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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

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Skema Instrumen HPLC
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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
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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).

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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 column’s
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.

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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 µm

 Materials of construction for the tubing


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

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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 µm, 3.5 µm and 1.8 µm
 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 µm, 5 µm or 3
µm 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
matrices to extremely low Mobile phase
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  Does not work with


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

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