高等食品分析(advanced food analysis) vi. plane...

高等食品分析(Advanced Food Analysis)

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VI. PLANE CHROMATOGRAPHY

*Plane, planar, plate or two-dimensional chromatography:

1. Thin-layer chromatography (TLC) 2. Paper chromatography (PC) 3. Electrophoresis, electrochromatography (EC)

Stationary phase: Flat, relatively thin layer of material that is self-supporting or is coated on a glass, plastic or metal surface.

Mobile phase: Liquid, moves through by capillary action, sometimes assisted by gravity or electrical potential.

1. Thin-layer chromatography (TLC): Detection limit: 10-9 g. Small amounts of adsorbent and minute samples are needed. Separated spots are located on the plate using visualization

techniques in common with PC. Preparative separations are achieved by increasing the thickness of

adsorbent layer, and using higher loading of sample. Use of authentic substances: For tentative identification. The area containing the analyte is scraped from the plate with a

razor or spatula, and the contents are transferred to a test tube. Analyte is dissolved with suitable solvent and separated by

centrifugation and filtration. Further identification: MS, NMR and IR. Apparatus: A chamber or tank, glass or Al plates, an applicator (or

using precoated plates), small pipettes, and a device for spraying the plates.

*Preparative TLC: For isolation of 10 - 100 mg of components. Using plates with fluorescence indicator to facilitate non-

destructive location of the components.

*TLC vs. Adsorption column chromatography: Same adsorbent. Conventional column chromatography is a fairly slow process that

requires large amounts of adsorbent and sample. Disadvantages of CC: Speed, scale and characterization.


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*TLC vs. HPLC:

Multiple samples can be analyzed simultaneously in TLC. Multiple development (two dimensional). All components may be located, and spots can be removed and

purified.

HPLC HPTLC Simplicity * Efficiency * Cost-initial * Cost-analysis * *(disposable plates) Detection * * Quantitation ** Automation *

*TLC vs. Paper chromatography (PC):

TLC: Great speed and better resolution. => 10 cm run in TLC: 20 - 30 min; in PC: 2 hrs. => Adsorbent in TLC: Higher capacity and very small particle

size of adsorbent compared to large cellulose fibers of paper matrix.

Advantage of adsorption system: The separation of hydrophobic substances such as lipids and hydrocarbons.

Location of separated substances on TLC is done in the same way as it is on paper, but more reactive reagents, e.g. concentrated sulfuric acid, can be applied on thin-layer of silica gel or alumina.

*Retardation factor or retention factor (Rf): Rf = dR/dM (1)

The measure of migration of a component. Where Rf: Vary from 1 to solutes that are retarded to 0. (1> Rf >0) dR: Distance of spot from origin. dM: Distance of solvent front from origin.


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=> dR/ = tM/(tM + tR) and dM/ = tM (2)(3) Where : Linear velocity of solvent.

=> Capacity factor, k = (dM - dR)/dR = (1 -Rf)/Rf (4)

Capacity factor obtained by TLC is usually simpler and more rapid than that from column, and can be used for method development in column chromatography.

=> Theoretical plates, N = 16(dR/W)2 (5) => Plate heights, H = dR/N (6) H = A[r]1/3 + B/ + C (7)

All contributing in TLC, A[r]1/3 being the big number, where r: Diameter of particles.

*Factors affecting Rf:

Thickness and activity of the adsorbent; Polarity of the solvent system; Moisture content of stationary and mobile phases; Degree of saturation and temperature of the tank; Amount and purity of the sample; and Distance of solvent traveled.


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*Relative retention factor, Rx: To standardize Rf variation. Rx = Travel distance of analyte/travel distance of standard

*Adsorbents for TLC: Solid Used to separate Silica gel A A’s, alkaloids, sugars, F A’s, lipids, essential oils inorganic anions and cations, steroids, terpenoids Alumina Alkaloids, food dyes, phenols, steroids, vit’s, carotenes, A A’s Kieselguhr Sugars, oligosaccharides, dibasic acids, F A’s, A A’s TG’s, steroids Celite Steroids, inorganic cations Cellulose powder A A’s, food dyes, alkaloids, nucleotides Ion-exchange cellulose Nucleotides, halide ions Starch A A’s Polyamide powder Anthocyanins, aromatic acids, antioxidants, flavonoids, proteins. Sephadex Nucleotides, proteins, metal complexes.

