ANALISIS HPLC
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Transcript of ANALISIS HPLC
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ANALISIS HPLC dan METODE DERIVATISASI
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INTERAKSI SOLUT-FASE GERAK-FASE DIAM
ADSORPSI PARTISI
PENUKAR ION SIZE EXCLUSION
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PASANGAN ION
INTERAKSI SOLUT-FASE GERAK-FASE DIAM
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SEC
GFC
LSCRPC
RPCBPC
RPCBPCLSC
IECIPC
LARUT DALAM PELARUT ORGANIK
LARUT DALAM AIR
NON POLAR
POLAR
NON IONIK
IONIK
LARUT DALAM PELARUT ORGANIK
LARUT DALAM AIR
BM>2000
BM<2000
SAMPEL
SKEMA PEMILIHAN SISTEM KROMATOGRAFI
Keterangan :SEC : Size Exclusion ChromatographyGFC : Gel Filtration ChromatographyLSC : Liquid Solid ChromatographyRPC : Reverse Phase ChromatographyBPC : Bonded Phas ChromatographyIEC : Ion Exchange ChromatographyIPC : Ion Pair Chromatography
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SKEMA HPLC
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Ada banyak faktor yang mempengaruhi efisiensi kolom sehingga harus dioptimasi agar diperoleh pemisahan yang baik, yaitu :•Kecepatan alir fase gerak, kecepatan alir yang sangat lambat akan menyebabkan terjadinya difusi longitudinal, sedangkan bila terlalu cepat akan menyebabkan terjadinya transfer masa non ekuilibrium, sehingga terjadi pelebaran pita kromatogram•Ukuran partikel fase diam, semakin kecil ukuran partikel maka efisiensi semakin baik Tetapi menyebabkan tekanan dalam kolom semakin besra sehingga dibutuhkan kekuatan pompa yang semakin besar.•Panjang kolom, semakin panjang akan semakin besar harga efisiensi kolom, tetapi dapat menyebabkan terjadinya pelebaran pita.•Viskositas fase gerak, semakin kecil harga viskositas fase gerak maka efisiensi kolom semakin besar.•Temperatur, semakin tinggi temperatur maka viskositas semakin rendah dan efisiensi kolom menjadi lebih besar.•Volume ekstra kolom, semakin besar volume ekstra kolom maka kemungkinan terjadinya pelebaran pita semakin besar, sehingga efisiensi semakin berkurang.•Jumlah sampel dan volume sampel, bila jumlah maupun volume sampel sangat besar (overload) maka kemungkinan terjadinya pelebaran pita semakin besar, sehingga efisiensi semakin berkurang.
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Agar diperoleh hasil pemisahan yang baik, maka perlu dilakukan evaluasi kolom secara berkala. Evaluasi terhadap kelayakan kolom dapat dipantau dengan mengevaluasi harga beberapa parameter di bawah ini secara berkala :
•Faktor kapasitas berkisar 2 – 10• Jumlah lempeng teoritik tidak mengalami perubahan yang signifikan
•Faktor resolusi (RS) harus > 1.5•Peak asimetri < 1.2•Tekanan kolom dalam kisaran normal (dapat dikerjakan oleh pompa dengan ringan)
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VARIABEL PEPTIDA PROTEIN
Kolom
Bonded phase
Dimensi
Partikel
C18 atau C8
0.46 X 15 atau 25 cm
3.5-10 m (diameter)
80-300 A ()
C4, C3, CN
0.46 x 5-15 cm
3.5-10 m
(diameter)
300 A ()
Fase gerak
Solven A
Solven B
Gradien
0.12% TFA/air
0.10%TFA/air
0-60% B/60 menit
0.12% TFA/air
0.10%TFA/air
0-60% B/60 menit
Temperatur 40 – 80 C 40 – 80 C
Kecepatan alir 0.5 – 2 ml/menit 0.5 – 2 ml/menit
Ukuran sampel
Volume
Berat
10-50 L
1-100 g
10-50 L
1-100 g
Contoh Aplikasi Pemisahan Peptida Dan Protein Dengan Rp-HPLC
