Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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Detection

0039 Detection is generally carried out by measuring the

ultraviolet or visible absorbance at the end of the

capillary. Other detection systems such as laser-

induced fluorescence, mass spectrometry, and con-

ductivity detection have also been employed.

Fundamentals

0040 The basic phenomena in capillary electrophoresis are

electroosmotic flow (EOF) and electrophoretic flow

). Electroosmotic flow is bulk flow of a solvent in

a capillary under an applied potential. Electrophor-

etic flow is the flow of ions due to a certain charge,

with cations being attracted to the cathode and

anions being attracted to the anode.

0041 The magnitude of the EOF can expressed by

EOF

¼ðezÞ=, ð9Þ

where m

EOF

¼ EOF mobility; e ¼ dielectric constant;

z ¼zeta potential; Z ¼bulk viscosity.

0042According to eqn (9), the mobility is independent

of the applied electric field. The zeta potential is

essentially determined by the surface charge on the

capillary wall. Since it is strongly pH-dependent, the

magnitude of the EOF varies with pH. Depending on

the specific conditions, the EOF can vary by more

than an order of magnitude between pH 2 and 12.

The zeta potential is also dependent on the ionic

strength of the buffer.

0043The separation parameters of capillary electro-

phoresis can be described in similar terms to those

for GC and HPLC.

0044The time required for a solute to migrate to the

point of detection is called the migration time, and is

given by the quotient of migration and velocity. The

migration time and other experimental parameters can

be used to calculate the apparent solute mobility, using

¼ 1=tE ¼ 1L=tV ð10Þ

and

¼ m

þ m

EOF

, ð11Þ

(a)

Capillary zone electrophoresis (CZE)

(b)

Capillary gel electrophoresis

(c)

Micellar electrokinetic chromotography (MEKC)

(d)

Capillary isotachophoresis

(e)

Capillary isoelectric focusing

t = 0

t = t'

t = 2t'

t = 0

t = t'

t = 2t'

−

+ V

−

C H

/(1

)]V

fig0009 Figure 9 Different modes of capillary electrophoresis. In (a), V

is the electromigration velocity, and V

is the electroosmotic

velocity. In (b), there are obstructing strands of gel, and large ions move more slowly through the pores than the small ions. In (c), V

the electromigration velocity of the solute, V

is the electroosmotic velocity, V

is the migration velocity of the micelle, and K

is the

phase capacity ratio. In (d), L is the leading electrolyte, and T is the terminating electrolyte.

1266 CHROMATOGRAPHY/Principles

where l is the effective capillary length (to the de-

tector), L is the total length, t is the migration time,

and E is the electric field. In the presence of EOF, the

measured mobility is called the apparent mobility

). The effective mobility (m

) can be calculated

from the apparent mobility by independently measur-

ing the EOF using a neutral marker that moves at a

velocity equal to the EOF.

See also: Chromatography: Thin-layer Chromatography;

High-performance Liquid Chromatography; Gas

Chromatography; Combined Chromatography and Mass

Spectrometry

Further Reading

Cserha

ti T and Forga

cs E (1999) Chromatography in Food

Science and Technology. Lancaster, PA: Technomic.

Forga

cs E and Cserha

ti T (1997) Molecular Basis of Chro-

matographic Separation. Boca Raton, FL: CRC Press.

Grob RL (1995) Modern Practice of Gas Chromatography.

New York: John Wiley.

Grossmann PD and Colburn JC (eds) (1992) Capillary

Electrophoresis – Theory and Practice. San Diego, CA:

Academic Press.

Hill HH and McMinn DG (1995) Detectors for Capillary

Chromatography. New York: John Wiley.

Jennings W (1987) Analytical Gas Chromatography.

Orlando, FL: Academic Press.

Jinno K (1992) Hyphenated Techniques in SFC and Extrac-

tion. New York: Elsevier Science.

Li SFY (1992) Capillary Electrophoresis – Principles, Prac-

tice and Applications, Journal of Chromatography

Library. Amsterdam: Elsevier Scientific.

Maguire KL and Denyszyn RB (1985) In: White CM (ed.)

Modern Supercritical Fluid Chromatography, p. 45.

New York: Springer.

Sherma J and Fried B (eds) (1991) Handbook of Thin Layer

Chromatography, vol. 55. New York: Marcel Dekker.

Snyder LR (1968) Principles of Adsorption Chromatog-

raphy. New York: Marcel Dekker.

Snyder LR, Glajch JL and Kirkland JJ (1988) Practical

HPLC Method Development. New York: John Wiley.

Vindevogel J and Sandra P (1992) Introduction to Micellar

Electrokinetic Chromatography. Heidelberg: Hu

rtig.