Two important factors for resolution and separating efficiency: Particle size (1-25 m) and its homogeneity (distribution). Fine material in CC => Unacceptably slow flow rate. while in TLC => Faster and more even flow rate.

1) Silica gel: Most commonly used adsorbent. Prepared by hydrolysis of sodium silicate to polysilicic acid, and

condensation and polymerization of it to yield silica gel with a binder added at ca. 10% w/w.

G: Denote silica gel with a gypsum binder, calcium sulfate hemi- hydrate (CaSO4•0.5H2O).

Particle size: Mean: 15 m with a range of 5-40 m. For precoated plates: Mean 10 m, for HPTLC: 5-6 m. F254: Fluorescent indicator phosphor which emit green

fluorescence at 254 nm UV light.


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1) Silica gel: Argentation TLC: Adding silver nitrate to silica gel to increase

separation of unsaturated compounds, especially alkenes. Reverse-phase plate: Impregnating a long chain hydrocarbon into

the support, such as octadecylsilane (ODS) modified silica for analysis of lipophilic substances, fats and waxes, steroids, fat-soluble vitamins and dyes.

2) Silanized silica gel: Use in reverse-phase chromatography. Prepared by treating silica gel with silanizing agent, e.g.

dimethyldichlorosilane to transform silanol groups to dimethylsilyl with little adsorptive capacity.

OHOH Si

CH

CH

Cl

Cl

Silanol groups on silica

Dimethyl dichlorosilane

3

3

Silyl ether linkages

O

OSi

CH

CH

3

3

3) Kieselguhr (Celite): Diatomaceous earths with very little

adsorptive property and used primarily as a support for the stationary phase in partition chromatography.

Adsorptive capacity further reduced by treatment with acids or alkali or by silanizing.

4) Alumina (aluminum oxide): Strong adsorbent and amphoteric ion exchanger depending on the

surface and the solvent. In aqueous or aqueous-alcoholic solution: Anionic exchanger for

basic dyes, basic amino acids and inorganic cations. Basic with organic eluants: Aromatic and unsaturated

hydrocarbons, carotenoids, steroids, alkaloids and other natural products.

Neutral: Substances that are labile or bound to strong alkalis. Acidic: Neutral or acid materials that are not acid-labile.


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5) Cellulose powders: Containing water by hydrogen bonding, thus separation via a

partition mechanism for hydrophilic substances such as amino acids and sugars.

Eluants used similar to those used for PC. Diethylaminoethyl (DEAE)-cellulose with positive charge at

neutral and acidic pH: For negatively charged molecules such as proteins and nucleic acids by ion-exchange chromatography.

6) Molecular sieve layers: Sephadex gel chromatography media with a particle size of 10 - 40 m.

The layers of 0.4 to 1.0 mm slower and more troublesome than other forms in TLC, but faster and more convenient than gel chromatography in a column.

7) PEI-cellulose: Prepared by impregnating cellulose with polyethyleneimine (PEI).

Strong anion exchanger with high capacity for nucleotides, nucleosides, nucleobases and sugar phosphates.

PEI: Prepared by copolymerization of aziridine in the presence of acid catalyst and resultant product is highly branched and have primary, secondary and tertiary amino groups.

Layers can be obtained with fluorescence indicator, and should be stored at 0-5°C to prevent deterioration.

8) Polyamide layers: e.g. Polycaprolactam and Nylon 6.6 (polyhexamethylenediaminoadipate) for closely related phenols that react with layers by hydrogen bonding.

Desorptive power of commonly used solvents: Dimethylformamide > formamide > acetone > methanol > water.

