Beberapa Istilah Dalam Kimia Bioanalitik An assay is a method for the determination of the level of a given analyte in a given sample or set of samples An analysis is determination of the level of a given analyte in a given sample or set of sample An analyte is a substance (element, ion or compound) being analyzed The analyte matrix is the chemical environment of the samples that contains the analyte

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Specific questions in Bio-analytics Is the analyte in the sample? What is the level of the analyte in the sample? Does this sample have a concentration of the analyte higher than (lower than, equal to that sample? Does the product meet the specification for the level of the analyte? Does the treatment alter the sample, and so on? Bioanalytical Chemistry: Interface between Biology and Chemistry Biology is concerned with stimulus and respons of living organism Chemistry is concerned with chemical structures and reactions at the molecular level2012/6/6 Mudasir 2

Suatu respon dapat ditimbulkan oleh > 1 senyawa kimia. Sebaliknya suatu senyawa kimia kadang-kadang dapat menyebabkan respon biologis yang berganda. contoh: aktivitas vitamin B6: 6 senyawa kimia aktivitas vitamin A: > 20 senyawa kimia Therefore, For Chemical assay of vitamin B6 activity, one would have to assay 6 different chemical compounds

Analit Struktur Kimia dan sifat-sifat fisika analit merupakan faktor yang menentukan teknik pengukuran apa yang sebaiknya digunakan dalam penentuan level analit dalam suatu sampel. contoh: Protein mempunyai gugus kimia dan sifat fisika sebagaimana disajikan dalam tabel berikut yang dapat dijadikan sebagai dasar identifikasi level analit.2012/6/6 Mudasir 3

Table Chemical Features of Protein Peptide Bonds Amino groups Carboxyl group Aromatic groups Phenyl Indole Imino groups Alcohol groups Phenol groups Size Sulfohydrile groups Imidazole groups Hydrophobic area Enzymatic activity Immunological sites Bound chromophores Other bound chemical compounds (e.g. metals) Shape ChargeMudasir 4

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Biological Matrices Properties of the matrix and the concentration of the analyte in that matrix determine which measurement may not be used to assay the analyte in the matrixAnalyte & The concentrationChemical matrices

Method is not okay

Method is Okay

Chemical methods

Fig. Selection of appropriate assay methodologies

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Which assay method should be used Chemical Analysis of biological system are done to answer specific questions: - What is the problem that needs these analyses? - What is the analyte? - What is the sample matrix? - What are the possibilities that the other components of the matrix will interfere with the analysis? - What assay methodology characteristics are required to provide the results needed to solve the problem The key to a successful solution in the selection of the best assay methodology is the evaluation of - Problem at hand Mungkin dihasilkan > 1 assay - The sample matrices Dengan tingkat presisi dan - The available assay methodology akurasi yang memadai - And the resources of the laboratory - Cost and laboratory resources2012/6/6 Mudasir 6

Analytical Process Defining the problem Making the measurements: Sample selection Sample pretreatment Measurement Identify Quantify Computing the results Reporting the results

A holistic step: The actions at any one of the steps will influence the results of the later steps.

Mechanism for the determination of an analyte concentration The amount of analyte is proportional to: - The magnitude of the signal or - The number of the signal or events, or - The amount of the reagent required to react completely with the analyte - The rate of change in the concentration of a compound2012/6/6 Mudasir 7

Sample Matrix Characteristics Analyte concentration in samples Concentration ranges in routine samples Maximum known ranges in all samples Natural variability of concentrations Probability of sample contamination Probability of interfering materials in sample matrices Probability of modifications of analyte with storage and/or processing Physical state, particle size and homogeneity of sample Toxicity of sample Availability and cost of sample Use of sample after analysis

Method validationAnalysis of standards Analysis of SRMs Analysis of pool samples Method of standard additions Comparison with accepted methods Review

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Accuracy: selectivity Precision Figures of merit: correlation coefficient (R2), sensitivity, linear range, limit of detection Level of method validation: In house, published in peer-review literature, published in method book, published by trade, professional or standard organization Fragility / ruggedness: cost factors, peer acceptance, safety factors, time factors

Method Characteristics

Table Approximate Chemical Composition of a Typical Mammalian Cell Component Percent Component Percent H 2O 70 RNA 1,1 Inorganic ions (Na+, K+, 1 DNA 0,25 Mg2+, Ca2+, Cl-) Phospolipids 3 Miscellaneous small 3 other lipids 2 metabolites Polysaccarides 2 Proteins 182012/6/6 Mudasir 9

Sampel dalam BioanalitikSampel sangat bervariasi, biasanya: - mikroorganisma - Jaringan hewan (animal tissues) - Jaringan tumbuhan (plant tissues) The choice of the samples depend on the goal of the study The components or molecules (analytes) might be from some specific part of an animal or plant: Animal Plant - Rabbit Skeletal muscle - leaves - rat liver - Steams (batang) - Bovine or porcine heart, pancreas, Brain - Flowers - Human blood - Roots - Saliva - Tubers (umbi-umbian) - Seeds In most cases, It would be necessary to separate the cells by centrifugation or filtration The species and the types of organ determine the kind of procedure usedMudasir 10

