4. analisis karbohidrat
DESCRIPTION
anal.TRANSCRIPT
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CARBOHYDRATE ANALYSIS
Nia Kristiningrum, M.Farm., Apt.
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Karbohidrat adalah polihidroksi aldehid atau polihidroksi
keton dan meliputi kondensat polimer-polimernya
yang terbentuk.
KARBOHIDRAT
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INTRODUCTION
• Classification of carbohydrates (CHO)
• Monosaccarides
• Disaccharides
• Oligosaccharides
• Polysaccharides
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Jenis-jenis Karbohidrat
amilosa
sukrosa
amilopektin
glukosa
Oligosakarida
Polisakarida
fruktosa galaktosa
maltosa
laktosa rafinosa
Monosakarida
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Tujuan analisis karbohidrat
• Menentukan komposisi makanan
• Menentukan sifat fisika dan kimia dalam kaitannya
dengan pembentukan kekentalan, stabilitas larutan
dan tekstur hasil olahannya
• Kaitan dg ilmu gizi peranan membentuk kalori
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Sample Preparation for mono-, di- and oligosaccharides
• Need to isolate the CHO first before the measurement is made.
• Preliminary method commonly used to many isolation techniques
1. Food are dried under vacuum to prevent thermal degradation.
2. Ground to a fine powder to enhance solvent extraction.
3. Defatted by solvent extraction.
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• Most commonly used method for isolation
Boil a defatted sample with an 80% alcohol solution to extract low molecular weight CHO from foods.
Mono-, di- and oligosaccharides are soluble in alcoholic solution while the other components not.
The soluble part are separated from the insoluble part by filtration method.
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Treating the filtrate with clarifying agents or by passing it through one or more ion-exchange resins – to remove minor components such as amino acids;
Clarifying agents:
Example: heavy metal salts (Ba(OH)2 ;
ZnSO4 ; TCA)
Function: to form insoluble complexes with interfering substances that can be removed by filtration or centrifugation.
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Analisis Kualitatif Karbohidrat
• Uji Molisch
• Uji seliwanoff
• Uji Anthrone
• Uji Benedict
• Uji Barfoed
• Uji Iodin
• Uji Fehling
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Uji molisch
• + H2SO4 + -naftol
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Uji seliwanoff
• + HCl 12% + resorcinol
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Uji Anthrone
• + H2SO4 + anthrone (9,10 dihidro 9-oxoanthracene)
kompleks biru kehijauan
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Uji Benedict
Gula reduksi +Larutan benedict (camp. kuprisulfat+Na
sitrat+Na karbonat)
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Uji Barfoed
• Gula reduksi + larutan Barfoed (kupri asetat + asam asetat) endapan merah
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Uji Iodin
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Uji Fehlings
• Gula reduksi + lar. Fehlings (kupri sulfat + Na-K tartrat +
NaOH) → dipanaskan → endapan hijau, kuning-orange
atau merah
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ANALISIS KUANTITATIF KARBOHIDRAT
• CARA KIMIAWI
- METODE OKSIDASI DENGAN CUPRI
- METODE OKSIDASI DENGAN LARUTAN
FERRISIANIDA ALKALIS
- METODE IODOMETRI
• CARA ENZIMATIS
• CARA KROMATOGRAFI
• CARA OPTIC
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Metode oksidasi dg kupri (Luff schoorl)
Dasar : reduksi CuO Cu2O oleh gula reduksi
Reagen :
- Reagen Luff (campuran CuSO4, Na2CO3, dan asam sitrat)
Yang ditentukan adalah CuO sebelum dan sesudah
direaksikan dengan gula reduksi
(titrasi blanko – titrasi sampel)
Analisis Kuantitatif Karbohidrat (cara kimiawi)
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Analisis Kuantitatif Karbohidrat
Metode oksidasi dg kupri (Luff schoorl)
• R-COH + 2CuO Cu2O + R-COOH
• H2SO4 + CuO CuSO4 + H2O
• CuSO4 + 2KI CuI2+ K2SO4
• 2CuI2 Cu2I2 + I2
• I2 + Na2S2O3 Na2S3O6 + NaI
• Indikator amilum (I2+amilum =biru)
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Analisis Kuantitatif Karbohidrat
Metode oksidasi dg Ferrisianida alkalis
• Prinsip ferriasianida tereduksi menjadi ferrosianida karena