*Preparation of plates: Plates: 20 × 5 or 20 × 20 cm Adsorbent thickness: 0.2 to 0.5 mm; standard: 0.25 mm. For preparative separation: 0.5 to 2.0 mm.


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*Procedure: 1) Adding 1.5 -2.0 parts of distilled or deionized water to 1 part

of adsorbent and mixing to form a slurry. 2) 50 g adsorbent are required to coat 5 plates (20 × 20 cm) to a

thickness of 0.25 mm. 3) After coating using an applicator, the plate should be air-dried

for ca. 45 min, and activated in an oven at 110°C for at least 1 hr, and then stored in a desiccator.

*Sample application: Sample is dissolved in a solvent (e.g. benzene) and applied with a

small pipette in such a way that the sample is concentrated in a small spot (ca. 2-5 mm in diameter).

Spots should be separated more than 1 cm and at least 2 cm from the bottom of the plate to avoid sample dissolving into the solvent, and 1.5 cm from two sides.

*Eluting solvent System: Elutropic values of solvents indicating their increasing polarity are

typically expressed by their dielectric constant.

Eluting solvent series: In the order of increasing eluting power. n-Pentane (least), hexane, heptane, cyclohexane, carbon

tetrachloride, benzene, chloroform, diethyl ether, ethyl acetate, pyridine, acetone, methanol, water (greatest).

Acetic acid is added in small amount (ca. 1%) to reduce sample spreading during TLC development.

*Chromatographic development: To saturate the chamber or tank with solvent vapor to avoid edge

effect resulted from evaporation of solvent from the plate, it is lined with filter paper or shaken with solvent for a while.

200 ml of solvent is necessary for proper development. A suitable solvent should give Rf values of 0.3 to 0.7 for components of interest.


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1) Ascending development: Moving force: Capillary action. The paper is suspended vertically in a chamber with the spotted

end down, and allowed to dip into 0.5 to 1 cm. The plate is positioned in the tank against the wall.

2) Descending development: Capillary action with gravity. Solvent is fed from trough via a wick into the plate with spotted

end up.


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3) Continuous development: Continuous solvent flow. The eluant is allowed to wash off the plates. For resolution of compounds using low polarity solvents.

4) Stepwise development: After development, plate is removed from the tank, dried and then

developed using different solvent system with increasing or decreasing elutropic strength.

5) Multiple or repeated development: Stepwise development with the same solvent system used successively due to poor separation obtained.

6) Two-dimensional development: Two solvent systems used. After first development, plate is dried and then turned 90°, and

developed again using a second solvent.

7) Radial or horizontal development: For components of low Rf with linear development. Rf = [RRf]2 Where RRf: Radial Rf (8)

*Solvent system examples: Coating Substances separated Solvent system Silica Amino acids BuOH/AcOH/H2O(4:1:1) PhOH/H2O(3:1) Fatty acids Pet. ether/Et2O Lipids Pet. ether/Et2O/AcOH (80:20:1) Sterols CHCl3/Acetone (95:5) Sugars EtOAc/AcOH/MeOH/H2O(60:15:15:10)


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*Detection: For colored compounds, such as dyes and plant

pigments. => No need for special visualizing reagents. For colorless compounds:

1) Chemical methods:

Reagent Color of spots Component detected I2 Vapor Brown General organic, unsaturated 50% H2SO4 Black Organic 2,7-Fluorescein Yellow-green Most organic Ninhydrin Pink/purple AA’s and amines 2,4-DNP Orange/red Ketones and aldehydes Antimony chlorides Varied Steroids, alicyclic vit’s, carotenoids Bromophenol blue Yellow Carboxylic acids or bromocresol green Phosphomolybdic acid Reducible compounds Diphenylcarbazide Varied Metals

After spraying, it is necessary to heat the plate to accelerate chemical reaction between reagent and components.


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2) Physical Methods:

a. UV detection: Using adsorbent containing fluorescence indicator phosphor at 254 nm or fluoresce at 370 nm.

