MATA KULIAH ANALISIS FARMASI

3. European Pharmacopoeia

Undang-undang Nomor 23 Tahun 1992 tentang Kesehatan

(Lembaran Negara Republik Indonesia tahun 1992 Nomor
100, Tambahan Lembaran Negara Nomor 3495)

Undang-undang Nomor 8 Tahun 1999 tentang Perlindungan

Konsumen; (Lembaran Negara Republik Indonesia Tahun 1999
Nomor 42, Tambahan Lembaran Negara Nomor 3821 );

Keputusan Kepala Badan Pengawas Obat Dan Makanan

Republik Indonesia Nomor : Hk.00.05.4.3870 tahun 2003
tentang Pedoman Cara Pembuatan KosmetikYang Baik

Kosmetik adalah bahan atau sediaan yang dimaksudkan untuk

digunakan pada bagian luar tubuh manusia (epidermis, rambut,
kuku, bibir, organ genital bagian luar) atau gigi dan mukosa mulut
terutama untuk membersihkan, mewangikan, mengubah
penampilan, dan atau memperbaiki bau badan atau melindungi
atau memelihara tubuh pada kondisi baik.

Chromatographic separation techniques are multi-stage separation

methods in which the components of a sample are distributed
between 2 phases, one of which is stationary,
while the other is mobile.
The stationary phase may be a solid or a liquid supported on a solid
or a gel. The stationary phase may be packed in a column, spread
as a layer, or distributed as a film, etc.
The mobile phase may be gaseous or liquid or supercritical fluid.

The separation may be based on adsorption, mass distribution

(partition), ion exchange, etc., or may be based on differences in
the physico-chemical properties of the molecules such as size,
mass, volume, etc.

A chromatogram is a graphical or other

representation of detector response, effluent
concentration or other quantity used as a
measure of effluent concentration, versus
time, volume or distance.
Idealised chromatograms are represented as a
sequence of gaussian peaks on a baseline.

Thin-layer chromatography (TLC), discovered

in 1938 by Izmailov and Schraiber and
standardized by Stahl, is still regarded as one
of the most effective techniques for isolation,
identification, and quantitative analyses of
inorganic and organic compounds.

TLC offers several other advantages such as:

minimal sample clean up,
wide choice of mobile phases,
flexibility in sample detection,
high sample-loading capacity,
easy accessibility,
open and disposable natureof TLC plates,
low solvent consumption,
comparatively low operational cost, and
relatively little need for modern laboratory

facilities.

The spotting of sample mixture (5-10 /L for conventional TLC ) about 1.52 cm above the lower edge of the layer;
Drying the spot completely at room temperature or at an elevated
temperature;
Developing the plate, usually by one-dimensional ascending technique in a
closed chamber (cylindrical or rectangular) to a distance of 8-10 cm;
Removing the plate from the developing chamber;
Removing mobile phase from the layer by drying;
Detecting spots on the plate using a suitable detection reagent and
procedure;
Measuring the Rf values of resolved spots; and
Determining the separated analyte.

The differential migration of components in a mixture is due to

varying degrees of affinity of the components for the stationary and
mobile phases.

In a TLC system, the Rf coefficient is a basic quantity used to

express the exact position of the solute on the developed
chromatogram.

It is calculated as the ratio

The Rf value varies from 0 (solute remains on the point of

application) to 0.999 (solute migrates up with the solvent
front).

The value of Rs serves to define the separation

of components from the mixture.
For Rs = 1, the two components are reasonably
well separated;
for Rs > 1, the two components are perfectly
separated, and
for Rs < 1, the two components are poorly
separated or not separated at all.

Improved resolution can thus be achieved

either by decreasing the average diameter of
the two spots or by increasing the distance
between them.

Resolution (Rs), which determines the

separation efficiency of ions, is defined as the
ratio of the center-to-center distance (x)
between the two components (A and B)
and the average diameter of the two spots

Thin-layer chromatography (TLC) remains a popular

method for the analysis of natural pigments.

Stationary Phase:
A wide variety of TLC plates are commercially
available, and many of these have found
applications in the analysis of pigments. In this
chapter, individual sorbents are discussed under
specific pigment groups.

a. Silica Layers.

Silica gel G (30 g) is mixed with water (60 mL), and the slurry is transferred
to the TLC plates with a commercial spreader. The plates are allowed to dry for 30
min followed by activation at 120C for 1-2 h.

b. MgO Layers.

MgO (10 g) and kieselguhr G (10 g) are passed through a 60 mesh sieve followed
by mixing with 80 mL of distilled water. The slurry is transferred to the plates with
a commercial spreader. The plates are allowed to dry for 12 h.

c. Cellulose Layers.

