High Performance

Liquid Chromatography

Chem. 331
 Introduction
 HPLC adalah suatu cara kromatografi cair yg bisa
digunakan untuk memisahkan senyawa target dari
campurannya yg larut berupa larutan . HPLC adalah
rangkaian alat yg terdiri dar reservoir fase gerak, pompa,
injektor, kolom separator, dan detektor .

 Senyawa yang dipisahkan diinjeksikan dalam bentuk

sampel campuran beberapa senyawa ke dalam kolom.
Senyyawa yg berbeda di dalam campuran akan lewatt
melalui kolom pada waktu yg berbeda disebabkan oleh
perbedaan partisinya diantara fasa gerak dan fasa diam.
Fasa gerak dipaksa untuk mengeluarkan gelembng
udara. .
HPLC system
 FOUR TYPES OF LIQUID
CHROMATOGRAPHY
 Kromatografi partisi

 Adsorpsi, or cair-padat

 Kromatografi

 Kromatografi penukar ion

 Size exclusion, or kromatografi gel

 COMPOSITION OF A LIQUID
CHROMATOGRAPH SYSTEM
 eluen
 eluen dialirkan dg sistem pompa (Pump)
 Injector
 Sample
 kolom
 Detektor (Diode Array)
 penampung luaran
 perekam (himpunan data)
Rangkaian alat HPLC
 Kegunaan HPLC
 Teknik ini digunakan oleh ahli kimia atau biokimia untuk
penelitian analisa campuran komplek, pemurnian senyawa
kimia, pengembangan proses sinntesa senyawa kimia,
pemishan campuran produk, atau untuk menentukan sifat
fisika. Mastikan kemurnian bahan baku, untuk mengendalikan
dan HPLC juga digunakan untuk pengendalian mutu untuk
memastikan kemurnian bahan baku, untuk mengontrol dan
memperbaiki yield proses, emonitor degradasi molekul.
 Selanjutnya HPLC juga bisa digunnakan untuk menentukan
indek pencemaran udara dan air, untuk monitoring bahan yg
digunakan dalam pelaksanaan keselamatan dan kesehatan.
Dan memonitor pencemaran petisida terhadap lingkungan .
Badan pengawasan obat dan makanan mnggunakan HPLC
untuk menguji produk makanan dan obat, identifikasi
kandungan narrkotika, dan memeriksa label kadaluarsa produk
yang berddar.
HPLC Chromatograph injectors
 The function of the injector is to place the sample into
the high-pressure flow in as narrow volume as possible
so that the sample enters the column as a
homogeneous, low-volume plug. To minimize spreading
of the injected volume during transport to the column, the
shortest possible length of tubing should be used from
the injector to the column.

 When an injection is started, an air actuator rotates the

valve: solvent goes directly to the column; and the
injector needle is connected to the syringe. The air
pressure lifts the needle and the vial is moved into
position beneath the needle. Then, the needle is lowered
to the vial.
 HPLC columns
 The column is one of the
most important components
of the HPLC chromatograph
because the separation of
the sample components is
achieved when those
components pass through
the column. The High
performance liquid
chromatography apparatus
is made out of stainless
steel tubes with a diameter
of 3 to 5mm and a length
ranging from 10 to 30cm.
 Normally, columns are filled
with silica gel because its
particle shape, surface
properties, and pore structure
help to get a good separation.
Silica is wetted by nearly every
potential mobile phase, is inert
to most compounds and has a
high surface activity which can
be modified easily with water
and other agents. Silica can be
used to separate a wide variety
of chemical compounds, and its
chromatographic behavior is
generally predictable and
reproducible.
Picture of an HPLC column
 WHAT AFFECTS SYSTEM
Column Parameters Instrument Parameters

 Column Material  Temperature

 Deactivation  Flow
 Stationary Phase  Signal
 Coating Material  Sample Sensitivity
 Detector
 WHAT AFFECTS SYSTEM
Sample Parameters

