2 Kromatografi Kolom Sederhana Bergerak / aliran karena gaya grafitasi↓Pemilihan fase diam + fase gerakKepolaranPita-pita kromatogramTerbentuk fraksi-fraksiDianalisis dengan KLT / KK↓
3 Pengisian Kolom Tehnis : fase diam + pelarut → bubur (f. gerak) pengisian kolom homogenfase diam ukuran samafase diam bentuk homogenbebas gelembung udaraFase diamPasirKapas / glass woolTehnis : fase diam + pelarut → bubur(f. gerak)
4 Klasifikasi Sistem Kromatografi UmumTehnik SpesifikFase diamKeseimbangan1. K. Cair(LC)LLCLSCIECCair pd padatanPadatanResinPartisiAbsorbsiTukar ion2. K. Gas(GC)GLCGSCGas terikatP / A
5 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.
14 Aliran fase gerak pada kolom GravitasiPressure/tekananVacumpompa
15 How does reverse phase chromatography to normal phase chromatography ? compareto normal phase chromatography ?
16 Normal Phase Column Chromatography … The stationary phase is POLARThe more polar component interacts more strongly with the stationary phaseThe more polar component moves more slowly.The non-polar component moves more rapidly.
17 Reverse Phase Chromatography… Silica is alkylated with long chain hydrocarbon groups, using 18 carbons long. This is usually referred to as C-18 silica.
18 Reverse Phase Column Chromatography…. The stationary phase (column packing) is now NON-POLARNon-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 chromatographyUsed mainly in columns, such as HPLC
22 LIQUID SOLID CHROMATOGRAPHY Normal phase LSReverse phase LSd- d+Si - O - H30 mSilica GelThe 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.
26 LIQUID-LIQUID CHROMATOGRAPHY ODPN (oxydipropionylnitrile)Normal Phase LLCReverse Phase LLCNCCH3CH2OCHCN(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.
27 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).
29 Types of Ion Exchange Resins Type of ExchangerFunctional Exchanger GroupTrade NameCationStrong AcidSulfonic acid (-SO3-H+)Dowex 50; Amberlite IR 120Weak acidCarboxyclic acid (-CO2-H+)Amberlite IRC 50AnionStrong baseQuaternary ammonium ion (-NR3+OH-)Dowex 1; Amberlite IRA 400Weak baseAmine group (-NH3+OH-)Dowex 3; Amberlite IR 45
30 Chromatography Conditions associated with each kind of chromatography Ion exchange chromatographyOrganic cation exchange resins involve crosslinked polystyrene containing either SO3- or COO- functional groups with an associated cationOrganic anion exchange resin involvecrosslinked polystyrene containing NH3+functional groups with an associated anionThe affinity of dissolved ions for the resin varies with the ion and the composition of the solution
34 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 waterConcentrationsThe concentration of trace elements in seawater.Analytical SeparationsSeparations of metal ions and amino acid or halide ions
35 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.
36 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.
40 PARAMETER PEMISAHAN DALAM KROMATOGRAFI KOLOM 1. KAPASITASKapasitas menggambarkan kemampuan fase diam dalam menahan analit. Jika waktu tambat lama, berarti kapasitasnya besar.2. SELEKTIVITASSelektivitas menggambarkan kemampuan fase diam untuk dapat memisahkan suatu campuran senyawa. Semakin besar nilai , campuran senyawa semakin terpisah.3. RESOLUSIResolusi menggambarkan kemampuan kolom dalam memisahkan campuran senyawa4. JUMLAH PLATE TEORITISJml pelat teoritis N, dalam kolom dapat diketahui dari hasil kromatogram.5. TINGGI PLATE TEORITIS = HHETP = High Equivalent to A Teoritical PlateAdl : ukuran yang menunjukkan ruang yg ditempati oleh setiap pelat teoritis.panjang kolom
42 Adjusted Retention Time tr = retention timetm = min. time for unretained mobile phase to travel through columnIn GC tm is the time CH4 takes to travel through the columnRelative RetentionCapacity Factor
44 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 – t1kolom diperpanjangjumlah fase diam diperbesarmanipulasi faktor pemisahanpengoptimalan suhupilih f.d dan f.g yang cocok2. 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.
45 Resolutionw = peak width at the baseline between tangents drawn to the steepest parts of the peakw1/2 = measured at ½ the peak height
46 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.
47 Chromatography Chromatographic column theory of packed columns The effect of column efficiency and column selectivity on resolutionPoor resolution because of poor column efficiencyGood resolution because of good column efficiency, although column selectivity is not greatGood resolution because of good column selectivity, although column efficiency is poorPoor resolution because of poor column selectivity, although column efficiency is good
48 Theories of Elution Chromatography some zone broadeningzone separation
50 Plate height: constant of proportionality between the variance (s2) of the band and the distance traveled (x)Smaller plate height = narrow peaks = better separationsPlate height (H)Number of plates (N)
51 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
52 N is the number of theoretical plates a 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
53 Remember that variance is additive but standard deviation is not
54 Applications of Chromatography Qualitative AnalysisQuantitative AnalysisAnalyses Based on Peak HeightAnalyses Based on Peak AreasCalibration and StandardsThe Internal Standard MethodThe Area Normalization Method
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