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Two‐Dimensional Thin‐Layer ChromatographySimion Gocan aa Department of Analytical Chemistry Babescedil‐Bolyai University Cluj‐Napoca RomaniaPublished online 22 Aug 2007

To cite this article Simion Gocan (2005) Two‐Dimensional Thin‐Layer Chromatography Journal of Liquid Chromatographyamp Related Technologies 276 1105-1113 DOI 101081JLC-120030182

To link to this article httpdxdoiorg101081JLC-120030182

PLEASE SCROLL DOWN FOR ARTICLE

Taylor amp Francis makes every effort to ensure the accuracy of all the information (the ldquoContentrdquo) containedin the publications on our platform However Taylor amp Francis our agents and our licensors make norepresentations or warranties whatsoever as to the accuracy completeness or suitability for any purpose ofthe Content Any opinions and views expressed in this publication are the opinions and views of the authorsand are not the views of or endorsed by Taylor amp Francis The accuracy of the Content should not be reliedupon and should be independently verified with primary sources of information Taylor and Francis shallnot be liable for any losses actions claims proceedings demands costs expenses damages and otherliabilities whatsoever or howsoever caused arising directly or indirectly in connection with in relation to orarising out of the use of the Content

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Two-Dimensional Thin-LayerChromatography

Simion Gocan

Department of Analytical Chemistry Babes-Bolyai University

Cluj-Napoca Romania

ABSTRACT

The two-dimensional thin-layer chromatography (2D TLC) technique is

one of the more versatile methods of TLC development The first

application of the two-dimensional chromatographic method to paper

chromatography was reported in 1944 by Consden Gordon and Martin

[Nyiredy Sz Dallenbach-Tolke K Sticher O J Planar Chromatogr

1988 1 336] Since that time this method has been mostly used for the

separation of a large number of compounds that cannot be separated in a

single dimension TLC experiment In 2D TLC separation is on one

surface spread along the entire area of the plate The resolving power of

the 2D TLC method has great application especially in the areas of

1105

DOI 101081JLC-120030182 1082-6076 (Print) 1520-572X (Online)

Copyright 2004 by Marcel Dekker Inc wwwdekkercom

Reprinted from the Encyclopedia of Chromatography ( 2003) Marcel Dekker Inc

URL httpwwwdekkercomservletproductproductidE-ECHRCorrespondence Simion Gocan Department of Analytical Chemistry Babes-Bolyai

University Cluj-Napoca Romania E-mail mgocanxnetro

JOURNAL OF LIQUID CHROMATOGRAPHY amp RELATED TECHNOLOGIESw

Vol 27 No 6 pp 1105ndash1113 2004

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biochemistry biology natural products pharmaceuticals and environ-

mental analysis

Key Words TLC 2D Prisma system UDM AMD Peptides

TWO-DIMENSIONAL DEVELOPMENT

Two-dimensional thin-layer chromatography (2D TLC) is performed by

spotting the sample in one corner of a square thin-layer plate and developing in

the usual manner with the first eluent The chromatographic plate is then

removed from the developing chamber and the solvent is allowed to evapo-

rate from the layer Then the plate is placed in the second eluent so that

development can take place in a second direction which is perpendicular to

that of the first development direction In 2D TLC usually the layer is of

continuous composition but two different eluents must be employed to obtain

a better separation of a mixture The success of the separation will depend

on the ability to modify the selectivity of the second eluent compared to the

selectivity of the first eluent Figure 1 shows the scheme of spot distribution

on a 2D TLC plate following two developments for a theoretical case In 2D

TLC any spot can be identified by a pair of xi and yi coordinates or

respectively Rfi1 and Rfi2 where xi divided by Zf1 is equal to Rfi1 for the first

eluent and yiZf2 is equal Rfi2 for the second eluent The final position of the

spot can be determined by the coordinates (xi yi) in which Rfi2D can be

expressed as (Rfi1 Rfi2)

A very good method for selection of the appropriate mobile phase for 2D

TLC separations is with the use of the ldquoPrismardquo system[1]

The indole group of compounds is conveniently divided into the so-called

ldquosimplerdquo indole derivatives and the indole alkaloids which often have

complicated structures and indole dyes Thus it was demonstrated that not

all compounds are completely separated by either the basic eluent methyl

acetatendashisopropanolndash25 ammonia (45 thorn 35 thorn 20 vv) or the acidic eluent

chloroformndash96 acetic acid (95 thorn 5 vv) Therefore one combines the

effects of both of these eluent systems in the 2D TLC method and in this

way 14 simple indole derivatives and anthranilic acid can be separated

Compounds are separated into groups according to their polarities[2]

The 2D TLC was successfully applied to the separation of amino acids as

early as the beginning of thin-layer chromatography Separation efficiency

is by far best with chloroformndashmethanolndash17 ammonium hydroxide

(40 thorn 40 thorn 20 vv) n-butanolndashglacial acetic acidndashwater (80 thorn 20 thorn 20 vv) in combination with phenolndashwater (75 25 gg)[3] A novel 2D TLC

Gocan1106

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method has been elaborated and found suitable for the chromatographic

identification of 52 amino acids This method is based on three 2D TLC

developments on cellulose (CMN 300 50m) using the same solvent system I

for the first dimension and three different systems (IIndashIV) of suitable

properties for the second dimension System I n-butanolndashacetonendash

diethylaminendashwater (10 10 2 5 vv) system II 2-propanolndashformic

acidndashwater (40 2 10 vv) system III sec-butanolndashmethyl ethyl ketonendash

dicyclohexylaminendashwater (10 10 2 5 vv) and system IV phenolndashwater

(75 25 gg) (thorn75 mg Na-cyanide) with 3 ammonia With this technique

all amino acids can be differentiated and characterized by their fixed posi-

tions and in addition by some color reactions Also the relative merits of

cellulose and silica gel are discussed in relation to separation efficiency

reproducibility and detection sensitivity[4] The 2D TLC separation of a

performic acid oxidized mixture of 20 protein amino acids plus b-alanine and

g-amino-n-butyric acid was performed in the first direction with chloroformndash

methanolndashammonia (17) (40 40 20 vv) and in the second direction with

Figure 1 Scheme of spot distribution on a 2D TLC plate

Two-Dimensional Thin Layer Chromatography 1107

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phenolndashwater (75 25 gg) Detection was performed by means of ninhidrin

reagent spray[5]

The thin-layer chromatographic method was developed for amino acids

and therefore in principle it is equally applicable to peptides Peptides

like amino acids are generally hydrophilic There are however limits to

this analogy Dinitrophenylamino acid derivatives (DNP-amino acids) and

phenylthiohydantoin derivatives (PTH-amino acids) are obtained when reac-

tion of peptides or proteins with dinitrofluorobenzene or phenyl mustards

are properly degraded[6] Their separation from reaction mixtures and their

identification are considerable practical importance because they constitute

essential steps in the process of sequential analysis of peptide and protein

structures Generally the run in the first direction is done in a toluene system

For the run in the second direction there are many eluent systems that may be

selected The 2D TLC was used to perform separation of DNP-amino acids

using toluene as eluent for first direction and chloroformndashbenzylalcoholndash

glacial acetic acid (70 thorn 30 thorn 3 vv)[7] chloroformndashmethanolndashglacial

acetic acid (95 thorn 5 thorn 1 vv) or benzenendashpyridinendashglacial acetic acid

(80 thorn 20 thorn 2 vv) as eluent for the second direction[8] The majority of

DNP-amino acids are colored yellow

The 2D TLC separation of PTH-amino acids may be performed in the

first development with chloroformndashmethanol (90 thorn 10 vv) as eluent and

chloroformndashformic acid (100 thorn 5 vv) as eluent for the second develop-

ment[9] There are many other systems of eluents that can be used to obtain

good resolution The chlorinetolidine reagent is very useful for detection of

PTH-amino acids An alternative is offered by fluorescence quenching

A very complex mononucleotide mixture can be separated by 2D TLC on

poly(ethyleneimine)ndashcellulose anionexchange thin-layers After applying the

nucleotide solution at the starting point the chromatogram is developed in a

closed rectangular jar by stepwise elution with LiCl solution of 02 M 10 M

and finally with 16 M In order to remove LiCl the plate is placed in a flat dish

filled with anhydrous methanol Then the chromatogram is developed in the

second direction with formic acidndashsodium formate buffers pH 3ndash4 by a

stepwise elution with buffers of 05 20 and 40 M The complete resolution

of a model mixture containing DPA TPN six nucleotide sugars and fourteen

common nucleoside-50-mono- di- and triphosphates takes less than 3 hr[10]

The separation and identification of plant phospholipids and glycolipids

was performed by 2D TLC silica gel using chloroformndashmethanolndashwater

(65 25 4 vv) as eluent for the first dimension and diisobutyl ketonendashacetic

acidndashwater (80 50 10 vv) for the second direction[11]

Amino-modified HPTLC silica gel layers (NH2 F254s HPTLC) was used for

identification of 13 amphetamine derivatives by uni-dimensional multiple

development (UDM) in the 2D TLC mode The mobile phases used were

Gocan1108

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ethanolndashtriethylaminendashn-hexane (15 thorn 9 thorn 76 vv) as mobile phase in

the first direction and acetonendashtriethylaminendashn-hexane (23 thorn 9 thorn 68 vv) as

mobile phase in the second direction Because one-dimensional HP-TLC was

not effective UDM with the same solvent system and development distance and

two development steps was investigated The resolution of separation was

higher in the low-hRf compared to that predicted by the UDM theory[12]

In the first direction the development is isocratic and in the second

direction it is automated multiple development (AMD) The instrumental

chromatographic method AMD provides a mean of normal phase (NP)

gradient developed in HPTLC A maximum of 25 steps can be used to form an

AMD gradient The development distances increase as the solvent polarity is

reduced The plates are dried by vacuum between consecutive steps The

bands on the plate will be compressed by repeated developments resulting in

increased sensitivity and resolution For the functional principle of the AMD

device one should consult Ref[13] The developed distance was 8 cm Isocratic

TLC was performed with one typical eluent as mobile phase in normal

chambers previously equilibrated with the mobile phase The AMD system

was performed in a Camag device (Muttenz Switzerland) but the gradient

systems must have a different selectivity than with isocratic developement

The number of compounds separated by this method was greater than with

isocratic TLC or with one-dimensional AMD[14] The use of 2D AMD with

two gradients of different selectivities is a powerful method that can improve

the separation of samples containing an unknown number of constituents in

hydroalcoholic plant extracts[15] The plant extract ldquofingerprintrdquo is better for

authenticity certification of the respectively extract

The 2D TLC is a simple method that makes possible the rapid recognition

of changes in individual components of a mixture For this purpose separations

must be carried out in both directions under exactly identical conditions In

such circumstances a slight increase in resolution might occur because of an

increase by a significant factor of the distance of migration of the zone which is

a diagonal line (Zf2) But this development method indicates alterations of

compounds during chromatography if the resulting spots do not lie on a

diagonal line bisecting the plate The compounds that between the first and

second development suffer several structural transformations under physical

agent (action of radiation) or from chemical reaction can be detected This

method can be used in photochemistry and stability control[3]

TWO-DIMENSIONAL DEVELOPMENT ON BILAYERS

There are many reports concerning the placement of two adsorbents on

the same plate

Two-Dimensional Thin Layer Chromatography 1109

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An apparatus for simultaneously coating a plate with two adjacent layers

of different adsorbents was accomplished by placing a plastic insert into a

commercial spreader thus forming two independent chambers For this

reason there were used combinations of two adsorbents such as cellulose

silica gel alumina charcoal silicic acid magnesium silicate etc as a function

of the sample Then the two eluents systems were optimized for the two

development directions For example two-dimensional separation of some

ketones on a bilayer (charcoalsilicic acid) with benzenendashetherndashacetic acid

(82 9 9 vv) in the first direction (on charcoal) and with benzenendashether

(85 15 vv) in the second direction (on silicic acid)[16] In another paper[17]

the first adsorbent was silica gel (air dried) and the second was deacti-

vated aluminum oxide The same solvent system toluenendashethyl acetate (3 1

vv) was used in the two directions In this condition a mixture of

24-dinitrophenylhydrazine derivatives of hydroxycarbonyl compounds was

resolved

Silver nitrate-impregnated silica gel has also been used successfully in

the separation of some classes of compounds that contain double bonds in

their structures Thus it was used in the separation of glycerides and

isomers of unsaturated fatty acid esters The 2D TLC on adjacent layers of

silica gel G and silica G impregnated with 5 of silver nitrate was used for

development The silica gel G layer covered a surface of 3 cm 20 cm and

the difference of the surface of 17 cm 20 cm was covered with silica gel G

impregnated with 5 AgNO3 The first direction was performed on silica

gel G with petroleum etherndashdiethyl ether (9 1 vv) as eluent The second

direction was done on the impregnated silica gel G and developed twice

with the same eluent as in the first direction After the first direction the

mixture was fractioned into four groups of compounds (trialkyl glyceryl

ethers dialkoxy glycerides alkoxy diglycerides and triglycerides) After

the second direction each group was fractioned for another period into three

or four compounds[18] So it the efficiency of the impregnated layer was

demonstrated

A method for the complete structural analysis of complex mixtures of

methyl esters of saturated and unsaturated fatty acids has been performed by

2D TLC Adsorbent silica gel impregnated with a 10 solution of dodecane

was used for the first direction and acetonitrilendashacetone (1 1 vv) as eluent

The silica gel for the second direction was impregnated with a 20 AgNO3

and dipropyl etherndashhexane (2 3 vv) was used as eluent In this way com-

plete separation of an standard mixture of the methyl esters of six saturated

nine monoethylenic and three polyenic esters were achieved[19]

