column chromatography
DESCRIPTION
Column Chromatography. Nina Salamah, MSc., Apt. Kromatografi Kolom Sederhana. Bergerak / aliran karena gaya grafitasi ↓ Pemilihan fase diam + fase gerak ↓ Kepolaran ↓ Pita-pita kromatogram ↓ Terbentuk fraksi-fraksi ↓ Dianalisis dengan KLT / KK↓. Pengisian Kolom. - PowerPoint PPT PresentationTRANSCRIPT
Column Chromatography
Nina Salamah, MSc., Apt
Kromatografi Kolom Sederhana
Bergerak / aliran karena gaya grafitasi↓
Pemilihan fase diam + fase gerak↓
Kepolaran↓
Pita-pita kromatogram↓
Terbentuk fraksi-fraksi↓
Dianalisis dengan KLT / KK↓
Pengisian Kolom
Fase diam
Pasir
Kapas / glass wool
• pengisian kolom homogen• fase diam ukuran sama• fase diam bentuk homogen• bebas gelembung udara
Tehnis : fase diam + pelarut → bubur (f. gerak)
Klasifikasi Sistem Kromatografi
Umum Tehnik Spesifik
Fase diam Keseimbangan
1. K. Cair
(LC)
LLC LSC IEC
Cair pd padatan
Padatan
Resin
Partisi
Absorbsi
Tukar ion
2. K. Gas
(GC)
GLC GSC Gas terikat
Cair pd padatan
Padatan
Padatan
Partisi
Absorbsi
P / A
LIQUID COLUMN CHROMATOGRAPHY
A sample mixture is passed through a column packed with solid particles which may or may not be coated with another liquid.
With the proper solvents, packing conditions, some components in the sample will travel the column more slowly than others resulting in the desired separation.
Principles of Separation on a column
Principles of Separation
Principles of Separation
Principles of Separation
Principles of Separation
Principles of Separation
Principles of Separation
Gravitasi Pressure/tekanan Vacum pompa
How does reverse phase chromatography
compare to normal phase chromatography ?
Normal Phase Column Chromatography …
The stationary phase is POLAR The more polar component
interacts more strongly with the stationary phase
The more polar component moves more slowly.
The non-polar component moves more rapidly.
Reverse Phase Chromatography…
O
Si
O
O
Si
OO
O
O
Si
OO
O
Si
OO
O
Si
O OO
Si
OO
Si
OO
Si
OO
Si
O OO
Si
OO
Si
OO
Si
O OO
CH2
CH3
17Si
CH3
CH3
CH2
CH3
17Si
CH3
CH3
SiCH3)3SiCH3)3
SiCH3)3
Silica is alkylated with long chain hydrocarbon groups, using 18 carbons long. This is usually referred to as C-18 silica.
Reverse Phase Column Chromatography…. The stationary phase (column packing) is
now NON-POLAR Non-polar compounds will move more
slowly because they are attracted to the column packing.
The more polar component moves more quickly down the column.
Polar solvents, such as water and methanol are used in reverse phase chromatography
Used mainly in columns, such as HPLC
A + B + C
OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOO OOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOO OOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOO OOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO OOOOOOOOOO OOOOOOOOOOO OOOOOOOOOOO
Sample (A+B+C)
Column
Solid Particles(packing material- stationary phase)
Eluant (eluate)
DIAGRAM OF SIMPLE LIQUID COLUMN CHROMATOGRAPHY
A
B
C
Solvent(mobile or moving phase)
Diagram of Simple Liquid Column ChromatographyDiagram of Simple Liquid Column Chromatography
The 4 basic liquid chromatography modes are named according
to the mechanism involved:
1. Liquid/Solid Chromatography (adsorption chromatography)
A. Normal Phase LSC
B. Reverse Phase LSC
2. Liquid/Liquid Chromatography (partition chromatography)
A. Normal Phase LLC
B. Reverse Phase LLC
3. Ion Exchange Chromatography
4. Gel Permeation Chromatography (exclusion chromatography)
FOUR BASIC LIQUID CHROMATOGRAPHY
Types of Chromatography
LIQUID SOLID CHROMATOGRAPHY
Si - O - H
Normal phase LS Reverse phase LS
Silica Gel
The separation mechanism in LSC is based on the competition of the components of the mixture sample for the active sites on an absorbent such as Silica Gel.
