ANALISIS KUALITATIF DAN KUANTITATIF SENYAWA FITOKIMIA
DIVISI KIMIA ANALITIK 2015
Metode Analisis Tumbuhan Kualitatif
Analisis Lanjutan
Kuantitatif
Bioasai
Analisis Kualitatif dan Kuantitatif Senyawa Fitokimia
Kualitatif
Kuantitatif
• Pereaksi pewarna • Spektrum UV, IR, massa, RMI
• Titrimetri dlsb • Spektrofotometri UV-Vis, IR • KCKT, KG • Teknik tandem
Analisis Kualitatif Senyawa Fitokimia Karbohidrat, protein, lipid
Fitokimia
Fenol: flavonoid, tanin, fenilpropanoid, kuinon, antosianin Glikosidanya Terpenoid (monoterpenoid, seskuiterpenoid, triterpenoid, karotenoid), steroid, saponin Alkaloid
Analisis Kualitatif Karbohidrat Uji Molisch • Dikembangkan oleh ahli botani Austria Hans Molisch, uji kimia sensitif untuk karbohidrat • Berdasarkan dehidrasi karbohidrat oleh asam sulfat/asam klorida → aldehida yang dapat berkondensasi dengan 2 molekul fenol yaitu α-naftol yang akan menghasilkan komponen berwarna merah/ungu (α-naftol dapat diganti dengan resorsinol atau timol)
Analisis Kualitatif Karbohidrat Uji Molisch reaksinya:
Analisis Kualitatif Karbohidrat Uji Benedict
• Dikembangkan oleh ahli kimia AS, Stanley Rossiter Benedict • Digunakan untuk mendeteksi keberadaan gula pereduksi (monosakarida dan disakarida), akan tetapi senyawa pereduksi lainnya juga akan memberikan hasil yang positif
Analisis Kualitatif Karbohidrat Uji Benedict • Pereaksi Benedict dapat mendeteksi adanya aldehida dan -hidroksi keton (termasuk sebagai ketosa). Hasil positif ditunjukkan dengan perubahan warna dari biru menjadi endapan merah bata • Reaksinya:
negatif
positif
Analisis Kualitatif Karbohidrat
? Mengapa sukrosa menunjukkan hasil negatif? Sedangkan fruktosa menunjukkan hasil positif
Analisis Kualitatif Karbohidrat Uji Fehling
Justus Liebigs Annalen der Chemie Volume 72, Issue 1, pages 106– 113, 1849
• Uji Fehling merupakan uji kimia yang dapat digunakan dalam membedakan karbohidrat larut air dan gugus fungsi keton dan untuk monosakarida. Uji ini dikembangkan oleh ahli kimia Jerman Hermann von Fehling di 1849
Analisis Kualitatif Karbohidrat Uji Fehling • Larutan Fehling selalu disiapkan terlebih dahulu sebelum pengujian. Larutan ini terdiri atas larutan Fehling A yang berisi tembaga (II) sulfat berwarna biru dan larutan Fehling B yang berisi kalium natrium tartrat (garam Rochelle) dan suatu basa kuat (umumnya NaOH) • Reaksinya:
negatif
positif
Analisis Kualitatif Karbohidrat Uji Barfoed Zeitschrift für analytische Chemie, 1873, Volume 12, Issue 1, pp 27-32
• Dikembangkan oleh ahli kimia Denmark, Christen Thomsen Barfoed • Uji ini merupakan uji untuk mengetahui adanya suatu monosakarida yang berdasarkan reduksi tembaga (ii) asetat menjadi tembaga (I) oksida (Cu2O) → endapan merah bata
Analisis Kualitatif Karbohidrat Uji Barfoed • Reaksinya: RCHO + 2Cu2+ + 2H2O → RCOOH + Cu2O↓ + 4H+
• Disakarida juga dapat bereaksi tetapi lebih lambat. • Reaksi pada uji ini mirip dengan uji Fehling. Gugus aldehida pada monosakarida (hemiasetal siklik) dioksidasi menjadi karboksilat . negatif
positif
