This airy comment by a practical pharmacist is true in principle, but is nevertheless a bold generalization in view of the many possibilities presented by the ca. 23 different types of sorbent and ca. 140 different commercial grades of precoated layers currently marketed by Merck alone. As other manufacturers such as Macherey-Nagel, What- man or Analtech offer other products with their own names and product numbers it is essential to study the catalogs of the manufacturers of precoated layers in great detail.
TLC products are primarily classified according to sorption agents and secondarily ac- cording to particle size, any fluorescence indicator present, supports and other charac- teristic properties. For documentation in compliance with GMP/GLP, the item number of the manufacturer is a characteristic that is important for the user, there being no overlapping of item numbers worldwide.
Table 3 shows the most important commercially available precoated layers and some typical examples of their use. In the choice of the “hardware” for TLC it cannot be as- sumed that nominally “identical” sorbents from different manufacturers will lead to equal separations [13]. It follows that trade names and item numbers used in publica- tions or testing procedures should not be regarded simply as surreptitious advertising, but are absolutely essential if reproducible results are to be obtained. This is especially true for validated methods. An example is given in Fig. 6, which shows separations of peppermint oils on TLC plates whose sorbent specifications in the catalogs of different manufacturers are identical. When the development heights are equal, the variation in separation efficiency is clearly visible, and different hRf values are obtained for the same substances (see Table 4).
Figure 6:see Photograph Section.
21 2.3 What Types of Precoated Layers Are There?
Table 3a: Important commercially available precoated layers and examples of typical applicationsa) Sorbent material Chromatographic principle Typical applications
Aluminum oxide Adsorption chromatography due to polar interactions
Alkaloids, steroids, terpenes, aliphatic, aromatic and basic compounds
Cellulose
Unmodified cellulose Partition chromatography due to polar interactions
Amino acids and other carboxylic acids as well as carbohydrates Acetylated cellulose Depending on acetyl con-
tent transition from normal phase to reversed phase chromatography
Anthraquinones, antioxidants, polycyclic aromatics, carboxylic acids, nitrophenols, sweeteners Cellulose ion exchangers Anion exchange Amino acids, peptides, enzymes,
nucleic acids constituents (nucleotides, nucleosides) etc.
Mixed layers
Cellulose DEAE/cellulose HR
Ion exchange Mono- and oligonucleotides in nucleic acid hydrolyzates Ionex ion exchangers Cation and anion exchange Amino acids, nucleic acid hydro-
lyzates, amino sugars, antibiotics, inorganic phosphates, cations;
racemate separation in peptide synthesis
Kieselguhr Commonly impregnated for
reversed phase separations
Aflatoxins, herbicides, tetracyclines
Polyamide Partition chromatography
due to polar interactions (e.g. hydrogen bonds)
Phenolic and polyphenolic natural substances
Silica
Unmodified silica gel Standard and nano silica gel, also with concentrating zone
Normal phase chromatography
Most frequent application of all TLC layers
High purity silica gel 60 Aflatoxins
Silica gel G, impregnated with ammonium sulfate
Surfactants, lipids
(neonatal respiratory syndrome) Silica gel 60, impregnated
with caffeine for PAH determination
Charge transfer complexes Polycyclic aromatic hydrocarbons (PAH) acc. to German drinking water specification (TVO) Chemically modified layers:
CHIRalplate
Enantiomer separation based on ligand exchange chromatography
Chiral amino acids, -hydroxy- carboxylic acids and other com- pounds which can form chelate complexes with Cu(II) ions 22 2 Precoated Layers
Table 3a: Continued
Sorbent material Chromatographic principle Typical applications Cyano-modified layer CN Normal phase and reversed
phase chromatography
Pesticides, phenols, preservatives, steroids
DIOL-modified layer Steroids, hormones
Amino-modified layer NH2 Anion exchange, normal phase and reversed phase chromatography
Nucleotides, pesticides, phenols, purine derivates, steroids, vitamins, sulfonic acids, carboxylic acids, xanthines
RP layers:
RP-2, RP-8, RP-18 Nonpolar substances
(lipids, aromatics)
Silica gel 60 silanized Polar substances (basic and acidic pharmaceutical active ingredients) RP-18 W/UV254, wettable Normal phase and reversed
phase chromatography
Aminophenols, barbiturates, preservatives, nucleobases, PAH, steroids, tetracyclines, phthalates Spherical silica gel
LiChrospher®Si 60 Normal phase chromato- graphy
Pesticides, phytopharmaceuticals Mixed layers
Aluminum oxide/acetylated cellulose
Normal phase and reversed phase chromatography
Polycyclic aromatic hydrocarbons (PAH)
Cellulose/silica gel Normal phase chromato- graphy
Preservatives Kieselguhr/silica gel Normal phase chromato-
graphy, reduced Adsorption capacity compared to silica gel
Carbohydrates, antioxidants, steroids, photographic developer substances
a)Following Macherey-Nagel’s and Merck’s catalog Table 3b: New precoated layers
Name of the plate Particularities Typical applications Adamant®(Macherey-Nagel)
Lux®(Merck)
Both:
Increased amount of fluo- rescence indicator
Universal
UTLC(Merck) Ultra thin monolithic
silica gel
Steroids, azepams, amino acids, phthalates and phenols ProteoChrom®(Merck) a) HPTLC silica gel 60 F254s,
20 × 10 cm glass plate
Amino acids, peptides (from protein digest) b) HPTLC cellulose,
10 × 10 cm
Amino acids, peptides, 2D-TLC
aluminum sheet HPTLC Premium Purity Plate
(Merck)
Wrapped in a special plastic-coated foil
All pharmacopoeia applica- tions
23 2.3 What Types of Precoated Layers are there?
