identification & quantitative analysis of colouring materials,

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Identification & quantitative determination of coloring materials ,emulsifiers & stabilizers:

Identification & quantitative determination of coloring materials ,emulsifiers & stabilizers By Radhika tekula ( 11FJ1S0410 ) M.Pharmacy (pharmaceutical analysis & quality assurance) SSJ college of pharmacy.

Coloring agents :

Coloring agents are mainly used to impart a distinctive appearance to the pharmaceutical dosage forms. The main categories of dosage form that are colored are: tablets (either the core itself or the coating.) hard or soft gelatin capsules : (the capsule shell or coated beads). oral liquids, topical creams . toothpastes, ointments and salves . Coloring agents

Pharmaceutical preparations are colored mainly for:

Increases acceptability For identification of products the stability of light-sensitive active materials patient compliance Pharmaceutical preparations are colored mainly for

Classification According to their solubility:

Classification According to their solubility

According to the nature:

According to the nature


Colors for clear liquid preparations Examples of organic dyes: Sunset Yellow FCF (FD&C name - Yellow 6 ) Fast green FCF (FD&C name – green no.3) Indigotine (FD&C name – blue no.2) Examples of natural dyes: TURMERIC, SAFFRON SAFFLOWER, ANNATO Dyes


Hydro soluble Eg : Carminic acid, Caramel, D&C Orange No. 4. Liposoluble Eg : Carotene,D&C Red No. 1,D&C Yellow No.11 Organic pigments : 3 types Lakes : Organic or “carbon-based” pigments are sometimes combined with inorganic substances like alumina (aluminum hydroxide) to create “lakes”; Eg : hydrated aluminium , titanium dioxide & aluminium benzoate. Pigments

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2) Toners water-soluble dyes that are precipitated as metal salts The most used metals are calcium and barium 3) True pigments Insoluble compounds which contain no metal ions. Eg : D&C Red No. 36 and D&C Red No. 30 Mineral pigments Iron oxides, Titanium dioxide

Identification of coloring agents:

Dyes converted to the corresponding aluminium lake. soluble in water & sparingly soluble in methanol Method of assay : titration with TITANOUS CHLORIDE Sunset Yellow FCF : Weight of sample: 0.5-0.6 g Buffer: 10 g sodium citrate Weight (D) of colouring matters equivalent to 1.00 ml of 0.1 N TiCl3:11.31 mg Identification of coloring agents

Identification of coloring agents:

paper chromatography Thin layer chromatography Uv/visible spectroscopy Reverse–phase HPLC voltammetry these methods are used to identify the mixture of coloring agents Identification of coloring agents

Quantitative determination of coloring agents:

1)Inorganic coloring agents: X-ray fluorescence method - for the determination of chromium & manganese in colorants. This method widely used to measure the elemental composition of materials. cold-vapor atomic absorption spectrometry- uses microwave digestion of the sample. determination of mercury in colour additives Quantitative determination of coloring agents

Quantitative determination of coloring agents:

2)Organic components: capillary-electrophoresis (CE) method Determination of the main component and two subsidiary colours in FD&C Red No. 3 Reference materials used for that method, 2,4,5-triiodofluorescein, 2,4,7-triiodofluorescein, and 2,4,5,7-tetraiodofluorescein. Quantitative determination of coloring agents

B. Reversed-phase liquid chromatography: :

Reversed-phase LC uses columns packed with short-chain alkyl-bonded silica (e.g. octyl (C8), octadecyl (C18)), amino-bonded, and cyano -bonded phases , or cross-linked polystyrene- divinyl benzene copolymer packing materials . Sample preparation: Heat sample with DME +diluted with 0.1M tetra butyl ammonium hydroxide & extracted twice with chloroform. C18 bonded silica-packed column Detection – UV/Vis diode array detector. B. Reversed-phase liquid chromatography:

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Ion pair reversed phase LC method: Mobile phase : 3 solvents 1) citrate buffer containing tetra butyl ammonium hydroxide as the ion-pairing reagent. 2) Acetonitrile 3) Tetra hydro furan Column - Polymeric Material & gradient elution The UV/VIS spectra were recorded with a DAD.

C)Thin layer chromatography::

The colouring agents were separated in two steps : 1)Elution with methylene chloride - fats and oils to the top of the plate & enables separation of the oil-soluble dyes and leaves the water-soluble dyes and the pigments at the origin; 2)Then the water-soluble dyes were separated from each other by elution with ethylacetate :methanol:8.7% ammonium hydroxide (15:3:3). a reversed-phase TLC on octa decyl silica (C18) gel method that complementarily employs four solvent systems to separate 45 water-soluble dyes C)Thin layer chromatography:

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Silica gel is the most widely used adsorbent , followed by alumina and microcrystalline cellulose. Quantified spectrophotometrically( uv / vis ). Solvent for water soluble dyes: ethyl acetate:methanol:8.7% ammonium hydroxide (15:3:3) Reversed-phase TLC – separate water-soluble dyes

D) Liquid chromatography:

LC combined with UV/VIS detection is the most used analytical technique for the determination of dyes and pigments. Ion-exchange LC uses strong anion-exchange columns (or weak anion-exchange columns for separation of azo dyes ) and gradient elution with buffered eluants . The preferred method of detection is with a UV/VIS diode-array detector (DAD) , which has the capability of simultaneously recording absorbance data from 190 to 800 nm D) Liquid chromatography