LIQUID SOLID CHROMATOGRAPHY
Si - OH
HEXANE
OH
C-CH3
CH3
CH3 - C
CH3
CH3
OH
OH
CH3
CH3
WATER-SOLUBLE VITAMINS
1. Niacinamide 2. Pyridoxine
N
CONH2
N
CH2OH
CH2OH
HO
H3C
3. Riboflavin N
NNH
N
CH2
HOCH
HOCH
HOCH
CH2OH
O
OH3C
H3C
ClN
S
N
NH3C
CH2
NH2
CH3
CH2CH2OH
4. Thiamin
WATER-SOLUBLE VITAMINS
0 5 10 15 20
Column: u Bondapak C18 Solvent: MeOH Sample: Water-Soluble Vitamins
Inject1
2
3
4
LIQUID-LIQUID CHROMATOGRAPHY
ODPN (oxydipropionylnitrile)
Normal Phase LLC Reverse Phase LLC
NCCH3CH2OCH2CH2CN(Normal)CH3(CH2)16CH3 (Reverse)
The stationary solid surface is coated with a 2nd liquid (the Stationary Phase) which is immiscible in the solvent (Mobile) phase. Partitioning of the sample between 2 phases delays or retains some components more than others to effect separation.
ION-EXCHANGE CHROMATOGRAPHY
SO3-Na+
Separation in Ion-exchange Chromatography is based on the competition of different ionic compounds of the sample for the active sites on the ion-exchange resin (column-packing).
Types of Ion Exchange Resins
Type of Exchanger
Functional Exchanger Group Trade Name
Cation
Strong Acid Sulfonic acid (-SO3-H+) Dowex 50;
Amberlite IR 120
Weak acid Carboxyclic acid (-CO2-H+) Amberlite IRC 50
Anion
Strong base Quaternary ammonium ion (-NR3
+OH-)Dowex 1; Amberlite IRA 400
Weak base Amine group (-NH3+OH-) Dowex 3;
Amberlite IR 45
ChromatographyConditions associated with each kind of chromatography
•Ion exchange chromatography•Organic cation exchange resins involve crosslinked polystyrene
containing either SO3- or COO- functional groups with an
associated cation
•Organic anion exchange resin involve
•crosslinked polystyrene containing NH3+
•functional groups with an associated anion
C
H
C
H
H
C
H
C
H
H
Na+SO3
- SO3-Na
+
C
H
C
H
H
C
H
C
H
H
Cl-NH3 NH3
+Cl
-
The affinity of dissolved ions for the resin varies with the ion and the composition of the
solution
nRzSO3–H+ + Mn+ (RzSO3)nM + nH+
nRzCO2–H+ + Mn+ (RzCO2)nM + nH+
nRzNR3+OH-+ An- (RzNR3)nA + nOH-
MECHANISM OF ION-EXCHANGE CHROMATOGRAPHY OF AMINO ACIDS
SO3-
SO3-
Na+
COO-
H3 N+
Na+
COOHH3 N
+
pH2
pH4.5
Ion-exchange Resin
H3N
+
SO3-
SO3-
SO3-
SO3-
SO3-
SO3-
H3N+
COOH
OH
COOH
COOH
H3N+
H3N+
OH
COO-
Na+
H3N+
COO-
Na+
Na+
H+ OH
- = H2O
H+ OH
- = H2O
Na+
Na+
pH3.5
Mobile PhaseStationary Phase
Exchange Resin
pH4.5
Chromatography of Amino AcidsChromatography of Amino Acids
Some Applications of Ion Exchange Chromatography
Purificationsa mixed bed cation-anion exchanger remove salts (ex CaCl2) from water by exchanging them for H2O :Deionization of water
ConcentrationsThe concentration of trace elements in seawater.
Analytical SeparationsSeparations of metal ions and amino acid or halide ions
SIZE EXCLUTION CHROMATOGRAPHY
Gel-Permeation Chromatography is a mechanical sorting of molecules based on the size of the molecules in solution. Small molecules are able to permeate more pores and are, therefore, retained longer than large molecules.
SIZE EXCLUTION
CHROMATOGRAPHY Molecules that can penetrate the gel particles are
separated based on size and shape. Others pass straight through the column.
Gel filtration chromatography : mobile phase is water.
