KROMATOGRAFI

KROMATOGRAFI
Syarif Hamdani
http://www.catatankimia.com
Kromatografi
Adalah teknik pemisahan fisik suatu
campuran zat-zat kimia yang berdasar pada
perbedaan migrasi dari masing-masing
komponen campuran yang terpisah pada
fase diam di bawah pengaruh pergerakan
fase gerak

Sejarah kromatografi
Pertama kali diperkenalkan oleh W. Ramsey
pada tahun 1905
Istilah kromatografi (artinya penulisan warna)
pertama kali diberikan oleh Mikhail
Semenovic Tswett pada tahun 1908
KLT diperkenalkan oleh Izamailov dan
Shraiber pada tahun 1938
Asas dan Dasar-dasar
Kromatografi
1. Kromatografi dengan asas adsorpsi, memakai fase
diam padat dan fase gerak cair atau gas
2. Kromatografi dengan asas partisi, memakai fase
diam cair dan fase gerak cair
3. Kromatografi dengan asas fitrasi, memakai fase
diam padat yang mempunyai sifat fitrasi dan fase
gerak cairan
4. Kromatografi dengan asas suhu kritik, memakai
CO
2
dalam keadaan superkritik
Klasifikasi kromatografi
kromatografi
gas SFC liquid
GSC GLC colomn planar
TLC
PC
LSC
LLC
BPC
IEC
EC
GPC
GFC
Kromatografi Gas
Macamnya :
Kromatografi gas padat : fase diam adalah
butiran-butiran adsorben dan fase gerak
adalah gas
Kromatografi gas cair : fase diam adalah
cairan yang disalutkan pada permukaan tipis
butiran padat dan fase gerak adalah gas
Kromatografi gas
Sampel berupa gas. Untuk senyawa yang
bukan gas dibuat turunan esternya.
Gas pembawa : He, Ar, N
2
atau campuran
He dan CH
4

Detektor terdiri dari berbagai macam
tergantung keperluan diantaranya : TCD,
FID,ECD, NPD, FPD, IRD, TID, dll.
Analisis kualitatif dibandingkan terhadap
BANK DATA
Kromatografi Lapis Tipis
Keuntungan :
Digunakan untuk tujuan analitik
Identifikasi komponen dapat dilakukan dengan
pereaksi warna, fluoresensi atau pemadaman
fluoresensi, radiasi UV
Dapat dilakukan elusi dengan mekanik (ascending)
atau menurun (descending) atau dengan cara elusi
2 dimensi
Ketepatan penentuan kadar akan lebih baik karena
komponen yang ditentukan merupakan noda yang
tidak bergerak
Kromatografi Lapis Tipis
Identitas komponen dijabarkan dalam harga
Rf (retardation factor) yang dalam penentuan
kualitatif dibandingkan dengan standar
Untuk tujuan kuantitatif digunakan KLT
preparatif (dikerok lalu senyawa diisolasi
dalam pelarutnya)

Kromatografi Kertas
Prinsip sama dengan KLT
Fase diam adalah air yang didukung oleh pelat serat
selulosa, fase mobil air dicampur pelarut organik
Lebih banyak digunakan untuk pemisahan senyawa
non polar, karena selulosa (kertas) bersifat polar
Banyak digunakan untuk pemisahan senyawa
bahan alam
Kekurangan : lebih lama karena panjang kertas bisa
sampai 50 cm.
Kromatografi Kolom
Digunakan untuk isolasi senyawa dari sample
Merupakan kelanjutan dari KLT
Prinsip pemisahan, fase diam dan fase gerak
sama dengan KLT

Kromatografi Cair Kinerja
Tinggi
Dimaksudkan untuk mendapatkan
pemisahan dan hasil analisa kuantitatif yang
baik dengan waktu singkat
Pelarut pengembang harus dipilih dengan
seksama
Kolom harus sesuai
Detektor harus memadai
Sample berupa larutan

HPLC
Liquid
Chromatography
The original development of HPLC used higher pressures
than previously used ----High Pressure Liquid Chromatography

However, over the years the preferred term has become:

High Performance Liquid Chromatography
Advantages of HPLC
High resolution
Speed
Re-usable columns
Great reproducibility
Control of physical parameters
flow rate, polarity, packing efficiency, and particle size.
Easy automation of instrument and data analysis.

HPLC Chromatograph of
Muscadine Grape Juice
SOLVENTS
Includes both liquid phase
and solid materials (Buffers)
dissolved in the liquid.
Solvent properties affecting detection
Solvent properties affecting separation
Solvent properties affecting flow
Viscosity
Miscibility
UV Cutoff -Solvent may interfere with detection

For peptide analysis UV = 215 nm. Solvents that
absorb UV at this wavelength would not be good
candidates for the mobile phase.