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Problem yang mungkin muncul :•Bentuk pita yang jelek : melebar, tailing•Recovery rendah•Timbul pita yang misterius•Pita ganda untuk satu jenis analit•Performance kolom berubah, tr tidak reprodusibel
Faktor penyebab :•Kolom rusak, terlalu asam, terlalu hidrofob, terlalu kecil ukuran pori-porinya•Denaturasi sampel•Isomerisasi (cis ke trans)
Pengatasannya :•Pemisahan pada pH rendah (fase gerak 0.1 % Tetra fluoro Acid)•Gunakan asetonitril sebagai solven organik. Untuk sampel yang hidrofob gunakan propanol•Analisis dikerjakan pada temperatur kolom 50 - 80c•Gunakan zwitterionic detergent
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VARIABEL PROTEIN ASAM
(ANION EXCHANGE)
PROTEIN BASA
(KATION EXCHANGE)
Kolom
Bonded phase
Ukuran
DEAE, SAX,
5-25 x 0.46cm
CM, SP
5-25 x 0.46 cm
Fase gerak
Solven A
Solven B
Gradien
10mm tris atau phosphat (pH 8)
Solvent A + 0.5m NaCl atau Na-
asetat
0-100% B dalam 30 menit
10mm bis-tris atau phosphat (pH
6)
Solvent A + 0.5m NaCl atau Na-
asetat
0-100% B dalam 30 menit
Temperatur 35 c 35 c
Kecepatan alir 1.0ml/menit 1.0ml/menit
Ukuran sampel
Volume
Berat
10-50 l
1-100 g
10-50 l
1-100 g
Contoh aplikasi pemisahan peptida dan protein dengan ion exchange HPLC
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Keuntungan :• Konformasi protein tetap terjaga, • Kemungkinan denaturasi kecil, • Dapat digunakan untuk isolasi dan purifikasi protein dengan tetap berbentuk bioaktif
• Protein basa biasa memakai kation exchange, dengan ph 3
Problem yang sering muncul :• Recovery rendah dan dapat diatasi dengan menggunakan gradien elusi dengan fase gerak mengandung garam.
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Detector, hendaknya memiliki kriteria : sensitivitasnya tinggi, batas deteksi rendah, linearitas respon tinggi dan reprodusibilitasnya tinggi. Ada banyak detektor yang dapat diaplikasikan, yaitu :• Detektor spektrofotometer UV/Vis, merupakan detektor universal dan dapat diaplikasikan pada semua analit yang dapat menyerap sinar uv/vis.
• Detektor spektrofluorometer, merupakan detektor yang lebih selektif dan lebih sensitif.
• Detektor indeks bias, tergantung pada perubahan harga indeks bias fase gerak oleh karena adanya analit atau solut.
• Detektor elektrokimia, ada banyak jenisnya antara lain : detektor konstanta dielektrika, detektor konduktometer, detektor amperometer.
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DERIVATISASI
ANALIT SENYAWA YANG SENSITIF DITANGKAP DETEKTOR
KOMPLEX
PEMISAHAN LEBIH BAGUSLEBIH SENSITIF
FLUORESCENT
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DERIVATIZATION……
• Derivatization, prior to chromatographic analysis, can be used for a number of reasons in sample preparation, such as.– TO IMPROVE PERFORMANCE OF COMPOUNDS
and ENHANCED DETECTABILITY– TO IMPROVE SENSITIVITY– TO IMPROVE SEPARATION
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DERIVATIZATION…….• Liquid Chromatography:
– fluorescent derivatives can be prepared to render the substances specifically detectable at high sensitivity. dansyl chloride (5-dimethyl aminonaphthalene-1-sulphonyl chloride) is strongly recommended for phenols, and primary and secondary amines. 4-chloro-7-nitrobenz-2,1,3-oxadiazole (NBD chloride) which provides highly fluorescent derivatives of primary and secondary amines but aromatic amines, phenols and thiols only yield weakly or non fluorescent derivatives.