The 2D TLC on bilayers silica gel for NP and reversed phase (RP) offer

the special possibility to carry out two different separation processes on

the same plate Normal phase chromatography can be performed in the first

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direction on silica gel after the evaporation of the solvent the plate was

impregnated with paraffin oil dissolved in petroleum ether After removal of

the excess solvent the plate was RP developed in the second direction

Another approach may also be performed by transferring the spots from

one plate to another plate[16] In such circumstances the development in the

first direction was carried out on a narrow 2 cm strip After drying it was

clipped face to face to a 20 cm 20 cm or 10 cm 10 cm plate for develop-

ment in the second direction Close contact has to be maintained between the

two layers for a proper development In this case the first plate can be used

with a silica gel layer and the second plate with silica gel RP-C18 or vice-

versa[20] In this way the analysis of saponins in Silene vulgaris Gracke

was done in the first direction on RP-18W HPTLC plates with 1 aqueous

formic acidndashmethanol (30 thorn 70 vv) as mobile phase and in the second

perpendicular direction on silica gel Si 60 HPTLC with chloroformndash

methanolndashformic acidndashwater (100 thorn 40 thorn 10 thorn 10 vv) as mobile phase

By the use 2D TLC bilayers the saponin mixture could be separated into

18 components whereas conventional TLC separated the mixture into only

9 components[21]

TWO-DIMENSIONAL SEPARATION BY

TLCELECTROPHORESIS

Peptide maps of the tryptic digest of myosin have been performed on

thin-layer plates (20 cm 20 cm) by successive TLC and electrophoresis In

the first dimension TLC with chloroformndashmethanolndashammonium hydroxide

34 (40 40 20 vv) as eluent the time was as long as 60 min The second

dimension electrophoresis with a buffer pyridinendashglacial acetic acidndashwater

(1 10 489 vv) current 980 V 30 mA the time was 1 hr The peptide

mixture is applied in amounts of 005ndash05 mg per peptide map[22] In

another paper[23] electrophoresis was applied in one direction on buf-

fered adsorbents followed by chromatography in the second dimension In

this circumstance phosphate esters were separated by TLC development

twice with n-propanolndashammonium hydroxidendashwater (60 30 1 vv) and

electrophoresis was carried out in 028 M acetate buffer (pH 36) 1000 V

35 mA and 16 min

CHROMATOGRAM EVALUATION

The best 2D TLC separation is when all the components are separated and

distributed on the entire surface of the chromatographic plate The estimation

Two-Dimensional Thin Layer Chromatography 1111

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ORDER REPRINTS

of this separation can be made by an objective function Generally a good

agreement between visual evaluation of a chromatogram and computer

evaluation using these objective functions has been noticed[24] On the other

hand a function is needed that can predict Rf value of the one-component

function of the composition from the mobile phase

There are programs for simulated chromatograms which are compar-

able to that obtained with experimental chromatograms[24] Other details

concerning the strategy of optimization of the mobile phase can be found

in Ref[20]

REFERENCES

1 Nyiredy Sz Dallenbach-Tolke K Sticher O J Planar Chromatogr

1988 1 336

2 Stahl E Kaldewey H Hoppe-Seylers Z Physiol Chem 1961 323

182

3 Stahl E Thin-Layer Chromatopgraphy Springer-Verlag 1965

4 von Arx E Neher R J Chromatogr 1963 12 329

5 Hirs CHW J Biol Chem 1956 219 611

6 Biserte G Holleman JW Holleman-Dehove J Sautiere P

J Chromatogr 1959 2 225 J Chromatogr 1960 3 85

7 Brenner M Pataki G Experientia 1961 17 145

8 Brenner M Niederwieser A Experientia 1961 17 237

9 Pataki G Thesis Basel University Basel Switzerland 1962

10 Randerath E Randerath K J Chromatogr 1964 16 126

11 Lepage M J Chromatogr 1964 13 99

12 Fater ZS Tasi G Szabady B Nyiredy Sz J Planar Chromatogr

1998 11 225

13 Cazes J Encyclopedia of Chromatography Cazes J Ed Marcel

Dekker Inc New York 2001 533

14 Muresan L Thesis Babes-Bolyai University Cluj-Napoca 1998

15 Olah N-K Muresan L Cımpan G Gocan S J Planar Chromatogr

1998 11 361

16 Kirchner JG Thin-Layer Chromatography 2nd Ed John Wiley amp Sons

New York 1978

17 Anet EFLJ J Chromatogr 1962 9 291

18 Schmid HHO Baumann WJ Cubero JM Mangold HK Biochem

Biophys Acta 1966 125 189

19 Bergelson D Dyatlovitskaya EV Voronkova VV J Chromatogr

1964 15 191

Gocan1112

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20 Nyredy Sz Ed Planar Chromatography A Retrospective View for the

Third Millennium Springer 2001

21 Glensk M Cisowski W J Planar Chromatogr 2000 13 9

22 Ritschard WJ J Chomatogr 1964 16 327

23 Bieleski RL Anal Biochem 1965 12 230

24 Grinberg N Modern Thin-Layer Chromatography Grinberg N Ed

Marcel Dekker Inc New York 1990

Received October 29 2003

Accepted November 18 2003

Manuscript 6253

Two-Dimensional Thin Layer Chromatography 1113

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All information and materials found in this article including but not limited to text trademarks patents logos graphics and images (the Materials) are the copyrighted works and other forms of intellectual property of Marcel Dekker Inc or its licensors All rights not expressly granted are reserved

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biochemistry biology natural products pharmaceuticals and environ-

mental analysis

Key Words TLC 2D Prisma system UDM AMD Peptides

TWO-DIMENSIONAL DEVELOPMENT

Two-dimensional thin-layer chromatography (2D TLC) is performed by

spotting the sample in one corner of a square thin-layer plate and developing in

the usual manner with the first eluent The chromatographic plate is then

removed from the developing chamber and the solvent is allowed to evapo-

rate from the layer Then the plate is placed in the second eluent so that

development can take place in a second direction which is perpendicular to

that of the first development direction In 2D TLC usually the layer is of

continuous composition but two different eluents must be employed to obtain

a better separation of a mixture The success of the separation will depend

on the ability to modify the selectivity of the second eluent compared to the

selectivity of the first eluent Figure 1 shows the scheme of spot distribution

on a 2D TLC plate following two developments for a theoretical case In 2D

TLC any spot can be identified by a pair of xi and yi coordinates or

respectively Rfi1 and Rfi2 where xi divided by Zf1 is equal to Rfi1 for the first

eluent and yiZf2 is equal Rfi2 for the second eluent The final position of the

spot can be determined by the coordinates (xi yi) in which Rfi2D can be

expressed as (Rfi1 Rfi2)

A very good method for selection of the appropriate mobile phase for 2D

TLC separations is with the use of the ldquoPrismardquo system[1]

The indole group of compounds is conveniently divided into the so-called

ldquosimplerdquo indole derivatives and the indole alkaloids which often have

complicated structures and indole dyes Thus it was demonstrated that not

all compounds are completely separated by either the basic eluent methyl

acetatendashisopropanolndash25 ammonia (45 thorn 35 thorn 20 vv) or the acidic eluent

chloroformndash96 acetic acid (95 thorn 5 vv) Therefore one combines the

effects of both of these eluent systems in the 2D TLC method and in this

way 14 simple indole derivatives and anthranilic acid can be separated

Compounds are separated into groups according to their polarities[2]

The 2D TLC was successfully applied to the separation of amino acids as

early as the beginning of thin-layer chromatography Separation efficiency

is by far best with chloroformndashmethanolndash17 ammonium hydroxide

(40 thorn 40 thorn 20 vv) n-butanolndashglacial acetic acidndashwater (80 thorn 20 thorn 20 vv) in combination with phenolndashwater (75 25 gg)[3] A novel 2D TLC

Gocan1106

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ded

by [

Mos

kow

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te U

niv

Bib

liote

] at

06

16 0

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2013

ORDER REPRINTS

method has been elaborated and found suitable for the chromatographic

identification of 52 amino acids This method is based on three 2D TLC

developments on cellulose (CMN 300 50m) using the same solvent system I

for the first dimension and three different systems (IIndashIV) of suitable

properties for the second dimension System I n-butanolndashacetonendash

diethylaminendashwater (10 10 2 5 vv) system II 2-propanolndashformic

acidndashwater (40 2 10 vv) system III sec-butanolndashmethyl ethyl ketonendash

dicyclohexylaminendashwater (10 10 2 5 vv) and system IV phenolndashwater

(75 25 gg) (thorn75 mg Na-cyanide) with 3 ammonia With this technique

all amino acids can be differentiated and characterized by their fixed posi-

tions and in addition by some color reactions Also the relative merits of

cellulose and silica gel are discussed in relation to separation efficiency

reproducibility and detection sensitivity[4] The 2D TLC separation of a

performic acid oxidized mixture of 20 protein amino acids plus b-alanine and

g-amino-n-butyric acid was performed in the first direction with chloroformndash

methanolndashammonia (17) (40 40 20 vv) and in the second direction with

Figure 1 Scheme of spot distribution on a 2D TLC plate

Two-Dimensional Thin Layer Chromatography 1107

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ded

by [

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te U

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ORDER REPRINTS

phenolndashwater (75 25 gg) Detection was performed by means of ninhidrin

reagent spray[5]

The thin-layer chromatographic method was developed for amino acids

and therefore in principle it is equally applicable to peptides Peptides

like amino acids are generally hydrophilic There are however limits to

this analogy Dinitrophenylamino acid derivatives (DNP-amino acids) and

phenylthiohydantoin derivatives (PTH-amino acids) are obtained when reac-

tion of peptides or proteins with dinitrofluorobenzene or phenyl mustards

are properly degraded[6] Their separation from reaction mixtures and their

identification are considerable practical importance because they constitute

essential steps in the process of sequential analysis of peptide and protein

structures Generally the run in the first direction is done in a toluene system

For the run in the second direction there are many eluent systems that may be

selected The 2D TLC was used to perform separation of DNP-amino acids

using toluene as eluent for first direction and chloroformndashbenzylalcoholndash

glacial acetic acid (70 thorn 30 thorn 3 vv)[7] chloroformndashmethanolndashglacial

acetic acid (95 thorn 5 thorn 1 vv) or benzenendashpyridinendashglacial acetic acid

(80 thorn 20 thorn 2 vv) as eluent for the second direction[8] The majority of

DNP-amino acids are colored yellow

The 2D TLC separation of PTH-amino acids may be performed in the

first development with chloroformndashmethanol (90 thorn 10 vv) as eluent and

chloroformndashformic acid (100 thorn 5 vv) as eluent for the second develop-

ment[9] There are many other systems of eluents that can be used to obtain

good resolution The chlorinetolidine reagent is very useful for detection of

PTH-amino acids An alternative is offered by fluorescence quenching

A very complex mononucleotide mixture can be separated by 2D TLC on

poly(ethyleneimine)ndashcellulose anionexchange thin-layers After applying the

nucleotide solution at the starting point the chromatogram is developed in a

closed rectangular jar by stepwise elution with LiCl solution of 02 M 10 M

and finally with 16 M In order to remove LiCl the plate is placed in a flat dish

filled with anhydrous methanol Then the chromatogram is developed in the

second direction with formic acidndashsodium formate buffers pH 3ndash4 by a

stepwise elution with buffers of 05 20 and 40 M The complete resolution

of a model mixture containing DPA TPN six nucleotide sugars and fourteen

common nucleoside-50-mono- di- and triphosphates takes less than 3 hr[10]

The separation and identification of plant phospholipids and glycolipids

was performed by 2D TLC silica gel using chloroformndashmethanolndashwater

(65 25 4 vv) as eluent for the first dimension and diisobutyl ketonendashacetic

acidndashwater (80 50 10 vv) for the second direction[11]