Thin-layer Chromatography

D R Tocher, University of Stirling, Stirling, UK

Introduction

0001 Thin-layer chromatography (TLC) is a type of liquid

chromatography performed on a stationary phase in

the form of a thin layer usually on a glass, plastic, or

aluminum support. A solution of sample is applied as

a small spot or narrow band to the thin layer of

adsorbent that has been spread uniformly over the

support. The mobile phase (a solvent mixture) then

passes through the adsorbent by capillary action, and

the sample is resolved into discrete components. The

separated components are then located by either

physical methods or chemical staining reagents after

the solvent has evaporated. Quantitation by methods

such as densitometry may also then be possible,

depending upon the precise conditions utilized.

Although high-performance liquid chromatography

(HPLC) and gas chromatography (GC) are often

regarded as superior, especially with regard to quan-

titation, TLC has many advantages including low

capital costs, simplicity of operation, the availability

of many sensitive and selective reagents for detection

and confirmation without interference of the mobile

phase, and in-system calibration for quantitative

analysis. Therefore, TLC should be regarded as

highly complementary to the other techniques and

particularly suited to low-cost screening of many

samples.

Apparatus

0002TLC is very cost-effective as the basic apparatus

required is simple and relatively cheap, and many

samples can be analyzed on a single plate with low

solvent usage. However, more sophisticated and

expensive apparatus are available particularly for

automated sample application and quantitation.

Adsorbents and Supports

0003The predominant adsorbent used in TLC is silicic

acid, a partially hydrated silicon dioxide, often

termed silica gel, but others are also available, includ-

ing cellulose, aluminum oxide, celite (kieselguhr),

magnesium oxide, and zeolites. Modified versions

of these include C

-orC

- (reverse-phase) and

3-aminopropyl-silica gel, polyamide- and polyethyl-

ene imine (PEI)-cellulose. The particle size of the

adsorbents is usually in the range 1–25 mm.

0004TLC plates were often made in the laboratory

using specific apparatus. However, home-made plates

could be variable, and commercially produced pre-

coated plates, which are available in various sizes and

supports with almost every adsorbent, are now the

first choice due to their much greater reproducibility

and consistent quality. Adherence of the adsorbent

to the support is often enhanced in precoated plates

by the inclusion of binders such as calcium phos-

phate (10% w/w) or organic binders like poly-

acrylic acid.

CHROMATOGRAPHY/Thin-layer Chromatography 1267

0005 Silica gel is the most commonly employed adsorb-

ent in TLC, particularly that with a pore size of 6 nm.

Precoated analytical plates frequently use silica gel of

particle size 10–12 mm coated in a layer of 0.25 mm

thickness. Plates coated with this type of silica pro-

vide 1000–2000 theoretical plates per 5 cm of migra-

tion. Modern high-performance TLC (HPTLC) uses

silica gel of particle size 5–7 mm in layers of 0.20 mm

thickness. The reduced particle size provides 5000–

10 000 theoretical plates per 5 cm of migration dis-

tance and increases the separation efficiency. In com-

parison with conventional TLC, HPTLC is a more

efficient, instrumentalized, and quantitative method.

The narrower particle size distribution, thinner layers,

and shorter development distances of HPTLC lead to

a greater separation efficiency, faster separations, and

enhanced sensitivity through improved detection

limits such that quantitative HPTLC can produce

results comparable with GC and HPLC when opti-

mally performed. HPTLC plates cannot be prepared

easily in the laboratory and are usually purchased

ready-made.

0006 The adsorption properties of silica gel can be

modified by impregnation with various complexing

reagents, including silver nitrate, urea, boric acid,

oxalic acid, and ethylenediaminetetraacetic acid

(EDTA) to aid the separation of particular classes of

compounds. Reverse-phase plates can be produced by

using silanized silica gel in which the surface silanol

groups of the silica are silylated with chlorosilanes.

Silanized silica has a low carrying capacity that can be

increased by impregnating the silanized silica plates

with hexadecane or paraffin oil. It is now more

common to utilize ready-made reverse-phase plates

with C

,orC

hydrocarbon chains, or diphenyl

groups chemically bonded to the silica. Aminopropyl

bonded silica gel plates are also available. Kieselguhr

or celite, a diatomous earth, is used less often than

synthetic silica gel on account of its natural variabil-

ity. Other adsorbents include cellulose and its deriva-

tives, such as diethylaminoethyl (DEAE)-cellulose for

ion-exchange chromatography, magnesium oxide,

and alumina (aluminum hydroxide), which is manu-

factured in three types, acidic, basic, and neutral,

according to its pH.

0007Glass is the support employed most commonly for

adsorbents on grounds of rigidity, flatness, and inert-

ness. Layers of adsorbents can also be obtained pre-

coated on flexible aluminum or plastic (polyester)

sheets, which have the advantage of being able to be

cut to smaller size, but care must be taken in insuring

their compatibility with solvent systems and subse-

quent detection reagents. The standard sizes of TLC

and HPTLC plates are 20 20 cm and 10 10 cm,

respectively, although other sizes such as 10 20 cm

are available (Figure 1).