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Decomposition of Biological Samples for Trace Metal Analysis Dekomposisi: proses pengubahan sampel ke dalam bentuk larutan dengan perlakuan tertentu Trace metal elements dalam sampel biologis dijumpai dalam berbagai variasi: - Kontaminan (biasanya dalam bentuk anorganik) dan tidak terkait langsung dengan matriks sampel. - Merupakan bagian yang terikat pada matriks sampel, atau - Terkait secara kuat dengan matriks sampel

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Hal-hal pokok yang harus terpenuhi dalam destruksi sampel organik/biologi Oksidasi bahan-bahan organik dalam sampel Mendapatkan nilai recovery logam yang memadai 1. no volatilization; 2. no retention by vessel, and 3. no reaction leading to incomplete solution of metal or to formation of species interfering in separation and determination reaction No contamination by the materials of the vessel Kemungkinan untuk dapat digunakan dalam destruksi sampel dengan jumlah yang banyak Keterpakainnya untuk destruksi berbagai jenis sampel (applicability to a wide range of materials) No method is wholly ideal, and in practice losses of metals and contamination will occur in greater or lesser degree Task of Analyst: To choose a method or so modify it that these errors will be within permissible limits

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Volatilization of elements from perchlorate and hydrofluoric acid solutionNo lossNa, K Cu, Ag, Au Be, Mg Ca, Sr, Ba Zn, Cd, Hg La, Ce Ti, Th Sn, Pb V, Bi Mo, W, U Fe, Co, Ni2012/6/6

Apparent lossB, 100% Si, 100% Ge, up to 10% As, 100% Sb, up to 10% Cr, varies greatly Se, varies greatly Mn, up to 3 % Re, varies greatly Evaporated in Pt to Strong fumes of HClO4 at 200 oC

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Klasifikasi Destruksi Sampel Biologi Oksidasi basah/ wet oxidation (hampir selalu dalam medium asam) Oksidasi kering (pengabuan/ ashing) a. Biasa (open system) b. Labu atau Bom oksigen c. Oksigen tereksitasi Peleburan oksidatif (Oxidative fussion)

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Destruksi Kering versus BasahDestruksi basah Destruksi kering

Recovery logam lebih terjamin, penguapan dapat dihindari dan retensi oleh wadah atau bahan yang tidak larut umumnya rendahPenggunaan reagen dalam jumlah besar Absobansi yang besar dalam lar. Blanko (akibat kontaminasi) penggunaan asam yang banyak memerlukan netralisasi Waktu yang diperlukan lebih singkat, tetapi diperlukan penanganan yg lbh hatihati Tingkat kemudahan oksidasi sampel bervariasi, tetapi umumnya dapat diperoleh destruksi yang sempurna dari sampel Resiko kontaminasi oleh atmosfer lebih rendah 2012/6/6

Sebagian besar logam dapat direcovery dengan baik apabila digunakan pemanasan secara hati-hatiHasil pengabuan biasanya dapat larut dalam sejumlah kecil larutan asam

Prosedur mudah tetapi memerlukan perhatian khusus pada tahap awal destruksi, Jika banyak bahan yang tidak mdh terabukan diperlukan ashing aids Beberapa sampel (spt jaringan hewan) tidak dapat diabukan dengan sempurna pada temp yang aman (mis. dari volatilisasi)

Sampel dalam jumlah besar dapat dikerjakan dengan lebih mudah Mudasir

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Conclusion Neither of these two general methods is so superior, and there is no procedure universal in the sense that it is always clearly superior to others However, wet oxidation is more reliable than dry ashing for most metals and for a wider variety of biological samples and it is usually adopted for more or less untested samples

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Determination of Metals after Oxidation of the Alga by Different Methods (using AAS)Elements Low temperature ashing 0.029 1.43 0.364 0.32 0.26 0.014 0.0026 0.024 0.0011 Muffle furnace Muffle furnace 400 C (in Pt 400 C (in crucible) Porcelain crucible) 0.031 1.47 0.354 0.36 0.24 0.015 0.0025 0.025 0.0010 0.030 1.41 0.354 0.34 0.23 0.015 0.0024 0.025 0.0011 Wet oxidation with HNO3HClO4 0.031 1.41 0.351 0.33 0.24 0.015 0.0027 0.026 0.0012

Na K Ca Mg Fe Mn Cu Zn Ni

Dry ashing is preferred because it is safe and simple Z. Anal. Chem., 260, 284 (1982).2012/6/6 Mudasir 17

Recovery of metals in Various Biometerials after Wet Oxidation (H2SO4: HClO4:HNO3=1:1:3Recovery (%) Urine Leaves Animal Tissue 94 95 92 100 77 65 92 95 85 87 45 30 95 94 94

Metals As Au Fe Hg Sb

Blood 93 77 98 24 99

Reflux condensor 101 100 100 100 101

Ag, Co, Cr, Cu, Mn, Mo, Pb, V, Zn: 98102 % for all samples (Anal. Chem., 58, 1084, 1986)

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Persen Recovery Logam pada Pengabuan Sampel Darah pada Berbagai SuhuRecovery (%)

Logam

400 C

500 C

700 C45 0 0 67 85 87 52 0 85 85 32 35 70 6919

Ag 65 67 As 23 0 Au 19 0,5 Co 98 75 Cr 100 99 Cu 100 98 Fe 86 82 Hg