Analisis Kualitatif Karbohidrat Uji Iodin • Uji iodin digunakan untuk deteksi pati yang akan membentuk senyawa kompleks berwarna ungu-hitam • Intensitas warna akan turun dengan naiknya suhu dan adanya pelarut organik yang larut air seperti etanol • Tidak dapat dilakukan pada pH rendah karena pati akan terhidrolisis • Reaksi:
Analisis Kualitatif Protein Uji Biuret • Uji ini digunakan untuk mendeteksi adanya ikatan peptida. Ion Cu2+ dengan adanya peptida akan membentuk senyawa kompleks berwarna ungu dalam larutan alkali • Reaksinya:
Uji Biuret juga akan bereaksi dengan senyawa yang memiliki dua atau lebih gugus fungsi berikut:
Analisis Kualitatif Protein Uji Ninhidrin
• Reaksi antara ninhidrin dengan peptida dan asam amino pertama kali dipelajari oleh Siegfried Ruhemann (1911) • Uji ini akan membentuk senyawa kompleks ungu biru ketika gugus -NH2 bebas ada dalam protein atau asam amino • Reaksinya:
-
+
Analisis Kualitatif Protein Reaksi ninhidrin dengan gugus –NH2
Analisis Kualitatif Protein Uji kualitatif protein lainnya: Uji xantoproteat: Phenyl rings containing an activating group can be nitrated producing a yellow product.
Uji Millon: to detect the presence of soluble proteins. A few drops of the reagent are added to the test solution, which is then heated gently. A reddish-brown coloration or precipitate indicates the presence of tyrosine residue which occur in nearly all protein
Analisis Kualitatif Lipid Uji Akrolein Ketika lipid/lemak/minyak dipanaskan dengan adanya suatu agen pengdehidrasi seperti Kalium bisulfat (KHSO4), bagian gliserol dari lipid akan terdehidrasi membentuk aldehida tak jenuh yaitu akrolein (CH2=CH–CHO) yang memiliki bau khas seperti minyak goreng yang dibakar Reaksinya:
Analisis Kualitatif Lipid Uji Sudan IV
Sudan IV merupakan pewarna nonpolar yang dapat berwarna merah-jingga jika terdapat lipid di dalam suatu sample, akan tetapi tidak untuk senyawa polar Uji lainnya: - Uji spot ---- minyak - Saponifikasi - Liebermann-Burchard ---- kolesterol
Analisis Kualitatif Flavonoid Uji Shinoda Serbuk Mg dan beberapa tetes HCl pekat diberikan ke dalam ekstrak sampel, terbentuknya warna merah (dapat pula hijau, jingga, merah muda) menandakan adanya flavonoid.
Analisis Kualitatif Flavonoid Uji menggunakan NaOH Penambahan larutan NaOH jika terdapat flavonoid akan menyebabkan timbulnya warna kuning dalam larutan sampel yang akan hilang jika ditambahkan dengan HCl Uji lainnya: • Pb-asetat ---- endapan warna kuning • FeCl3 ---- biru gelap • Gelatin ---- endapan putih
Analisis Kualitatif Flavonoid • solvent 1, 10% acetic acid in chloroform • solvent 2, 45% ethyl acetate in benzene; • 1, gallic acid; 2, 3,4dihydroxybenzoic acid; 3, 2,5dihydroxybenzoic acid; 4, rhododendrol; 5, hydroquinone; 6, orcinol; 7,p-hydroxybenzoic acid; 8, syringic acid; 9, vanillic acid; 10, salicylic acid.
Thin Layer Chromatography Separation on Silica Gel of Simple Plant Phenols
Compounds 1, 2 and 5 give a blue colour with Folin reagent. Compounds 3,4,6,7,8,9 and 10 give a blue colour with Folin reagent after fuming with ammonia. Compound 6 gives a pink colour with vanillin-HCI.
