analisis pewarna dalam kosmetik.pdf

8/19/2019 Analisis pewarna dalam kosmetik.pdf

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MATA KULIAH ANALISIS FARMASI


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1

2

3. European Pharmacopoeia


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Undang-undang Nomor 23 Tahun 1992 tentang Kesehatan(Lembaran Negara Republik Indonesia tahun 1992 Nomor100, Tambahan Lembaran Negara Nomor 3495)

Undang-undang Nomor 8 Tahun 1999 tentang PerlindunganKonsumen; (Lembaran Negara Republik Indonesia Tahun 1999Nomor 42, Tambahan Lembaran Negara Nomor 3821 );

Keputusan Kepala Badan Pengawas Obat Dan MakananRepublik Indonesia Nomor : Hk.00.05.4.3870 tahun 2003tentang Pedoman Cara Pembuatan KosmetikYang Baik


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Kosmetik adalah bahan atau sediaan yang dimaksudkan untukdigunakan pada bagian luar tubuh manusia (epidermis, rambut,kuku, bibir, organ genital bagian luar) atau gigi dan mukosa mulut

terutama untuk membersihkan, mewangikan, mengubahpenampilan, dan atau memperbaiki bau badan atau melindungiatau memelihara tubuh pada kondisi baik.


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Chromatographic separation techniques are multi-stage separationmethods in which the components of a sample are distributedbetween 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 solidor a gel. The stationary phase may be packed in a column, spreadas 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 inthe physico-chemical properties of the molecules such as size,mass, volume, etc.


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A chromatogram is a graphical or otherrepresentation of detector response, effluent

concentration or other quantity used as ameasure of effluent concentration, versustime, volume or distance.

Idealised chromatograms are represented as asequence of gaussian peaks on a baseline.


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Thin-layer chromatography (TLC), discoveredin 1938 by Izmailov and Schraiber and

standardized by Stahl, is still regarded as oneof the most effective techniques for isolation,identification, and quantitative analyses ofinorganic and organic compounds.


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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.

TLC offers several other advantages such as:


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The spotting of sample mixture (5-10 /μL for conventional TLC ) about 1.5-2 cm above the lower edge of the layer;

Drying the spot completely at room temperature or at an elevatedtemperature;

Developing the plate, usually by one-dimensional ascending technique in aclosed 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 andmobile phases.


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

express the exact position of the solute on the developedchromatogram.

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).


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The value of Rs serves to define the separationof components from the mixture.

For Rs = 1, the two components are reasonablywell separated;

for Rs > 1, the two components are perfectly

separated, and

for Rs < 1, the two components are poorlyseparated or not separated at all.

Improved resolution can thus be achievedeither by decreasing the average diameter ofthe two spots or by increasing the distancebetween them.


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Resolution (Rs), which determines theseparation 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


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Thin-layer chromatography (TLC) remains a popularmethod for the analysis of natural pigments.

Stationary Phase:A wide variety of TLC plates are commercially

available, and many of these have foundapplications in the analysis of pigments. In thischapter, individual sorbents are discussed underspecific pigment groups.


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

b. MgO Layers.MgO (10 g) and kieselguhr G (10 g) are passed through a 60 mesh sieve followedby mixing with 80 mL of distilled water. The slurry is transferred to the plates witha 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 commercialspreader 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 mixingwith 100 mL of petroleum ether (60-80°C). The slurry is transferred to the plates

with commercial spreading equipment. The plates are allowed to dry for 30 min.


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SolventThe choice of solvent is described under therespective pigment groups.

DevelopmentDevelopment is usually performed inrectangular glass tanks lined with filter paper. Aconvenient volume of solvent for a 21 x 21 x 6cm3 tank is 100 mL. After an equilibration periodof 20 min, the plate is placed in the tank andallowed to stand until the desired developingdistance is obtained.


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DetectionAlthough most pigments are immediately obvious on the

plate, the use of longwave ultraviolet light may improvedetection in some cases. Spray reagents have been used

extensively in the flavonoid and quinonoid groups, and asilver nitrate spray has been used to distinguish certaincarotenoid endgroups.

The temptation to believe that a compound that is purewith respect to all contamination should be resisted. Sprayreagents are valuable in identifying both the presence andidentity of colorless contaminants. Where specificnoncolored compounds are suspected, suitable reagents for

disclosing their presence should be used.


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QuantificationQuantification may be carried out by recovering the

separated zones from the plates and measuring them byspectrophotometry in solution. This has been successfullyshown for flavonoids, anthocyanins, photosyntheticpigments, and porphyrins.

Alternatively, densitometry may be used with visible,ultraviolet, and fluorescence detection. The results obtained

from optimum densitometric measurements have recentlybeen compared with those obtained by use of a videocamera equipped with a charge-coupled device (CCD). Theseresults are regarded as equivalent for the detection offlavonoids and other phenolics at wavelengths of 254 and

366 nm.


