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
HPLC = High Performance Liquid
Chromatography
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
Fase gerak
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
- Kran diputar (ke bawah) utk mengubah ke posisi injeksi
dan fasa gerak membawa sample ke dalam kolom
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 column

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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 C-2 Ethyl Silyl -Si-CH2-CH3
groups firmly linked
C-8 Octyl Silyl -Si-(CH2)7-CH3
(chemically bound) to the
C-18 Octadecyl -Si-(CH2)17-CH3
solid support. Silyl
Extremely stable CN Cyanopropyl -Si-(CH2)3-CN
Reproducible 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
Hingsensitivity & small
sample volume required
Can be used with gradient
elution
Is relatively cheap and not
sensitive to temperature
Sensitive to large number
of organic compounds
UV / Visible detector
suwedo hadiwiyoto
Disadvantages
Does not work with
compounds that do not
absorb light at this wavelength
region
Cannot be used with the
solvents having large
absorption in the UV region
Cannot be used for the
sample components which
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)