Analisis Kualitatif Flavonoid
Analisis Kualitatif Flavonoid
Analisis Kualitatif Flavonoid
Analisis Kualitatif Flavonoid
Analisis Kualitatif Tanin Uji Tanin • Menggunakan FeCl3 → hitam, hijau/biru tua • Uji gelatin --- terbentuknya endapan warna putih • Uji fenazon --- terbentuk endapan • Uji Gold beater’s skin --- terbentuknya warna hitam/coklat pada kulit
Analisis Kualitatif Terpenoid Deteksi kualitatif Triterpenoid dan Steroid Sampel 1 gram EtOH panas Saring
Filtrat dipanaskan hingga kering 1 ml Dietil eter
•
Homogenasikan •
+ 1 tetes H2SO4 pekat + 1 tetes anhidrida asetat Hijau/ Biru
+ steroid
Merah/ Ungu
N. C. Liebermann, Über das Oxychinoterpen, Ber., vol. 18, 1885, p. 1803 H. Burchard, Beitraegezur Kenntnis des Cholesterins, Chem. Zentralbl., vol. 61, 1890, p. 26
+ Triterpenoid
Analisis Kualitatif Terpenoid Pereaksi Liebermann-Burchard’s juga dapat digunakan untuk uji kualitatif saponin.
Caranya: - Campurkan 0,5 ml anhidrida asam asetat dan sampel lalu tambahkan 2-3 tetes asam sulfat pekat. Jika terbentuk warna di zona kontak maka saponin terdapat dalam contoh tersebut. Saponin steroid: violet biru terang hijau Saponin Triterpenoid: merah atau violet Selain itu dengan menggunakan pereaksi LB, beberapa triterpenoid dan turunan keton atau resin asam dapat membentuk warna kuning
Analisis Kualitatif Terpenoid Reaksi LB dengan triterpenoid/steroid
Analisis Kualitatif Saponin Uji pembentukan busa Larutan sampel dikocok dan jika terdapat saponin akan menyebabkan terbentuk busa yang stabil dengan tinggi sekitar 2 cm dan stabil selama minimal 10 menit
Analisis Kualitatif Alkaloid 1 gram sampel + beberapa tetes NH3 Haluskan + 5 ml CHCl3 Saring
Filtrat + H2SO4 2M
Lapisan asam dibagi 3 bagian
1. + Dragendrof 2. + Mayer 3. + Wagner (Untuk standar digunakan daun tapak dara)
jingga putih coklat
Culvenor & Fitzgerald (1963) J Pharm Sci 52:303-304
Analisis Kualitatif Alkaloid Pereaksi lainnya yang dapat digunakan: • Asam tanat, dapat mengendapkan hampir semua jenis alkaloid dan senyawa yang memiliki kemiripan dengannya dengan endapan yang terbentuk berwarna putih atau kekuningan. Dapat juga terlarut jika pengendap yang diberikan berlebih atau pada asam lainnya • Asam pikrat, membentuk endapan kuning (tidak dalam larutan), kadangkala membentuk kristal • Asam fosfomolibdat, mengendapkan alkaloid dan komponen nitogen lainnya dalam bantuk padatan berwarna kuning atau coklat yang dapat disaring. Selain itu larutan uji harus bebas dari alkalis lainnya dan juga karbonat
Analisis Kualitatif Alkaloid • Asam fosfotungstat, bertindak seperti fosfomolibdat pada banyak kasus
• Merkuri-kalium iodida, mengendapkan hampir seluruh alkaloid dalam bentuk garamklorida/sulfat dari larutannya sebagai endapan berwarna putih/kuning • Iod dalam bentuk kalium iodida, membentuk endapan coklat dengan larutan alkaloid.