Sample Applicators and Development Chambers

0008The application of the sample solution to the adsorb-

ent layer is usually performed manually using micro-

syringes or disposable glass capillaries. However, a

wide range of instruments, including microproces-

sor-controlled instruments, are available for the

automated application of samples either as spots or

streaks. Rectangular glass chambers with lids are

usually employed for the linear development of

chromatograms in solvent mixtures. Sizes are avail-

able for standard 20 20 cm plates and 10 10

fig0001 Figure 1 Typical apparatus used in TLC. (A) Applicator with micrometer-controlled syringe containing 10  20 cm HPTLC plate;

(B) drying/storage rack containing standard 20  20 cm TLC plates; (C)10 10 cm HPTLC plates in drying/storage rack; (D) atomizer

for spraying plates; (E)20 20 cm TLC plate being developed in a standard chamber; (F)10 10 cm HPTLC plate being developed in

HPTLC chamber; (G) glass microsyringe; (H) sample application template/R

calculator overlayed on the standard plate; (I) HPTLC

plate contained in the sample application template.

1268 CHROMATOGRAPHY/Thin-layer Chromatography

HPTLC plates. Chambers can be purchased with

vertical grooves in the side and end walls to allow

the simultaneous development of several plates

(Figure 1).

Equipment for Detection and Quantification of

Components

0009 Atomizers operated by compressed air or hand

bellows are required for spraying developed chro-

matograms evenly with detection reagents. An oven

is necessary for heating plates sprayed with certain

reagents. For the quantitation of separated compon-

ents on developed chromatograms, modern computer-

controlled scanning densitometers are available along

with sophisticated software packages for evaluation

and data storage. Densitometry is particularly useful

for one-dimensional chromatography, but programs

can be adapted for two-dimensional chromatograms.

The location and quantitation of radiolabelled com-

pounds can be achieved using specialized radioactiv-

ity detectors that can scan the whole chromatogram,

although these instruments are rather expensive.

However, less elaborate equipment is required for

the location of radiolabelled compounds on chro-

matograms by autoradiography. Fluorography can

be performed with similar equipment. New detection

apparatus that may prove useful include the charge

coupled device array detector for HPTLC and

matrix-assisted laser desorption/ionization mass

spectrometry.

Specialized Systems

0010 Several techniques that rely on the same chromato-

graphic principle as conventional TLC, but which

require the use of specialized equipment, have been

developed. The most durable of these specialized

systems is probably TLC with flame ionization detec-

tion (TLC–FID), which was developed to ally the

quantitative benefits of FID to TLC. In the ‘Iatroscan’

system, samples are applied to silica gel fused to

thin quartz rods, which are then developed conven-

tionally in mobile phase. After drying, the developed

‘chromarods’ are passed through a flame ionization

detector to determine the mass of individual compon-

ents by quantifying the amount of ionizable carbon.

The ‘Iatroscan’ has been relatively successful, particu-

larly in certain areas such as lipid metabolism.

Another reasonably successful system is automated

multiple development (AMD), which permits

the multiple development of TLC plates in the same

or different solvent systems. This repeated develop-

ment of TLC plates can improve the resolution of

components.

0011 Other specialized systems have not had such a

durable or widespread appeal but have had limited

use. In over-pressured TLC (OPTLC), the TLC plate

is held beneath a flexible membrane under hydro-

static pressure, and solvent is forced through the ad-

sorbent using a pump. This system allows compounds

to be eluted completely from the TLC plate for col-

lection or detection using detectors normally associ-

ated with HPLC. Centrifugal TLC also involves the

forced flow of solvent. In this case, solvent is fed into

the center of a rapidly rotating TLC or HPTLC plate

and is forced through the adsorbent by centrifugal

force. In radial-development TLC, the sample is ap-

plied to the center of a plate and the solvent supplied

through a hole in the plate via a wick that dips into

a solvent reservoir. Another system allowed the

application of solvent under pressure to HPTLC

plates allowing very short development times. (See

Chromatography: High-performance Liquid Chro-

matography.)

Methodology

Sample Application

0012In conventional TLC and HPTLC, the sample is

applied as a small volume of solution on to the

adsorbent near the bottom of the plate. The point

of application is termed the origin. A maximum of

about 80 or 10 mg for TLC and HPTLC, respectively,

can be applied as a spot on adsorbent layers of silica

gel G 0.20–0.25 mm thick. Samples can also be ap-

plied as a narrow streak along the origin. Standards

are applied as separate spots on the same plate.

