PRINSIP DASAR HPLC

PRINSIP DASAR HPLCBAGIAN IYUNIKA MAYANGSARI, S.Si., M.BiotechFasa diam dan fasa gerak (HPLC)Dalam HPLC, zat cair (liquid) digunakan sebagai fasa gerakKolom 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 kolomSehingga, agar zat cair dapat melewati kolom dengan cepat, dibutuhkan bantuan pompa bertekanan tinggiPersamaan dengan GC keluarannya berupa kromatogramKeuntungan dibanding pemakaian GC kemampuan menganalisis sampel yg unvolatile dan labil pada suhu tinggiAkan tetapi analisis HPLC lebih mahalHPLC = High Performance Liquid Chromatography

Advantages to HPLCHigher resolution and speed of analysisHPLC column can be reused without repacking or regenerationGreater reproducibility due to close control of the parameters affecting the effeciency separationEasy automation of the instrument operation and data anaysisAdaptability to large-scale, preparative procedursbasic principles of HPLC - 1suwedo hadiwiyoto4

Various instruments of HPLC systembasic principles of HPLC - 1suwedo hadiwiyoto5Skema Instrumen HPLC

basic principles of HPLC - 1suwedo hadiwiyoto7Prinsip kerja HPLCDengan 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 campuranSisitim HPLC dapat dihubungkan dengan software pada komputer dan dioperasikan secara computerize

basic principles of HPLC - 1suwedo hadiwiyoto10Instrumentasi HPLCFase gerakPompaSample injectorKolomDetektor

Fasa Gerak (MP) HPLC (Syarat fasa gerak)MP harus bertindak sbg pelarut yang baik utk sampel yang dianalisisMP harus murni sekali utk menghindari adanya impurities yang akan mengganggu intepretasi pada kromatogram dan mengotori kolomMP 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 beracunMP 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 gerakBerdasarkan kepolaran fasa diam dan fasa gerakFase 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 diamnyaPumpFlow rate range: 0.01 to 5 mL/minFlow 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/minNormal flow rates in HPLC are in the 1-2 ml/min rangeTypical pumps can reach pressures in the range until 600 psiDuring the cromatographic experiment, a pump can deliver a constant mobile phase composition (isocratic) or an increasing mobile phase composition (gradient).basic principles of HPLC - 116Sample InjectorInjeksi syringeSyringe diinjeksikan melalui septum (seal karet), injeksi dilakukan dengan konstan dan bebas gelembung udaraInjeksi stop-flowSaat injeksi pelarut dihentikan sementara. Sampel disuntikkan langsung pada ujung kolom.Kran sampelDisebut 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 columns 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 - 118HPLC columns.... proper choice of column is critical for success in HPLCTypes of columns in HPLC :Analytical : ID 1.0-4.6 mm ; length 15-250 mmPreparative : ID > 4.6 mm; length 50-250 mmCapillary : ID 0.1-1.0 mm; various lengthsNano : ID < 0.1 mm, or something stated as < 100 m

Materials of construction for the tubingStainless steel (most populer)Glass (mostly for biomolecules)PEEK polymes (biocompatible and chemically inert to most solvents)

basic principles of HPLC - 119PrecolumnPrecolumn (before injector) and Guard column (after injector) - Protects the analytical column:Remove impurities from solventMinimize the interferencesProlongs the life of the analytical columnSaturates mobile phase with liquid of stationary phase before the anlytical column

basic principles of HPLC - 120HPLC columns packing materialsToday, most packing fall into four classes : silica or alumina, bound phases on either alumina or silica, gels, controlled-pore glass or silicaColumns are packed with small diameter porous particles. The most popular size are 5 m, 3.5 m and 1.8 mColumns 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 columnbasic principles of HPLC - 1suwedo hadiwiyoto21Columns : bonded phasesSolid Support - Backbone for bonded phases. Usually 10 m, 5 m or 3 m silica or polymeric particles. Bonded Phases - Functional groups firmly linked (chemically bound) to the solid support.Extremely stableReproducibleBonded PhasesC-2Ethyl Silyl-Si-CH2-CH3C-8Octyl Silyl-Si-(CH2)7-CH3C-18Octadecyl Silyl-Si-(CH2)17-CH3CNCyanopropyl Silyl-Si-(CH2)3-CN

basic principles of HPLC - 1suwedo hadiwiyoto22Detektor (syarat)Cukup sensitifStabilitas dan reproducibility tinggiRespon linear terhadap solutWaktu respon pendek sehingga tidak bergantung flow rateMudah digunakanTidak merusak sampelJenis jenis Detektor HPLCDetektor FluorescenceDetektor UVRefractive Index DetectorFluorescene detectionCompared 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 fluoresceexcitationMobile phaseemissionbasic principles of HPLC - 1suwedo hadiwiyoto25

Ultraviolet (UV) absorptionAn ultraviolet light beam is directed through a flow cell and a sensor measures the light passing through the cellIf a compound elutes from the column that absorbs this light energy, it will change the amount of light energy falling on the sensorThe resulting change in this electrical signals is amplified and directed to recorder or data systemA UV spectrum is sometimes also obtained wich may aid in the identification of a compound or series of compoundsbasic principles of HPLC - 1suwedo hadiwiyoto26

UV / Visible detectorAdvantages Hingsensitivity & small sample volume requiredCan be used with gradient elutionIs relatively cheap and not sensitive to temperatureSensitive to large number of organic compoundsDisadvantages Does not work with compounds that do not absorb light at this wavelength regionCannot be used with the solvents having large absorption in the UV regionCannot be used for the sample components which cannot be absorbed in the UV regionbasic principles of HPLC - 1suwedo hadiwiyoto27

basic principles of HPLC - 128Refractive index (RI) detectionThe ability of a compound or solvent to deflect light provides a way to detect itThe RI is a measure of molecules ability to deflect light in a flowing mobile phase in a flow cell relative to a static mobile phase contained in a reference flow cellThe amount of deflection is proportional to concentrationThe RI detector is considered to be a universal detector but it is not very sensitive

basic principles of HPLC - 129LatihanJelaskan 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)