• Merkuri klorida, Platina klorida, Emas klorida, membentuk endapan putih/kuning (tidak dalam bentuk larutan). Jika alkaloid yang diuji dalam bentuk larutan maka hanya akan terbentuk koloid
Analisis Kualitatif Glikosida • • • • • •
Uji Uji Uji Uji Uji Uji
Legal Brontragers Balijet Raymond Keddii Keller-Killani
Tugas: Cari prinsip uji glikosida tersebut dan jenis senyawa glikosidanya
Analisis Kuantitatif • Menentukan kadar individu senyawa atau golongan senyawa (fenol total, flavonoid total dlsb) • Teknik yang digunakan mulai dari yang klasik (gravimetri, titrimetri) maupun instrumental (KCKT, KG, Spektrofotometer UV-Vis, Spektrofotometer IR, dan teknik tandemnya: KC-SM, KG-SM dlsb)
• Saat ini berkembang pula kajian yang menghubungkan dengan kadar senyawa bioaktif yang terdapat dalam tumbuhan dengan aktivitas biologis tertentu yang dimilikinya
Analisis Kuantitatif Karbohidrat - Ekstraksi: pelarut, hidrolisis? Jika ingin menganalisis penyusunnya - Jika dengan proses pelarutan atau presipitasi tidak dapat mengekstrak karbohidrat yang diinginkan → Perlukah dipisahkan? → Kromatografi eksklusi, pertukaran ion - Metode kuantitatif • Asai Anthron. Dengan pereaksi anthron dalam H2SO4; serapan diukur pada 620 nm; biru untuk heksosa, gula lainnya menjadi berwarna kehijauan, dapat menentukan free dan polymer-bound hexoses • Asai Orsinol. Dengan pereaksi orsinol dalam etanol dan FeCl3 dalam HCl; serapan diukur pada 665 nm; pentosa dari hijau ke biru; menentukan pentosa bebas dan yang terikat pada polimer
Analisis Kuantitatif - Metode kuantitatif • Phenol – sulfuric acid assay. with phenol and H2SO4; measurement at 485 nm; determination of all free and polymer-bound carbohydrates (soluble or insoluble polysaccharides) can also be performed on microplates • Biphenylol assay. with hydroxyl-biphenylol in NaOH and borax in H2SO4; measurement at 520 nm; uronic acids red to reddish blue; determination of free and polymer-bound uronic acids • Cystein/H2SO4 assay. with cysteine–HCl in H2O and H2SO4;measurement at 380, 396, and 427 nm; determination of free and polysaccharide-bound 6-deoxyhexoses
Analisis Kuantitatif - Metode kuantitatif • PAHBAH assay. with p-hydroxy-benzoic acid hydrazide in HCl and NaOH; measurement at 410 nm; determination of reducing carbohydrates • Updegraff assay. with HOAc/H2O/HNO3 (8 : 2 : 1) in H3PO4; determination of cellulose by hydrolysis of all noncellulosic polysaccharides and their removal by centrifugation followed by H2SO4 hydrolysis of remaining cellulose and Anthron test for glucose quantification • KLT • KCKT • KG --- derivatisasi • RMI dan SM
Analisis Kuantitatif Protein
• Metode Kjeldahl, dikembangkan tahun 1883 oleh Johann Kjeldahl. • A food is digested with a strong acid so that it releases nitrogen which can be determined by a suitable titration technique. The amount of protein present is then calculated from the nitrogen concentration of the food. • It is usually considered to be the standard method of determining protein concentration. • Because the Kjeldahl method does not measure the protein content directly a conversion factor (F) is needed to convert the measured nitrogen concentration to a protein concentration. A conversion factor of 6.25 (equivalent to 0.16 g nitrogen per gram of protein) is used for many applications, however, this is only an average value, and each protein has a different conversion factor depending on its amino-acid composition. • The Kjeldahl method can conveniently be divided into three steps: digestion, neutralization and titration.