Gel permeation chromatography : mobile phase is an organic solvent.
Sephadex is popular molecular-sieve material 4 the separation of proteins.
SOLVENTS
Polar Solvents
Water > Methanol > Acetonitrile > Ethanol > Oxydipropionitrile
Non-polar Solvents
N-Decane > N-Hexane > N-Pentane > Cyclohexane
Kekuatan elusi pelarut pada silika dan polaritas pelarut
Schematic of a chromatogram
PARAMETER PEMISAHAN DALAM KROMATOGRAFI KOLOM1. KAPASITAS
Kapasitas menggambarkan kemampuan fase diam dalam menahan analit. Jika waktu tambat lama, berarti kapasitasnya besar.
2. SELEKTIVITAS Selektivitas menggambarkan kemampuan fase diam untuk dapat memisahkan suatu campuran senyawa. Semakin besar nilai , campuran senyawa semakin terpisah.
3. RESOLUSI Resolusi menggambarkan kemampuan kolom dalam memisahkan campuran senyawa
4. JUMLAH PLATE TEORITIS Jml pelat teoritis N, dalam kolom dapat diketahui dari hasil kromatogram.
5. TINGGI PLATE TEORITIS = HHETP = High Equivalent to A Teoritical Plate Adl : ukuran yang menunjukkan ruang yg ditempati oleh setiap pelat teoritis.panjang kolom
MtMtRtK'
1K'2K'
α
)w(w0.5)t(tR
21
12
2Wt4N R
HETP = L/N
L = PANJANG KOLOM
Adjusted Retention Timetr = retention timetm = min. time for unretained mobile phase to travel through columnIn GC tm is the time CH4 takes to travel through the column
Relative Retention
Capacity Factor
mrtr ttt
'1
'2
r
r
t
t
1 so '1
'2 rr tt
m
mr
t
ttk
'
Resolution
Nilai R yg baik > 1,5. Jika R = 1 masih tjd tumpang tindih di antara kedua puncak 2%
Untuk memperbaiki R :1. memperbesar tR = t2 – t1
kolom diperpanjangjumlah fase diam diperbesarmanipulasi faktor pemisahanpengoptimalan suhu pilih f.d dan f.g yang cocok
2. Memperkecil lebar puncak, Wpilih ukuran fase diam kecil (halus) dan pengisian dalam kolom diperbaiki (seragam dan kompak.Kecepatan alir fase gerak optimumKurangi dead space dalam kolomKurangi jumlah sampelDiameter kolom diperkecil.
Resolution
av
r
av
t
av
r
w
t
w
V
w
t
2/1
589.0Resolution
w = peak width at the baseline between tangents drawn to the steepest parts of the peakw1/2 = measured at ½ the peak height
A peak with a retention time of 407 s has a width at the base of 13 s. A neighboring peak is eluted at 424 s with a width of 16 s. Find the resolution for these two components.
1.1)1613(2/1
407424Resolution
av
r
w
t
ChromatographyChromatographic column theory of packed columns
•The effect of column efficiency and column selectivity on resolution
Poor resolution because of poor column efficiency
Good resolution because of good column efficiency,
although column selectivity is not great
Good resolution because of good column selectivity,
although column efficiency is poor
Poor resolution because of poor column selectivity,
although column efficiency is good
Theories ofElution Chromatographysome zone broadeningzone separation
Plate height: constant of proportionality between the variance (2)of the band and the distance traveled (x)
Smaller plate height = narrow peaks = better separations
2
2
2
2
2
16
rr t
w
tN
xH Plate height (H)
Number of plates (N)
A solute with a retention time of 407 s has a width at the base of 13 s on a column 12.2 m long. Find the plate height and number of plates.
2
216
w
tN
N
LH
r
L = column length
mmm
N
LH
w
tN r
78.01057.1
2.12
1057.113
4071616
4
42
2
2
2
'
'2
1
1
4Resolution
avk
kN
N is the number of theoretical plates is the relative retention of two peaksk’2 is the capacity factor for the more retained componentk’av is the average capacity factor for both components
Remember that variance is additive but standard deviation is not
Applications of Chromatography
Qualitative Analysis Quantitative Analysis
Analyses Based on Peak HeightAnalyses Based on Peak AreasCalibration and StandardsThe Internal Standard MethodThe Area Normalization Method