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The 2-phenylbenzo-y-pyrone skeleton

is the parent nucleus of flavones and flavonols


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The 2-phenylbenzopyrylium ion,the skeleton of anthocyanidins


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


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Lipsticks are sometimes applied directly to TLC plates.Fat and fat-soluble colors are extracted from samplesof cosmetics with hexane, after which the organic

dyes are extracted with dimethylformamide in thepresence of H3PO4.

Cosmetics and food dyes are extracted from tabletcoating formulations, releasing the dyes from theirlakes by treatment with 85% H3PO4, then dissolvingin methanol and making alkaline with concentratedaqueous NH3 .


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Dyes are extracted from soaps by dissolution inmethanol or in CH2Cl2 and subsequent TLC.

Alternatively, the soaps are fused with formicacid and the fatty acids are extracted intoheptane. Oil-soluble dyes and some pigmentdyes are separated from fatty acids by back-

extraction into formic acid. After dilution of thefiltrate, 30% NaOH solution is added, themixture is extracted with CHCl3, and the extractis washed with H2O.


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Dyes can be extracted from mouthwashes andtoothpastes in either light petroleum or CH2Cl2

Nail lacquers are digested with ethyl acetate, and thedigest is extracted with aqueous 50%dimethylformamide. The lower dimethylformamidephase is separated and, after extraction with highpetroleum to remove fat, is mixed with polyamidepowder, which adsorbs the dye. The powder is packedin a column and washed with methanol. The dye isthen eluted with concentrated aqueous NH3-methanol (1:19)

Sili l 6 l t d t id tif li ti A i t f L


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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 hydroxide-

water (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), acetonitrile-

methanol-aqueous 5% sodium sulfate (1:1:1), and acetonitrile-CH2Cl2-

aqueous 5% sodium sulfate (10:1:5). The visible absorption spectra of the

dyes were measured by scanning densitometry at 370-700 nm.


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Rhodamine-B Naphtol yellow Tartrazine

Ponceau 3R Amaranth Indigo carmine


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ABSORPTION OF INFRARED LIGHTDUE TO MOLECULAR BOND VIBRATIONS.

KEY PARAMETERS:PEAK LOCATION [WAVE NUMBERS]PEAK SHAPE

PEAK STRENGTH [TRANSMITTANCE]


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Infrared radiation (10,000 – 100 cm-1) isabsorbed and converted by an organic

molecule into energy of molecularvibration. This absorption is alsoquantized, but vibrational spectra appearas bands rather than as lines because a

single vibrational energy change isaccompanied by a number of rotationalenergy changes.


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h

Low Energy High Energy

A disturbance of one of these massesalong the axis of the

spring results in avibration calleda simple harmonicmotion


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E = h = h k(m1 + m2)m1 m2

m2

m1

k2π


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

is inversely proportional to reduced mass:the lighter the reduced mass the greater thefrequency

Reduced mass = m1m2/m1+m2Consider C-H and C-D12x1/12+1 =12/13 12x2/12+2 = 24/14

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

So C=O 1700 cm-1

> C=C 1650-1600 cm-1


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There are two types of molecular vibration :

1. Stretchingis a rythmical movement

along the bond axis suchthat the interatomicdistance is increasing ordecreasing

2. Bendingmay consist of a changein bond angle betweenbonds with a common

atom


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A typical infrared spectrum can be divided into two regions.The left half, above 2000 cm-1, usually contains relatively fewpeaks:

Alkane C-H stretching absorptions below 3000 cm-1demonstrate 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, typicallyfrom alcohol, amine, amide or carboxylic acid groups

The frequencies from 2800 to 2000 cm-1 are normally void ofother absorptions, so the presence of alkyne or nitrile groups

can be easily seen here.


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In contrast, the right half of the spectrum, below 2000cm-1, normally contains many peaks of varyingintensities, many of which are not readily identifiable.

Two signals which can be seen clearly in this area is thecarbonyl group, which is a very strong peak around1700 cm-1, and the C-O bond with can be one or twostrong peaks around 1200 cm-1.

This complex lower region is also known as the

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


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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 amideC-O (1300-1000 cm-1)

indicates esterIf no OH, NH or C-O then look for a weakcarbonyl band around 1725-1705 cm-1indicates ketone


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2. If C=O is not present check for :OH (3600-3300 cm-1)indicates alcoholNH (3500 cm-1)

indicates amine3. If C=O & OH are not present check forC-O (1300 cm-1)

indicates etherC=C (1650-1450 cm-1) if it appears as medium

to strong absorption indicates aromatic


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The spectra of simple alkanes are characterized byabsorptions due to C –H stretching and bending (theC –C stretching and bending bands are either too weak

or of too low a frequency to be detected in IRspectroscopy) :

C –H stretch from 3000 –2850 cm-1

C –

H bend or scissoring from 1470-1450 cm-1

C –H rock, methyl from 1370-1350 cm-1

C –H rock, methyl, seen only in long chain alkanes,from 725-720 cm-1


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Alkenes are compounds that have a carbon-carbon doublebond, –C=C –. The stretching vibration of the C=C bond usuallygives rise to a moderate band in the region 1680-1640 cm-1.