Analisis Kuantitatif Digestion The food sample to be analyzed is weighed into a digestion flask and then digested by heating it in the presence of sulfuric acid (an oxidizing agent which digests the food), anhydrous sodium sulfate (to speed up the reaction by raising the boiling point) and a catalyst, such as copper, selenium, titanium, or mercury (to speed up the reaction). Digestion converts any nitrogen in the food (other than that which is in the form of nitrates or nitrites) into ammonia, and other organic matter to C02 and H20. Ammonia gas is not liberated in an acid solution because the ammonia is in the form of the ammonium ion (NH4+) which binds to the sulfate ion (SO42-) and thus remains in solution: N(food) → (NH4)2SO4 (1)
Analisis Kuantitatif Neutralization After the digestion has been completed the digestion flask is connected to a recieving flask by a tube. The solution in the digestion flask is then made alkaline by addition of sodium hydroxide, which converts the ammonium sulfate into ammonia gas: (NH4)2SO4 + 2 NaOH → 2NH3 + 2H2O + Na2SO4 (2)
The ammonia gas that is formed is liberated from the solution and moves out of the digestion flask and into the receiving flask - which contains an excess of boric acid. The low pH of the solution in the receiving flask converts the ammonia gas into the ammonium ion, and simultaneously converts the boric acid to the borate ion: NH3 + H3BO3 (boric acid) → NH4+ + H2BO3- (borate ion) (3)
Analisis Kuantitatif Titration The nitrogen content is then estimated by titration of the ammonium borate formed with standard sulfuric or hydrochloric acid, using a suitable indicator to determine the end-point of the reaction. H2BO3- + H+ → H3BO3 (4) The concentration of hydrogen ions (in moles) required to reach the end-point is equivalent to the concentration of nitrogen that was in the original food (Equation 3). The following equation can be used to determine the nitrogen concentration of a sample that weighs m grams using a xM HCl acid solution for the titration: Where vs and vb are the titration volumes of the sample and blank, and 14g is the molecular weight of nitrogen N. A blank sample is usually ran at the same time as the material being analyzed to take into account any residual nitrogen which may be in the reagents used to carry out the analysis. Once the nitrogen content has been determined it is converted to a protein content using the appropriate conversion factor: %Protein = F x %N
Analisis Kuantitatif Advantages and Disadvantages Advantages. The Kjeldahl method is widely used internationally and is still the standard method for comparison against all other methods. Its universality, high precision and good reproducibility have made it the major method for the estimation of protein in foods. Disadvantages. It does not give a measure of the true protein, since all nitrogen in foods is not in the form of protein. Different proteins need different correction factors because they have different amino acid sequences. The use of concentrated sulfuric acid at high temperatures poses a considerable hazard, as does the use of some of the possible catalysts The technique is time consuming to carry-out.
Analisis Kuantitatif Enhanced Dumas method Recently, an automated instrumental technique has been developed which is capable of rapidly measuring the protein concentration of food samples. This technique is based on a method first described by a scientist called Dumas over a century and a half ago. It is beginning to compete with the Kjeldahl method as the standard method of analysis for proteins for some foodstuffs due to its rapidness. General Principles A sample of known mass is combusted in a high temperature (about 900 oC) chamber in the presence of oxygen. This leads to the release of CO2, H2O and N2. The CO2 and H2O are removed by passing the gasses over special columns that absorb them. The nitrogen content is then measured by passing the remaining gasses through a column that has a thermal conductivity detector at the end. The column helps separate the nitrogen from any residual CO2 and H2O that may have remained in the gas stream. The instrument is calibrated by analyzing a material that is pure and has a known nitrogen concentration, such as EDTA (= 9.59%N). Thus the signal from the thermal conductivity detector can be converted into a nitrogen content. As with the Kjeldahl method it is necessary to convert the concentration of nitrogen in a sample to the protein content, using suitable conversion factors which depend on the precise amino acid sequence of the protein.
Analisis Kuantitatif Advantages and Disadvantages Advantages: It is much faster than the Kjeldahl method (under 4 minutes per measurement, compared to 1-2 hours for Kjeldahl). It doesn't need toxic chemicals or catalysts. Many samples can be measured automatically. It is easy to use. Disadvantages: High initial cost. It does not give a measure of the true protein, since all nitrogen in foods is not in the form of protein. Different proteins need different correction factors because they have different amino acid sequences. The small sample size makes it difficult to obtain a representative sample.
