two‐dimensional thin‐layer chromatography
TRANSCRIPT
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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
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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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te U
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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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liote
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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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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
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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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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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2013
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
Dow
nloa
ded
by [
Mos
kow
Sta
te U
niv
Bib
liote
] at
06
16 0
9 D
ecem
ber
2013
ORDER REPRINTS
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
Dow
nloa
ded
by [
Mos
kow
Sta
te U
niv
Bib
liote
] at
06
16 0
9 D
ecem
ber
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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kow
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te U
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liote
] at
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2013
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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kow
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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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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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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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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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ded
by [
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kow
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te U
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liote
] at
06
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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
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
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nloa
ded
by [
Mos
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
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ded
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kow
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te U
niv
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liote
] at
06
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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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ded
by [
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kow
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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 [
Mos
kow
Sta
te U
niv
Bib
liote
] at
06
16 0
9 D
ecem
ber
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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kow
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te U
niv
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liote
] at
06
16 0
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2013
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
Dow
nloa
ded
by [
Mos
kow
Sta
te U
niv
Bib
liote
] at
06
16 0
9 D
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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
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ded
by [
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kow
Sta
te U
niv
Bib
liote
] at
06
16 0
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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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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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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 [
Mos
kow
Sta
te U
niv
Bib
liote
] at
06
16 0
9 D
ecem
ber
2013
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
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
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nloa
ded
by [
Mos
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
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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
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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
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
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ded
by [
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kow
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te U
niv
Bib
liote
] at
06
16 0
9 D
ecem
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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
Dow
nloa
ded
by [
Mos
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 [
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 [
Mos
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
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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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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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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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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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liote
] at
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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
nloa
ded
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Mos
kow
Sta
te U
niv
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liote
] at
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2013
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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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liote
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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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Sta
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liote
] at
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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
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te U
niv
Bib
liote
] at
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ecem
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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
Dow
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liote
] at
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ecem
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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
nloa
ded
by [
Mos
kow
Sta
te U
niv
Bib
liote
] at
06
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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
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liote
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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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