E) Spectrophotometry:  :

Solid-phase spectrophotometry was applied to the simultaneous determination of Quinoline Yellow & Brilliant Blue FCF. Derivative spectrophotometry methods were used for the simultaneous determination of tartrazine & and Brilliant Blue FCF. UV/VIS spectrophotometry was applied to the simultaneous determination of Sunset Yellow, tartrazine, Brilliant Blue FCF & Quinoline Yellow. E) Spectrophotometry:

F)Other methods:

Micellar electrokinetic capillary chromatography (MEKC) : the samples are separated by differential partitioning between micelles (pseudo-stationary phase) and a surrounding aqueous buffer solution (mobile phase). Diode array uv detection. Electrophoretic method: Electrophoresis is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. F)Other methods

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surface-enhanced resonance Raman scattering (SERRS) spectroscopy: surface-sensitive technique that enhances Raman scattering by molecules adsorbed on rough metal surfaces. Qualitative in nature and was suggested to have potential for forensic and quality-control applications

The importance of determining colouring agents:  :

To ensure that only permitted colouring agents are added to the cosmetic product To ensure that the information on the label is complete and correct To determine the cause of allergic and dermatologic reactions Quality control The importance of determining colouring agents:


An emulsifier/ emulgent is a substance that stabilizes an emulsion by increasing its kinetic stability. Classification: according to chemical structure Synthetic natural finely dispersed solids auxiliary agents Emulsifiers

Synthetic emulsifiers:

Cationic: E.g: benzalkonium chloride, benzethonium chloride Anionic: E.g: alkali soaps (sodium or potassium oleate); amine soaps (triethanolamine stearate); detergents (sodium lauryl sulfate) Nonionic E.g: sorbitan esters (Span), (Tweens®), or glyceryl esters Synthetic emulsifiers

Natural Emulsifying Agents:

Vegetable derivatives E .g: acacia, tragacanth, agar, pectin, carrageenan, lecithin Animal derivatives E.g : gelatin, lanolin, cholesterol Semi-synthetic agents E .g : methylcellulose, carboxy methylcellulose Synthetic agents E .g : Carbopols Natural Emulsifying Agents

Quantitative analysis of emulsifiers: :

some chromatographic techniques, namely gas chromatography (GC), high performance liquid chromatography (HPLC), supercritical fluid chromatography (SFC), and planar chromatography for detection and quantification of emulsifiers. Quantitative analysis of emulsifiers:

High performance liquid chromatography (HPLC) :

Developed for the separation of mono-, di -, and triglycerides of fatty acid emulsifiers. The glycerides are eluted isocratically with iso -octane- isopropanol (95:5 v/v) within I0 min , and the components were detected using UV-absorption at 213 nm. HPLC with normal phase and ELSD to analyze the common MD and DG in vegetable oils and fats column of reversed-phase binary gradients consisting of ACN-water plus acetic acid (0.1%, v/v) and ACN-CH3Cl as the mobile phase. High performance liquid chromatography (HPLC )

Gas chromatography (GC) :

gas-liquid chromatography (GLC) has become an important tool for the analysis of many emulsifiers. The individual mono- and di -fatty acid esters and the polyols were analyzed using GLC as TMS(tri methyl silyl ) ethers. GC-based method has been adopted as a standard method for the determination of MG and DG in emulsifiers. Gas chromatography (GC)

Planar chromatography :

Planar chromatography refers to thin layer chromatography (TLC) and paper chromatography (PC). 1)Thin layer chromatography the MG and DG are separated from each other and then analyzed gravimetrically or densitometry. Separation of common polyglycerol products dissolving higher linear polyglycerols in EtOH and separated at ambient temperature with ethyl acetate-isopropylalcohol-acetone-methanol-H2O (50:15:15:4:16 v/v) on plates coated with a slurry of equal weight of Kieselguhr G . Planar chromatography

2)Paper chromatography (PC) :

Whatman No. 4 paper. PC is also used for analysis of poly oxy ethylene glycol (PEG 400) Rf values of some emulsifiers : Lecithin 0.20-0.25 Lyso lecithin 0.05-0.08 Ethanolamine 0.32-0.40 2)Paper chromatography (PC)


"Stabilizers are substances which make it possible to maintain the physico-chemical state of a food & drugs”. maintenance of a homogenous dispersion of two or more immiscible substances. Purpose of Stabilizers: They maintain the consistency stabilizers do not allow the separation of ingredients that are bound together by emulsifiers. stabilizers

Examples of stabilizers:

Lecithin and mono- and digycerides Pectin or calcium chloride Gelatin Agar agar Alginic acid Examples of stabilizers

Quantitative analysis of stabilizers:

Thin layer chromatography Paper chromatography Spectrometrical methods ( uv / vis ) GLC HPLC Quantitative analysis of stabilizers


Analysis of colorants , emulsifiers & stabilizers are important to ensure 1) Quality standards are maintained 2) Stability of the additives Conclusion:

References : prequal / trainingresources / pq_pres /Tanzania_Dec2007/ Ref_standards wp -content/uploads/2012/01/Review-article-Analysis-of-Emulsifier-in-Food-Using-Chromatographic-Techniques-A.- Rohman -et- al.pdf References

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