– UV chromaphores are often introduced into sample molecules to increase their sensitivity to UV absorption, for examples: benzoyl chloride, m-toluol chloride and p-nitrobenzoyl chloride are reagents that can add a benzene ring to a solute molecule and render it UV absorbing; 3,5-dinitrophenylhydrazine and p-nitrobenzylhydroxylamine are suitable for a solute containing carbonyl group
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• THERE ARE 3 TYPES OF DERIVATIZATION TECHNIQUE IN HPLC OR CE:– ON-COLUMN
after the HPLC column is equlibrated with mobile phase in the presence of the derivative reagent, the analyte is introduced and then eluated
– PRE COLUMNoften needs complicated procedures and can not handle unstable reaction compounds
– POST COLUMN
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DERIVATIZATION IN HPLC
DERIVATIZATION
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(Carbohydrates analysis by HPLC)….
SugarsPolyhydric alcohols
HPLCREFRACTIVE INDEXION-EXCHANGE
PROBLEM. INSUFFICIENT SENSITIVITY AND SPECIFITY
DERIVATIZATION
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• POST COLUMN:– POST COLUMN WITH P-AMINOBENZOIC ACID HYDRAZIDE
AND DETECTED USING SPECTRO VIS 410 NM– GLUCOSE USING IMMOBILZED (GOD), HYDROGEN
PEROXIDE IS DETECTED WITH ECD• PRE COLUMN– WITH P-NITROBENZOYL CHLORIDE (PNB-Cl), FOLLOWED BY
NORMAL HPLC AND SPECTRO UV 260 NM– WITH PHENYL ISOCYANANTE (PHI), FOLLOWED BY RP HPLC
USING SPECTRO UV 240 NM– WITH ISATOIC ANHYDRIDE TO FORM FLUORESCENT
ANTHRANILOYL, Λex 360 nm λem 420 nm, NORMAL AND RP HPLC
(Carbohydrates analysis by HPLC)….
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• TO IMPROVE UV DETECTION.– BORON IN THE ALKALINE MOBILE PHASE– ADDITION OF SORBIC ACID– DERIVATIZATION WITH 1-PHENYL-3-METHYL-5-
PYRAZOLONE,ETHYL-P-AMINOBENZOATE, 2-AMINOPYRIDINE, FOLLOWED BY UV 240-350 NM
• TO IMPROVE FLUORESCENT DETECTION– 5-AMINONAPHTHALENE-2-SULFONAT (Λex 325 nm; λem
475nm)– 9-AMINOPYRENE-1,4,6-TRISULFONATE λex 475 nm, λem
512nm)
(Carbohydrates analysis by CE)….
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DERIVATIZATION OF AMINO ACID
•WITH HEPTAFLUOROBUTYRIC ANHYDRIDE
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DERIVATIZATION AMINO ACID….. •WITH 6-aminoquinolyl-N-hydroxysccinimidyl carbamate (AQC)
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•WITH O-PHTHALALDEHYDE (OPA)
DERIVATIZATION AMINO ACID…..
OPA REACTS WITH PRIMARY AMINO GROUP IN THE PRESENCE OF THIOL REAGENTOPA DOES NOT REACT SECONDARY AMINO GROUP, SUCH AS PROLINE AND HYDROXYPROLINE
Λex 230 nm, λem 420 nm
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DERIVATIZATION AMINO ACID…..
• POST COLUMN WITH NINHYDRIN
Blue violet
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• WITH NITROBENZOFURAN CHLORIDE (NBD-Cl) FOR SECONDARY AMINO ACID
DERIVATIZATION AMINO ACID…..
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DERIVATIZATION AMINO ACID….. •WITH FMOC-Cl (Fluorenylmethyloxycarbonyl chloride) FOR SECONDARY AA
FMOC IS FLUOROGENIC. THE EXCESS OF FMOC MUST BE REMOVED BY EXTRACTION WITH PENTANEFMOC CAN FORM POLYMER, TO SOLVE THIS PROBLEM ALKALINE TREATMENT IS CARRIED OUT
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• FATTY ACIDS ARE AN IMPORTANT ENERGY SOURCE• ESSENTIAL FATTY ACIDS: LINOLEIC ACID AND
ARACHIDONIC ACID• FATTY ACIDS OFTEN NEED TO BE QUANTIFIED AS
INDICATORS OF RANCIDITY, FRESHNES AND ADULTERATIN
• DETERMINATION:– GC AND HPLC AFTER DERIVATIZATION (WITH
METHYLENE BLUE)– GC AFTER METHYL ESTERIFICATION
DERIVATIZATION FATTY ACID
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• DERIVATIZATION WITH METHYLENE BLUE: FATTY ACIDS ARE EXTRACTED AS ION-PAIRS WITH CHLOROFORM FROM AQUOEOUS ACETONITRILE MOBILE PHASE AFTER THE POST COLUMN ADDITION OF AQUEOUS METHYLENE BLUE SOLUTION
• DERIVATIZATION WITH 2-NITROPHENYLHYDRAZINE HCl (2-NPH) AND FOLLOWED BY RP-HPLC AND DTECTED AT 400NM.