Amino-modified HPTLC silica gel layers (NH2 F254s HPTLC) was used for

identification of 13 amphetamine derivatives by uni-dimensional multiple

development (UDM) in the 2D TLC mode The mobile phases used were

Gocan1108

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ORDER REPRINTS

ethanolndashtriethylaminendashn-hexane (15 thorn 9 thorn 76 vv) as mobile phase in

the first direction and acetonendashtriethylaminendashn-hexane (23 thorn 9 thorn 68 vv) as

mobile phase in the second direction Because one-dimensional HP-TLC was

not effective UDM with the same solvent system and development distance and

two development steps was investigated The resolution of separation was

higher in the low-hRf compared to that predicted by the UDM theory[12]

In the first direction the development is isocratic and in the second

direction it is automated multiple development (AMD) The instrumental

chromatographic method AMD provides a mean of normal phase (NP)

gradient developed in HPTLC A maximum of 25 steps can be used to form an

AMD gradient The development distances increase as the solvent polarity is

reduced The plates are dried by vacuum between consecutive steps The

bands on the plate will be compressed by repeated developments resulting in

increased sensitivity and resolution For the functional principle of the AMD

device one should consult Ref[13] The developed distance was 8 cm Isocratic

TLC was performed with one typical eluent as mobile phase in normal

chambers previously equilibrated with the mobile phase The AMD system

was performed in a Camag device (Muttenz Switzerland) but the gradient

systems must have a different selectivity than with isocratic developement

The number of compounds separated by this method was greater than with

isocratic TLC or with one-dimensional AMD[14] The use of 2D AMD with

two gradients of different selectivities is a powerful method that can improve

the separation of samples containing an unknown number of constituents in

hydroalcoholic plant extracts[15] The plant extract ldquofingerprintrdquo is better for

authenticity certification of the respectively extract

The 2D TLC is a simple method that makes possible the rapid recognition

of changes in individual components of a mixture For this purpose separations

must be carried out in both directions under exactly identical conditions In

such circumstances a slight increase in resolution might occur because of an

increase by a significant factor of the distance of migration of the zone which is

a diagonal line (Zf2) But this development method indicates alterations of

compounds during chromatography if the resulting spots do not lie on a

diagonal line bisecting the plate The compounds that between the first and

second development suffer several structural transformations under physical

agent (action of radiation) or from chemical reaction can be detected This

method can be used in photochemistry and stability control[3]

TWO-DIMENSIONAL DEVELOPMENT ON BILAYERS

There are many reports concerning the placement of two adsorbents on

the same plate

Two-Dimensional Thin Layer Chromatography 1109

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by [

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kow

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te U

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06

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2013

ORDER REPRINTS

An apparatus for simultaneously coating a plate with two adjacent layers

of different adsorbents was accomplished by placing a plastic insert into a

commercial spreader thus forming two independent chambers For this

reason there were used combinations of two adsorbents such as cellulose

silica gel alumina charcoal silicic acid magnesium silicate etc as a function

of the sample Then the two eluents systems were optimized for the two

development directions For example two-dimensional separation of some

ketones on a bilayer (charcoalsilicic acid) with benzenendashetherndashacetic acid

(82 9 9 vv) in the first direction (on charcoal) and with benzenendashether

(85 15 vv) in the second direction (on silicic acid)[16] In another paper[17]

the first adsorbent was silica gel (air dried) and the second was deacti-

vated aluminum oxide The same solvent system toluenendashethyl acetate (3 1

vv) was used in the two directions In this condition a mixture of

24-dinitrophenylhydrazine derivatives of hydroxycarbonyl compounds was

resolved

Silver nitrate-impregnated silica gel has also been used successfully in

the separation of some classes of compounds that contain double bonds in

their structures Thus it was used in the separation of glycerides and

isomers of unsaturated fatty acid esters The 2D TLC on adjacent layers of

silica gel G and silica G impregnated with 5 of silver nitrate was used for

development The silica gel G layer covered a surface of 3 cm 20 cm and

the difference of the surface of 17 cm 20 cm was covered with silica gel G

impregnated with 5 AgNO3 The first direction was performed on silica

gel G with petroleum etherndashdiethyl ether (9 1 vv) as eluent The second

direction was done on the impregnated silica gel G and developed twice

with the same eluent as in the first direction After the first direction the

mixture was fractioned into four groups of compounds (trialkyl glyceryl

ethers dialkoxy glycerides alkoxy diglycerides and triglycerides) After

the second direction each group was fractioned for another period into three

or four compounds[18] So it the efficiency of the impregnated layer was

demonstrated

A method for the complete structural analysis of complex mixtures of

methyl esters of saturated and unsaturated fatty acids has been performed by

2D TLC Adsorbent silica gel impregnated with a 10 solution of dodecane

was used for the first direction and acetonitrilendashacetone (1 1 vv) as eluent

The silica gel for the second direction was impregnated with a 20 AgNO3

and dipropyl etherndashhexane (2 3 vv) was used as eluent In this way com-

plete separation of an standard mixture of the methyl esters of six saturated

nine monoethylenic and three polyenic esters were achieved[19]

The 2D TLC on bilayers silica gel for NP and reversed phase (RP) offer

the special possibility to carry out two different separation processes on

the same plate Normal phase chromatography can be performed in the first

Gocan1110

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ded

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kow

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] at

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2013

ORDER REPRINTS

direction on silica gel after the evaporation of the solvent the plate was

impregnated with paraffin oil dissolved in petroleum ether After removal of

the excess solvent the plate was RP developed in the second direction

Another approach may also be performed by transferring the spots from

one plate to another plate[16] In such circumstances the development in the

first direction was carried out on a narrow 2 cm strip After drying it was

clipped face to face to a 20 cm 20 cm or 10 cm 10 cm plate for develop-

ment in the second direction Close contact has to be maintained between the

two layers for a proper development In this case the first plate can be used

with a silica gel layer and the second plate with silica gel RP-C18 or vice-

versa[20] In this way the analysis of saponins in Silene vulgaris Gracke

was done in the first direction on RP-18W HPTLC plates with 1 aqueous

formic acidndashmethanol (30 thorn 70 vv) as mobile phase and in the second

perpendicular direction on silica gel Si 60 HPTLC with chloroformndash

methanolndashformic acidndashwater (100 thorn 40 thorn 10 thorn 10 vv) as mobile phase

By the use 2D TLC bilayers the saponin mixture could be separated into

18 components whereas conventional TLC separated the mixture into only

9 components[21]

TWO-DIMENSIONAL SEPARATION BY

TLCELECTROPHORESIS

Peptide maps of the tryptic digest of myosin have been performed on

thin-layer plates (20 cm 20 cm) by successive TLC and electrophoresis In

the first dimension TLC with chloroformndashmethanolndashammonium hydroxide

34 (40 40 20 vv) as eluent the time was as long as 60 min The second

dimension electrophoresis with a buffer pyridinendashglacial acetic acidndashwater

(1 10 489 vv) current 980 V 30 mA the time was 1 hr The peptide

mixture is applied in amounts of 005ndash05 mg per peptide map[22] In

another paper[23] electrophoresis was applied in one direction on buf-

fered adsorbents followed by chromatography in the second dimension In

this circumstance phosphate esters were separated by TLC development

twice with n-propanolndashammonium hydroxidendashwater (60 30 1 vv) and

electrophoresis was carried out in 028 M acetate buffer (pH 36) 1000 V

35 mA and 16 min

CHROMATOGRAM EVALUATION

The best 2D TLC separation is when all the components are separated and

distributed on the entire surface of the chromatographic plate The estimation

Two-Dimensional Thin Layer Chromatography 1111

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ded

by [

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kow

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te U

niv

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liote

] at

06

16 0

9 D

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2013

ORDER REPRINTS

of this separation can be made by an objective function Generally a good

agreement between visual evaluation of a chromatogram and computer

evaluation using these objective functions has been noticed[24] On the other

hand a function is needed that can predict Rf value of the one-component

function of the composition from the mobile phase

There are programs for simulated chromatograms which are compar-

able to that obtained with experimental chromatograms[24] Other details

concerning the strategy of optimization of the mobile phase can be found

in Ref[20]

REFERENCES

1 Nyiredy Sz Dallenbach-Tolke K Sticher O J Planar Chromatogr

1988 1 336

2 Stahl E Kaldewey H Hoppe-Seylers Z Physiol Chem 1961 323

182

3 Stahl E Thin-Layer Chromatopgraphy Springer-Verlag 1965

4 von Arx E Neher R J Chromatogr 1963 12 329

5 Hirs CHW J Biol Chem 1956 219 611

6 Biserte G Holleman JW Holleman-Dehove J Sautiere P

J Chromatogr 1959 2 225 J Chromatogr 1960 3 85

7 Brenner M Pataki G Experientia 1961 17 145

8 Brenner M Niederwieser A Experientia 1961 17 237

9 Pataki G Thesis Basel University Basel Switzerland 1962

10 Randerath E Randerath K J Chromatogr 1964 16 126

11 Lepage M J Chromatogr 1964 13 99

12 Fater ZS Tasi G Szabady B Nyiredy Sz J Planar Chromatogr

1998 11 225

13 Cazes J Encyclopedia of Chromatography Cazes J Ed Marcel

Dekker Inc New York 2001 533

14 Muresan L Thesis Babes-Bolyai University Cluj-Napoca 1998

15 Olah N-K Muresan L Cımpan G Gocan S J Planar Chromatogr

1998 11 361

16 Kirchner JG Thin-Layer Chromatography 2nd Ed John Wiley amp Sons

New York 1978

17 Anet EFLJ J Chromatogr 1962 9 291

18 Schmid HHO Baumann WJ Cubero JM Mangold HK Biochem

Biophys Acta 1966 125 189

19 Bergelson D Dyatlovitskaya EV Voronkova VV J Chromatogr

1964 15 191

Gocan1112

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ORDER REPRINTS

20 Nyredy Sz Ed Planar Chromatography A Retrospective View for the

Third Millennium Springer 2001

21 Glensk M Cisowski W J Planar Chromatogr 2000 13 9

22 Ritschard WJ J Chomatogr 1964 16 327

23 Bieleski RL Anal Biochem 1965 12 230

24 Grinberg N Modern Thin-Layer Chromatography Grinberg N Ed

Marcel Dekker Inc New York 1990

Received October 29 2003

Accepted November 18 2003

Manuscript 6253

Two-Dimensional Thin Layer Chromatography 1113

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2013

Request PermissionOrder Reprints

Reprints of this article can also be ordered at

httpwwwdekkercomservletproductDOI101081JLC120030182

Request Permission or Order Reprints Instantly

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All information and materials found in this article including but not limited to text trademarks patents logos graphics and images (the Materials) are the copyrighted works and other forms of intellectual property of Marcel Dekker Inc or its licensors All rights not expressly granted are reserved

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ORDER REPRINTS

method has been elaborated and found suitable for the chromatographic

identification of 52 amino acids This method is based on three 2D TLC

developments on cellulose (CMN 300 50m) using the same solvent system I

for the first dimension and three different systems (IIndashIV) of suitable

properties for the second dimension System I n-butanolndashacetonendash

diethylaminendashwater (10 10 2 5 vv) system II 2-propanolndashformic

acidndashwater (40 2 10 vv) system III sec-butanolndashmethyl ethyl ketonendash

dicyclohexylaminendashwater (10 10 2 5 vv) and system IV phenolndashwater

(75 25 gg) (thorn75 mg Na-cyanide) with 3 ammonia With this technique

all amino acids can be differentiated and characterized by their fixed posi-

tions and in addition by some color reactions Also the relative merits of

cellulose and silica gel are discussed in relation to separation efficiency

reproducibility and detection sensitivity[4] The 2D TLC separation of a

performic acid oxidized mixture of 20 protein amino acids plus b-alanine and

g-amino-n-butyric acid was performed in the first direction with chloroformndash

methanolndashammonia (17) (40 40 20 vv) and in the second direction with

Figure 1 Scheme of spot distribution on a 2D TLC plate

Two-Dimensional Thin Layer Chromatography 1107

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ORDER REPRINTS

phenolndashwater (75 25 gg) Detection was performed by means of ninhidrin

reagent spray[5]

The thin-layer chromatographic method was developed for amino acids

and therefore in principle it is equally applicable to peptides Peptides

like amino acids are generally hydrophilic There are however limits to

this analogy Dinitrophenylamino acid derivatives (DNP-amino acids) and

phenylthiohydantoin derivatives (PTH-amino acids) are obtained when reac-

tion of peptides or proteins with dinitrofluorobenzene or phenyl mustards

are properly degraded[6] Their separation from reaction mixtures and their

identification are considerable practical importance because they constitute

essential steps in the process of sequential analysis of peptide and protein

structures Generally the run in the first direction is done in a toluene system

For the run in the second direction there are many eluent systems that may be

selected The 2D TLC was used to perform separation of DNP-amino acids

using toluene as eluent for first direction and chloroformndashbenzylalcoholndash

glacial acetic acid (70 thorn 30 thorn 3 vv)[7] chloroformndashmethanolndashglacial

acetic acid (95 thorn 5 thorn 1 vv) or benzenendashpyridinendashglacial acetic acid