This is a very useful tool for interpreting IR spectra: Onlyalkenes and aromatics show a C-H stretch slightly higher than3000 cm-1. Compounds that do not have a C=C bond show C-Hstretches only below 3000 cm-1.


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Alkynes are compounds that have a carbon-carbon triplebond ( –C≡C –). The –C≡C – stretch appears as a weak band

from 2260-2100 cm-1. This can be an importantdiagnostic tool because very few organic compoundsshow an absorption in this region.

A terminal alkyne (but not an internal alkyne) will show aC –H stretch as a strong, narrow band in the range 3330-3270 cm-1. (Often this band is indistinguishable from

bands resulting from other functional groups on the samemolecule which absorb in this region, such as the O-Hstretch)

A terminal alkyne will show a C –H bending vibration in

the region 700-610 cm-1.


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Alkyl halides are compounds that have a C –

X bond, where Xis a halogen: bromine, chlorine, fluorene, or iodine

In general, C –X vibration frequencies appear in the region850-515 cm-1, sometimes out of the range of typical IR

instrumentation. C –

Cl stretches appear from 850 –

550 cm-1,while C –Br stretches appear at slightly lower wavenumbersfrom 690-515 cm-1.

In terminal alkyl halides, the C –H wag of the –CH2X group isseen from 1300-1150 cm-1.

Complicating the spectra is a profusion of absorptionsthroughout the region 1250-770 cm-1, especially in thesmaller alkyl halides. Note that all of these bands are in thefingerprint region.


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Alcohols have characteristic IR absorptionsassociated with both the O-H and the C-Ostretching vibrations. When run as a thin liquid

film, or "neat", the O –

H stretch of alcoholsappears in the region 3500-3200 cm-1 and is avery intense, broad band. The C –O stretchshows up in the region 1260-1050 cm-1.

O –H stretch, hydrogen bonded 3500-3200 cm-1

C –O stretch 1260-1050 cm-1 (s)


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The carbonyl stretching vibration band C=Oof saturated aliphatic ketones appears at1715 cm-1. Conjugation of the carbonylgroup with carbon-carbon double bonds or

phenyl groups, as in alpha, beta-unsaturated aldehydes and benzaldehyde,shifts this band to lower wavenumbers,1685-1666 cm-1.

Summary: C=O stretch: aliphatic ketones 1715 cm-1

α, β-unsaturated ketones 1685-1666 cm-1


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The carbonyl stretch C=O of saturated aliphatic aldehydes appearsfrom 1740-1720 cm-1. As in ketones, if the carbons adjacent to thealdehyde group are unsaturated, this vibration is shifted to lowerwavenumbers, 1710-1685 cm-1.

Another useful diagnostic band for aldehydes is the O=C –Hstretch. This band generally appears as one or two bands of

moderate intensity in the region 2830-2695 cm-1

. Since the bandnear 2830 cm-1 is usually indistinguishable from other C –Hstretching vibration bands (recall that the C –H stretches ofalkanes appear from 3000-2850 cm-1), the presence of a moderateband near 2720 cm-1 is more likely to be helpful in determiningwhether 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 C –H stretches.

Summary: H –C=O stretch 2830-2695 cm-1 C=O stretch:

aliphatic aldehydes 1740-1720 cm-1

alpha, beta-unsaturated aldehydes 1710-1685 cm-1


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The N –H stretches of amines are in the region 3300-3000 cm-1.These bands are weaker and sharper than those of the alcoholO –H stretches which appear in the same region. In primary

amines (RNH2), there are two bands in this region, theasymmetrical N –H stretch and the symmetrical N –H stretch.

Secondary amines (R 2NH) show only a single weak band in the3300 3000 cm-1 region since they have only one N H bond


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3300-3000 cm 1 region, since they have only one N –H bond.Tertiary amines (R 3N) do not show any band in this region sincethey do not have an N –H bond.

(A shoulder band usually appears on the lower wavenumber sidein primary and secondary liquid amines arising from the overtoneof the N –H bending band: this can confuse interpretation. Note thespectrum of aniline, in the next slide.)

The N –H bending vibration of primary amines is observed in theregion 1650-1580 cm-1. Usually, secondary amines do not show aband in this region and tertiary amines never show a band in thisregion. (This band can be very sharp and close enough to thecarbonyl region to cause students to interpret it as a carbonylband.)

Another band attributed to amines is observed in the region 910-665 cm-1. This strong, broad band is due to N –H wag and observedonly for primary and secondary amines.

The C –N stretching vibration of aliphatic amines is observed asmedium 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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