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H P L C

H P L C. Ada bbrp ekspresi HPLC. H igh P ressure L iquid C hromatography H igh P erformance L iquid C hromatography H igh P rice L iquid C hromatography H igh P restige L iquid C hromatography H igh P eaks L iquid C hromatography

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H P L C

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  1. H P L C

  2. Ada bbrp ekspresi HPLC • High Pressure Liquid Chromatography • High Performance Liquid Chromatography • High Price Liquid Chromatography • High Prestige Liquid Chromatography • High Peaks Liquid Chromatography • High Profit Liquid Chromatography • High Propaganda Liquid Chromatography • High Promise Liquid Chromatography • High Philosophy Liquid Chromatography

  3. Lanjutan • High Problem Liquid Chromatography • High Phantasy Liquid Chromatography • High Polite Liquid Chromatography • High Pragmatic Liquid Chromatography • High Pleasure Liquid Chromatography .…… ada …... lagi……ada……lagi..... ………………?????…………………

  4. Review Bagaimana • memilih kolom • memilih fase gerak • menentukan kepolaran senyawa, pelarut/eluen • mekanisme pemisahan • memilih detektor

  5. Tujuan akhir HPLCadalahPemisahan

  6. Memilih Kolom HPLC

  7. Stationary Phase

  8. Polar Functional Groups

  9. Koef. Partisi o/w

  10. K.Partisi zatvs K.partisi hplc Log Kow = Log P = koef. partisi oktanol-air Log P Kekuatan retensi (pada kolom RP-HPLC)

  11. Menentukan Kepolaran / PolaritasSolvent atau Eluen

  12. Memilih fase gerak • Tunggal • Campuran • Berapakah kepolaran fase gerak campuran ?

  13. Kepolaran • Ada beberapa parameter kepolaran, a.l : - Momen dipol - Indeks polaritas - Eluotropic series - Konstante dielektirk - Elektronegativitas - Sifat asam/basa

  14. Momen dipol ( McMurry, 2004 )

  15. Kepolaran pelarut

  16. Eluotropics Series for Solvents

  17. Snyder dkk., 1997

  18. Elektronegativitas(EN) ( McMurry, 2004 )

  19. Susunan Berkala Unsur-unsur Kimia

  20. Pemisahan Bagaimana mekanisme pemisahan pada High Performance Liquid Chromatography

  21. Mekanisme Pemisahan

  22. Normal-Phase HPLC Fase diam > polard/pfasegerak • Senyawa polar terelusi belakangan (tR panjang) • Semakin non-polar fase gerak, tR seny. polar makin panjang • Solvents : Campuran metilen klorid, dietil eter, kloroform • Eluent strength dimodifikasi dengan n-heksan

  23. Reversed-Phase HPLC Fase diam > non-polar d/pfase gerak • Senyawa polar terelusi lebih dahulu • Semakin polar fase gerak, senyawa non- polar lebih kuat tertahan • Solvent : Campuran metanol, asetonitril, tetrahidrofuran • Eluent strength dimodifikasi dengan air (Kellner dkk., 1998)

  24. Detektor Bagaimana Memilih detektor dan Apa pertimbangannya ???

  25. Detektor • Bulk property detector : - mengukur sifat solute dan fase gerak contoh : detektor indeks bias ( RI ) • Solute property detector : - hanya mengukur sifat solute - 1000 kali ≥ sensitif dibanding BPD Contoh : detektor UV (Settle, 1997; Kellner dkk., 1998; Adamovics, 1997)

  26. Detektor Detektor lain untuk HPLC : Fluorescen Elektrokimia Konduktivitas Mass spectrometer (Settle, 1997; Kellner dkk., 1998; Adamovics, 1997)

  27. Detektor UVvs Fluorescen UV Fluorescen

  28. Luminescence Bagaimana terjadinya fluorescensi ??? Fluorescensi

  29. Noise and Drift

  30. S/N Ratio

  31. Validasi Metode

  32. Validasi …

  33. Derivatisasi Apa tujuannya…? Bagaimana caranya…? Post- / Pre-columns…!

  34. Derivatisasi dg Dansilklorid Dansil klorid Fluorescent derivative

  35. Catecholamine Catecholamine

  36. DPE-Catecholamine

  37. Captopril Captopril, BM = 217,3

  38. PK. Captopril (HPLC) • Parameter HPLC : (J.Pharm.Biomed.Anal., 1995, 13: 655-660) - Column : 250 x 4.6 mm, Kontron Analytical S5 ODS2 - Mobile phase : MeCN: 1% Acetic acid (60:4) - Flow rate : 1.3 mL/min. - Detector : UV 260 nm - Retention time : 4.0 min - Limit of detection : 15 ng/mL - Limit of quantitation : 30 ng/mL

  39. Derivatisasi dg p-Br fenacylbromide Captopril p -Br fenacylbromide

  40. Hasil derivatisasi : Captopril + p -Br Fenacylbromide Dapat dideteksi secara UV

  41. Peak Tailing Diatasi dengan : • Tambahkan 30 mM: - Trietilamin (utk seny. Basa) - Amonium asetat (utk seny. Asam) • Bila Tailing masih tetap ada, gantilah Trietil- amin dengan : - 10 mM dimetiloktilamin atau dimetiloktilamin asetat • Kurangi jumlah sampel hingga < 1 g

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