(80 thorn 20 thorn 2 vv) as eluent for the second direction[8] The majority of

DNP-amino acids are colored yellow

The 2D TLC separation of PTH-amino acids may be performed in the

first development with chloroformndashmethanol (90 thorn 10 vv) as eluent and

chloroformndashformic acid (100 thorn 5 vv) as eluent for the second develop-

ment[9] There are many other systems of eluents that can be used to obtain

good resolution The chlorinetolidine reagent is very useful for detection of

PTH-amino acids An alternative is offered by fluorescence quenching

A very complex mononucleotide mixture can be separated by 2D TLC on

poly(ethyleneimine)ndashcellulose anionexchange thin-layers After applying the

nucleotide solution at the starting point the chromatogram is developed in a

closed rectangular jar by stepwise elution with LiCl solution of 02 M 10 M

and finally with 16 M In order to remove LiCl the plate is placed in a flat dish

filled with anhydrous methanol Then the chromatogram is developed in the

second direction with formic acidndashsodium formate buffers pH 3ndash4 by a

stepwise elution with buffers of 05 20 and 40 M The complete resolution

of a model mixture containing DPA TPN six nucleotide sugars and fourteen

common nucleoside-50-mono- di- and triphosphates takes less than 3 hr[10]

The separation and identification of plant phospholipids and glycolipids

was performed by 2D TLC silica gel using chloroformndashmethanolndashwater

(65 25 4 vv) as eluent for the first dimension and diisobutyl ketonendashacetic

acidndashwater (80 50 10 vv) for the second direction[11]

Amino-modified HPTLC silica gel layers (NH2 F254s HPTLC) was used for

identification of 13 amphetamine derivatives by uni-dimensional multiple

development (UDM) in the 2D TLC mode The mobile phases used were

Gocan1108

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ded

by [

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] at

06

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2013

ORDER REPRINTS

ethanolndashtriethylaminendashn-hexane (15 thorn 9 thorn 76 vv) as mobile phase in

the first direction and acetonendashtriethylaminendashn-hexane (23 thorn 9 thorn 68 vv) as

mobile phase in the second direction Because one-dimensional HP-TLC was

not effective UDM with the same solvent system and development distance and

two development steps was investigated The resolution of separation was

higher in the low-hRf compared to that predicted by the UDM theory[12]

In the first direction the development is isocratic and in the second

direction it is automated multiple development (AMD) The instrumental

chromatographic method AMD provides a mean of normal phase (NP)

gradient developed in HPTLC A maximum of 25 steps can be used to form an

AMD gradient The development distances increase as the solvent polarity is

reduced The plates are dried by vacuum between consecutive steps The

bands on the plate will be compressed by repeated developments resulting in

increased sensitivity and resolution For the functional principle of the AMD

device one should consult Ref[13] The developed distance was 8 cm Isocratic

TLC was performed with one typical eluent as mobile phase in normal

chambers previously equilibrated with the mobile phase The AMD system

was performed in a Camag device (Muttenz Switzerland) but the gradient

systems must have a different selectivity than with isocratic developement

The number of compounds separated by this method was greater than with

isocratic TLC or with one-dimensional AMD[14] The use of 2D AMD with

two gradients of different selectivities is a powerful method that can improve

the separation of samples containing an unknown number of constituents in

hydroalcoholic plant extracts[15] The plant extract ldquofingerprintrdquo is better for

authenticity certification of the respectively extract

The 2D TLC is a simple method that makes possible the rapid recognition

of changes in individual components of a mixture For this purpose separations

must be carried out in both directions under exactly identical conditions In

such circumstances a slight increase in resolution might occur because of an

increase by a significant factor of the distance of migration of the zone which is

a diagonal line (Zf2) But this development method indicates alterations of

compounds during chromatography if the resulting spots do not lie on a

diagonal line bisecting the plate The compounds that between the first and

second development suffer several structural transformations under physical

agent (action of radiation) or from chemical reaction can be detected This

method can be used in photochemistry and stability control[3]

TWO-DIMENSIONAL DEVELOPMENT ON BILAYERS

There are many reports concerning the placement of two adsorbents on

the same plate

Two-Dimensional Thin Layer Chromatography 1109

Dow

nloa

ded

by [

Mos

kow

Sta

te U

niv

Bib

liote

] at

06

16 0

9 D

ecem

ber

2013

ORDER REPRINTS

An apparatus for simultaneously coating a plate with two adjacent layers

of different adsorbents was accomplished by placing a plastic insert into a

commercial spreader thus forming two independent chambers For this

reason there were used combinations of two adsorbents such as cellulose

silica gel alumina charcoal silicic acid magnesium silicate etc as a function

of the sample Then the two eluents systems were optimized for the two

development directions For example two-dimensional separation of some

ketones on a bilayer (charcoalsilicic acid) with benzenendashetherndashacetic acid

(82 9 9 vv) in the first direction (on charcoal) and with benzenendashether

(85 15 vv) in the second direction (on silicic acid)[16] In another paper[17]

the first adsorbent was silica gel (air dried) and the second was deacti-

vated aluminum oxide The same solvent system toluenendashethyl acetate (3 1

vv) was used in the two directions In this condition a mixture of

24-dinitrophenylhydrazine derivatives of hydroxycarbonyl compounds was

resolved

Silver nitrate-impregnated silica gel has also been used successfully in

the separation of some classes of compounds that contain double bonds in

their structures Thus it was used in the separation of glycerides and

isomers of unsaturated fatty acid esters The 2D TLC on adjacent layers of

silica gel G and silica G impregnated with 5 of silver nitrate was used for

development The silica gel G layer covered a surface of 3 cm 20 cm and

the difference of the surface of 17 cm 20 cm was covered with silica gel G

impregnated with 5 AgNO3 The first direction was performed on silica

gel G with petroleum etherndashdiethyl ether (9 1 vv) as eluent The second

direction was done on the impregnated silica gel G and developed twice

with the same eluent as in the first direction After the first direction the

mixture was fractioned into four groups of compounds (trialkyl glyceryl

ethers dialkoxy glycerides alkoxy diglycerides and triglycerides) After

the second direction each group was fractioned for another period into three

or four compounds[18] So it the efficiency of the impregnated layer was

demonstrated

A method for the complete structural analysis of complex mixtures of

methyl esters of saturated and unsaturated fatty acids has been performed by

2D TLC Adsorbent silica gel impregnated with a 10 solution of dodecane

was used for the first direction and acetonitrilendashacetone (1 1 vv) as eluent

The silica gel for the second direction was impregnated with a 20 AgNO3

and dipropyl etherndashhexane (2 3 vv) was used as eluent In this way com-

plete separation of an standard mixture of the methyl esters of six saturated

nine monoethylenic and three polyenic esters were achieved[19]

The 2D TLC on bilayers silica gel for NP and reversed phase (RP) offer

the special possibility to carry out two different separation processes on

the same plate Normal phase chromatography can be performed in the first

Gocan1110

Dow

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ded

by [

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kow

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te U

niv

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] at

06

16 0

9 D

ecem

ber

2013

ORDER REPRINTS

direction on silica gel after the evaporation of the solvent the plate was

impregnated with paraffin oil dissolved in petroleum ether After removal of

the excess solvent the plate was RP developed in the second direction

Another approach may also be performed by transferring the spots from

one plate to another plate[16] In such circumstances the development in the

first direction was carried out on a narrow 2 cm strip After drying it was

clipped face to face to a 20 cm 20 cm or 10 cm 10 cm plate for develop-

ment in the second direction Close contact has to be maintained between the

two layers for a proper development In this case the first plate can be used

with a silica gel layer and the second plate with silica gel RP-C18 or vice-

versa[20] In this way the analysis of saponins in Silene vulgaris Gracke

was done in the first direction on RP-18W HPTLC plates with 1 aqueous

formic acidndashmethanol (30 thorn 70 vv) as mobile phase and in the second

perpendicular direction on silica gel Si 60 HPTLC with chloroformndash

methanolndashformic acidndashwater (100 thorn 40 thorn 10 thorn 10 vv) as mobile phase

By the use 2D TLC bilayers the saponin mixture could be separated into

18 components whereas conventional TLC separated the mixture into only

9 components[21]

TWO-DIMENSIONAL SEPARATION BY

TLCELECTROPHORESIS

Peptide maps of the tryptic digest of myosin have been performed on

thin-layer plates (20 cm 20 cm) by successive TLC and electrophoresis In

the first dimension TLC with chloroformndashmethanolndashammonium hydroxide

34 (40 40 20 vv) as eluent the time was as long as 60 min The second

dimension electrophoresis with a buffer pyridinendashglacial acetic acidndashwater

(1 10 489 vv) current 980 V 30 mA the time was 1 hr The peptide

mixture is applied in amounts of 005ndash05 mg per peptide map[22] In

another paper[23] electrophoresis was applied in one direction on buf-

fered adsorbents followed by chromatography in the second dimension In

this circumstance phosphate esters were separated by TLC development

twice with n-propanolndashammonium hydroxidendashwater (60 30 1 vv) and

electrophoresis was carried out in 028 M acetate buffer (pH 36) 1000 V

35 mA and 16 min

CHROMATOGRAM EVALUATION

The best 2D TLC separation is when all the components are separated and

distributed on the entire surface of the chromatographic plate The estimation

Two-Dimensional Thin Layer Chromatography 1111

Dow

nloa

ded

by [

Mos

kow

Sta

te U

niv

Bib

liote

] at

06

16 0

9 D

ecem

ber

2013

ORDER REPRINTS

of this separation can be made by an objective function Generally a good

agreement between visual evaluation of a chromatogram and computer

evaluation using these objective functions has been noticed[24] On the other

hand a function is needed that can predict Rf value of the one-component

function of the composition from the mobile phase

There are programs for simulated chromatograms which are compar-

able to that obtained with experimental chromatograms[24] Other details

concerning the strategy of optimization of the mobile phase can be found

in Ref[20]

REFERENCES

1 Nyiredy Sz Dallenbach-Tolke K Sticher O J Planar Chromatogr

1988 1 336

2 Stahl E Kaldewey H Hoppe-Seylers Z Physiol Chem 1961 323

182

3 Stahl E Thin-Layer Chromatopgraphy Springer-Verlag 1965

4 von Arx E Neher R J Chromatogr 1963 12 329

5 Hirs CHW J Biol Chem 1956 219 611

6 Biserte G Holleman JW Holleman-Dehove J Sautiere P

J Chromatogr 1959 2 225 J Chromatogr 1960 3 85

7 Brenner M Pataki G Experientia 1961 17 145

8 Brenner M Niederwieser A Experientia 1961 17 237

9 Pataki G Thesis Basel University Basel Switzerland 1962

10 Randerath E Randerath K J Chromatogr 1964 16 126

11 Lepage M J Chromatogr 1964 13 99

12 Fater ZS Tasi G Szabady B Nyiredy Sz J Planar Chromatogr

1998 11 225

13 Cazes J Encyclopedia of Chromatography Cazes J Ed Marcel

Dekker Inc New York 2001 533

14 Muresan L Thesis Babes-Bolyai University Cluj-Napoca 1998

15 Olah N-K Muresan L Cımpan G Gocan S J Planar Chromatogr

1998 11 361

16 Kirchner JG Thin-Layer Chromatography 2nd Ed John Wiley amp Sons

New York 1978

17 Anet EFLJ J Chromatogr 1962 9 291

18 Schmid HHO Baumann WJ Cubero JM Mangold HK Biochem

Biophys Acta 1966 125 189

19 Bergelson D Dyatlovitskaya EV Voronkova VV J Chromatogr

1964 15 191

Gocan1112

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ded

by [

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te U

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] at

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ORDER REPRINTS

20 Nyredy Sz Ed Planar Chromatography A Retrospective View for the

Third Millennium Springer 2001

21 Glensk M Cisowski W J Planar Chromatogr 2000 13 9

22 Ritschard WJ J Chomatogr 1964 16 327

23 Bieleski RL Anal Biochem 1965 12 230

24 Grinberg N Modern Thin-Layer Chromatography Grinberg N Ed

Marcel Dekker Inc New York 1990

Received October 29 2003

Accepted November 18 2003

Manuscript 6253

Two-Dimensional Thin Layer Chromatography 1113

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ded

by [

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kow

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te U

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] at

06

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2013

Request PermissionOrder Reprints

Reprints of this article can also be ordered at

httpwwwdekkercomservletproductDOI101081JLC120030182

Request Permission or Order Reprints Instantly

Interested in copying and sharing this article In most cases US Copyright Law requires that you get permission from the articlersquos rightsholder before using copyrighted content

All information and materials found in this article including but not limited to text trademarks patents logos graphics and images (the Materials) are the copyrighted works and other forms of intellectual property of Marcel Dekker Inc or its licensors All rights not expressly granted are reserved

