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 • Titrimetri dlsb
• Spektrum UV, IR, • Spektrofotometri
massa, RMI UV-Vis, IR
• KCKT, KG
• Teknik tandem
 Analisis Kualitatif Senyawa Fitokimia

Karbohidrat, protein, lipid

Fenol: flavonoid, tanin,

fenilpropanoid, kuinon,
antosianin
Fitokimia 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)
negatif positif
• Reaksinya:
 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
Thin Layer Chromatography Separation on
Silica Gel of Simple Plant Phenols
• solvent 1, 10% acetic acid in
chloroform
• solvent 2, 45% ethyl acetate in
benzene;
• 1, gallic acid; 2, 3,4-
dihydroxybenzoic acid; 3, 2,5-
dihydroxybenzoic acid; 4,
rhododendrol; 5, hydroquinone; 6,
orcinol; 7,p-hydroxybenzoic acid; 8,
syringic acid; 9, vanillic acid; 10,
salicylic acid.
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 • N. C. Liebermann, Über das

Oxychinoterpen, Ber., vol.
Homogenasikan 18, 1885, p. 1803
• H. Burchard, Beitraegezur
+ 1 tetes H2SO4 pekat Kenntnis des Cholesterins,
+ 1 tetes anhidrida asetat Chem. Zentralbl., vol. 61,
1890, p. 26

Hijau/ Biru + steroid Merah/ Ungu + 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 jingga
Culvenor & Fitzgerald (1963) J Pharm
2. + Mayer putih Sci 52:303-304
3. + Wagner coklat

(Untuk standar digunakan daun tapak dara)

 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 Legal
• Uji Brontragers
• Uji Balijet
• Uji Raymond
• Uji Keddii
• Uji 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-deoxy-
hexoses
 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 15-
30 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 UV-
visible 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 yellow-
colored 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