DERIVATIZATION FATTY ACID……
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• VITAMIN B1 (THIAMINE)– POST AND PRE COLUMN RP-HPLC WITH THIOCHROME FLUORESCENE (λex 375
nm, λex 440 nm)
• VITAMIN B6 (PYRIDOXINE)– POST COLUMN WITH SULFUROUS ACID AFTER EXTRACTION WITH PERCHLORIC
ACID AND SEPARATION WITH ION-PAIRED RP-HPLC
• VITAMIN C (ASCORBIC ACID)– THE REDUCED AND OXIDIZED FORM OF VITAMIN C ARE REFFERED TO AS
ASCORBIC ACID (AA) AND DEHYDROASCORBIC ACID (DHAA). All of them are active
– OXIDIZATION OF AA TO DHAA FOLLOWED BY DERVATIZATION WITH DFQ 3-(1,2-DIHYDROXYETHYL)FLUORO[3,4-b]QUINOXALINE-1-ONE, AND THEN SEPARATION ON RP-HPLC AT λex 350 nm AND λem 430 nm
DERIVATIZATION VITAMINS
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ANALYSIS OF VITAMIN C
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FOLACIN (PTEROYLGLUTAMIC ACID)
DERIVATIZATION VITAMINS…..
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• BIOTIN (VITAMIN B7)– PRE COLUMN HPLC USING 4-BROMOMETHYL-7-
METHOXYCOUMARIN (Br-MMC)– POST COLUMN WITH CHLORINATION OF THE AMIDE
GROUP OF BIOTIN; OR INTRODUCTION OF THIAMINE TO FORM THIOCHROME AND DETECTION WITH FLUORESCENT.
DERIVATIZATION VITAMINS……
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• VITAMIN K– Naturally occuring vitamin K is vit K1
(phytonadione or phylloquinones) found in plants and vit K2 (menaquinones) found in bacteria and vitamin K3 (menadiones)
– POST COLUMN REDUCTION OF VITAMIN K WITH ZINC OR PLATINA TO FORM FLUORESCENCE PRODUCT (CORRESPONDING HYDROQUINONES) AND FLUORESCENT DETECTION
DERIVATIZATION VITAMINS……
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DERIVATIZATION VITAMINS……
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• SACCHARIN, GLYCYRRHIZIC ACID, ASPARTAME AND SORBITOL
• ASPARTAME:
DERIVATIZATION OF SWEETENERS
ASPARTAME CAN BE ANALYZED USING PRE COLUMN METHOD WITH FLUORESCAMINE TO FORM FLUORESCENT DERIVATIVES FOLLOWED BY RP-HPLC AND DETECTED AT λex 390 nm AND λem 480 nm
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ACESULFAME K
CYCLAMATE, SACCHARIN AND ACESULFAME K CAN BE DETECTED USING POST-COLUMN ION PAIR EXTRACTION AS DESCRIBED IN ANALYSIS FATTY ACID
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DERIVATIZATION OF EMULSIFIERS
• MONOGLYCERIDE (MGL), SUCROSE ESTER OF FATTY ACID (SuE), PROPYLENE GLYCOL ESTER OF FATTY ACID (PGE), SORBITAN ESTER OF FATTY ACID
• ANALYSIS OF MGL, SuE, PGE USES 3,5-DINITROBENZOIL CHLORIDE WITH PRE COLUMN METHOD TO FORM DNBC DERIVATIVES FOLLOWED BY SEPARATION WITH RP-HPLC AND UV DETECTION AT λmax 230 nm
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SUCROSE ESTER OF FATTY ACID