Get permission to lawfully reproduce and distribute the Materials or order reprints quickly and painlessly Simply click on the Request Permission Order Reprints link below and follow the instructions Visit the US Copyright Office for information on Fair Use limitations of US copyright law Please refer to The Association of American Publishersrsquo (AAP) website for guidelines on Fair Use in the Classroom

The Materials are for your personal use only and cannot be reformatted reposted resold or distributed by electronic means or otherwise without permission from Marcel Dekker Inc Marcel Dekker Inc grants you the limited right to display the Materials only on your personal computer or personal wireless device and to copy and download single copies of such Materials provided that any copyright trademark or other notice appearing on such Materials is also retained by displayed copied or downloaded as part of the Materials and is not removed or obscured and provided you do not edit modify alter or enhance the Materials Please refer to our Website User Agreement for more details

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ORDER REPRINTS

phenolndashwater (75 25 gg) Detection was performed by means of ninhidrin

reagent spray[5]

The thin-layer chromatographic method was developed for amino acids

and therefore in principle it is equally applicable to peptides Peptides

like amino acids are generally hydrophilic There are however limits to

this analogy Dinitrophenylamino acid derivatives (DNP-amino acids) and

phenylthiohydantoin derivatives (PTH-amino acids) are obtained when reac-

tion of peptides or proteins with dinitrofluorobenzene or phenyl mustards

are properly degraded[6] Their separation from reaction mixtures and their

identification are considerable practical importance because they constitute

essential steps in the process of sequential analysis of peptide and protein

structures Generally the run in the first direction is done in a toluene system

For the run in the second direction there are many eluent systems that may be

selected The 2D TLC was used to perform separation of DNP-amino acids

using toluene as eluent for first direction and chloroformndashbenzylalcoholndash

glacial acetic acid (70 thorn 30 thorn 3 vv)[7] chloroformndashmethanolndashglacial

acetic acid (95 thorn 5 thorn 1 vv) or benzenendashpyridinendashglacial acetic acid

(80 thorn 20 thorn 2 vv) as eluent for the second direction[8] The majority of

DNP-amino acids are colored yellow

The 2D TLC separation of PTH-amino acids may be performed in the

first development with chloroformndashmethanol (90 thorn 10 vv) as eluent and

chloroformndashformic acid (100 thorn 5 vv) as eluent for the second develop-

ment[9] There are many other systems of eluents that can be used to obtain

good resolution The chlorinetolidine reagent is very useful for detection of

PTH-amino acids An alternative is offered by fluorescence quenching

A very complex mononucleotide mixture can be separated by 2D TLC on

poly(ethyleneimine)ndashcellulose anionexchange thin-layers After applying the

nucleotide solution at the starting point the chromatogram is developed in a

closed rectangular jar by stepwise elution with LiCl solution of 02 M 10 M

and finally with 16 M In order to remove LiCl the plate is placed in a flat dish

filled with anhydrous methanol Then the chromatogram is developed in the

second direction with formic acidndashsodium formate buffers pH 3ndash4 by a

stepwise elution with buffers of 05 20 and 40 M The complete resolution

of a model mixture containing DPA TPN six nucleotide sugars and fourteen

common nucleoside-50-mono- di- and triphosphates takes less than 3 hr[10]

The separation and identification of plant phospholipids and glycolipids

was performed by 2D TLC silica gel using chloroformndashmethanolndashwater

(65 25 4 vv) as eluent for the first dimension and diisobutyl ketonendashacetic

acidndashwater (80 50 10 vv) for the second direction[11]

Amino-modified HPTLC silica gel layers (NH2 F254s HPTLC) was used for

identification of 13 amphetamine derivatives by uni-dimensional multiple

development (UDM) in the 2D TLC mode The mobile phases used were

Gocan1108

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ORDER REPRINTS

ethanolndashtriethylaminendashn-hexane (15 thorn 9 thorn 76 vv) as mobile phase in

the first direction and acetonendashtriethylaminendashn-hexane (23 thorn 9 thorn 68 vv) as

mobile phase in the second direction Because one-dimensional HP-TLC was

not effective UDM with the same solvent system and development distance and

two development steps was investigated The resolution of separation was

higher in the low-hRf compared to that predicted by the UDM theory[12]

In the first direction the development is isocratic and in the second

direction it is automated multiple development (AMD) The instrumental

chromatographic method AMD provides a mean of normal phase (NP)

gradient developed in HPTLC A maximum of 25 steps can be used to form an

AMD gradient The development distances increase as the solvent polarity is

reduced The plates are dried by vacuum between consecutive steps The

bands on the plate will be compressed by repeated developments resulting in

increased sensitivity and resolution For the functional principle of the AMD

device one should consult Ref[13] The developed distance was 8 cm Isocratic

TLC was performed with one typical eluent as mobile phase in normal

chambers previously equilibrated with the mobile phase The AMD system

was performed in a Camag device (Muttenz Switzerland) but the gradient

systems must have a different selectivity than with isocratic developement

The number of compounds separated by this method was greater than with

isocratic TLC or with one-dimensional AMD[14] The use of 2D AMD with

two gradients of different selectivities is a powerful method that can improve

the separation of samples containing an unknown number of constituents in

hydroalcoholic plant extracts[15] The plant extract ldquofingerprintrdquo is better for

authenticity certification of the respectively extract

The 2D TLC is a simple method that makes possible the rapid recognition

of changes in individual components of a mixture For this purpose separations

must be carried out in both directions under exactly identical conditions In

such circumstances a slight increase in resolution might occur because of an

increase by a significant factor of the distance of migration of the zone which is

a diagonal line (Zf2) But this development method indicates alterations of

compounds during chromatography if the resulting spots do not lie on a

diagonal line bisecting the plate The compounds that between the first and

second development suffer several structural transformations under physical

agent (action of radiation) or from chemical reaction can be detected This

method can be used in photochemistry and stability control[3]

TWO-DIMENSIONAL DEVELOPMENT ON BILAYERS

There are many reports concerning the placement of two adsorbents on

the same plate

Two-Dimensional Thin Layer Chromatography 1109

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by [

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te U

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2013

ORDER REPRINTS

An apparatus for simultaneously coating a plate with two adjacent layers

of different adsorbents was accomplished by placing a plastic insert into a

commercial spreader thus forming two independent chambers For this

reason there were used combinations of two adsorbents such as cellulose

silica gel alumina charcoal silicic acid magnesium silicate etc as a function

of the sample Then the two eluents systems were optimized for the two

development directions For example two-dimensional separation of some

ketones on a bilayer (charcoalsilicic acid) with benzenendashetherndashacetic acid

(82 9 9 vv) in the first direction (on charcoal) and with benzenendashether

(85 15 vv) in the second direction (on silicic acid)[16] In another paper[17]

the first adsorbent was silica gel (air dried) and the second was deacti-

vated aluminum oxide The same solvent system toluenendashethyl acetate (3 1

vv) was used in the two directions In this condition a mixture of

24-dinitrophenylhydrazine derivatives of hydroxycarbonyl compounds was

resolved

Silver nitrate-impregnated silica gel has also been used successfully in

the separation of some classes of compounds that contain double bonds in

their structures Thus it was used in the separation of glycerides and

isomers of unsaturated fatty acid esters The 2D TLC on adjacent layers of

silica gel G and silica G impregnated with 5 of silver nitrate was used for

development The silica gel G layer covered a surface of 3 cm 20 cm and

the difference of the surface of 17 cm 20 cm was covered with silica gel G

impregnated with 5 AgNO3 The first direction was performed on silica

gel G with petroleum etherndashdiethyl ether (9 1 vv) as eluent The second

direction was done on the impregnated silica gel G and developed twice

with the same eluent as in the first direction After the first direction the

mixture was fractioned into four groups of compounds (trialkyl glyceryl

ethers dialkoxy glycerides alkoxy diglycerides and triglycerides) After

the second direction each group was fractioned for another period into three

or four compounds[18] So it the efficiency of the impregnated layer was

demonstrated

A method for the complete structural analysis of complex mixtures of

methyl esters of saturated and unsaturated fatty acids has been performed by

2D TLC Adsorbent silica gel impregnated with a 10 solution of dodecane

was used for the first direction and acetonitrilendashacetone (1 1 vv) as eluent

The silica gel for the second direction was impregnated with a 20 AgNO3

and dipropyl etherndashhexane (2 3 vv) was used as eluent In this way com-

plete separation of an standard mixture of the methyl esters of six saturated

nine monoethylenic and three polyenic esters were achieved[19]

The 2D TLC on bilayers silica gel for NP and reversed phase (RP) offer

the special possibility to carry out two different separation processes on

the same plate Normal phase chromatography can be performed in the first

Gocan1110

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2013

ORDER REPRINTS

direction on silica gel after the evaporation of the solvent the plate was

impregnated with paraffin oil dissolved in petroleum ether After removal of

the excess solvent the plate was RP developed in the second direction

Another approach may also be performed by transferring the spots from

one plate to another plate[16] In such circumstances the development in the

first direction was carried out on a narrow 2 cm strip After drying it was

clipped face to face to a 20 cm 20 cm or 10 cm 10 cm plate for develop-

ment in the second direction Close contact has to be maintained between the

two layers for a proper development In this case the first plate can be used

with a silica gel layer and the second plate with silica gel RP-C18 or vice-

versa[20] In this way the analysis of saponins in Silene vulgaris Gracke

was done in the first direction on RP-18W HPTLC plates with 1 aqueous

formic acidndashmethanol (30 thorn 70 vv) as mobile phase and in the second

perpendicular direction on silica gel Si 60 HPTLC with chloroformndash

methanolndashformic acidndashwater (100 thorn 40 thorn 10 thorn 10 vv) as mobile phase

By the use 2D TLC bilayers the saponin mixture could be separated into

18 components whereas conventional TLC separated the mixture into only

9 components[21]

TWO-DIMENSIONAL SEPARATION BY

TLCELECTROPHORESIS

Peptide maps of the tryptic digest of myosin have been performed on

thin-layer plates (20 cm 20 cm) by successive TLC and electrophoresis In

the first dimension TLC with chloroformndashmethanolndashammonium hydroxide

34 (40 40 20 vv) as eluent the time was as long as 60 min The second

dimension electrophoresis with a buffer pyridinendashglacial acetic acidndashwater

(1 10 489 vv) current 980 V 30 mA the time was 1 hr The peptide

mixture is applied in amounts of 005ndash05 mg per peptide map[22] In

another paper[23] electrophoresis was applied in one direction on buf-

fered adsorbents followed by chromatography in the second dimension In

this circumstance phosphate esters were separated by TLC development

twice with n-propanolndashammonium hydroxidendashwater (60 30 1 vv) and

electrophoresis was carried out in 028 M acetate buffer (pH 36) 1000 V

35 mA and 16 min

CHROMATOGRAM EVALUATION

The best 2D TLC separation is when all the components are separated and

distributed on the entire surface of the chromatographic plate The estimation

Two-Dimensional Thin Layer Chromatography 1111

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te U

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2013

ORDER REPRINTS

of this separation can be made by an objective function Generally a good

agreement between visual evaluation of a chromatogram and computer

evaluation using these objective functions has been noticed[24] On the other

hand a function is needed that can predict Rf value of the one-component

function of the composition from the mobile phase

There are programs for simulated chromatograms which are compar-

able to that obtained with experimental chromatograms[24] Other details

concerning the strategy of optimization of the mobile phase can be found

in Ref[20]

REFERENCES

1 Nyiredy Sz Dallenbach-Tolke K Sticher O J Planar Chromatogr

1988 1 336

2 Stahl E Kaldewey H Hoppe-Seylers Z Physiol Chem 1961 323

182

3 Stahl E Thin-Layer Chromatopgraphy Springer-Verlag 1965

4 von Arx E Neher R J Chromatogr 1963 12 329

5 Hirs CHW J Biol Chem 1956 219 611

6 Biserte G Holleman JW Holleman-Dehove J Sautiere P

J Chromatogr 1959 2 225 J Chromatogr 1960 3 85

7 Brenner M Pataki G Experientia 1961 17 145

8 Brenner M Niederwieser A Experientia 1961 17 237

9 Pataki G Thesis Basel University Basel Switzerland 1962

10 Randerath E Randerath K J Chromatogr 1964 16 126

11 Lepage M J Chromatogr 1964 13 99

12 Fater ZS Tasi G Szabady B Nyiredy Sz J Planar Chromatogr

1998 11 225

13 Cazes J Encyclopedia of Chromatography Cazes J Ed Marcel

Dekker Inc New York 2001 533

14 Muresan L Thesis Babes-Bolyai University Cluj-Napoca 1998

15 Olah N-K Muresan L Cımpan G Gocan S J Planar Chromatogr

1998 11 361

16 Kirchner JG Thin-Layer Chromatography 2nd Ed John Wiley amp Sons

New York 1978

17 Anet EFLJ J Chromatogr 1962 9 291

18 Schmid HHO Baumann WJ Cubero JM Mangold HK Biochem

Biophys Acta 1966 125 189

19 Bergelson D Dyatlovitskaya EV Voronkova VV J Chromatogr

1964 15 191

Gocan1112

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] at

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ORDER REPRINTS

20 Nyredy Sz Ed Planar Chromatography A Retrospective View for the

Third Millennium Springer 2001

21 Glensk M Cisowski W J Planar Chromatogr 2000 13 9

22 Ritschard WJ J Chomatogr 1964 16 327

23 Bieleski RL Anal Biochem 1965 12 230

24 Grinberg N Modern Thin-Layer Chromatography Grinberg N Ed

Marcel Dekker Inc New York 1990

Received October 29 2003

Accepted November 18 2003

Manuscript 6253

Two-Dimensional Thin Layer Chromatography 1113

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te U

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] at

06

16 0

9 D

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ber

2013

Request PermissionOrder Reprints

Reprints of this article can also be ordered at

httpwwwdekkercomservletproductDOI101081JLC120030182

Request Permission or Order Reprints Instantly

Interested in copying and sharing this article In most cases US Copyright Law requires that you get permission from the articlersquos rightsholder before using copyrighted content

