PRINSIP DASAR

HPLC
BAGIAN I
YUNIKA MAYANGSARI, S.Si., M.Biotech
Fasa diam dan fasa gerak (HPLC)
Dalam HPLC, zat cair (liquid) digunakan
sebagai fasa gerak
Kolom berisi serbuk halus yang dipadatkan
(sebagai fasa diam)
The stationary phase is defined as the
immobile packing material in the column.
Dapat dibayangkan betapa sulitnya zat cair
mengalir melalui fasa diam di dalam kolom
Sehingga, agar zat cair dapat melewati
kolom dengan cepat, dibutuhkan bantuan
pompa bertekanan tinggi
Persamaan dengan GC
keluarannya berupa
kromatogram
Keuntungan dibanding
pemakaian GC kemampuan
menganalisis sampel yg
unvolatile dan labil pada suhu
tinggi
Akan tetapi analisis HPLC lebih
mahal
Advantages to HPLC
Higher resolution and speed of analysis
HPLC column can be reused without
repacking or regeneration
Greater reproducibility due to close
control of the parameters affecting the
effeciency separation
Easy automation of the instrument
operation and data anaysis
Adaptability to large-scale, preparative
procedurs

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Various instruments of HPLC system

suwedo hadiwiyoto basic principles of HPLC - 1 5

Skema Instrumen HPLC
suwedo hadiwiyoto basic principles of HPLC - 1 7
Prinsip kerja HPLC
Dengan bantuan pompa fase
gerak dialirkan melalui kolom ke
detektor. Sampel yang dilarutkan
dalam solvent, dimasukkan ke
dalam aliran fasa gerak dengan
cara injeksi. Di dalam kolom
terjadi pemisahan komponen2
campuran perbedaan kekuatan
interaksi anatara analat (solut-
solut) dengan stationary phase
pada kolom.
Prinsip kerja HPLC (lanj)
Solut-solut yang kurang kuat interaksinya
dengan fase diam akan keluar dari kolom
terlebih dahulu. Sebaliknya, solut2 yang kuat
berinteraksi dengan fasa diam maka solut2 tsb
akan keuar dari kolom lebih lama. Setiap
komponen campuran yang keluar dari kolom
dideteksi oleh detektor kemudian direkam
dalam bentuk kromatogram.
Kromatogram HPLC serupa dengan
kromatogram GC jumlah peak menyatakan
jumlah komponen; luas area peak menyatakan
konsentrasi dalam campuran
Sisitim HPLC dapat dihubungkan dengan
software pada komputer dan dioperasikan
secara computerize
suwedo hadiwiyoto basic principles of HPLC - 1 10
Instrumentasi HPLC
Fasegerak
Pompa
Sample injector
Kolom
Detektor
Fasa Gerak (MP) HPLC (Syarat
fasa gerak)
MP harus bertindak sbg pelarut yang
baik utk sampel yang dianalisis
MP harus murni sekali utk menghindari
adanya impurities yang akan
mengganggu intepretasi pada
kromatogram dan mengotori kolom
MP harus jernih sekali utk
menghindarkan kolom kotor dan
tersumbat. Pelarut harus disaring dan
didegas. Saringan digunakan nilon
diameter 0.45 mikrometer.
MP mudah diperoleh, tidak
mudah terbakar, dan tidak
beracun
MP tidak viskous. Kekentalan
tidak melebihi 0,5 cP (centiPoise)
MP sesuai dengan detektornya.
Untuk detektor RI (refractive
index) pelarut harus mempunyai
indeks bias yang berbeda dengan
solut; utk det. UV, pelarut tdk
boleh mnyerap cahaya pada
gelombang cahaya yg dipakai.
Jenis fasa gerak
Berdasarkan kepolaran fasa diam dan fasa
gerak
Fase Normal (Normal Phase)
Kombinasi antara fase diam polar dan fase
gerak non-polar (misal: fase diam: silika
atau alumina, fase gerak: heksana atau i-
propileter)
Fase Terbalik (Reversed Phase)
Fase diam non-polar dan fase geraknya polar
(air, metanol, asetonitril) kepolaran fase
gerak lebih tinggi dibanding fase diamnya
Pump
Flow rate range: 0.01 to 5
mL/min
Flow rate stability: not more than
1%
For flow rate stability should be
less than 0.2%
It is desirable to have an
integrated degassing system,
either helium purging, or
membrane filtering.
Pump
The role of the pump is to force a liquid
(called mobile phase) through the liquid
chromatograph at a specific flow rate,
expressed in ml/min
Normal flow rates in HPLC are in the 1-2 ml/min
range
Typical pumps can reach pressures in the range
until 600 psi
During the cromatographic experiment, a
pump can deliver a constant mobile phase
composition (isocratic) or an increasing
mobile phase composition (gradient).

basic principles of HPLC - 1 16

Sample Injector
Injeksi syringe
Syringe diinjeksikan melalui septum (seal
karet), injeksi dilakukan dengan konstan dan
bebas gelembung udara
Injeksi stop-flow

Saat injeksi pelarut dihentikan sementara.