Cellulose powder (15 g) is mixed with distilled water (100 mL) and homogenized in
a blender for 30 s. The slurry is transferred to the plates with a commercial
spreader unit. The plates are allowed to dry for 6 h.

d. Sucrose Layers.

Sucrose (65 g) is passed through a 60 mesh sieve followed by mixing

with 100 mL of petroleum ether (60-80C). The slurry is transferred to the plates
with commercial spreading equipment. The plates are allowed to dry for 30 min.

 Solvent

The choice of solvent is described under the

respective pigment groups.

Development

Development is usually performed in

rectangular glass tanks lined with filter paper. A
convenient volume of solvent for a 21 x 21 x 6
cm3 tank is 100 mL. After an equilibration period
of 20 min, the plate is placed in the tank and
allowed to stand until the desired developing
distance is obtained.

 Detection

Although most pigments are immediately obvious on the

plate, the use of longwave ultraviolet light may improve
detection in some cases. Spray reagents have been used
extensively in the flavonoid and quinonoid groups, and a
silver nitrate spray has been used to distinguish certain
carotenoid endgroups.
The temptation to believe that a compound that is pure
with respect to all contamination should be resisted. Spray
reagents are valuable in identifying both the presence and
identity of colorless contaminants. Where specific
noncolored compounds are suspected, suitable reagents for
disclosing their presence should be used.

 Quantification

Quantification may be carried out by recovering the

separated zones from the plates and measuring them by
spectrophotometry in solution. This has been successfully
shown for flavonoids, anthocyanins, photosynthetic
pigments, and porphyrins.
Alternatively, densitometry may be used with visible,
ultraviolet, and fluorescence detection. The results obtained
from optimum densitometric measurements have recently
been compared with those obtained by use of a video
camera equipped with a charge-coupled device (CCD). These
results are regarded as equivalent for the detection of
flavonoids and other phenolics at wavelengths of 254 and
366 nm.

The 2-phenylbenzo-y-pyrone skeleton

is the parent nucleus of flavones and flavonols

The 2-phenylbenzopyrylium ion,

the skeleton of anthocyanidins

TLC separation on cellulose (0.1 mm, Merck) of the six common

anthocyanidins using concentrated hydrochloric acid-formic acid-water
(7:51:42) as mobile phase. Band identities:
(A) pelargonidin, (B) peonidin, (C) malvidin, (D) mixture of pigments A-C and
E-G, (E) cyanidin, (F) petunidin, (G) delphinidin.

The dyes are classified based primarily on characteristic structural

units :

The azo group

The anthraquinone nucleus,
The heterocyclic ring systems (e.g., pyrazolone, thiazole,
acridine, thiazine, oxazine)
The nitro dyes is restricted to the nitrophenols and the
nitroarylamines, in which the nitro group is a vital factor in the
production of color and dyeing properties.
Dyes that do not justify treatment in separate chemical classes
on account of their limited numbers and indefinite constitution
have been classified as miscellaneous dyes.

Lipsticks are sometimes applied directly to TLC plates.

Fat and fat-soluble colors are extracted from samples
of cosmetics with hexane, after which the organic
dyes are extracted with dimethylformamide in the
presence of H3PO4.

Cosmetics and food dyes are extracted from tablet

coating formulations, releasing the dyes from their
lakes by treatment with 85% H3PO4, then dissolving
in methanol and making alkaline with concentrated
aqueous NH3 .

Dyes are extracted from soaps by dissolution in

methanol or in CH2Cl2 and subsequent TLC.
Alternatively, the soaps are fused with formic
acid and the fatty acids are extracted into
heptane. Oil-soluble dyes and some pigment
dyes are separated from fatty acids by backextraction into formic acid. After dilution of the
filtrate, 30% NaOH solution is added, the
mixture is extracted with CHCl3, and the extract
is washed with H2O.

Dyes can be extracted from mouthwashes and

toothpastes in either light petroleum or CH2Cl2

Nail lacquers are digested with ethyl acetate, and the

digest is extracted with aqueous 50%
dimethylformamide. The lower dimethylformamide
phase is separated and, after extraction with high
petroleum to remove fat, is mixed with polyamide
powder, which adsorbs the dye. The powder is packed
in a column and washed with methanol. The dye is
then eluted with concentrated aqueous NH3methanol (1:19)

