BAGIAN I YUNIKA MAYANGSARI, S.Si., M.Biotech PRINSIP DASAR HPLC BAGIAN I YUNIKA MAYANGSARI, S.Si., M.Biotech

Fasa diam dan fasa gerak (HPLC) Dalam HPLC, zat cair (liquid) digunakan sebagai fasa gerak Kolom berisi serbuk halus yang dipadatkan (sebagai fasa diam) The stationary phase is defined as the immobile packing material in the column. Dapat dibayangkan betapa sulitnya zat cair mengalir melalui fasa diam di dalam kolom Sehingga, agar zat cair dapat melewati kolom dengan cepat, dibutuhkan bantuan pompa bertekanan tinggi

Persamaan dengan GC  keluarannya berupa kromatogram Keuntungan dibanding pemakaian GC  kemampuan menganalisis sampel yg unvolatile dan labil pada suhu tinggi Akan tetapi analisis HPLC lebih mahal HPLC = High Performance Liquid Chromatography

Advantages to HPLC Higher resolution and speed of analysis HPLC column can be reused without repacking or regeneration Greater reproducibility due to close control of the parameters affecting the effeciency separation Easy automation of the instrument operation and data anaysis Adaptability to large-scale, preparative procedurs suwedo hadiwiyoto basic principles of HPLC - 1

Various instruments of HPLC system suwedo hadiwiyoto basic principles of HPLC - 1

Skema Instrumen HPLC

suwedo hadiwiyoto basic principles of HPLC - 1

Prinsip kerja HPLC Dengan bantuan pompa fase gerak dialirkan melalui kolom ke detektor. Sampel yang dilarutkan dalam solvent, dimasukkan ke dalam aliran fasa gerak dengan cara injeksi. Di dalam kolom terjadi pemisahan komponen2 campuran  perbedaan kekuatan interaksi anatara analat (solut-solut) dengan stationary phase pada kolom.

Prinsip kerja HPLC (lanj) Solut-solut yang kurang kuat interaksinya dengan fase diam akan keluar dari kolom terlebih dahulu. Sebaliknya, solut2 yang kuat berinteraksi dengan fasa diam maka solut2 tsb akan keuar dari kolom lebih lama. Setiap komponen campuran yang keluar dari kolom dideteksi oleh detektor kemudian direkam dalam bentuk kromatogram. Kromatogram HPLC serupa dengan kromatogram GC  jumlah peak menyatakan jumlah komponen; luas area peak menyatakan konsentrasi dalam campuran Sisitim HPLC dapat dihubungkan dengan software pada komputer dan dioperasikan secara computerize

suwedo hadiwiyoto basic principles of HPLC - 1

Instrumentasi HPLC Fase gerak Pompa Sample injector Kolom Detektor

Fasa Gerak (MP) HPLC (Syarat fasa gerak) MP harus bertindak sbg pelarut yang baik utk sampel yang dianalisis MP harus murni sekali utk menghindari adanya impurities yang akan mengganggu intepretasi pada kromatogram dan mengotori kolom MP harus jernih sekali utk menghindarkan kolom kotor dan tersumbat. Pelarut harus disaring dan didegas. Saringan digunakan nilon diameter 0.45 mikrometer.

MP mudah diperoleh, tidak mudah terbakar, dan tidak beracun MP tidak viskous. Kekentalan tidak melebihi 0,5 cP (centiPoise) MP sesuai dengan detektornya. Untuk detektor RI (refractive index) pelarut harus mempunyai indeks bias yang berbeda dengan solut; utk det. UV, pelarut tdk boleh mnyerap cahaya pada gelombang cahaya yg dipakai.

Jenis fasa gerak Berdasarkan kepolaran fasa diam dan fasa gerak Fase Normal (Normal Phase) Kombinasi antara fase diam polar dan fase gerak non-polar (misal: fase diam: silika atau alumina, fase gerak: heksana atau i- propileter) Fase Terbalik (Reversed Phase) Fase diam non-polar dan fase geraknya polar (air, metanol, asetonitril)  kepolaran fase gerak lebih tinggi dibanding fase diamnya

Pump Flow rate range: 0.01 to 5 mL/min Flow rate stability: not more than 1% For flow rate stability should be less than 0.2% It is desirable to have an integrated degassing system, either helium purging, or membrane filtering.