Analisis Kuantitatif Direct measurement at 280nm • Tryptophan and tyrosine absorb ultraviolet light strongly at 280 nm. The tryptophan and tyrosine content of many proteins remains fairly constant, and so the absorbance of protein solutions at 280nm can be used to determine their concentration. • The advantages of this method are that the procedure is simple to carry out, it is nondestructive, and no special reagents are required. The major disadvantage is that nucleic acids also absorb strongly at 280 nm and could therefore interfere with the measurement of the protein if they are present in sufficient concentrations. • Even so, methods have been developed to overcome this problem, e.g., by measuring the absorbance at two different wavelengths.
Analisis Kuantitatif Biuret Method • A violet-purplish color is produced when cupric ions (Cu2+) interact with peptide bonds under alkaline conditions. • It is mixed with a protein solution and then allowed to stand for 1530 minutes before the absorbance is read at 540 nm. • The major advantage of this technique is that there is no interference from materials that adsorb at lower wavelengths, and the technique is less sensitive to protein type because it utilizes absorption involving peptide bonds that are common to all proteins, rather than specific side groups. • However, it has a relatively low sensitivity compared to other UVvisible methods.
Analisis Kuantitatif Lowry Method The Lowry method combines the biuret reagent with another reagent (the Folin-Ciocalteau phenol reagent) which reacts with tyrosine and tryptophan residues in proteins. This gives a bluish color which can be read somewhere between 500 - 750 nm depending on the sensitivity required. There is a small peak around 500 nm that can be used to determine high protein concentrations and a large peak around 750 nm that can be used to determine low protein concentrations. This method is more sensitive to low concentrations of proteins than the biuret method. Metode Coomassie with Coomassie Brillant Blue in H3PO4; measurement at 595 and 465 nm; determination of soluble proteins
Analisis Kuantitatif Lipid
• The most common approach is a macro-gravimetric method in which lipids are extracted from a sample, the extraction solvent is evaporated and the retained material is measured as the lipid content [8, 9]. • This traditional gravimetrical method requires a relatively large quantity of sample and is time-consuming and labor-intensive when analysis of many samples is needed. • Spectrofluorometric analysis of lipid, which uses the fluorescent dye Nile red, was originally developed by Greenspan et al (1985) Greenspan P, Fowler S (1985) J Lipid Res 26(7):781–789
Analisis Kuantitatif
A colorimetric sulfo-phospho-vanillin (SPV) method was developed for high throughput analysis of total lipids. The developed method uses a reaction mixture that is maintained in a 96-well microplate throughout the entire assay. The new assay provides the following advantages over other methods of lipid measurement: (1) background absorbance can be easily corrected for each well, (2) there is less risk of handling and transferring sulfuric acid contained in reaction mixtures, (3) color develops more consistently providing more accurate measurement of absorbance, and (4) the assay can be used for quantitative measurement of lipids extracted from a wide variety of sources. Unlike other spectrophotometric approaches that use fluorescent dyes, the optimal spectra and reaction conditions for the developed assay do not vary with the sample source. The developed method was used to measure lipids in extracts from four strains of microalgae. No significant difference was found in lipid determination when lipid content was measured using the new method and compared to results obtained using a macro-gravimetric method.
Analisis Kuantitatif
Analisis Kuantitatif Flavonoid
Analisis Kuantitatif Alkaloid
A rapid, easy, and simple spectrophotometric method was developed for the estimation of total alkaloids precipitated by Dragendorff’s reagent (DR) in plant materials. It is based on the formation of yellow bismuth complex in nitric acid medium with thiourea. The yellowcolored complex formed obeys Lambert-Beer’s law in the concentration range of 0.06–50 g/mL with max at 435 nm. Using this method, the alkaloidal percentage of certain alkaloids (ajamalicine, papaverine, cinchonine, piperine, berberine) and some plant materials containing alkaloids (Berberis aristata, Solanum nigrum, and Piper longum) were determined. The method was compared with other methods. It can be used for routine analysis of commercial samples by industries dealing with herbal drugs for standardization of plant materials containing alkaloids and for alkaloid-containing pharmaceutical products.
Analisis Kuantitatif
Analisis Kuantitatif