Silica gel 60 plates were used to identify lip cosmetics . A mixture of 15 mL

of ethyl acetate, 3 mL of methanol, and 3 mL of ammonium hydroxidewater (3:7) solvent (a) and dichloromethane solvent (b) were used as the
mobile phases.
The shiny surface from the rounded end of the lipstick was removed with
tissue, and the lipstick was weighed. The TLC plate was activated, and
10-20 mg of lipstick was applied directly to the plate. The plate was
developed in two separate steps: oil-soluble, unsulfonated colors (D&C
Orange 17 and D&C Red 36) were separated using dichloromethane (b),
and other colors were separated using solvent (a).
Mikami and coworkers analyzed coal tar dyes used in the cosmetics and
food industries by TLC. The dyes were spotted on reversed-phase RP-18
F254 S plates, and the plates were developed in four solvent systems:
acetonitrile-methanol-5% sodium sulfate solution (3:3:10), methyl ethyl
ketone-methanol-aqueous 5% sodium sulfate (1:1:1), acetonitrilemethanol-aqueous 5% sodium sulfate (1:1:1), and acetonitrile-CH2Cl2aqueous 5% sodium sulfate (10:1:5). The visible absorption spectra of the
dyes were measured by scanning densitometry at 370-700 nm.

Rhodamine-B

Ponceau 3R

Naphtol yellow

Amaranth

Tartrazine

Indigo carmine

ABSORPTION OF INFRARED LIGHT

DUE TO MOLECULAR BOND VIBRATIONS.
KEY PARAMETERS:
PEAK LOCATION [WAVE NUMBERS]
PEAK SHAPE
PEAK STRENGTH [TRANSMITTANCE]

Infrared radiation (10,000 100 cm-1) is

absorbed and converted by an organic
molecule into energy of molecular
vibration. This absorption is also
quantized, but vibrational spectra appear
as bands rather than as lines because a
single vibrational energy change is
accompanied by a number of rotational
energy changes.

Low Energy

High Energy

A disturbance of
one of these masses
along the axis of the
spring results in a
vibration called
a simple harmonic
motion

m2
E=h =h
2

k(m1 + m2)
m1 m2

k
m1

1/2
is directly proportional to bond strength:
the stronger the bond the greater the frequency

So C=O 1700 cm-1 > C=C 1650-1600 cm-1

is inversely proportional to reduced mass:
the lighter the reduced mass the greater the
frequency
Reduced mass = m1m2/m1+m2

Consider C-H and C-D

12x1/12+1 =12/13

12x2/12+2 = 24/14

So C-H vibrates at a higher frequency than C-D

There are two types of molecular vibration :

1. Stretching
is a rythmical movement
along the bond axis such
that the interatomic
distance is increasing or
decreasing

2. Bending
may consist of a change
in bond angle between
bonds with a common
atom

A typical infrared spectrum can be divided into two regions.

The left half, above 2000 cm-1, usually contains relatively few
peaks:

Alkane C-H stretching absorptions below 3000 cm-1

demonstrate the presence of saturated carbons

Signals just above 3000 cm-1 demonstrate unsaturation.

A very broad peak in the region between 3100 and 3600 cm-1
indicates the presence of exchangeable protons, typically
from alcohol, amine, amide or carboxylic acid groups

The frequencies from 2800 to 2000 cm-1 are normally void of

other absorptions, so the presence of alkyne or nitrile groups
can be easily seen here.

In contrast, the right half of the spectrum, below 2000

cm-1, normally contains many peaks of varying
intensities, many of which are not readily identifiable.

Two signals which can be seen clearly in this area is the

carbonyl group, which is a very strong peak around
1700 cm-1, and the C-O bond with can be one or two
strong peaks around 1200 cm-1.

This complex lower region is also known as the

"fingerprint region" because almost every organic
compound produces a unique pattern in this area -Therefore identity can often be confirmed by
comparison of this region to a known spectrum.

1. Look for C=O peak (1820-1660 cm-1)

If C=O is present check for :
OH (3400-2400 cm-1)
indicates carboxylic acid

NH (3500 cm-1)

indicates amide

C-O (1300-1000 cm-1)

indicates ester

If no OH, NH or C-O then look for a weak

carbonyl band around 1725-1705 cm-1
indicates ketone

If C=O is not present check for :

OH (3600-3300 cm-1)
indicates alcohol
NH (3500 cm-1)
indicates amine
3. If C=O & OH are not present check for
C-O (1300 cm-1)
indicates ether
C=C (1650-1450 cm-1) if it appears as medium
to strong absorption indicates aromatic
2.

The spectra of simple alkanes are characterized by

absorptions due to CH stretching and bending (the
CC stretching and bending bands are either too weak
or of too low a frequency to be detected in IR
spectroscopy) :

CH stretch from 30002850 cm-1

CH bend or scissoring from 1470-1450 cm-1
CH rock, methyl from 1370-1350 cm-1
CH rock, methyl, seen only in long chain alkanes,
from 725-720 cm-1

Alkenes are compounds that have a carbon-carbon double

bond, C=C. The stretching vibration of the C=C bond usually
gives rise to a moderate band in the region 1680-1640 cm-1.