Pump The role of the pump is to force a liquid (called mobile phase) through the liquid chromatograph at a specific flow rate, expressed in ml/min Normal flow rates in HPLC are in the 1-2 ml/min range Typical pumps can reach pressures in the range until 600 psi During the cromatographic experiment, a pump can deliver a constant mobile phase composition (isocratic) or an increasing mobile phase composition (gradient). basic principles of HPLC - 1

Sample Injector Injeksi syringe Syringe diinjeksikan melalui septum (seal karet), injeksi dilakukan dengan konstan dan bebas gelembung udara Injeksi ‘stop-flow’ Saat injeksi pelarut dihentikan sementara. Sampel disuntikkan langsung pada ujung kolom. Kran sampel Disebut juga “loop” dan paling banyak digunakan. - Sejumlah volume sample (dalam solvent) disuntikkan ke dalam loop dalam posisi “load”, sampel masih berada di dalam loop - Kran diputar (ke bawah) utk mengubah ke posisi “injeksi” dan fasa gerak membawa sample ke dalam kolom

Column .... within the column is where separation occurs Considered the ‘heart of the chromatograph” the column’s stationary phase separates the sample components of the interest using various physical and chemical parameters. The small particles inside the column are what cause the high backpressure at normal flow rates. The pump must push hard to move the mobile phase through the column and this resistance causes a high pressure within the chromatograph. basic principles of HPLC - 1

HPLC columns .... proper choice of column is critical for success in HPLC Types of columns in HPLC : Analytical : ID 1.0-4.6 mm ; length 15-250 mm Preparative : ID > 4.6 mm; length 50-250 mm Capillary : ID 0.1-1.0 mm; various lengths Nano : ID < 0.1 mm, or something stated as < 100 µm Materials of construction for the tubing Stainless steel (most populer) Glass (mostly for biomolecules) PEEK polymes (biocompatible and chemically inert to most solvents) basic principles of HPLC - 1

Precolumn Precolumn (before injector) and Guard column (after injector) - Protects the analytical column: Remove impurities from solvent Minimize the interferences Prolongs the life of the analytical column Saturates mobile phase with liquid of stationary phase before the anlytical column basic principles of HPLC - 1

HPLC columns packing materials Today, most packing fall into four classes : silica or alumina, bound phases on either alumina or silica, gels, controlled- pore glass or silica Columns are packed with small diameter porous particles. The most popular size are 5 µm, 3.5 µm and 1.8 µm Columns are packed using high pressure to ensure that they are stable during use. These porous particles in the column usually have a chemically bonded phase on their surface which interacts with the sample components to separate them from one another (for example, C18 is a popular bonded phase) The process of retention of the sample components (often called analytes) is determined by the choice column packing and the selection of the mobile phase to phase the anlytes through the packed column suwedo hadiwiyoto basic principles of HPLC - 1

Columns : bonded phases Solid Support - Backbone for bonded phases. Usually 10 µm, 5 µm or 3 µm silica or polymeric particles. Bonded Phases - Functional groups firmly linked (chemically bound) to the solid support. Extremely stable Reproducible Bonded Phases C-2 Ethyl Silyl -Si-CH2-CH3 C-8 Octyl Silyl -Si-(CH2)7-CH3 C-18 Octadecyl Silyl -Si-(CH2)17-CH3 CN Cyanopropyl Silyl -Si-(CH2)3-CN suwedo hadiwiyoto basic principles of HPLC - 1

Detektor (syarat) Cukup sensitif Stabilitas dan reproducibility tinggi Respon linear terhadap solut Waktu respon pendek sehingga tidak bergantung flow rate Mudah digunakan Tidak merusak sampel

Jenis – jenis Detektor HPLC Detektor Fluorescence Detektor UV Refractive Index Detector

Fluorescene detection excitation Mobile phase emission Compared to UV-VIS detectors fluorescence detectors offer a higher sensitivity and selectivity that allows to quantify and identify compounds and impurities in complex matrices to extremely low concentration levels (trace level analysis) Fluorescence detectors sense only those substances that fluoresce suwedo hadiwiyoto basic principles of HPLC - 1

Ultraviolet (UV) absorption An ultraviolet light beam is directed through a flow cell and a sensor measures the light passing through the cell If a compound elutes from the column that absorbs this light energy, it will change the amount of light energy falling on the sensor The resulting change in this electrical signals is amplified and directed to recorder or data system A UV spectrum is sometimes also obtained wich may aid in the identification of a compound or series of compounds suwedo hadiwiyoto basic principles of HPLC - 1

UV / Visible detector Advantages Disadvantages Hingsensitivity & small sample volume required Can be used with gradient elution Is relatively cheap and not sensitive to temperature Sensitive to large number of organic compounds Does not work with compounds that do not absorb light at this wavelength region Cannot be used with the solvents having large absorption in the UV region Cannot be used for the sample components which cannot be absorbed in the UV region UV / Visible detector suwedo hadiwiyoto basic principles of HPLC - 1

basic principles of HPLC - 1

Refractive index (RI) detection The ability of a compound or solvent to deflect light provides a way to detect it The RI is a measure of molecule’s ability to deflect light in a flowing mobile phase in a flow cell relative to a static mobile phase contained in a reference flow cell The amount of deflection is proportional to concentration The RI detector is considered to be a universal detector but it is not very sensitive basic principles of HPLC - 1

Latihan Jelaskan mengapa fasa gerak cair harus disaring dengan saringan sangat halus sebelum dilalirkan? Mengapa HPLC menggunakan pompa bertekanan tinggi, sedangkan GC tidak? Jelaskan fungsi “guard column”? PR : - Sebutkan contoh2 aplikasi penggunaan detector HPLC (IR, FRLD, UV/UV Vis/PDA)