GC dan HPLC

Lilis Febriyanti, M. Farm.

Outline

• Pendahuluan
• Jenis-jenis Prinsip Pemisahan pada Kromatograf
• HPLC
• GC
Pendahuluan

Chromatographic techniques involve two phases of which at

least one is mobile and the other most often is stationary. The
two phases move relative to each other and in most cases the
mobile phase moves through a bed of a stationary phase.
When a mixture of analytes is introduced into these systems a
number of separation mechanisms influence the partition of
analytes between the two phases
 Prinsip Fasa
Normal

Prinsip Fasa
Terbalik
Jenis Kromatograf
berdasar prinsip
pemisahan

Prinsip Pertukaran
Ion
Prinsip Ekslusi
Ukuran
1. Prinsip Fasa Normal

• In normal phase chromatography the stationary phase Is more

polar than the mobile phase. The stationary phase consists of
porous particles with polar groups on the surface, and the
mobile phase is an organic solvent.

• Aplikasi prinsip mendasari Teknik TLC dan HPLC

Fasa Diam (Polar)
• Silika • Alumina dan Magnesium
Karakteristik adsorptivitas yang tinggi. Silikat
Oleh karena itu KFN disebut sebagai
Kroma adsorpsi. Gugus silanol pada silikatnya
Proses sintesis: Pengasaman larutan bisa diderivatisasi dengan
NaSilikat (purifkasi-pulverisasi- ligan yang mengandung
pengeringan-fraksinansi). Luas gugus polar lain seperti diol,
permukaan 200-800m2/g berpengaruh
terhadap besaran dan jumlah pori CN, dan NH2.
yang dibutuhkan pada saat proses
elusi analit dan fasa gerak.
Situs aktif: gugus silanon (-OH) asam
lemah
Fasa gerak (nonpolar)

• The strength of the mobile phase is decided by the polarity of the

solvent used, and solvents can be ranked according to their solvent
strength. Solvent strength increases with increasing polarity. Increased
solvent strength of mobile phase decreases the retention of the
substances.
• In normal phase chromatography the mobile phase seldom consists of
a single solvent. Usually it consists of a mixture of two or three
solvents.
• Retention decreases with increasing solvent strength and a reasonable
choice of e in the mobile phase ensures that drug substances receive
adequate retention. However, not all
Interaksi Fasa Diam dengan Analit
1. Dispersion interactions
Dispersion gives weak interactions and occurs when the silanol
groups induce temporary dipoles in an analyte molecule. For
example, found in the aromatics.
2. Dipole–dipole interactions
Analytes having permanent dipoles may induce dipole interactions.
Examples of groups that provide permanent dipoles are: CN > NO2 >
C¼O, CHO > COOR > halogen > OH > COOH > –O– > NH2
3. Hydrogen bonding interactions
Proton donors and proton acceptors provide hydrogen bonding
interactions. The more acidic the proton donor and the more alkaline
the acceptor, the stronger is the interaction.
4. Ionic interactions
Ionic interactions can occur between basic analytes and the acidic
silanol groups.
2. Prinsip Fasa Terbalik

Fasa diam (Hidrofobik)

• Silika diderivatisasi dengan
reagen agar membentuk
permukaan yang kurang lebih
hidrofobik dengan partikel silika.
• Biasanya klorosilan atau reagen
silan organic lain. Reagen
derivatisasi silan ini memiliki
gugus alkil yang terhubung
dengan atom silisium sehingga
membentuk gugus yang bulki,
• Contohnya: oktadesil atau
oktadesilsilan
• Activated Carbon. Recently
also diamond, which is also
made of carbon, has been
introduced as a column
packing material for
reversed phase
chromatography. This latter
material is however not
often used in
pharmaceutical analysis.
Fasa Gerak (polar)

• consist of mixtures of water and one or more organic solvents that

must be miscible with water. The organic solvents used are called
organic modifers as they modify the strength of the mobile phase.
Increased content of organic modifer increases the strength of the
mobile phase and retention of analytes decreases
• The solvent strength of methanol is somewhat weaker than that of
acetonitrile which is weaker than tetrahydrofuran
3. Prinsip Ekslusi Ukuran
• In SEC the analytes are separated according to their molecular size in
solution.
• Separation occurs when analytes penetrate into the pores of the matrix.
• The matrix particles for SEC are manufactured with controlled pore sizes.
The pore size is chosen dependent on the molecular range to be
investigated
• Packing materials can be soft or hard. Soft materials are made of
polysaccharides such as dextrans, polyacrylamide or polystyrene. Soft
gels are used with gravity flow (flow driven only by gravity), where the
mobile phase flows through the column as a function of gravity only. For
HPLC rigid packing materials that resist the higher pressure are used.
Rigid packaging materials are made of silica or a highly crosslinked
organic polymer like polystyrene–divinylbenzene copolymer. Silica is
used with aqueous mobile phases, while polystyrene–divinylbenzene
often is used with organic solvents as mobile phases.
4. Prinsip Pertukaran Ion
• Ion exchange chromatography is a technique that allows the
separation of ions based on their charge. It can be used for
almost any kind of charged molecules, including large proteins,
nucleotides and amino acids.
• The ionic analytes are retained by ionic interaction between
the analytes and ionic sites of the opposite charge placed on the
stationary
 HPL
GC
C
HPLC (High Performance Liquid
Chromatography)
Sampel Injektor
• A prerequisite for the • The injection process can be
samples to be analyzed by automated using an
autosampler. The process can be
HPLC is that they are
controlled by the HPLC software
soluble in the so that analysis can take place
mobile phase without supervision, running for
24 h or more. Autosamplers can
also be equipped with a
refrigerator for the cooling of
sensitive samples so that they
do not decompose before
analysis.
 Pump