Development

0013The choice of solvent system depends on the compon-

ents to be resolved and the type of adsorbent utilized

(see Table 1). Regardless of the components, in

conventional TLC, the solvent mixture is placed in

the development chamber. In the case of solvent

systems containing large proportions of polar solv-

ents, the chamber can be lined with filter paper to aid

saturation of the atmosphere. The plate is placed

within the chamber so that the bottom edge is im-

mersed in the solvent mixture, and the chamber is

sealed with a lid (Figure 1). When the solvent has

migrated to within 1 cm of the top edge of the plate,

the plate is removed from the chamber. Although

ascending chromatography, as described above, is

the most common form of TLC, chromatograms can

also be developed by descending chromatography

using a wick to feed the solvent on to the adsorbent

layer.

0014TLC plates can also be subjected to multiple

development in which the plate is developed par-

tially in one solvent system and removed from the

CHROMATOGRAPHY/Thin-layer Chromatography 1269

tbl0001 Table 1 Substances separated by selected one-dimensional TLC systems

AdsorbentSubstancesseparatedSolventsystem

Detectionreagent

Alumina Alkaloids CHCl

Dragendorff reagent

Amino acids (dicarboxylic) 2

M HAc Ninhydrin

Aromatic hydrocarbons CCl

10% tetracyanoethylene in C

Carotenes Hexane/C

/EtOH (100:100:1) None required

Food dyes (fat-soluble) Hexane/EtAc (98:2) None required

Food dyes (water-soluble) H

O/EtOH/n-BuOH (1:1:5) None required

Neutral lipids Hexane/Et

O/HAc (94.5:5:0.5) 0.05% 2

dichlorofluorescein in

MeOH

Sterols C

/EtOH (95:2) 10% phosphotungstic acid in 90%

EtOH

Steroids CHCl

/EtOH (99:1) Anisaldehyde/H

/HAc

(1:2:100), UV

Vitamins (fat-soluble) Toluene 1.3% FeCl

in 2 M HCl/0.7%

FeCN

(1:1)

Cellulose Amino acids n-BuOH/Me

CO/Et

NH/H

(10:10:2:5)

Ninhydrin

Antibiotics (water-soluble) PrOH/pyridine/HAc/H

(15:10:3:12)

Ninhydrin

Food dyes (fat-soluble) n-PrOH/EtAc/H

O (6:1:3) None required

Nucleotides n-BuOH/Me

CO/HAc/5% NH

OH/

O (4.5:1.5:1:1:2)

20% SbCl

in CCl

Nucleosides and free bases H

O 20% SbCl

in CCl

DEAE cellulose Amino acids n-BuOH/HAc/H

O (4:1:5) Ninhydrin

Nucleotides Isobutyric acid/NH

OH/H

(33:1:16)

20% SbCl

in CCl

PEI cellulose Nucleotides 0.25, 1.0, 1.6 M LiCl in H

O (in

successive developments)

20% SbCl

in CCl

Celite (Kieselguhr) Sugars EtAc/iso-PrOH/H

O (130:57:23) 0.2% naphthoresorcinol in EtOH/

10% H

Oligosaccharides Iso-PrOH/EtAc (65:35) 0.2% naphthoresorcinol in EtOH/

10% H

Food dyes (fat-soluble) Cyclohexane None required

Magnesium oxide Carotenoids Pet. ether/C

(3:1) or pet. ether/

CO (97:3)

None required

Polyamine Anthocyanins HAc/HCl/H

O (10:1:3) 10% oxalic acid in Me

CO/H

(1:1), UV

Antioxidants MeOH/Me

CO/H

O (6:1:3) 10% phosphomolybdic acid in

EtOH

Flavonoids Me

CO/95% EtOH/H

O (2:1:2) 25% PbAc in basic aqueous

solution

Sephadex Proteins 0.5 M NaCl 1% naphthalene black in MeOH/

O/HAc (5:4:1)

Silica gel Alkaloids CHCl

/Me

CO/Et

NH (5:4:1) Dragendorf reagent

Amino acids n-BuOH/HAc/H

O (4:1:1) Ninhydrin

Antibiotics

Macrolides CHCl

/MeOH/H

O (80:20:2.5) 20% phosphomolybdic acid in

EtOH

Penicillins Me

CO/MeOH (1:1) 20% phosphomolybdic acid in

EtOH

Streptomycins H

O/Na citrate/citric acid

(100:20:5)

20% phosphomolybdic acid in

EtOH

Tetracyclines BuOH/HAc/H

O (2:1:1) 20% phosphomolybdic acid in

EtOH

Antioxidants CHCl

20% phosphomolybdic acid in

EtOH

Bile acids Me

pentane/iso-PrOH/HAc

(60:20:0.5)

5% phosphomolybdic acid in

EtOH/Et

O (1:1)

Food dyes (fat-soluble) Hexane/Et

O/HAc (70:30:1) None required

Continued

1270 CHROMATOGRAPHY/Thin-layer Chromatography

chamber. After evaporation of solvent from the

developed plate, the plate is developed fully or

partially in another solvent system. Multiple devel-

opments enable improved resolution of compon-

ents. Development of the chromatogram with the

mobile phase in one direction only is known

as one-dimensional TLC (Figure 2). In instances

where one-dimensional TLC/HPTLC does not

allow the complete resolution of all the compon-

ents of a mixture, improved separation can be

achieved with two-dimensional TLC/HPTLC. In this

technique, the sample is applied as a spot at one

corner and developed fully in one direction in the

first solvent system. The plate is removed from

the chamber and, after evaporation of the solvent, is

then developed in a second solvent system in a

direction at 90



to that of the first development

(Figure 3).