adanya gula pereduksi
• Jml ferrosianida yg terbtk setara dg jml gula pereduksi
• Pereaksi : K4Fe(CN)6
• Jumlah ferrosianida yang terbentuk ekivalen dg jumlah
gula reduksi dalam sampel
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Analisis Kuantitatif Karbohidrat
Metode oksidasi dg ferrisianida alkalis
• R-COH + K4Fe(CN)6 K3Fe(CN)6 + R-COOH
• 2K3Fe(CN)6 + 2KI 2K4Fe(CN)6 + I2
• I2 + Na2S2O3 Na2S3O6 + NaI
• Indikator amilum (I2+amilum =biru)
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Analisis Kuantitatif Karbohidrat
Metode Oksidasi dg Hipoiodida (Iodometri)
• Prinsip iodin dlm alkalis mjd hipoiodida. Hipoiodida dpt
mengoksidasi aldosa
• Titrasi sampel
• R-COH + I2 + 3NaOH 2NaI + 2H2O + R-COONa
• I2 (sisa) + Na2S2O3 Na2S3O6 + 2NaI
• Titrasi blangko
• I2 (total) + Na2S2O3 Na2S3O6 + 2NaI
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Titration Methods
• Disadvantages:1. The results depend on the precise reaction times, temp
and reagent conc. → must be carefully controlled2. Cannot distinguishing bet different types of reducing
sugar3. Cannot directly determine of non-reducing sugars4. Susceptible to interference from other types of
molecules that act as reducing agents
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Analisis Kuantitatif Karbohidrat
Metode Enzimatis (penentuan glukosa)
• Glukosa + Adenosin-5-trifosfat (ATP) G-6-P + ADP
• G-6-P + NADP G6P-DH Glukonat-6-P + NADPH + H+
NADPH diukur dg spektro panj gel 334 atau 340 atau 365nm
(Nikotinamid adenin dinukleotida)
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Analisis Kuantitatif Karbohidrat
Metode Enzimatis (Penentuan Laktosa/galaktosa)
• Laktosa + H2O -galaktosidase Glukosa + galaktosa
• galaktosa + NAD GAL-DH as. galatonat + NADH + H+
• NADH diukur dg spektro panj gel 334,340,365nm
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Analisis Kuantitatif Karbohidrat
Penentuan karbohidrat dg Polarimeter
• karbohidrat bersifat optis aktif : dpt dianalisis scr polarimetri
karena molekul penyusun karbohidrat mempunyai susunan
asimetris shg mempunyai kemampuan memutar bidang sinar
terpolarisasi
• Putaran optik diukur dg polarimeter yaitu instrumen pengukur
arah getaran atau vibrasi sinar polarisasi
• Keuntungan sampel tdk rusak dan dpt dilakukan cepat
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Analisis Kuantitatif Karbohidrat
Penentuan karbohidrat dg Polarimeter
• Hukum Biot = besar rotasi tiap individu gula sebanding dengan
konsentrasi larutan dan panjang cairan dalam tabung.
[] D t = 100
I. C
[] : putaran/rotasi spesifik
t : suhu pengukuran
D : sinar Na (589nm)
: sudut putar yg diamati
C : konsentrasi %b/v
I : panjang tabung (dm)
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Analisis Kuantitatif Karbohidrat
Penentuan karbohidrat dg Polarimeter
• Hal yg perlu diperhatihan pd pengukuran dg polarimater :
1. Lar jernih & tdk brwarna
2. Lar tdk mengandung bahan lain yg brsifat optis
aktif
3. Konsentrasi sampel pd daerah optimum dari alat,
tdk terlalu pekat/encer
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Analisis Kuantitatif Karbohidrat
Penentuan karbohidrat dg Spektrofotometer
• Karbohidrat (aldosa) + pararosanilin ungu shg bisa
diukur pd panjang gel
• Karbohidrat (aldosa) + asam 3,5 dinitrosalisilat merah-
coklat shg bisa diukur pd panjang gel 540nm
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Chromatographic Methods
1. HPLC • Advantages: rapid, can tolerate a wide range of sample
conc., precise and accurate and requires no prior derivatization
• Disadvantages: require micron-filter filtration prior to injection
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2. GC
• Most be converted to volatile derivatives
• Involve two preparation steps Reduction of aldehyde groups to primary
alcohol groups Conversion of the reduced sugar into a volatile
derivative These steps must be complete 100% to ensure
the measurement is accurate and precise.
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Cara Optic (Fisis)
• Penentuan indeks bias karbohidrat dg refraktometer
• Tiap jenis gula mempunyai indeks bias ttt
• Indeks bias zat dipengaruhi oleh panjang gelombang sinar
dan suhu pengukuran
• N20 = pengukuran indeks bias pd suhu 20o dengan sinar Na D
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