• The pumps deliver the mobile phase

at a constant flow rate through the
column. The pumps can be
constructed in different ways, but a
piston pump is the most common.
• When the pumping system delivers a
mobile phase with a constant
composition to the column it is called
isocratic elution.
• The change of the composition of the
mobile phase during chromatography
is called gradient elution.
Kolom (Fasa Diam)
• The typical analytical HPLC
column has been a 15–25 cm
long steel tube packed with
5 mm particles. Inner diameter
of the tube has been 4.6 mm
Fasa gerak
The following requirements for liquids to be used for mobile phases have to
be considered:
• The solvents should not give any response in the detector used.
• The solvents must have a satisfactory degree of purity.
• The solvents should have low viscosity to provide as low a back pressure
as possible in the HPLC system.
• The solvents should have low toxicity, preferable be inflammable and
nonreactive, and they must be suitable for disposal after use
• Degassing can be performed by ultrasound treatment, by purging with
helium or by vacuum treatment.
• The mobile phase is contained in a bottle.
Detector
The LC detector gives a response for the analyte that is converted
into an electrical signal. The detectors can be
divided into two types:
(i) general detectors that measure any change in the mobile
phase
(ii) specifc detectors that respond only to substances with
specifc properties.
GC (Gas Chromatography)
Temperatur
The temperature of the column is
the main parameter that controls
retention in GC analysis. The
substances are distributed
between the gas and liquid
phases, but they only move
forward through the column when
they are in the gas phase.
Gas (Fasa gerak)
The carrier gas is an inert
transport medium for the
substances. Gasses like nitrogen,
helium and hydrogen can be
used. Short analysis times are
desirable, and this is achieved by
using a high gas velocity through
the column. However, it turns out
that the peaks become broader
both if the gas speed is too low as
well as if it is too high. Highest
efficiency is achieved by an
optimum gas velocity. The
optimum gas velocity is different
for nitrogen, helium and
hydrogen
Fasa Diam

• Stationary phases in GC are temperature-stable liquids having very low

vapor pressure. Many different (hundreds) have been suggested through
the years, but if GC columns are to be used at temperatures up to or
even above 300 C, the choice of appropriate substances is limited. Only a
number of polymers possess the characteristics to be liquid at or close to
room temperature, to be stable and to have a low vapor pressure at high
temperature.
• For examples; The polydimethylsiloxane polymer, Polysiloxanes, and
Polyethylene glycols
Selektivitas pada GC
• On apolar stationary phases • Retention depends on both
analytes are separated the substances’ boiling
according to their boiling point and solubility in
points, and analytes with stationary phase.
low boiling points are eluted
before substances with
higher boiling points
Capillary Column
Capillary columns can be made of
a number of materials like metal
or fused silica. The fused silica
capillary is preferred due to its
robustness and inactivity. Glass
columns are fragile and metal
columns may contribute to
analyte decomposition. The outer
surface of the fused silica
capillary is coated with a
polyimide layer to improve the
mechanical stability.
Packed Column
The most common support
materials are made of
diatomaceous earth, which is the
skeleton of single-celled siliceous
diatoms. The solid support is
purifed and pretreated to make it
suitable for GC. The amount of
stationary phase on the support
material is given as a percentage,
and the normal range is 1–10%
Injection System
In GC, it is common to inject the
substances dissolved in a volatile
solvent. The Solvent and the substances
evaporate in the injector and the gas
mixture is brought to the column by the
carrier gas.
The chemicals are injected with a
syringe, and regular injection volumes
are in the range 0.5 to 2.0 ml. It is
important that the sample evaporates
instantly and that the
volume brought to the column does not
overload the column. Otherwise peak
broadening
will occur.
Flame Ionization Detector
Nitrogen-Phospherous Detector
Thermal Conductivity Detector
Mass Spectrophotometer Detector

• A mass spectrometer (MS) is used both to quantify substances and to

provide structural information about the substances. In the mass
spectrometer the analytes are ionized under vacuum, usually by
being bombarded by an electron beam. Normally the energy of the
electron is controlled at 70 eV
• Therefore, GC-MS is a standard method for drug analysis and forensic
science. Identifcations are based upon a comparison of retention
data from the GC separation and from the mass spectra of the
substances.
Terima Kasih