Identification of Separated Components

0015Colored compounds are visible on developed chro-

matograms, but colorless compounds require detec-

tion by physical or chemical means. Detection by

chemical means usually involves spraying the adsorb-

ent layer of the developed chromatogram with a deri-

vatizing reagent that reacts with the separated

components to produce a colored derivative in situ.

Nonspecific reagents such as sulfuric acid and iodine

will detect a wide range of compounds. Specific re-

agents react with specific functional groups and will

AdsorbentSubstancesseparatedSolventsystem

Detectionreagent

Food dyes (water-soluble) HAc/iso-BuOH/H

O (2:5:2) None required

Gangliosides CHCl

/MeOH/0.3% KCl (30:18:4)

to purify CHCl

/MeOH/0.25%

CaCl

(55:45:10) to resolve

classes

0.5% orcinol in 20% sulfuric acid

0.5% orcinol in 20% sulfuric

acid

Insecticide (Imidacloprid) CHCl

/Me

CO/MeOH (23:1:1) UV

Lipids (neutral) Hexane/Et

O/HAc (80:20:2) 3% CuAc in 8% H

Lipids (polar) MeAc/iso-PrOH/CHCl

/MeOH/

0.25% aq. KCl (25:25:25:10:9)

3% CuAc in 8% H

Lipopolysaccharides iso-PrOH/H

O/CHCl

/NH

NH (120:60:16:4:1)

0.1% orcinol in 30% aq. MeOH

Mycotoxins CHCl

/Me

CO (90:10) p-Anisaldehyde/MeOH/HAc/

(0.5:70:10:5)

Opiates (acetate or methoximine

derivatives)

/iso-PrOH (88:12) þ 1.5%

Conc. Sulfuric acid

Plasticizers CH

4 M H

/20% resorcinol (1:1)

Mono-, di-, and trisaccharides MeCN/H

O (85:15) 0.5% KMnO

Polysaccharides n-BuOH/MeOH/H

O (50:25:20) 0.5% KMnO

Shellfish toxin (domoic acid) BuOH/HAc/H

O (3:1:1) UV and ninhydrin

Steroids CHCl

/EtOH (92:8) 10% phosphomolybdic acid in

EtOH

Synthetic sweeteners CHCl

/HAc (90:10) 0.2% 2

dichlorofluorescein in

EtOH

Te r p e n e a l c o h ol s CH

0.06% diphenylpicryl hydrazyl in

CHCl

Terpene aldehydes CHCl

10% SbCl

in CCl

Vitamins (fat-soluble) Cyclohexane/EtAc (75:25) UV

Vitamins (water-soluble) HAc/Me

CO/MeOH/C

(5:5:20:70)

3-Aminopropyl silica Oligosaccharides MeCN/10 m

M Me

Ac (3:2) Fluorography after spraying with

4-methylenaphthalene

enhancer

8/18

-silica (reverse-phase) Alkaloids

Antifungals MeOH/H

O (90:10) UV

Carotenoids Pet.ether/MeCN/MeOH (2:4:4) None required

Quinones Me

CO/H

O (19:1) 10% phosphomolybdic acid in

EtOH

Vitamin K CH

/MeOH(70:30) UV

Solvent proportions by volume.

Ac, sub>/sub>CO



; Bu, C

-;Et, C

-; Me, CH

-; Pr, C

-; DEAE cellulose, diethylaminoethyl cellulose; PEI cellulose, polyethylene imine cellulose;

UV, ultraviolet. For health and safety reasons, C

can usually be replaced with toluene, and hexane can be replaced with iso-hexane.

Table 1 Continued

CHROMATOGRAPHY/Thin-layer Chromatography 1271

only detect compounds containing the group, e.g.,

ninhydrin for amino groups. The use of specific re-

agents can be used as an aid to identification and

characterization of components.

0016 Physical methods of location commonly involve

ultraviolet light. Commercial plates are available

coated with adsorbents containing an indicator that

absorbs light at 254 nm and reemits or fluoresces light

at the green end of the spectrum. When separated

on these plates, compounds that absorb ultraviolet

light appear as dark spots on a green fluorescent

background. Plates with adsorbents not containing

fluorescent indicator can be sprayed with 2

dichlorofluorescein with similar results when viewed

under ultraviolet light. Radiolabelled compounds can

be located on TLC plates physically by autoradiog-

raphy or in situ measurement of radioactivity using a

radioscanner.