Component spot present quenches the fluorescence and shows a dark spot against the green background.

Non-destructive, and allowing isolation for subsequent analysis. b. Densitometer: Spectrodensitometers or TLC scanners based

on transmittance, fluorescence or reflectance intensity for compounds which absorb UV or visible light.

c. Fluorescence measurements: For compounds, which emit fluorescence after irradiation?

d. Radiochemical detection: Using autoradiography, liquid scintillation counting and in situ measurement of radioactivity for radiolabeled compounds.

*Quantitative analysis: Comparing the area of spot with that of standard. Scraping off the plate and measuring by suitable physical or

chemical methods.

Spraying visualization

UV light on plate with fluorescence indicator

Using scanning densitometer to measure the radiation emitted from the spot by fluorescence or reflection.

*Documentation: Plate chromatograms should be recorded using photograph or photocopy.


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*High performance TLC (HPTLC):

Normal TLC: Adsorbent of 5-40 m particle size distribution => N: 1000 - 2000/5 cm migration. HPTLC: 5 m silica gel in TLC with sample loading of 5-100 ng => N: 5000 - 10,000/5 cm migration. Advantages: Improved detection due to smaller zone diffusion,

reduced solvent consumption, and higher efficiency. Plates coated with microcrystalline cellulose and RP materials on

HPLC packings when used with in situ detection (densitometry) are very powerful tool of extreme sensitivity.


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2. Paper chromatography (PC)

*Papers: Standard filter papers made from cotton cellulose, composed of (1–>4) linked glucose residues.

Cellulose contains up to 3 hydroxyl groups per glucose monomer, during processing many of them may oxidize to carbonyl or carboxyl functional groups.

Cellulose absorbs 5 - 20% of water depending on the nature of paper, humidity and temperature.

About 7% of water is strongly held via H-bonding probably in the crystalline region, while the remainder is present as more loosely bound surface water.

*Sorption mechanism:

1) Partition with bound water acting as the stationary phase. 2) Affinity for more polar molecules due to hydrophilic surface

of hydroxyl groups. 3) Ion-exchange effects due to carboxyl groups.

*Ion-exchange papers:

1) Chemical modification of -OH for separation of cations, amines and amino acids.

2) Treatment with aqueous NaOH followed by chloroacetic acid converts -OH to ethoxy acid -O-CH2-COOH.

3) Other treatments such as phosphoric acid by phosphorous oxychloride.

*Reverse-phase methods:

Impregnating paper with non-aqueous media such as rubber latex, olive oil and silicones, to act as stationary phase.

In normal chromatography, the stationary phase, being aqueous, is more polar than the mobile phase.

In reverse-phase chromatography, mobile phase is more polar.


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Whatman modified cellulose ion-exchange materials

No. Type Name Group Flow rate* Capacity**

P81 S acid Cellulose -O-PO3H 125 18.0 phosphate CM82 W acid Carboxymethyl -CO2H 110 2.5 cellulose DE81 S basic Diethylamino- -C2H4NEt2 95 3.5 ethyl cellulose ET81 W basic Ecteola cellulose tert-amino 125 2.0

*Flow rate: water ascending, mm/30 min; **Capacity: mmol H+/cm2.

*Solvents:

For polar organic substances: Water: Cause those substances to migrate. Butan-1-ol: Has to be saturated with water. Acetic acid: Increases the solubility of amino acids. Ammonia: Increases the solubility of acidic materials.

For hydrophobic stationary phases: Various mixtures of benzene, cyclohexane and chloroform.

*Chromatographic development:

Same as those used in TLC, such as ascending, descending, stepwise, multiple, two-dimensional and radial or development methods.

*Detection: 100-fold less sensitive than TLC. (PC: 10-7 g)

Similar to those used in TLC, such as chemical derivatization (excluding corrosive agents, e.g. H2SO4), physical methods, e.g. UV, fluorescence, densitometer techniques and radiochemical methods.

高等食品分析(advanced food analysis) vi. plane...

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