This is a very useful tool for interpreting IR spectra: Only

alkenes and aromatics show a C-H stretch slightly higher than
3000 cm-1. Compounds that do not have a C=C bond show C-H
stretches only below 3000 cm-1.

Alkynes are compounds that have a carbon-carbon triple

bond (CC). The CC stretch appears as a weak band
from 2260-2100 cm-1. This can be an important
diagnostic tool because very few organic compounds
show an absorption in this region.

A terminal alkyne (but not an internal alkyne) will show a

CH stretch as a strong, narrow band in the range 33303270 cm-1. (Often this band is indistinguishable from
bands resulting from other functional groups on the same
molecule which absorb in this region, such as the O-H
stretch)

A terminal alkyne will show a CH bending vibration in

the region 700-610 cm-1.

Alkyl halides are compounds that have a CX bond, where X

is a halogen: bromine, chlorine, fluorene, or iodine

In general, CX vibration frequencies appear in the region

850-515 cm-1, sometimes out of the range of typical IR
instrumentation. CCl stretches appear from 850550 cm-1,
while CBr stretches appear at slightly lower wavenumbers
from 690-515 cm-1.

In terminal alkyl halides, the CH wag of the CH2X group is

seen from 1300-1150 cm-1.

Complicating the spectra is a profusion of absorptions

throughout the region 1250-770 cm-1, especially in the
smaller alkyl halides. Note that all of these bands are in the
fingerprint region.

Alcohols have characteristic IR absorptions

associated with both the O-H and the C-O
stretching vibrations. When run as a thin liquid
film, or "neat", the OH stretch of alcohols
appears in the region 3500-3200 cm-1 and is a
very intense, broad band. The CO stretch
shows up in the region 1260-1050 cm-1.

OH stretch, hydrogen bonded 3500-3200 cm-1

CO stretch 1260-1050 cm-1 (s)

The carbonyl stretching vibration band C=O

of saturated aliphatic ketones appears at
1715 cm-1. Conjugation of the carbonyl
group with carbon-carbon double bonds or
phenyl groups, as in alpha, betaunsaturated aldehydes and benzaldehyde,
shifts this band to lower wavenumbers,
1685-1666 cm-1.

Summary:
C=O stretch:

aliphatic ketones 1715 cm-1

, -unsaturated ketones 1685-1666 cm-1

The carbonyl stretch C=O of saturated aliphatic aldehydes appears

from 1740-1720 cm-1. As in ketones, if the carbons adjacent to the
aldehyde group are unsaturated, this vibration is shifted to lower
wavenumbers, 1710-1685 cm-1.

Another useful diagnostic band for aldehydes is the O=CH

stretch. This band generally appears as one or two bands of
moderate intensity in the region 2830-2695 cm-1. Since the band
near 2830 cm-1 is usually indistinguishable from other CH
stretching vibration bands (recall that the CH stretches of
alkanes appear from 3000-2850 cm-1), the presence of a moderate
band near 2720 cm-1 is more likely to be helpful in determining
whether or not a compound is an aldehyde.

If you suspect a compound to be an aldehyde, always look for a

peak around 2720 cm-1; it often appears as a shoulder-type peak
just to the right of the alkyl CH stretches.

Summary:
HC=O stretch 2830-2695 cm-1
C=O stretch:
aliphatic aldehydes 1740-1720 cm-1
alpha, beta-unsaturated aldehydes 1710-1685 cm-1

The NH stretches of amines are in the region 3300-3000 cm-1.

These bands are weaker and sharper than those of the alcohol
OH stretches which appear in the same region. In primary
amines (RNH2), there are two bands in this region, the
asymmetrical NH stretch and the symmetrical NH stretch.

Secondary amines (R2NH) show only a single weak band in the

3300-3000 cm-1 region, since they have only one NH bond.
Tertiary amines (R3N) do not show any band in this region since
they do not have an NH bond.

(A shoulder band usually appears on the lower wavenumber side

in primary and secondary liquid amines arising from the overtone
of the NH bending band: this can confuse interpretation. Note the
spectrum of aniline, in the next slide.)

The NH bending vibration of primary amines is observed in the

region 1650-1580 cm-1. Usually, secondary amines do not show a
band in this region and tertiary amines never show a band in this
region. (This band can be very sharp and close enough to the
carbonyl region to cause students to interpret it as a carbonyl
band.)

Another band attributed to amines is observed in the region 910665 cm-1. This strong, broad band is due to NH wag and observed
only for primary and secondary amines.

The CN stretching vibration of aliphatic amines is observed as

medium or weak bands in the region 1250-1020 cm-1. In aromatic
amines, the band is usually strong and in the region 1335-1250
-1

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