食品分析
9. Paper and thin-layer chromatography
Chromatography is a method in which the components of a
mixture are separated on an adsorbent column in a
flowing system. —Tswett (1906)
Chromatography: Greek: Chroma (color) + graphein (write)
Classified based on the stationary phase type:
1. Column chromatography: GC, LC, SFC.
2. Plane, planar, plate or two-dimensional chromatography:
a. Paper chromatography (PC).
b. Thin-layer chromatography (TLC).
c. Electrophoresis, electrochromatography (EC)
Stationary phase: Flat, relatively thin layer of material that is
self-supporting or coated on a glass, plastic or metal
surface.
Mobile phase: Liquid, moves through by capillary action,
sometimes assisted by gravity or electrical potential.
Retention factor (Rf): a measure of migration of a component.
Rf = dR/dM
(1)
Where Rf: Vary from 1 to 0 for solutes that are retarded.
(1 > Rf > 0)
dR: Distance of spot from origin.
dM: Distance of solvent front from origin.
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食品分析
9. Paper and thin-layer chromatography
*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 absorbent 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 dissolve 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.
*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.
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食品分析
9. Paper and thin-layer chromatography
*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,
AA’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.
*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.
*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 a 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).
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食品分析
9. Paper and thin-layer chromatography
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 ether, 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.
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.
3) Continuous development: Continuous solvent flow.
The eluant is allowed to wash off the plates.
For resolution of compounds using low polarity solvents.
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食品分析
9. Paper and thin-layer chromatography
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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食品分析
9. Paper and thin-layer chromatography
*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
I2 Vapor
Brown
Black
50% H2SO4
2,7-Fluorescein Yellow-green
Ninhydrin
Pink/purple
2,4-DNP
Orange/red
Antimony chlorides Varied
Bromophenol blue Yellow
or bromocresol green
Phosphomolybdic acid
Diphenylcarbazide Varied
Component detected
General organic, unsaturated
Organic
Most organic
AA’s and amines
Ketones and aldehydes
Steroids, alicyclic vit’s,
carotenoids
Carboxylic acids
Reducible compounds
Metals
After spraying, it is necessary to heat the plate to accelerate
chemical reaction between reagent and components.
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食品分析
9. Paper and thin-layer chromatography
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.
UV light on plate with
Spraying visualization
fluorescence indicator
Using scanning densitometer to measure the radiation emitted
from the spot by fluorescence or reflection.
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食品分析
9. Paper and thin-layer chromatography
*Documentation: Plate chromatograms should be recorded
using photograph or photocopy.
*Paper chromatography (PC)
Papers: Standard filter papers made from cotton cellulose,
composed of β(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.
Characteristic of Whatman papers: fast: Nos. 4, 54, 540;
medium, Nos. 1, 7; and slow: Nos. 2, 20.
*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.
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食品分析
9. Paper and thin-layer chromatography
*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.
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 times less sensitive than TLC.
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.
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