All information and materials found in this article including but not limited to text trademarks patents logos graphics and images (the Materials) are the copyrighted works and other forms of intellectual property of Marcel Dekker Inc or its licensors All rights not expressly granted are reserved

Get permission to lawfully reproduce and distribute the Materials or order reprints quickly and painlessly Simply click on the Request Permission Order Reprints link below and follow the instructions Visit the US Copyright Office for information on Fair Use limitations of US copyright law Please refer to The Association of American Publishersrsquo (AAP) website for guidelines on Fair Use in the Classroom

The Materials are for your personal use only and cannot be reformatted reposted resold or distributed by electronic means or otherwise without permission from Marcel Dekker Inc Marcel Dekker Inc grants you the limited right to display the Materials only on your personal computer or personal wireless device and to copy and download single copies of such Materials provided that any copyright trademark or other notice appearing on such Materials is also retained by displayed copied or downloaded as part of the Materials and is not removed or obscured and provided you do not edit modify alter or enhance the Materials Please refer to our Website User Agreement for more details

Dow

nloa

ded

by [

Mos

kow

Sta

te U

niv

Bib

liote

] at

06

16 0

9 D

ecem

ber

2013

ORDER REPRINTS

ethanolndashtriethylaminendashn-hexane (15 thorn 9 thorn 76 vv) as mobile phase in

the first direction and acetonendashtriethylaminendashn-hexane (23 thorn 9 thorn 68 vv) as

mobile phase in the second direction Because one-dimensional HP-TLC was

not effective UDM with the same solvent system and development distance and

two development steps was investigated The resolution of separation was

higher in the low-hRf compared to that predicted by the UDM theory[12]

In the first direction the development is isocratic and in the second

direction it is automated multiple development (AMD) The instrumental

chromatographic method AMD provides a mean of normal phase (NP)

gradient developed in HPTLC A maximum of 25 steps can be used to form an

AMD gradient The development distances increase as the solvent polarity is

reduced The plates are dried by vacuum between consecutive steps The

bands on the plate will be compressed by repeated developments resulting in

increased sensitivity and resolution For the functional principle of the AMD

device one should consult Ref[13] The developed distance was 8 cm Isocratic

TLC was performed with one typical eluent as mobile phase in normal

chambers previously equilibrated with the mobile phase The AMD system

was performed in a Camag device (Muttenz Switzerland) but the gradient

systems must have a different selectivity than with isocratic developement

The number of compounds separated by this method was greater than with

isocratic TLC or with one-dimensional AMD[14] The use of 2D AMD with

two gradients of different selectivities is a powerful method that can improve

the separation of samples containing an unknown number of constituents in

hydroalcoholic plant extracts[15] The plant extract ldquofingerprintrdquo is better for

authenticity certification of the respectively extract

The 2D TLC is a simple method that makes possible the rapid recognition

of changes in individual components of a mixture For this purpose separations

must be carried out in both directions under exactly identical conditions In

such circumstances a slight increase in resolution might occur because of an

increase by a significant factor of the distance of migration of the zone which is

a diagonal line (Zf2) But this development method indicates alterations of

compounds during chromatography if the resulting spots do not lie on a

diagonal line bisecting the plate The compounds that between the first and

second development suffer several structural transformations under physical

agent (action of radiation) or from chemical reaction can be detected This

method can be used in photochemistry and stability control[3]

TWO-DIMENSIONAL DEVELOPMENT ON BILAYERS

There are many reports concerning the placement of two adsorbents on

the same plate

Two-Dimensional Thin Layer Chromatography 1109

Dow

nloa

ded

by [

Mos

kow

Sta

te U

niv

Bib

liote

] at

06

16 0

9 D

ecem

ber

2013

ORDER REPRINTS

An apparatus for simultaneously coating a plate with two adjacent layers

of different adsorbents was accomplished by placing a plastic insert into a

commercial spreader thus forming two independent chambers For this

reason there were used combinations of two adsorbents such as cellulose

silica gel alumina charcoal silicic acid magnesium silicate etc as a function

of the sample Then the two eluents systems were optimized for the two

development directions For example two-dimensional separation of some

ketones on a bilayer (charcoalsilicic acid) with benzenendashetherndashacetic acid

(82 9 9 vv) in the first direction (on charcoal) and with benzenendashether

(85 15 vv) in the second direction (on silicic acid)[16] In another paper[17]

the first adsorbent was silica gel (air dried) and the second was deacti-

vated aluminum oxide The same solvent system toluenendashethyl acetate (3 1

vv) was used in the two directions In this condition a mixture of

24-dinitrophenylhydrazine derivatives of hydroxycarbonyl compounds was

resolved

Silver nitrate-impregnated silica gel has also been used successfully in

the separation of some classes of compounds that contain double bonds in

their structures Thus it was used in the separation of glycerides and

isomers of unsaturated fatty acid esters The 2D TLC on adjacent layers of

silica gel G and silica G impregnated with 5 of silver nitrate was used for

development The silica gel G layer covered a surface of 3 cm 20 cm and

the difference of the surface of 17 cm 20 cm was covered with silica gel G

impregnated with 5 AgNO3 The first direction was performed on silica

gel G with petroleum etherndashdiethyl ether (9 1 vv) as eluent The second

direction was done on the impregnated silica gel G and developed twice

with the same eluent as in the first direction After the first direction the

mixture was fractioned into four groups of compounds (trialkyl glyceryl

ethers dialkoxy glycerides alkoxy diglycerides and triglycerides) After

the second direction each group was fractioned for another period into three

or four compounds[18] So it the efficiency of the impregnated layer was

demonstrated

A method for the complete structural analysis of complex mixtures of

methyl esters of saturated and unsaturated fatty acids has been performed by

2D TLC Adsorbent silica gel impregnated with a 10 solution of dodecane

was used for the first direction and acetonitrilendashacetone (1 1 vv) as eluent

The silica gel for the second direction was impregnated with a 20 AgNO3

and dipropyl etherndashhexane (2 3 vv) was used as eluent In this way com-

plete separation of an standard mixture of the methyl esters of six saturated

nine monoethylenic and three polyenic esters were achieved[19]

The 2D TLC on bilayers silica gel for NP and reversed phase (RP) offer

the special possibility to carry out two different separation processes on

the same plate Normal phase chromatography can be performed in the first

Gocan1110

Dow

nloa

ded

by [

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kow

Sta

te U

niv

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liote

] at

06

16 0

9 D

ecem

ber

2013

ORDER REPRINTS

direction on silica gel after the evaporation of the solvent the plate was

impregnated with paraffin oil dissolved in petroleum ether After removal of

the excess solvent the plate was RP developed in the second direction

Another approach may also be performed by transferring the spots from

one plate to another plate[16] In such circumstances the development in the

first direction was carried out on a narrow 2 cm strip After drying it was

clipped face to face to a 20 cm 20 cm or 10 cm 10 cm plate for develop-

ment in the second direction Close contact has to be maintained between the

two layers for a proper development In this case the first plate can be used

with a silica gel layer and the second plate with silica gel RP-C18 or vice-

versa[20] In this way the analysis of saponins in Silene vulgaris Gracke

was done in the first direction on RP-18W HPTLC plates with 1 aqueous

formic acidndashmethanol (30 thorn 70 vv) as mobile phase and in the second

perpendicular direction on silica gel Si 60 HPTLC with chloroformndash

methanolndashformic acidndashwater (100 thorn 40 thorn 10 thorn 10 vv) as mobile phase

By the use 2D TLC bilayers the saponin mixture could be separated into

18 components whereas conventional TLC separated the mixture into only

9 components[21]

TWO-DIMENSIONAL SEPARATION BY

TLCELECTROPHORESIS

Peptide maps of the tryptic digest of myosin have been performed on

thin-layer plates (20 cm 20 cm) by successive TLC and electrophoresis In

the first dimension TLC with chloroformndashmethanolndashammonium hydroxide

34 (40 40 20 vv) as eluent the time was as long as 60 min The second

dimension electrophoresis with a buffer pyridinendashglacial acetic acidndashwater

(1 10 489 vv) current 980 V 30 mA the time was 1 hr The peptide

mixture is applied in amounts of 005ndash05 mg per peptide map[22] In

another paper[23] electrophoresis was applied in one direction on buf-

fered adsorbents followed by chromatography in the second dimension In

this circumstance phosphate esters were separated by TLC development

twice with n-propanolndashammonium hydroxidendashwater (60 30 1 vv) and

electrophoresis was carried out in 028 M acetate buffer (pH 36) 1000 V

35 mA and 16 min

CHROMATOGRAM EVALUATION

The best 2D TLC separation is when all the components are separated and

distributed on the entire surface of the chromatographic plate The estimation

Two-Dimensional Thin Layer Chromatography 1111

Dow

nloa

ded

by [

Mos

kow

Sta

te U

niv

Bib

liote

] at

06

16 0

9 D

ecem

ber

2013

ORDER REPRINTS

of this separation can be made by an objective function Generally a good

agreement between visual evaluation of a chromatogram and computer

evaluation using these objective functions has been noticed[24] On the other

hand a function is needed that can predict Rf value of the one-component

function of the composition from the mobile phase

There are programs for simulated chromatograms which are compar-

able to that obtained with experimental chromatograms[24] Other details

concerning the strategy of optimization of the mobile phase can be found

in Ref[20]

REFERENCES

1 Nyiredy Sz Dallenbach-Tolke K Sticher O J Planar Chromatogr

1988 1 336

2 Stahl E Kaldewey H Hoppe-Seylers Z Physiol Chem 1961 323

182

3 Stahl E Thin-Layer Chromatopgraphy Springer-Verlag 1965

4 von Arx E Neher R J Chromatogr 1963 12 329

5 Hirs CHW J Biol Chem 1956 219 611

6 Biserte G Holleman JW Holleman-Dehove J Sautiere P

J Chromatogr 1959 2 225 J Chromatogr 1960 3 85

7 Brenner M Pataki G Experientia 1961 17 145

8 Brenner M Niederwieser A Experientia 1961 17 237

9 Pataki G Thesis Basel University Basel Switzerland 1962

10 Randerath E Randerath K J Chromatogr 1964 16 126

11 Lepage M J Chromatogr 1964 13 99

12 Fater ZS Tasi G Szabady B Nyiredy Sz J Planar Chromatogr

1998 11 225

13 Cazes J Encyclopedia of Chromatography Cazes J Ed Marcel

Dekker Inc New York 2001 533

14 Muresan L Thesis Babes-Bolyai University Cluj-Napoca 1998

15 Olah N-K Muresan L Cımpan G Gocan S J Planar Chromatogr

1998 11 361

16 Kirchner JG Thin-Layer Chromatography 2nd Ed John Wiley amp Sons

New York 1978

17 Anet EFLJ J Chromatogr 1962 9 291

18 Schmid HHO Baumann WJ Cubero JM Mangold HK Biochem

Biophys Acta 1966 125 189

19 Bergelson D Dyatlovitskaya EV Voronkova VV J Chromatogr

1964 15 191

Gocan1112

Dow

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ded

by [

Mos

kow

Sta

te U

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liote

] at

06

16 0

9 D

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2013

ORDER REPRINTS

20 Nyredy Sz Ed Planar Chromatography A Retrospective View for the

Third Millennium Springer 2001

21 Glensk M Cisowski W J Planar Chromatogr 2000 13 9

22 Ritschard WJ J Chomatogr 1964 16 327

23 Bieleski RL Anal Biochem 1965 12 230

24 Grinberg N Modern Thin-Layer Chromatography Grinberg N Ed

Marcel Dekker Inc New York 1990

Received October 29 2003

Accepted November 18 2003

Manuscript 6253

Two-Dimensional Thin Layer Chromatography 1113

Dow

nloa

ded

by [

Mos

kow

Sta

te U

niv

Bib

liote

] at

06

16 0

9 D

ecem

ber

2013

Request PermissionOrder Reprints

Reprints of this article can also be ordered at

httpwwwdekkercomservletproductDOI101081JLC120030182

Request Permission or Order Reprints Instantly

Interested in copying and sharing this article In most cases US Copyright Law requires that you get permission from the articlersquos rightsholder before using copyrighted content