0017 A useful aid to the identification of separated com-

ponents is the R

value, which is defined as the ratio of

the distance traveled by the compound to the distance

traveled by the solvent. Although the R

value of

a given compound in a defined mobile phase and

adsorbent is very characteristic, many factors,

including the thickness and moisture content of the

adsorbent and development distance, affect the repro-

ducibility of the value. For this reason, the R

value is

only an indication of the identity of a compound, and

confirmation of identity should be obtained by other

means. Identification of separated components can be

aided by comparison with authentic standards run

alongside the samples, if such standards are available,

and by staining patterns to different reagents.

Quantification of Separated Components

0018Once located, it is often useful and necessary to quan-

tify the components in a mixture, and this has been

the most difficult aspect of TLC. However, various

methods can be employed for the quantitation of

separated components.

0019Areas of adsorbent that contain components can be

scraped from the support and the compounds eluted

from the adsorbent with suitable solvents. The

recovered compounds can be estimated gravimetri-

cally after evaporation of the solvent, or by using a

FAME

TAG

FFA

DAG

MAG

FAI

Pigm-

MGDG

DGDG

SQDG

LPC

123

SF2

FFA

FAI

MAG

SF1

LPC

TAG

Pigm

MGDG

DGDG

SQDG

12 3

fig0002 Figure 2 HPTLC chromatograms of lipid mixtures developed in various systems. (A) Single development in hexane/Et

O/HAc

(80:20:2 by volume): 1, zooplankton total lipid; 2, standard authentic mixture, all based on oleic acid. (B) Single development in MeAc/

iso-PrOH/CHCl

/MeOH/0.25% aqueous KCl (25:25:25:10:9, by volume): 1, cod roe total lipid; 2, rat brain total lipid; 3, algal (Croomonas

salina) total lipid. (C) Double-development with development to half final distance in solvent system of (B) followed by full development

in solvent system of (A): 1, Zooplankton total lipid; 2, rat brain total lipid; 3, algal (C. salina) total lipid. All chromatograms were stained

by charring after spraying with 3% copper acetate in 8% orthophosphoric acid. C, cholesterol; CB, cerebrosides; CE, cholesteryl ester;

DAG, diacylglycerol; DGDG, digalactosyl diacylglycerol; FAI, fatty alcohol; FAME, fatty acid methyl ester; FFA, free fatty acid; GL,

glycolipid; LPC, lyso-phosphatidylcholine; MAG, monoacylglycerol; MGDG, monogalactosyl diacylglycerol; NL, neutral lipid; O, origin;

PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; Pigm, pigment; PL, polar

lipid; PS, phosphatidylserine; SF, solvent front; SM, sphingomyelin; SQDG, sulfoquinovosyldiacylglycerol; St, sterol; TAG, triacylgly-

cerol; U, unknown; WE, wax ester.

1272 CHROMATOGRAPHY/Thin-layer Chromatography

specific assay such as the measurement of phosphorus

or sugar content.

0020 Developed chromatograms can be subjected to

scanning densitometry and the separated components

quantified on the basis of transmittance, reflectance

intensity, or fluorescence, depending on the nature of

the compounds and the detection reagent/stain used.

Modern techniques of imaging in conjunction with

sophisticated computer software packages for image

analysis and data processing have increased the accur-

acy of these techniques. As well as locating radio-

labeled compounds, radioscanners can also quantify

the amount of radioactivity present in individual com-

ponents. Alternatively, bands of adsorbent that con-

tain compounds can be scraped from the support into

scintillation vials and measured directly for radio-

active content by liquid scintillation counting after

addition of suitable scintillation fluid.

Preparative TLC

0021 Although TLC is mostly used as an analytical tech-

nique, one-dimensional TLC can be scaled up to a

preparative scale. To this end, sample can be applied

as a streak across the origin of an analytical plate, the

length of the streak being determined by the amount

of sample. Special preparative TLC plates coated with

thicker layers of adsorbent can be obtained, which

can accommodate relatively large amounts of sample.

However, these plates lack the resolving power of the

analytical plates. After nondestructive visualization

of the separated components, the bands of adsorbent

containing the compound or compounds of interest

are scraped from the support and eluted with suitable

solvents.

Modes of Chromatography

0022TLC conforms to the basic principles of liquid chro-

matography. The most common mode of chromatog-

raphy used in TLC is adsorption chromatography,

although other modes of chromatography can be

employed. With silica, celite, keiselghur, and cellu-

lose, the mechanism is by adsorption chromatog-

raphy if the adsorbent on the plate is completely free

of water, and the solvent system is a nonpolar mix-

ture. However, if water is present on the adsorbent or

if the mobile phase contains a highly polar solvent,

then separation will be by partition chromatography.

