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Dasar Dasar Kromatografi: Rinaldi Idroes, Prof., DR - Rer.nat., S.Si. Lely Fajry, DR., M.Si., S.Si

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Dasar Dasar Kromatografi

Oleh :
Rinaldi Idroes, Prof., Dr.rer.nat., S.Si.
Lely Fajry, Dr., M.Si., S.Si

Jurusan Farmasi
Fakultas Matematika dan Ilmu Pengetahuan Alam
Universitas Syiah Kuala
1. Solvent Extraction

Liquid-liquid extraction is a useful method to


separate components (compounds) of a mixture
Let's see an example.

Suppose that you have a mixture of sugar in vegetable oil


(it tastes sweet!) and you want to separate the sugar
from the oil.

You observe that the sugar particles are too tiny to


filter and you suspect that the sugar is partially
dissolved in the vegetable oil.

What will you do?


How about shaking the mixture with
water
Will it separate the sugar from the oil?
Sugar is much more soluble in water
than in vegetable oil, and, as you know,
water is immiscible (=not soluble) with
oil.

Did you see the result? The water phase


is the bottom layer and the oil phase is
the top layer, because water is denser
than oil.

*You have not shaken the mixture yet, so


sugar is still in the oil phase.
By shaking the layers (phases) well, you
increase the contact area between the two
phases. The sugar will move to the phase in
which it is most soluble: the water layer

Now the water phase tastes


sweet,
because the sugar is moved to the
water phase upon shaking.**You extracted
sugar from the oil with water.**

In this example, water was the


extraction solvent ;the original oil-
sugar mixture was the solution to be
extracted; and sugar was the compound
extracted from one phase to another.
Separating the two layers accomplishes
the separation of the sugar from the
vegetable oil
Did you get it? .....the concept of liquid-liquid extraction?

Liquid-liquid extraction is based on the transfer of a solute


substance from one liquid phase into another liquid phase
according to the solubility.

Extraction becomes a very useful tool if you choose a suitable


extraction solvent.
You can use extraction to separate a
substance selectively from a mixture, or to remove unwanted
impurities from a solution. In the practical use, usually one
phase is a water or water-based (aqueous) solution and the
other an organic solvent which is immiscible with water.

The success of this method depends upon the difference in


solubility of a compound in various solvents. For a given
compound, solubility differences between solvents is
quantified as the "distribution coefficient"
Distribution Coefficient Kd
When a compound is shaken in a separatory funnel with two immiscible
solvents, the compound will distribute itself between the two solvents.

Normally one solvent is water and the other solvent is a water-immiscible


organic solvent.

Most organic compounds are more soluble in organic solvents, while some
organic compounds are more soluble in water.
Here is the universal rule:

At a certain temperature, the ratio of concentrations of a solute


in each solvent is always constant.ハAnd this ratio is called the
distribution coefficient, K.

(when solvent1 and solvent2 are immiscible liquids

For example,Suppose the


compound has a distribution
coefficient K = 2 between
solvent1 and solvent2

By convention the organic


solvent is (1) and water is (2)
Nerst Partition Coefficient
Same analogy with distribution coefficient, but ….
In this case, the distribution of analyte occurs between stationary
and mobile phase.
Stationary phase, mobile phase, & analyte form a ternary system.

Each analyte is distributed between the two phases (in


equilibrium) :
Partition Coefficient
– CS: concentration of analyte on the stationary phase
– CM: concentration of analyte on the mobile phase
Migrasi dan Retensi
 Dalam sistem kromatografi, sampel (terdiri dari beberapa
analit) dimasukkan kedalam kolom melalui injektor.

 Fase gerak akan mengangkut sampel sepanjang kolom.


Didalam kolom, sampel akan melakukan kontak/interaksi
dengan fase diam.

 Pergerakan analit dalam fase gerak disebut dengan migrasi,


sedangkan tertahannya analit akibat interaksi dengan fase
diam disebut dengan retensi.

 Kecepatan migrasi analit tergantung pada kekuatan retensi


analit tersebut.

 Semakin besar nilai K, maka analit semakin lama di fase


diam, maka semakin lama retensi, semakin lambat migrasi
Prinsip Pemisahan pada kromatografi

 Setiap analit memiliki nilai K yang spesifik (tiap analit berbeda)

 Perbedaan nilai K membuat retensi dan migrasi tiap analit


berbeda-beda.

 Analit-analit yang diinjeksi kedalam sistem fase gerak & fase


diam secara bersamaan, akan bermigrasi dengan waktu yang
berbeda-beda.

 Perbedaan waktu migrasi akan membuat tiap analit mencapai


terminal sistem pemisahan dengan waktu berbeda, Fenomena
ini yang menyebabkan analit tersebut terpisah.
Prototype Chromatogram
Kurva Gaussian
Puncak elusi sempurna sebuah kromatogram
distandarisasi terhadap bentuk representasi grafik hukum
distribusi normal error random atau dikenal dengan Kurva
Gaussian.
Puncak elusi ideal dideskripsikan dengan fungsi probability
density disamping:

Dimana µ adalah representasi dari waktu retensi, σ adalah standar deviasi puncak,
σ2 adalah varian dan y adalah sinyal sebagai fungsi waktu x dari detektor.
Terima Kasih

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