All information and materials found in this article including but not limited to text trademarks patents logos graphics and images (the Materials) are the copyrighted works and other forms of intellectual property of Marcel Dekker Inc or its licensors All rights not expressly granted are reserved

Get permission to lawfully reproduce and distribute the Materials or order reprints quickly and painlessly Simply click on the Request Permission Order Reprints link below and follow the instructions Visit the US Copyright Office for information on Fair Use limitations of US copyright law Please refer to The Association of American Publishersrsquo (AAP) website for guidelines on Fair Use in the Classroom

The Materials are for your personal use only and cannot be reformatted reposted resold or distributed by electronic means or otherwise without permission from Marcel Dekker Inc Marcel Dekker Inc grants you the limited right to display the Materials only on your personal computer or personal wireless device and to copy and download single copies of such Materials provided that any copyright trademark or other notice appearing on such Materials is also retained by displayed copied or downloaded as part of the Materials and is not removed or obscured and provided you do not edit modify alter or enhance the Materials Please refer to our Website User Agreement for more details

Dow

nloa

ded

by [

Mos

kow

Sta

te U

niv

Bib

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] at

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ORDER REPRINTS

An apparatus for simultaneously coating a plate with two adjacent layers

of different adsorbents was accomplished by placing a plastic insert into a

commercial spreader thus forming two independent chambers For this

reason there were used combinations of two adsorbents such as cellulose

silica gel alumina charcoal silicic acid magnesium silicate etc as a function

of the sample Then the two eluents systems were optimized for the two

development directions For example two-dimensional separation of some

ketones on a bilayer (charcoalsilicic acid) with benzenendashetherndashacetic acid

(82 9 9 vv) in the first direction (on charcoal) and with benzenendashether

(85 15 vv) in the second direction (on silicic acid)[16] In another paper[17]

the first adsorbent was silica gel (air dried) and the second was deacti-

vated aluminum oxide The same solvent system toluenendashethyl acetate (3 1

vv) was used in the two directions In this condition a mixture of

24-dinitrophenylhydrazine derivatives of hydroxycarbonyl compounds was

resolved

Silver nitrate-impregnated silica gel has also been used successfully in

the separation of some classes of compounds that contain double bonds in

their structures Thus it was used in the separation of glycerides and

isomers of unsaturated fatty acid esters The 2D TLC on adjacent layers of

silica gel G and silica G impregnated with 5 of silver nitrate was used for

development The silica gel G layer covered a surface of 3 cm 20 cm and

the difference of the surface of 17 cm 20 cm was covered with silica gel G

impregnated with 5 AgNO3 The first direction was performed on silica

gel G with petroleum etherndashdiethyl ether (9 1 vv) as eluent The second

direction was done on the impregnated silica gel G and developed twice

with the same eluent as in the first direction After the first direction the

mixture was fractioned into four groups of compounds (trialkyl glyceryl

ethers dialkoxy glycerides alkoxy diglycerides and triglycerides) After

the second direction each group was fractioned for another period into three

or four compounds[18] So it the efficiency of the impregnated layer was

demonstrated

A method for the complete structural analysis of complex mixtures of

methyl esters of saturated and unsaturated fatty acids has been performed by

2D TLC Adsorbent silica gel impregnated with a 10 solution of dodecane

was used for the first direction and acetonitrilendashacetone (1 1 vv) as eluent

The silica gel for the second direction was impregnated with a 20 AgNO3

and dipropyl etherndashhexane (2 3 vv) was used as eluent In this way com-

plete separation of an standard mixture of the methyl esters of six saturated

nine monoethylenic and three polyenic esters were achieved[19]

The 2D TLC on bilayers silica gel for NP and reversed phase (RP) offer

the special possibility to carry out two different separation processes on

the same plate Normal phase chromatography can be performed in the first

Gocan1110

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ORDER REPRINTS

direction on silica gel after the evaporation of the solvent the plate was

impregnated with paraffin oil dissolved in petroleum ether After removal of

the excess solvent the plate was RP developed in the second direction

Another approach may also be performed by transferring the spots from

one plate to another plate[16] In such circumstances the development in the

first direction was carried out on a narrow 2 cm strip After drying it was

clipped face to face to a 20 cm 20 cm or 10 cm 10 cm plate for develop-

ment in the second direction Close contact has to be maintained between the

two layers for a proper development In this case the first plate can be used

with a silica gel layer and the second plate with silica gel RP-C18 or vice-

versa[20] In this way the analysis of saponins in Silene vulgaris Gracke

was done in the first direction on RP-18W HPTLC plates with 1 aqueous

formic acidndashmethanol (30 thorn 70 vv) as mobile phase and in the second

perpendicular direction on silica gel Si 60 HPTLC with chloroformndash

methanolndashformic acidndashwater (100 thorn 40 thorn 10 thorn 10 vv) as mobile phase

By the use 2D TLC bilayers the saponin mixture could be separated into

18 components whereas conventional TLC separated the mixture into only

9 components[21]

TWO-DIMENSIONAL SEPARATION BY

TLCELECTROPHORESIS

Peptide maps of the tryptic digest of myosin have been performed on

thin-layer plates (20 cm 20 cm) by successive TLC and electrophoresis In

the first dimension TLC with chloroformndashmethanolndashammonium hydroxide

34 (40 40 20 vv) as eluent the time was as long as 60 min The second

dimension electrophoresis with a buffer pyridinendashglacial acetic acidndashwater

(1 10 489 vv) current 980 V 30 mA the time was 1 hr The peptide

mixture is applied in amounts of 005ndash05 mg per peptide map[22] In

another paper[23] electrophoresis was applied in one direction on buf-

fered adsorbents followed by chromatography in the second dimension In

this circumstance phosphate esters were separated by TLC development

twice with n-propanolndashammonium hydroxidendashwater (60 30 1 vv) and

electrophoresis was carried out in 028 M acetate buffer (pH 36) 1000 V

35 mA and 16 min

CHROMATOGRAM EVALUATION

The best 2D TLC separation is when all the components are separated and

distributed on the entire surface of the chromatographic plate The estimation

Two-Dimensional Thin Layer Chromatography 1111

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te U

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ORDER REPRINTS

of this separation can be made by an objective function Generally a good

agreement between visual evaluation of a chromatogram and computer

evaluation using these objective functions has been noticed[24] On the other

hand a function is needed that can predict Rf value of the one-component

function of the composition from the mobile phase

There are programs for simulated chromatograms which are compar-

able to that obtained with experimental chromatograms[24] Other details

concerning the strategy of optimization of the mobile phase can be found

in Ref[20]

REFERENCES

1 Nyiredy Sz Dallenbach-Tolke K Sticher O J Planar Chromatogr

1988 1 336

2 Stahl E Kaldewey H Hoppe-Seylers Z Physiol Chem 1961 323

182

3 Stahl E Thin-Layer Chromatopgraphy Springer-Verlag 1965

4 von Arx E Neher R J Chromatogr 1963 12 329

5 Hirs CHW J Biol Chem 1956 219 611

6 Biserte G Holleman JW Holleman-Dehove J Sautiere P

J Chromatogr 1959 2 225 J Chromatogr 1960 3 85

7 Brenner M Pataki G Experientia 1961 17 145

8 Brenner M Niederwieser A Experientia 1961 17 237

9 Pataki G Thesis Basel University Basel Switzerland 1962

10 Randerath E Randerath K J Chromatogr 1964 16 126

11 Lepage M J Chromatogr 1964 13 99

12 Fater ZS Tasi G Szabady B Nyiredy Sz J Planar Chromatogr

1998 11 225

13 Cazes J Encyclopedia of Chromatography Cazes J Ed Marcel

Dekker Inc New York 2001 533

14 Muresan L Thesis Babes-Bolyai University Cluj-Napoca 1998

15 Olah N-K Muresan L Cımpan G Gocan S J Planar Chromatogr

1998 11 361

16 Kirchner JG Thin-Layer Chromatography 2nd Ed John Wiley amp Sons

New York 1978

17 Anet EFLJ J Chromatogr 1962 9 291

18 Schmid HHO Baumann WJ Cubero JM Mangold HK Biochem

Biophys Acta 1966 125 189

19 Bergelson D Dyatlovitskaya EV Voronkova VV J Chromatogr

1964 15 191

Gocan1112

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te U

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] at

06

16 0

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2013

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20 Nyredy Sz Ed Planar Chromatography A Retrospective View for the

Third Millennium Springer 2001

21 Glensk M Cisowski W J Planar Chromatogr 2000 13 9

22 Ritschard WJ J Chomatogr 1964 16 327

23 Bieleski RL Anal Biochem 1965 12 230

24 Grinberg N Modern Thin-Layer Chromatography Grinberg N Ed

Marcel Dekker Inc New York 1990

Received October 29 2003

Accepted November 18 2003

Manuscript 6253

Two-Dimensional Thin Layer Chromatography 1113

Dow

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ded

by [

Mos

kow

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te U

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] at

06

16 0

9 D

ecem

ber

2013

Request PermissionOrder Reprints

Reprints of this article can also be ordered at

httpwwwdekkercomservletproductDOI101081JLC120030182

Request Permission or Order Reprints Instantly

Interested in copying and sharing this article In most cases US Copyright Law requires that you get permission from the articlersquos rightsholder before using copyrighted content

All information and materials found in this article including but not limited to text trademarks patents logos graphics and images (the Materials) are the copyrighted works and other forms of intellectual property of Marcel Dekker Inc or its licensors All rights not expressly granted are reserved

Get permission to lawfully reproduce and distribute the Materials or order reprints quickly and painlessly Simply click on the Request Permission Order Reprints link below and follow the instructions Visit the US Copyright Office for information on Fair Use limitations of US copyright law Please refer to The Association of American Publishersrsquo (AAP) website for guidelines on Fair Use in the Classroom

The Materials are for your personal use only and cannot be reformatted reposted resold or distributed by electronic means or otherwise without permission from Marcel Dekker Inc Marcel Dekker Inc grants you the limited right to display the Materials only on your personal computer or personal wireless device and to copy and download single copies of such Materials provided that any copyright trademark or other notice appearing on such Materials is also retained by displayed copied or downloaded as part of the Materials and is not removed or obscured and provided you do not edit modify alter or enhance the Materials Please refer to our Website User Agreement for more details

Dow

nloa

ded

by [

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kow

Sta

te U

niv

Bib

liote

] at

06

16 0

9 D

ecem

ber

2013

ORDER REPRINTS

direction on silica gel after the evaporation of the solvent the plate was

impregnated with paraffin oil dissolved in petroleum ether After removal of

the excess solvent the plate was RP developed in the second direction

Another approach may also be performed by transferring the spots from

one plate to another plate[16] In such circumstances the development in the

first direction was carried out on a narrow 2 cm strip After drying it was

clipped face to face to a 20 cm 20 cm or 10 cm 10 cm plate for develop-

ment in the second direction Close contact has to be maintained between the

two layers for a proper development In this case the first plate can be used

with a silica gel layer and the second plate with silica gel RP-C18 or vice-

versa[20] In this way the analysis of saponins in Silene vulgaris Gracke

was done in the first direction on RP-18W HPTLC plates with 1 aqueous

formic acidndashmethanol (30 thorn 70 vv) as mobile phase and in the second

perpendicular direction on silica gel Si 60 HPTLC with chloroformndash

methanolndashformic acidndashwater (100 thorn 40 thorn 10 thorn 10 vv) as mobile phase

By the use 2D TLC bilayers the saponin mixture could be separated into

18 components whereas conventional TLC separated the mixture into only

9 components[21]

TWO-DIMENSIONAL SEPARATION BY

TLCELECTROPHORESIS

Peptide maps of the tryptic digest of myosin have been performed on

thin-layer plates (20 cm 20 cm) by successive TLC and electrophoresis In

the first dimension TLC with chloroformndashmethanolndashammonium hydroxide

34 (40 40 20 vv) as eluent the time was as long as 60 min The second

dimension electrophoresis with a buffer pyridinendashglacial acetic acidndashwater