Sampel disuntikkan langsung pada ujung
kolom.
Kran sampel

Disebut juga loop dan paling banyak

digunakan.
- Sejumlah volume sample (dalam solvent)
disuntikkan ke dalam loop dalam posisi
load, sampel masih berada di dalam loop
Column
.... within the column is where separation
occurs
Considered the heart of the
chromatograph the columns
stationary phase separates the
sample components of the interest
using various physical and chemical
parameters.
The small particles inside the column
are what cause the high backpressure at
normal flow rates.
The pump must push hard to move the
mobile phase through the column and
this resistance causes a high pressure
within the chromatograph.

basic principles of HPLC - 1 18

HPLC columns
.... proper choice of column is critical for success in
HPLC
Types of columns in HPLC :
Analytical : ID 1.0-4.6 mm ; length 15-250
mm
Preparative : ID > 4.6 mm; length 50-250
mm
Capillary : ID 0.1-1.0 mm; various lengths
Nano : ID < 0.1 mm, or something stated
as < 100 m

Materials of construction for the

tubing
Stainless steel (most populer)
Glass (mostly for biomolecules)
PEEK polymes (biocompatible and
chemically inert to most solvents)

basic principles of HPLC - 1 19

 Precolumn
Precolumn (before
injector) and Guard
column (after injector) -
Protects the analytical
column:
Remove impurities from
solvent
Minimize the interferences
Prolongs the life of the
analytical column
Saturates mobile phase
with liquid of stationary
phase before the anlytical basic principles of HPLC - 1 20
HPLC columns packing materials
Today, most packing fall into four classes : silica or
alumina, bound phases on either alumina or silica, gels,
controlled-pore glass or silica
Columns are packed with small diameter porous particles.
The most popular size are 5 m, 3.5 m and 1.8 m
Columns are packed using high pressure to ensure that
they are stable during use.
These porous particles in the column usually have a
chemically bonded phase on their surface which interacts
with the sample components to separate them from one
another (for example, C18 is a popular bonded phase)
The process of retention of the sample components (often
called analytes) is determined by the choice column
packing and the selection of the mobile phase to phase
the anlytes through the packed column

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 Columns : bonded
phases
Solid Support - Backbone
for bonded phases.
Usually 10 m, 5 m or
3 m silica or polymeric
particles.
Bonded Phases - Bonded Phases
Functional groups firmly C-2 Ethyl Silyl -Si-CH2-CH3
linked (chemically bound) C-8 Octyl Silyl -Si-(CH2)7-CH3
to the solid support. C-18 Octadecyl -Si-(CH2)17-CH3
Extremely stable Silyl
Reproducible CN Cyanoprop -Si-(CH2)3-CN
yl Silyl

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Detektor (syarat)
Cukup sensitif
Stabilitas dan reproducibility
tinggi
Respon linear terhadap solut
Waktu respon pendek sehingga
tidak bergantung flow rate
Mudah digunakan
Tidak merusak sampel
Jenis jenis Detektor
HPLC
Detektor Fluorescence
Detektor UV
Refractive Index Detector
 Fluorescene detection
Compared to UV-VIS
detectors fluorescence
detectors offer a higher
sensitivity and selectivity excitation emission
that allows to quantify and
identify compounds and
impurities in complex Mobile phase
matrices to extremely low
concentration levels (trace
level analysis)
Fluorescence detectors
sense only those
substances that fluoresce

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Ultraviolet (UV) absorption
An ultraviolet light beam is directed through a flow cell and a sensor
measures the light passing through the cell
If a compound elutes from the column that absorbs this light energy, it will
change the amount of light energy falling on the sensor
The resulting change in this electrical signals is amplified and directed to
recorder or data system
A UV spectrum is sometimes also obtained wich may aid in the identification
of a compound or series of compounds

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Advantages Disadvantages

Hingsensitivity &
small sample volume
required Does not work with
Can be used with compounds that do not
absorb light at this
gradient elution wavelength region
Is relatively cheap and Cannot be used with the
not sensitive to solvents having large
temperature absorption in the UV region
Cannot be used for the
Sensitive to large
sample components which
number of organic
UV / Visible detector
compounds
cannot be absorbed in the
UV region

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basic principles of HPLC - 1 28
 Refractive index (RI)
detection
The ability of a compound or
solvent to deflect light provides
a way to detect it
The RI is a measure of
molecules ability to deflect
light in a flowing mobile phase
in a flow cell relative to a static
mobile phase contained in a
reference flow cell
The amount of deflection is
proportional to concentration
The RI detector is considered to
be a universal detector but it is
not very sensitive

basic principles of HPLC - 1 29

Latihan
Jelaskan mengapa fasa gerak cair harus
disaring dengan saringan sangat halus
sebelum dilalirkan?
Mengapa HPLC menggunakan pompa
bertekanan tinggi, sedangkan GC tidak?
Jelaskan fungsi guard column?

PR : - Sebutkan contoh2 aplikasi

penggunaan detector HPLC (IR, FRLD,
UV/UV Vis/PDA)