Refractive Index of Solvent vs Sample for
Refractive Index detection (Carbohydrates)

Volatility needed for HPLC Mass Spectrometry
(trifluoroacetic acid is a typical volatile buffer)
Solvent Properties Affecting Detection
BUFFERS
1)Buffers are needed to control the pH differences caused by
the sample matrix.

2)Buffers are used to control the ionization of compounds
and therefore their retention by the column.
Retention Time and pH in Reversed Phase
3 4 5 6 7 8 9
pH
R
e
l
a
t
i
v
e

R
e
t
e
n
t
i
o
n

T
i
m
e

pK
a
partially charged
fully charged
not charged
When an acid or a base is ionized it becomes much less
hydrophobic and will elute much earlier. Acids lose a
proton and become ionized (negative charge) as pH increases.
Bases on the other hand, gain a proton and acquire a positive
charge as pH decreases.
Basic
Compound
SOLVENT SELECTIVITY
The less time a compound spends in the stationary phase,
the faster it will move through the column (less retention time).
If two compounds are added to the column, the ratio of their
retention times is called the selectivity.

The higher the selectivity, the better the separation.

Selectivity can be increased by adjustment of the mobile and
stationary phases.
Solvent Selectivity Triangle
Representing 3 Polarity factors
1) Each dot in the triangle
represent a different solvent
2) Solvents can be grouped
based on their type of polarity
3) Solvents and solvent mixtures
are available for just about any
separation you may desire.
Viscosity - resistance to flow
Difficult to force high viscosity solvents through
the column.

Mixing solvents can drastically change viscosity
Viscosity of Water-Organic Solvent Mixtures
Viscosity vs. Pressure
The higher the solvent viscosity, the harder it is for the
solvent to move through a column, and the more pressure in
required to move the solvent at a specific velocity. The pressure
required to move a solvent through a column can be estimated by
the following formula:
P = 250 L F / D
p
2
D
c
2
Where P = pressure drop in psi. F = flow rate (mL/min)
L = column length (cm) D
p
= particle diameter (m)
= solvent viscosity (cP) D
c
= column diameter (cm)
P = 250 L F / D
p
2
D
c
2
EXAMPLE
column length = 15 cm, column diameter =.5 cm,
particle diameter = 5 m, flowrate = 2.0 mL/min

For water n = 1.0 250 x 15 x 1.0 x 2 / 5
2
x .5
2
= 7125/6.25 = 1200 psi
For methanol n = 0.54 250 x 15 x .54 x 2 / 5
2
x .5
2
= 2025/6.25 = 648 psi
For 60% water n = 1.9 250 x 15 x 1.9 x 2 / 5
2
x .5
2
= 7125/6.25 = 2280 psi
40% methanol
SOLVENTS
Water 190 1.00 10.2 ---
Methanol 205 0.55 5.1 Y
Tetrahydrofuran 212 0.55 4.0 Y
Propanol 210 2.3 4.0 Y
Acetonitrile 190 0.38 5.8 Y
Hexane 195 0.31 0.1 N
Ethyl Acetate 256 0.45 4.4 N
Chloroform 245 0.57 4.1 N
UV
Cutoff
Viscosity Polarity
Miscible
with
Water?
Toxicity
Flammability
Reactivity solvent should not react with sample
Cost
Disposal can be more than purchase cost

Peripheral Properties
Geometry of HPLC Columns

Diameter
Length
Particle Size
What is the effect on pressure?
P = 250 L F / D
p
2
D
c
2
Where P = pressure drop in psi. F = flow rate (mL/min)
L = column length (cm) D
p
= particle diameter (m)
= solvent viscosity (cP) D
c
= column diameter (cm)
Geometry of HPLC Columns

Diameter
Length
Particle Size
What is the effect on Theoretical Plates?
What is the effect of column geometry
on Theoretical Plates?
N=L/H
where N is number of plates, H is plate height
and L is Column Length
Remember that separation is best on columns with high
number of theoretical plates.
Therefore, doubling the column length will double N
but this will double analysis time and pressure!
Decreasing column diameter by half
halving the column diameter can also increase N slightly
For comparison purposes, lets keep the mobile phase
velocity constant. Therefore, flow would be reduced 4X
and analysis wont take any longer!
This reduces the amount of solvent used by 4X but also
reduces the amount of sample that can be injected by 4X.
Decreasing particle size by half
However, halving the particle size can double N
Will increase pressure by 4X
Decreasing particle size and making the column half as long
will keep N the same but cut sample time in half and solvent
use in half.
In general small diameter columns with
small particles are best for rapid separation,

.but require higher pressures, smaller samples, and can plug easier.

The problem with plugging should not be underestimated
and care should be exercised in keeping the sample, mobile
phases, and columns CLEAN!

KROMATOGRAFI

Uploaded by

Document Information

Original Description:

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

KROMATOGRAFI

Uploaded by

Copyright:

Available Formats

KROMATOGRAFI

You might also like