(1 10 489 vv) current 980 V 30 mA the time was 1 hr The peptide

mixture is applied in amounts of 005ndash05 mg per peptide map[22] In

another paper[23] electrophoresis was applied in one direction on buf-

fered adsorbents followed by chromatography in the second dimension In

this circumstance phosphate esters were separated by TLC development

twice with n-propanolndashammonium hydroxidendashwater (60 30 1 vv) and

electrophoresis was carried out in 028 M acetate buffer (pH 36) 1000 V

35 mA and 16 min

CHROMATOGRAM EVALUATION

The best 2D TLC separation is when all the components are separated and

distributed on the entire surface of the chromatographic plate The estimation

Two-Dimensional Thin Layer Chromatography 1111

Dow

nloa

ded

by [

Mos

kow

Sta

te U

niv

Bib

liote

] at

06

16 0

9 D

ecem

ber

2013

ORDER REPRINTS

of this separation can be made by an objective function Generally a good

agreement between visual evaluation of a chromatogram and computer

evaluation using these objective functions has been noticed[24] On the other

hand a function is needed that can predict Rf value of the one-component

function of the composition from the mobile phase

There are programs for simulated chromatograms which are compar-

able to that obtained with experimental chromatograms[24] Other details

concerning the strategy of optimization of the mobile phase can be found

in Ref[20]

REFERENCES

1 Nyiredy Sz Dallenbach-Tolke K Sticher O J Planar Chromatogr

1988 1 336

2 Stahl E Kaldewey H Hoppe-Seylers Z Physiol Chem 1961 323

182

3 Stahl E Thin-Layer Chromatopgraphy Springer-Verlag 1965

4 von Arx E Neher R J Chromatogr 1963 12 329

5 Hirs CHW J Biol Chem 1956 219 611

6 Biserte G Holleman JW Holleman-Dehove J Sautiere P

J Chromatogr 1959 2 225 J Chromatogr 1960 3 85

7 Brenner M Pataki G Experientia 1961 17 145

8 Brenner M Niederwieser A Experientia 1961 17 237

9 Pataki G Thesis Basel University Basel Switzerland 1962

10 Randerath E Randerath K J Chromatogr 1964 16 126

11 Lepage M J Chromatogr 1964 13 99

12 Fater ZS Tasi G Szabady B Nyiredy Sz J Planar Chromatogr

1998 11 225

13 Cazes J Encyclopedia of Chromatography Cazes J Ed Marcel

Dekker Inc New York 2001 533

14 Muresan L Thesis Babes-Bolyai University Cluj-Napoca 1998

15 Olah N-K Muresan L Cımpan G Gocan S J Planar Chromatogr

1998 11 361

16 Kirchner JG Thin-Layer Chromatography 2nd Ed John Wiley amp Sons

New York 1978

17 Anet EFLJ J Chromatogr 1962 9 291

18 Schmid HHO Baumann WJ Cubero JM Mangold HK Biochem

Biophys Acta 1966 125 189

19 Bergelson D Dyatlovitskaya EV Voronkova VV J Chromatogr

1964 15 191

Gocan1112

Dow

nloa

ded

by [

Mos

kow

Sta

te U

niv

Bib

liote

] at

06

16 0

9 D

ecem

ber

2013

ORDER REPRINTS

20 Nyredy Sz Ed Planar Chromatography A Retrospective View for the

Third Millennium Springer 2001

21 Glensk M Cisowski W J Planar Chromatogr 2000 13 9

22 Ritschard WJ J Chomatogr 1964 16 327

23 Bieleski RL Anal Biochem 1965 12 230

24 Grinberg N Modern Thin-Layer Chromatography Grinberg N Ed

Marcel Dekker Inc New York 1990

Received October 29 2003

Accepted November 18 2003

Manuscript 6253

Two-Dimensional Thin Layer Chromatography 1113

Dow

nloa

ded

by [

Mos

kow

Sta

te U

niv

Bib

liote

] at

06

16 0

9 D

ecem

ber

2013

Request PermissionOrder Reprints

Reprints of this article can also be ordered at

httpwwwdekkercomservletproductDOI101081JLC120030182

Request Permission or Order Reprints Instantly

Interested in copying and sharing this article In most cases US Copyright Law requires that you get permission from the articlersquos rightsholder before using copyrighted content

All information and materials found in this article including but not limited to text trademarks patents logos graphics and images (the Materials) are the copyrighted works and other forms of intellectual property of Marcel Dekker Inc or its licensors All rights not expressly granted are reserved

Get permission to lawfully reproduce and distribute the Materials or order reprints quickly and painlessly Simply click on the Request Permission Order Reprints link below and follow the instructions Visit the US Copyright Office for information on Fair Use limitations of US copyright law Please refer to The Association of American Publishersrsquo (AAP) website for guidelines on Fair Use in the Classroom

The Materials are for your personal use only and cannot be reformatted reposted resold or distributed by electronic means or otherwise without permission from Marcel Dekker Inc Marcel Dekker Inc grants you the limited right to display the Materials only on your personal computer or personal wireless device and to copy and download single copies of such Materials provided that any copyright trademark or other notice appearing on such Materials is also retained by displayed copied or downloaded as part of the Materials and is not removed or obscured and provided you do not edit modify alter or enhance the Materials Please refer to our Website User Agreement for more details

Dow

nloa

ded

by [

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kow

Sta

te U

niv

Bib

liote

] at

06

16 0

9 D

ecem

ber

2013

ORDER REPRINTS

of this separation can be made by an objective function Generally a good

agreement between visual evaluation of a chromatogram and computer

evaluation using these objective functions has been noticed[24] On the other

hand a function is needed that can predict Rf value of the one-component

function of the composition from the mobile phase

There are programs for simulated chromatograms which are compar-

able to that obtained with experimental chromatograms[24] Other details

concerning the strategy of optimization of the mobile phase can be found

in Ref[20]

REFERENCES

1 Nyiredy Sz Dallenbach-Tolke K Sticher O J Planar Chromatogr

1988 1 336

2 Stahl E Kaldewey H Hoppe-Seylers Z Physiol Chem 1961 323

182

3 Stahl E Thin-Layer Chromatopgraphy Springer-Verlag 1965

4 von Arx E Neher R J Chromatogr 1963 12 329

5 Hirs CHW J Biol Chem 1956 219 611

6 Biserte G Holleman JW Holleman-Dehove J Sautiere P

J Chromatogr 1959 2 225 J Chromatogr 1960 3 85

7 Brenner M Pataki G Experientia 1961 17 145

8 Brenner M Niederwieser A Experientia 1961 17 237

9 Pataki G Thesis Basel University Basel Switzerland 1962

10 Randerath E Randerath K J Chromatogr 1964 16 126

11 Lepage M J Chromatogr 1964 13 99

12 Fater ZS Tasi G Szabady B Nyiredy Sz J Planar Chromatogr

1998 11 225

13 Cazes J Encyclopedia of Chromatography Cazes J Ed Marcel

Dekker Inc New York 2001 533

14 Muresan L Thesis Babes-Bolyai University Cluj-Napoca 1998

15 Olah N-K Muresan L Cımpan G Gocan S J Planar Chromatogr

1998 11 361

16 Kirchner JG Thin-Layer Chromatography 2nd Ed John Wiley amp Sons

New York 1978

17 Anet EFLJ J Chromatogr 1962 9 291

18 Schmid HHO Baumann WJ Cubero JM Mangold HK Biochem

Biophys Acta 1966 125 189

19 Bergelson D Dyatlovitskaya EV Voronkova VV J Chromatogr

1964 15 191

Gocan1112

Dow

nloa

ded

by [

Mos

kow

Sta

te U

niv

Bib

liote

] at

06

16 0

9 D

ecem

ber

2013

ORDER REPRINTS

20 Nyredy Sz Ed Planar Chromatography A Retrospective View for the

Third Millennium Springer 2001

21 Glensk M Cisowski W J Planar Chromatogr 2000 13 9

22 Ritschard WJ J Chomatogr 1964 16 327

23 Bieleski RL Anal Biochem 1965 12 230

24 Grinberg N Modern Thin-Layer Chromatography Grinberg N Ed

Marcel Dekker Inc New York 1990

Received October 29 2003

Accepted November 18 2003

Manuscript 6253

Two-Dimensional Thin Layer Chromatography 1113

Dow

nloa

ded

by [

Mos

kow

Sta

te U

niv

Bib

liote

] at

06

16 0

9 D

ecem

ber

2013

Request PermissionOrder Reprints

Reprints of this article can also be ordered at

httpwwwdekkercomservletproductDOI101081JLC120030182

Request Permission or Order Reprints Instantly

Interested in copying and sharing this article In most cases US Copyright Law requires that you get permission from the articlersquos rightsholder before using copyrighted content

All information and materials found in this article including but not limited to text trademarks patents logos graphics and images (the Materials) are the copyrighted works and other forms of intellectual property of Marcel Dekker Inc or its licensors All rights not expressly granted are reserved

Get permission to lawfully reproduce and distribute the Materials or order reprints quickly and painlessly Simply click on the Request Permission Order Reprints link below and follow the instructions Visit the US Copyright Office for information on Fair Use limitations of US copyright law Please refer to The Association of American Publishersrsquo (AAP) website for guidelines on Fair Use in the Classroom

The Materials are for your personal use only and cannot be reformatted reposted resold or distributed by electronic means or otherwise without permission from Marcel Dekker Inc Marcel Dekker Inc grants you the limited right to display the Materials only on your personal computer or personal wireless device and to copy and download single copies of such Materials provided that any copyright trademark or other notice appearing on such Materials is also retained by displayed copied or downloaded as part of the Materials and is not removed or obscured and provided you do not edit modify alter or enhance the Materials Please refer to our Website User Agreement for more details

Dow

nloa

ded

by [

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kow

Sta

te U

niv

Bib

liote

] at

06

16 0

9 D

ecem

ber

2013

ORDER REPRINTS

20 Nyredy Sz Ed Planar Chromatography A Retrospective View for the

Third Millennium Springer 2001

21 Glensk M Cisowski W J Planar Chromatogr 2000 13 9

22 Ritschard WJ J Chomatogr 1964 16 327

23 Bieleski RL Anal Biochem 1965 12 230

24 Grinberg N Modern Thin-Layer Chromatography Grinberg N Ed

Marcel Dekker Inc New York 1990

Received October 29 2003

Accepted November 18 2003

Manuscript 6253

Two-Dimensional Thin Layer Chromatography 1113

Dow

nloa

ded

by [

Mos

kow

Sta

te U

niv

Bib

liote

] at

06

16 0

9 D

ecem

ber

2013

Request PermissionOrder Reprints

Reprints of this article can also be ordered at

httpwwwdekkercomservletproductDOI101081JLC120030182

Request Permission or Order Reprints Instantly

Interested in copying and sharing this article In most cases US Copyright Law requires that you get permission from the articlersquos rightsholder before using copyrighted content

All information and materials found in this article including but not limited to text trademarks patents logos graphics and images (the Materials) are the copyrighted works and other forms of intellectual property of Marcel Dekker Inc or its licensors All rights not expressly granted are reserved

Get permission to lawfully reproduce and distribute the Materials or order reprints quickly and painlessly Simply click on the Request Permission Order Reprints link below and follow the instructions Visit the US Copyright Office for information on Fair Use limitations of US copyright law Please refer to The Association of American Publishersrsquo (AAP) website for guidelines on Fair Use in the Classroom

The Materials are for your personal use only and cannot be reformatted reposted resold or distributed by electronic means or otherwise without permission from Marcel Dekker Inc Marcel Dekker Inc grants you the limited right to display the Materials only on your personal computer or personal wireless device and to copy and download single copies of such Materials provided that any copyright trademark or other notice appearing on such Materials is also retained by displayed copied or downloaded as part of the Materials and is not removed or obscured and provided you do not edit modify alter or enhance the Materials Please refer to our Website User Agreement for more details

Dow

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ded

by [

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kow

Sta

te U

niv

Bib

liote

] at

06

16 0

9 D

ecem

ber

2013

Request PermissionOrder Reprints

Reprints of this article can also be ordered at

httpwwwdekkercomservletproductDOI101081JLC120030182

Request Permission or Order Reprints Instantly

Interested in copying and sharing this article In most cases US Copyright Law requires that you get permission from the articlersquos rightsholder before using copyrighted content

All information and materials found in this article including but not limited to text trademarks patents logos graphics and images (the Materials) are the copyrighted works and other forms of intellectual property of Marcel Dekker Inc or its licensors All rights not expressly granted are reserved

Get permission to lawfully reproduce and distribute the Materials or order reprints quickly and painlessly Simply click on the Request Permission Order Reprints link below and follow the instructions Visit the US Copyright Office for information on Fair Use limitations of US copyright law Please refer to The Association of American Publishersrsquo (AAP) website for guidelines on Fair Use in the Classroom

The Materials are for your personal use only and cannot be reformatted reposted resold or distributed by electronic means or otherwise without permission from Marcel Dekker Inc Marcel Dekker Inc grants you the limited right to display the Materials only on your personal computer or personal wireless device and to copy and download single copies of such Materials provided that any copyright trademark or other notice appearing on such Materials is also retained by displayed copied or downloaded as part of the Materials and is not removed or obscured and provided you do not edit modify alter or enhance the Materials Please refer to our Website User Agreement for more details

Dow

nloa

ded

by [

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kow

Sta

te U

niv

Bib

liote

] at

06

16 0

9 D

ecem

ber

2013