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 CO2 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, N2 atau campuran He dan CH4
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
The original development of HPLC used higher pressuresthan 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.
•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 thatabsorb UV at this wavelength would not be goodcandidates for the mobile phase.
Refractive Index of Solvent vs Sample forRefractive 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 bythe sample matrix.
2)Buffers are used to control the ionization of compoundsand therefore their retention by the column.
Retention Time and pH in Reversed Phase
3 4 5 6 7 8 9pH
Rel
ativ
e R
eten
tio
n T
ime
pKa
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.
BasicCompound
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 theirretention 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 TriangleRepresenting 3 “Polarity” factors 1) Each dot in the triangle
represent a different solvent2) Solvents can be groupedbased on their type of polarity3) Solvents and solvent mixturesare available for just about anyseparation you may desire.
Viscosity - resistance to flow
Difficult to force high viscosity solvents throughthe column.
Mixing solvents can drastically change viscosity
Viscosity vs. PressureThe higher the solvent viscosity, the harder it is for the
solvent to move through a column, and the more pressure inrequired to move the solvent at a specific velocity. The pressurerequired to move a solvent through a column can be estimated bythe following formula:
P = 250 L F / Dp2 Dc
2
Where P = pressure drop in psi. F = flow rate (mL/min)L = column length (cm) Dp = particle diameter (m)= solvent viscosity (cP) Dc = column diameter (cm)
P = 250 L F / Dp2 Dc
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 / 52 x .52 = 7125/6.25 = 1200 psiFor methanol n = 0.54 250 x 15 x .54 x 2 / 52 x .52 = 2025/6.25 = 648 psiFor 60% water n = 1.9 250 x 15 x 1.9 x 2 / 52 x .52 = 7125/6.25 = 2280 psi 40% methanol
SOLVENTS
Water 190 1.00 10.2 ---Methanol 205 0.55 5.1 YTetrahydrofuran 212 0.55 4.0 YPropanol 210 2.3 4.0 YAcetonitrile 190 0.38 5.8 YHexane 195 0.31 0.1 NEthyl Acetate 256 0.45 4.4 NChloroform 245 0.57 4.1 N
UVCutoff Viscosity Polarity
Misciblewith
Water?
– ToxicityToxicity
– FlammabilityFlammability– Reactivity Reactivity solvent should not react with samplesolvent should not react with sample
– Cost Cost – Disposal Disposal can be more than purchase costcan be more than purchase cost
Peripheral Properties
P = 250 L F / Dp2 Dc
2
Where P = pressure drop in psi. F = flow rate (mL/min)L = column length (cm) Dp = particle diameter (m)= solvent viscosity (cP) Dc = column diameter (cm)
What is the effect of column geometryon 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 highnumber of theoretical plates.
Therefore, doubling the column length will double Nbut this will double analysis time and pressure!
What is the effect of column geometryon Theoretical Plates?
Decreasing column diameter by half
halving the column diameter can also increase N slightly
For comparison purposes, let’s keep the mobile phasevelocity constant. Therefore, flow would be reduced 4Xand analysis won’t take any longer!
This reduces the amount of solvent used by 4X but alsoreduces the amount of sample that can be injected by 4X.
What is the effect of column geometryon Theoretical Plates?
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 solventuse in half.
In general small diameter columns withsmall particles are best for rapid separation,
….but require higher pressures, smaller samples, and can plug easier.
The problem with plugging should not be underestimatedand care should be exercised in keeping the sample, mobilephases, and columns CLEAN!