column chromatography

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Column Chromatography Nina Salamah, MSc., Apt

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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 Presentation

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Page 1: Column Chromatography

Column Chromatography

Nina Salamah, MSc., Apt

Page 2: Column Chromatography

Kromatografi Kolom Sederhana

Bergerak / aliran karena gaya grafitasi↓

Pemilihan fase diam + fase gerak↓

Kepolaran↓

Pita-pita kromatogram↓

Terbentuk fraksi-fraksi↓

Dianalisis dengan KLT / KK↓

Page 3: Column Chromatography

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)

Page 4: Column Chromatography

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

Page 5: Column Chromatography

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.

Page 6: Column Chromatography

Principles of Separation on a column

Page 7: Column Chromatography

Principles of Separation

Page 8: Column Chromatography

Principles of Separation

Page 9: Column Chromatography

Principles of Separation

Page 10: Column Chromatography

Principles of Separation

Page 11: Column Chromatography

Principles of Separation

Page 12: Column Chromatography

Principles of Separation

Page 13: Column Chromatography
Page 14: Column Chromatography

Gravitasi Pressure/tekanan Vacum pompa

Page 15: Column Chromatography

How does reverse phase chromatography

compare to normal phase chromatography ?

Page 16: Column 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.

Page 17: Column Chromatography

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.

Page 18: Column Chromatography

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

Page 19: Column Chromatography

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

Page 20: 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

Page 21: Column Chromatography

Types of Chromatography

Page 22: Column 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.

Page 23: Column Chromatography

LIQUID SOLID CHROMATOGRAPHY

Si - OH

HEXANE

OH

C-CH3

CH3

CH3 - C

CH3

CH3

OH

OH

CH3

CH3

Page 24: Column Chromatography

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

Page 25: Column Chromatography

WATER-SOLUBLE VITAMINS

0 5 10 15 20

Column: u Bondapak C18 Solvent: MeOH Sample: Water-Soluble Vitamins

Inject1

2

3

4

Page 26: Column Chromatography

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.

Page 27: Column Chromatography

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).

Page 28: Column Chromatography
Page 29: Column Chromatography

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

Page 30: Column Chromatography

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

Page 31: Column Chromatography

nRzSO3–H+ + Mn+ (RzSO3)nM + nH+

nRzCO2–H+ + Mn+ (RzCO2)nM + nH+

nRzNR3+OH-+ An- (RzNR3)nA + nOH-

Page 32: Column Chromatography

MECHANISM OF ION-EXCHANGE CHROMATOGRAPHY OF AMINO ACIDS

SO3-

SO3-

Na+

COO-

H3 N+

Na+

COOHH3 N

+

pH2

pH4.5

Ion-exchange Resin

Page 33: Column Chromatography

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

Page 34: Column Chromatography

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

Page 35: Column Chromatography

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.

Page 36: Column Chromatography

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.

Page 37: Column Chromatography

SOLVENTS

Polar Solvents

Water > Methanol > Acetonitrile > Ethanol > Oxydipropionitrile

 Non-polar Solvents

N-Decane > N-Hexane > N-Pentane > Cyclohexane

Page 38: Column Chromatography

Kekuatan elusi pelarut pada silika dan polaritas pelarut

Page 39: Column Chromatography

Schematic of a chromatogram

Page 40: Column Chromatography

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

Page 41: Column Chromatography

MtMtRtK'

1K'2K'

α

)w(w0.5)t(tR

21

12

2Wt4N R

HETP = L/N

L = PANJANG KOLOM

Page 42: Column Chromatography

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

'

Page 43: Column Chromatography

Resolution

Page 44: Column Chromatography

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.

Page 45: Column Chromatography

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

Page 46: Column Chromatography

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

Page 47: Column Chromatography

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

Page 48: Column Chromatography

Theories ofElution Chromatographysome zone broadeningzone separation

Page 49: Column Chromatography
Page 50: Column Chromatography

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)

Page 51: Column Chromatography

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

Page 52: Column Chromatography

'

'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

Page 53: Column Chromatography

Remember that variance is additive but standard deviation is not

Page 54: Column Chromatography

Applications of Chromatography

Qualitative Analysis Quantitative Analysis

Analyses Based on Peak HeightAnalyses Based on Peak AreasCalibration and StandardsThe Internal Standard MethodThe Area Normalization Method

Page 55: Column Chromatography