 Concentration
 Matrix
 Solvent Effect
 Sample Effect
 Several column types
(can be classified as )

 Normal phase

 Reverse phase

 Size exclusion

 Ion exchange
 Normal phase

 In this column type, the retention is

governed by the interaction of the polar
parts of the stationary phase and solute.
For retention to occur in normal phase, the
packing must be more polar than the
mobile phase with respect to the sample
 Reverse phase
 In this column the packing material is relatively
nonpolar and the solvent is polar with respect to
the sample. Retention is the result of the
interaction of the nonpolar components of the
solutes and the nonpolar stationary phase.
Typical stationary phases are nonpolar
hydrocarbons, waxy liquids, or bonded
hydrocarbons (such as C18, C8, etc.) and the
solvents are polar aqueous-organic mixtures
such as methanol-water or acetonitrile-water.
 Size exclusion
 In size exclusion the HPLC column is
consisted of substances which have
controlled pore sizes and is able to be
filtered in an ordinarily phase according to
its molecular size. Small molecules
penetrate into the pores within the packing
while larger molecules only partially
penetrate the pores. The large molecules
elute before the smaller molecules.
 Ion exchange
 Pada tipe kolm ini komponen sampel
dipisahkan berdasar gaya tarik ion
diantara dua molekul yg membawa
muatan gugus yang berlawanan terhadap
matan fasa diam. Pemisahan dilakukan
antara dua fasa gerak cair bermuatan,
biasanya air yang mengandung garam
atau sedikit akohol, dan fase stasioer yg
mengandung gugus asam atau basa.
basic fixed sites.
 Selectivity Factor

 Nilai K’ menerangkan tentang relatifitas

pita2 elusi terhadap volume rongga fasa
diam. Nilai pita elusi tidak dipengaruhi
oleh laju alir fluida dan dimensi kolom.
 Nilai semua parameter relatif terhadap yg
lainnya. Nilai pita elusi ini menunnjukkan
faktor selektifitas atau faktor pemisahan
(mencakup semua faktor kimia).
 Types of Liquid Column
Chromatography
(LCC)
 LLC
(KROMATOGRAFI 
CAIR-CAIR) GLC( KROMATOGR
 LSC (KROMATOGRAFI AFI GAS CAIR)
ADSORPSI PADAT CAIR) GSC(KROMATOGR
 SEC (KROMATOGRAFI AFI PADAT CAIR)
PERBEDAAN UKURAN
MOLEKUL)  SFC
(KROMATOGRAFI
FLUIDA
SUPERKRITIS)
 Types of Detectors
 Absorbance (UV  Evaporative Light
with Filters, UV with Scattering Detector
Monochromators) (ELSD)

 IR Absorbance  Electrochemical

 Fluorescence  Mass-
Spectrometric
 Refractive-Index
 Photo-Diode Array
 EVALUASI PARAMETERS
 EFISIENSI
 RESOLUSI
 KETAHANAN
 INDEK RETENSI
 KEBANJIRAN KOLOM
 FAKTOR KAPASITAS
 References
 http://192.215.107.101/ebn/942/tech/techfocus/1071main.html
 http://www.chem.usu.edu/~sbialk/Classes/565/opamps/opamps.ht
ml
 Skoog, Holler, and Neiman. Principles of Instrumental Analysis.
5th ed. Orlando: Harcourt Brace & Co., 1998.
 http://weather.nmsu.edu
 http://elchem.kaist.ac.kr/vt/chem-ed/sep/lc/hplc.htm
 http://www.chemistry.nmsu.edu/Instrumentation/Lqd_Chroma.html
 http://weather.nmsu.edu/Teaching_Material/SOIL698/Student_Mat
erial/HPLCHP1090/HPLCINJ.HTM
 http://test-
equipment.globalspec.com/LearnMore/Labware_Scientific_Instru
ments/Analytical_Instruments/Chromatographs/HPLC_Columns
 http://www.chemistry.adelaide.edu.au/external/soc-rel/content/lc-
col.htm