In addition, reverse-phase TLC plates also separate

by partition chromatography. TLC plates are also

available precoated with reverse-phase silica gel that

has been impregnated with a chiral reagent and

copper ions. These can be used to separate optically

active isomers, e.g., amino acids, by chiral chroma-

tography on the basis of ligand exchange.

0023Alumina separates components by adsorption but,

depending upon the nature of the surface and the

solvent system, can also function as an ion exchanger.

SF1

SF2

SF1

SF2

MGDG

DGDG

SQDG

fig0003 Figure 3 Two-dimensional HPTLC of total lipid from (A) rat brain and (B) the alga C. salina. Plates were first developed in direction

1 with MeAc/iso-PrOH/CHCl

/MeOH/0.25% aqueous KCl (25:25:25:10:9, by volume) and then in direction 2 with CHCl

/MeOH/7M

OH (65:35:5, by volume). Developed chromatograms were stained by charring after spraying with 3% copper acetate in 8%

orthophosphoricacid.Abbreviations:asin Figure 2.

CHROMATOGRAPHY/Thin-layer Chromatography 1273

Modified cellulose such as DEAE-cellulose can be

used for ion-exchange separations on TLC. Separ-

ation by size-exclusion chromatography can be

carried out with TLC plates coated with Sephadex

gel but is slower and significantly more difficult

than other forms of TLC. Polyamides such as

polyhexamethylenediaminoadipate can be used as

the adsorbent to separate components that interact

with it by hydrogen bonding. (See Chromatography:

Principles.)

Applications

0024 The actual method employed for preparing samples

from foodstuffs for analysis by TLC depends on the

nature of the substances being investigated. Many

methods involve the extraction of the food with a

suitable solvent followed by precipitation and filtra-

tion steps to remove classes of compounds that are

not of interest. (See Analysis of Food.)

0025 The choice of adsorbent and solvent system used in

TLC is dictated by the nature of the sample to be

analyzed. Examples of broad groups of substances

that have been successfully resolved by TLC methods

are shown in Table 1, along with suggested solvent

systems and detection reagents commonly used.

However, for most substances, there can be a variety

of different solvent systems and detection reagents

that are suitable, depending on the precise compos-

ition of the samples to be resolved.

See also: Chromatography: Principles; High-

performance Liquid Chromatography; Gas

Chromatography

Further Reading

Ackman RG (1982) Flame ionization detection applied to

thin-layer chromatography on coated quartz rods.

Methods in Enzymology 72: 205–252.

Henderson RJ and Tocher DR (1992) Thin-layer chroma-

tography. In: Hamilton RJ and Hamilton S (eds) Lipid

Analysis: A Practical Approach, pp. 65–111. Oxford:

Oxford University Press.

Jork J, Funk G, Fischer G and Wimmer CJ (1989) Thin-

layer Chromatography: Reagents and Detection

Methods. Cambridge: VCH.

Kirchner JG (1978) Thin-layer Chromatography. Tech-

niques of Chemistry, vol. XIV. New York: Wiley.

Sherma J (1996) Thin layer chromatography in food

analysis. Food Testing and Analysis 2: 12–15.

Touchstone JC (1988) Instrumentation for thin-layer chro-

matography: a review. Journal of Chromatographic

Science 26: 645–649.

Wilson ID (1996) Thin layer chromatography: a neglected

technique. Therapeutic Drug Monitoring 18: 484–492.

High-performance Liquid

Chromatography

M V Moreno-Arribas and M C Polo, Instituto de

Fermentaciones Industriales (CSIC), Madrid, Spain

Introduction

0001High-performance liquid chromatography (HPLC) is

an instrumental form of liquid chromatography that

employs stationary phases consisting of small par-

ticles, thereby achieving more efficient separations

than those used in conventional liquid chromatog-

raphy. Since its origin in the late 1960s, it has been

known by several different names, including high-

pressure liquid chromatography, because of the

high pressures required to force the mobile phase or

solvent through the stationary phase, and high-

resolution liquid chromatography, because of the

good resolution achieved using this technique.

0002This article describes the equipment needed to

carry out HPLC and summarizes some of the app-

lications of this technique in food analysis. Two

techniques related to HPLC, fast-protein liquid chro-

matography (FPLC) and supercritical fluid chroma-

tography (SFC), are also considered. The basic theory

of the chromatographic process and the factors

that affect separation efficiency are discussed in

the section on Chromatography. (See Chromatog-

raphy: Principles; Thin-layer Chromatography;

High-performance Liquid Chromatography; Gas

Chromatography; Supercritical Fluid Chromatog-

raphy; Combined Chromatography and Mass Spec-

trometry.)

