Thin Layer Chromatography.ppt
Transcript of Thin Layer Chromatography.ppt
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Thin Layer Chromatography 1
Thin Layer Chromatography
Henry K.S.,M.Si.,Apt.
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Thin Layer Chromatography 2
Thin Layer Chromatography
TLC ?????
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Thin Layer Chromatography 3
Metode TLC merupakan metode yang banyak digunakan untuk pemisahan dan identifikasi obat/zat
Penggunaan TLC:
Senyawa obat murni (uji kemurnian)
Hasil ekstraksi produk obat jadi
Senyawa2 yang tidak diperkenankan dalam sediaan (mis: bahan kimia dalam jamu)
Sampel biologis
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Thin Layer Chromatography 4
Dibandingkan dengan HPLC dan GC, TLC memiliki keunggulan:SederhanaMurahDapat langsung digunakan tanpa menunggu waktu flushing/pemanasan (warm-up time)Preparasi sampel sederhana (tdk perlu clean up)Dapat digunakan untuk analisa beberapa sampel sekaligusFleksibilitas dalam pemilihan fase diam dan fase gerak Analit yang dipisahkan tidak hilang
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Thin Layer Chromatography 5
Kekurangan TLC dibanding HPLC / GC:
Sensitivitas lebih rendah
Resolusi / pemisahan lebih rendah
HPTLC
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Thin Layer Chromatography 6
Thin Layer Chromato-graphy / Kromatografi Lapis Tipis merupakan metode kromatografi / pemisahan di mana fase gerakfase gerak bergerak berdasarkan daya kapilaritas melalui fase fase diamdiam (adsorben) yang dilekatkan pada suatu lapisan tipis.
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Thin Layer Chromatography 7
Mekanisme pemisahan
Pemisahan dipengaruhi:
Kelarutan analit
Harga pKa
Daya adsorpsi
Ikatan hidrogen
Polaritas
Molecular weight
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Thin Layer Chromatography 8
Fase Diam
Plate TLC dapat terbuat dari:- Kaca- Aluminium- Plastik
Fase diam diikatkan pada plate TLC dengan menggunakan “binding agent” (mis: kalsium sulfat)
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Thin Layer Chromatography 9
Fase diam:
Silica gel (silica gel G/GF254)
Alumina (aluminium oksida)
Kieselguhr
Magnesium silicate (florisil)
Pemisahan terjadi karena interaksi analit dengan permukaan fase diam
}
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Fase diam lain:Cellulose mekanisme pemisah-an: partisi Reversed Phase plate silica yang ditambah dengan minyak atau parafin cairNormal PhaseResin Penukar IonImpregnated plate
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Fase gerak
Fase gerak dapat berupa pelarut tunggal atau campuran dan bergantung pada zat yang akan dipisahkan dan fase diam yang digunakan.
Dapat pula ditambahkan larutan dapar untuk pemisahan senyawa berdasarkan pertukaran ion.
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Syarat fase gerak
MurahMudah didapatStabil di udara luar, dalam campuran asam atau basaMudah dihilangkan dari plate setelah selesai proses eluasiTidak bereaksi dengan analit yang akan dipisahkan
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Thin Layer Chromatography 13
Teknik pelaksanaan
Penyiapan plate
Penotolan sampel
Proses eluasi
Proses deteksi
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Thin Layer Chromatography 14
Penyiapan plate
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Thin Layer Chromatography 15
Penotolan sampel
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Thin Layer Chromatography 16
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Thin Layer Chromatography 17
Nanomat
Linomat
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Thin Layer Chromatography 18
Automatic TLC Sampler
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Thin Layer Chromatography 19
Proses eluasi
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Thin Layer Chromatography 20
Pour solvent into the beaker to a depth of just less than 0.5 cm.
To aid in the saturation of the TLC chamber with solvent vapors, line part of the inside of the beaker with filter paper
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Thin Layer Chromatography 21
Cover the beaker with a watch glass, swirl it gently, and allow it to stand while you prepare your TLC plate.
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Thin Layer Chromatography 22
5’ 10’ drying
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Thin Layer Chromatography 23
Deteksi
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Thin Layer Chromatography 24
Metode deteksi
Destruktif (disemprot penampak noda)
(misal: asam sulfat, iodine, dragendorf, dll)
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Thin Layer Chromatography 25
Non destruktif (dengan sinar tampak untuk zat berwarna dan sinar UV untuk zat tidak berwarna)
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Thin Layer Chromatography 26
TLC Scanner (Densitometri)
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Performance TLC
Mi g
rati
on d
ista
nce
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Retardation Factor
Rf =Jarak migrasi sampel
Jarak migrasi pelarut pengembang
hRf = 100 Rf
Bgm kalau noda tidak bulat ?
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Thin Layer Chromatography 29
Faktor yang mempengaruhi reprodusibilitas Rf
Fase diam
Fase gerak
Kejenuhan chamber
Suhu
Jarak eluasi
Jumlah sampel
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Flow ConstantThe flow constant or velocity constant (K) is a measure
of the migration rate of the solvent front. It is an important parameter for TLC users and can be used to calculate,
The flow constant is given by the following equation:
K =ZF2/twhere
K= flow constant [mm2/s]
ZF = distance between the solvent front and the solvent level [mm]
t = development time [s]
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Thin Layer Chromatography 31
Start line
Solvent level
ZF
Zo
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The following example illustrates the usefulness of the flow constant in laboratory work. In a TLC, if the development time for a migration distance of 10 cm was 30 min and the Z0 distance is 5 mm, the K value is 6.125 mm2/s.
