kromofor,
5. Elektrokimia
 Kromatografi Cair Kinerja Tinggi (KCKT)
High Performance Liquid Chromatography (HPLC)
Analisis obat antihistamin
8
dan dekongestan
HPLC of Orange Juice Compounds