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

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
Persamaan

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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basic principles of HPLC - 1

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 (solutsolut) 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 ipropileter)
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

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.
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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 m
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

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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 Functional groups firmly C-2
linked (chemically bound) C-8
to the solid support.
C-18
Extremely stable
CN
Reproducible

Bonded Phases
Ethyl Silyl

-Si-CH2-CH3

Octyl Silyl

-Si-(CH2)7-CH3

Octadecyl
Silyl

-Si-(CH2)17-CH3

Cyanoprop
yl Silyl

-Si-(CH2)3-CN

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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
that allows to quantify and
identify compounds and
impurities in complex
matrices to extremely low
concentration levels (trace
level analysis)
Fluorescence detectors
sense only those
substances that fluoresce

excitation

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emission

Mobile phase

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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
Can be used with
gradient elution
Is relatively cheap and
not sensitive to
temperature
Sensitive to large
number of organic
compounds

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

UV / Visible detector
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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

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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)