Question: How much time is required to develop a 15-cm migration distance if the sorbent,solvent system, Z0 and laboratory temperature remain constant?
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Thin Layer Chromatography 33
Faktor retensi
k’ =1 – dR/dM
dR/dM
k’ =1 - Rf
Rf
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Thin Layer Chromatography 34
Separation Number (SN)
Separation Number or Spot capacity, indicates the maximum number of spots that can be separated with Rs=1 over a given separation distance, between Rf = 0 and Rf = 1.
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Thin Layer Chromatography 35
Z = The migration distance from the origin to the solvent frontb0 = width of spot with Rf = 0b1 = width of spot with Rf = 1Widths are measured at half height from a densitogram of a mixture of substances having different Rf values spread over the entire chromatogram
110
bb
ZSN
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Thin Layer Chromatography 36
Aplikasi TLC
Preparatif
Kualitatif
Kuantitatif
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Thin Layer Chromatography 37
TLC preparatif
Digunakan untuk isolasi komponen dari campuran agar dapat diperoleh senyawa murni.
Plate yang digunakan lebih tebal dibandingkan plate untuk analisis (Tebal: 0,5 – 2 mm; plate untuk analisis: 100 – 250 µm)
Jumlah sampel: 10 – 100 mg
Sampel ditotolkan dalam bentuk band
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Thin Layer Chromatography 38
Kalau ditotol bentuk spot bagaimana?
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Thin Layer Chromatography 39
Deteksi : harus dengan metode non destruktif
Hasil pemisahan di ambil dan diekstraksi untuk memisahkan analit dengan fase diam
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Thin Layer Chromatography 40
High Performance Thin layer Chromatography (HPTLC)
Lebih cepat
Lebih reprodusibel
Lebih sensitif
Lebih akurat
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Thin Layer Chromatography 41
Typical condition TLC HPTLC
Plate dimension (cm)
Particle size (µm)
Layer thickness (µm)
Sample volume (µl)
Spot (d) before running (mm)
Spot (d) after running (mm)
Running distance (cm)
Running time (min)
Number of sample/plate
Detection limit (absorption)(ng)
Detection limit (fluorescence)(ng)
Reproducibility of Rf values
Reproducibility of quantification
20x20
20
100-250
1-10
< 4
5-10
10-15
30-120
10-15
10-100
0.1-1
3%
5%
10x10
5
150-200
< 0.1
< 1.5
2-5
3-6
5-15
30-40
0.5-5
0.01-0.1
1%
2-3%
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Thin Layer Chromatography 42
TLC 2 dimensi
Sol
vent
A
Solvent B
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Thin Layer Chromatography 43
Uji Kualitatif
Berdasarkan harga RF
Butuh standar
Dikombinasi dengan spektrum UV, IR, MS atau NMR
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Thin Layer Chromatography 44
Uji Kuantitatif
Hasil pemisahan diambil dan diekstraksi serta dianalisa dengan metode tertentu.
Dilakukan scanning dengan densito-meter (TLC Scanner)
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Thin Layer Chromatography 45
TLC Scanner (Densitometer)
Butuh standar zat yang dianalisa
Untuk analisa kuantitatif dapat digunakan tinggi puncak atau luas area puncak
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Latihan soal
1. Pada penetapan kadar campuran parasetamol dan ibuprofen dalam sampel, mula-mula ditimbang 100 mg sampel dan diekstraksi dengan 4 ml methanol sebanyak 2 kali. Hasil ekstraksi dimasukkan labu takar 10 ml dan ditambahkan methanol sampai garis tanda. Hasil ekstraksi ditotolkan sebanyak 2µl pada plate KLT dan dieluasi dengan fase gerak tertentu.
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Thin Layer Chromatography 47
Hasil pemisahan diamati dengan densito-meter dan diperoleh data sbb: Luas area parasetamol = 2150, luas area ibuprofen = 725. Sebagai larutan baku dibuat campuran parasetamol dan ibuprofen dg konsentrasi tertentu dan ditotol 2 µl serta dieluasi dengan fase gerak yang sama dengan sampel, diperoleh hasil sebagai berikut:
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Thin Layer Chromatography 48
Kons parac
(µg/ml)
Area parac Kons ibup
(µg/ml)
Area ibup
300 525 100 340
600 1045 200 695
900 1580 300 1025
1200 2140 400 1330
1500 2675 500 1690
Hitung kadar parasetamol dan ibuprofen dalam sampel !
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Thin Layer Chromatography 49
2. Pada penetapan kadar zat X dalam sirup, mula-mula dipipet 5 ml sampel dan diekstraksi dengan 5 ml etil asetat, hasil ekstraksi dimasukkan labu takar dan di tambah etil asetat hingga tepat 5 ml. Dipipet 1 ml dan diencerkan hingga tepat 5 ml dengan etil asetat. Kemudian ditotolkan sebanyak 4 µl pada plate KLT dan dieluasi dengan fase gerak tertentu.
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Hasil eluasi diamati dengan densitome-ter dan didapat luas area noda = 1045. Untuk larutan baku dibuat larutan zat x dalam etil asetat dengan konsentrasi 250 µg/ml dan ditotolkan pada plate KLT dengan volume tertentu, kemudian dieluasi dengan fase gerak tertentu dan diamati luas area nodanya dengan densitometer, diperoleh hasil sbb:
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Volume totolan baku
(µl)
Luas Area
2 420
4 850
6 1275
8 1690
10 2125
Hitunglah kadar zat X dalam sampel !