Separation Modes

0003There are many different stationary and mobile

phases that can be used in HPLC, and for this reason

there is a great variety of separation mechanisms.

The treatises on HPLC use different criteria in their

attempt to classify the modes of this technique:

type of stationary phase, predominant separation

mechanism, type of groups of compounds it is

aimed at. These criteria sometimes overlap; that is

to say, it is possible to work in several different chro-

matographic methods with the same stationary

phase, and the same group of compounds can be

separated using several different stationary phases,

and for this reason it is not easy to classify HPLC

techniques into specific groups. Generally speaking,

it can be said that there are three large groups

1274 CHROMATOGRAPHY/High-performance Liquid Chromatography

that dominate the field of chromatographic separ-

ations: reversed-phase chromatography, ion exchange

chromatography and size exclusion chromatography.

There are also other types of chromatography that

are rapidly gaining in importance and cannot be in-

cluded exactly in any of these three groups: ion pair

chromatography, affinity chromatography, and chiral

separations.

Reversed-Phase Chromatography

0004 This is the most commonly used method for chroma-

tographic separations, so it could in fact be called

‘normal-phase’ except that it was developed later

than the latter. In reversed-phase chromatography,

the stationary phase is of a nonpolar nature and the

mobile phase is more polar than the stationary one.

Among the most widely used stationary phases are

silica-based ones with cyano, phenyl, or alkyl func-

tionality and those of a polymeric nature, above all

polystyrene-divinylbenzene (PSDVB). The mobile

phase is usually water with an organic modifier

(methanol, acetonitrile, or tetrahydrofuran). Ternary

or quaternary mixtures of these components can be

used to improve selectivity.

0005 There is controversy over the mechanism that

controls reversed-phase separations. The three basic

models are the partitioning model, the solvophobic

effect, and the adsorption model. It is also necessary

to consider the possibility that a mixed mechanism

might exist.

0006 In the partitioning mechanism it is accepted that

the retention of a solute in reversed-phase liquid

chromatography is due to its partitioning between

the mobile phase and the stationary phase. This

mechanism assumes that the stationary phase behaves

in a similar way to a liquid, a situation that may occur

in certain conditions, such as when working with

monomeric phases and high percentages of water in

the mobile phase, and the size of the hydrocarbonated

chains is large enough for them to be associated. The

adsorption mechanism accepts that retention takes

place through the interaction of the solute to the

organic molecules bonded to the surface of the

packing matrix. The solvophobic effect theory

accepts that the solute is expelled from the mobile

phase towards the stationary phase.

Ion Exchange Chromatography

0007 This is used for the analysis of electrically charged

substances in working conditions. Stationary phases

with electrically charged functional groups are used

for this method of chromatography. The support can

be silica or a polymer. The mobile phase contains a

buffer which controls the loading of the solutes, and a

salt that competes with the solutes in their interaction

with the charges of the stationary phase. The charges

permanently bonded to the chromatographic support

are compensated by counterions, that is to say, by free

ions with the opposite charge. Passage through the

column of a sample or of the mobile phase with ions

of the same sign as the counterions of the column may

result in the displacement of the counterions and the

retention of the ions of the sample or of the stationary

phase.

0008The ion exchangers can be classified as cationics

and anionics. Within each group it is possible to

differentiate strong and weak exchangers. Strong

cation exchangers (SCX) usually contain sulfonic

groups, while weak ones have carboxylic groups.

Quaternary ammonium salts are the most usual func-

tional groups in strong anion exchangers (SAX), and

primary amines are used in weak anion exchangers.

Size Exclusion Chromatography

0009This separation method is based on the size of the

molecules. For many years it was known as gel

permeation chromatography (GPC) or gel filtration

chromatography (GFC). The retention of the mol-

ecules depends on their size: the larger molecules

that do not enter the pores of the packing elute first,

while the smaller molecules, whose diameter makes it

possible for them to enter and exit the pores of the

chromatographic packing, take a longer time.

Ion Pair Chromatography

0010This chromatographic method enables the separation

of solutes that are ionized in separation conditions.

On most occasions the separations are carried out on

the same columns as those used in reversed-phase

separations without the formation of ion pairs. The

mobile phase is made up of a water buffer and an

organic modifier, to which mixture is added a coun-

terion with an opposite charge to that of the solute.

Quaternary ammonium salts are often used as

counterions for the analysis of anion substances,

while n-alkyl sulfate salts are among the most com-

monly used for the analysis of cation substances.

Affinity Chromatography

0011This is based on the specific interaction between a

molecule, or group of molecules, and a ligand,

which is a molecule that is attached to the station-

ary phase to interact with those of the solute. The

growing importance of affinity chromatography is

due to the development of bioaffinity chromatog-

raphy in which the interactions between ligands and

molecules are based on recognitions of a biological

type.

CHROMATOGRAPHY/High-performance Liquid Chromatography 1275