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 Tehnis : fase diam + pelarut → bubur (f. gerak) pengisian kolom homogen fase diam ukuran sama fase diam bentuk homogen bebas gelembung udara Fase diam Pasir Kapas / glass wool 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 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

Pemisahan pada Kromatografi Kolom

Aliran fase gerak pada kolom Gravitasi Pressure/tekanan Vacum pompa

How does reverse phase chromatography to normal 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… 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

Diagram of Simple Liquid Column Chromatography

FOUR BASIC LIQUID 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)

Types of Chromatography

LIQUID SOLID CHROMATOGRAPHY Normal phase LS Reverse phase LS d- d+ Si - O - H 30 m 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 OH HEXANE Si - OH OH OH CH CH 3 3 CH - C C-CH 3 3 CH CH 3 3 CH 3

WATER-SOLUBLE VITAMINS

WATER-SOLUBLE VITAMINS

LIQUID-LIQUID CHROMATOGRAPHY ODPN (oxydipropionylnitrile) Normal Phase LLC Reverse Phase LLC NCCH 3 CH 2 OCH CN(Normal) (CH ) 16 (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 SO 3 - 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

Chromatography Conditions 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 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 + + SO 3 Na H N 3 COOH Ion-exchange Resin - + SO H N 3 3 - COO pH4.5 + Na

Chromatography of Amino Acids

Some Applications of Ion Exchange Chromatography Purifications a mixed bed cation-anion exchanger remove salts (ex CaCl2) from water by exchanging them for H2O :Deionization of water Concentrations The concentration of trace elements in seawater. Analytical Separations Separations 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 KOLOM 1. 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 = H HETP = High Equivalent to A Teoritical Plate Adl : ukuran yang menunjukkan ruang yg ditempati oleh setiap pelat teoritis.panjang kolom

HETP = L/N L = PANJANG KOLOM

Adjusted Retention Time tr = retention time tm = min. time for unretained mobile phase to travel through column In GC tm is the time CH4 takes to travel through the column Relative Retention Capacity Factor

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 diperpanjang jumlah fase diam diperbesar manipulasi faktor pemisahan pengoptimalan suhu pilih f.d dan f.g yang cocok 2. Memperkecil lebar puncak, W pilih ukuran fase diam kecil (halus) dan pengisian dalam kolom diperbaiki (seragam dan kompak. Kecepatan alir fase gerak optimum Kurangi dead space dalam kolom Kurangi jumlah sampel Diameter kolom diperkecil.

Resolution w = peak width at the baseline between tangents drawn to the steepest parts of the peak w1/2 = 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 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.

Chromatography Chromatographic 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 of Elution Chromatography some zone broadening zone separation

Plate height: constant of proportionality between the variance (s2) of the band and the distance traveled (x) Smaller plate height = narrow peaks = better separations 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. L = column length

N is the number of theoretical plates a is the relative retention of two peaks k’2 is the capacity factor for the more retained component k’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 Height Analyses Based on Peak Areas Calibration and Standards The Internal Standard Method The Area Normalization Method

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