Table 4: Peppermint oil
DAB 10 Pachaly TLC-
Atlas
Alternative I Alternative II
Sample solution 0,1 g/ml toluene 10 g/ml toluene 10 l/ml toluene 10 l/ml toluene Sorbent Prescribed:
silica gel G F254
used:
TLC silica gel 60 F254
(Merck 1.05715)
TLC silica gel 60 F254
20 × 20 cm (Merck 1.05715)
TLC-silica gel 60 F254
20 × 20 cm (Merck 1.05715)
Durasil-25 UV 254 nm 20 × 20 cm (Machery-Nagel 812008) Solvent system Toluene + ethyl
acetate
Toluene + ethyl acetate
Toluene + ethyl acetate
Toluene + ethyl acetate
(95 + 5) (93 + 7) (93 + 7) (93 +7)
Applied sample volume
20 l 15 l 10 l 10 l
20 × 3 mm 15 × 3 mm 10 mm bandwise 10 mm bandwise Chamber
saturation
Yes Yes Yes Yes
Migration distance 15 cm 15 cm 13 cm 13 cm
Development time 35 min 39 min 27 min 25 min
Derivatization reagent
Anisaldehyde- sulfuric acid
Anisaldehyde- sulfuric acid
Vanillin-sulfuric acid
Vanillin-sulfuric acid
Color of substances after derivatization:
Acc. to literature Acc. to literature
Menthol Bluish violet Bluish violet Bluish violet Bluish violet
Menthone Grayish blue Yellowish green Green Green
Menthyl acetate Bluish violet Bluish violet Blue Blue Menthofuran Brownish yellow Orange + violet
overlap
Brownish orange Orange
hRf-values: Acc. to literature
Menthol 11–15 20 18–22 28–32
Menthone 37–40 46 46–49 56–60
Menthyl acetate 40–45 51 50–54 61–64
Menthofuran 64–68 66 66–70 70–73
24 2 Precoated Layers
Since the publishing of the 1st edition of this book, some new precoated layers have been introduced. To simplify the visual evaluation for the user, Macherey-Nagel and Merck have developed precoated silica gel 60 layers with an increased amount of fluo- rescence indicator that go by the trade names of Adamant®- and Lux®-plates. Further- more these plates contain a higher amount of binder which improves the abrasion re- sistance. Figure 7 shows the comparison at UV-light 254 nm between a normal TLC- and a Lux®-plate.
Figure 7:see Photograph Section.
Following the common trend towards miniaturization also in analytical techniques, Merck has developed an UTLC-plate (UTLC: ultra thin-layer chromatography). Its precoated layer consists of a monolithic, porous silica gel. This non-particular ready- to-use plate contains no binder. With a layer thickness of only 10 m these plates show very fast separations in combination with an extremely low consumption of solvents.
Only very small sample volumes have to be applied on the layer, e.g. 20 nl solution of dyes (see Fig. 8).
Figure 8:see Photograph Section.
To improve analytical tools within the life sciences, Merck has introduced a new prod- uct line with two new ready-to-use plates called “ProteoChrom”:
ProteoChrom®HPTLC silica gel glass plate ProteoChrom®HPTLC cellulose aluminium sheet
Figures 9 a and b show the separation of peptides obtained by tryptic digest of Cyto- chrome C. For more information see Table 3b.
Figures 9 a and b:see Photograph Section.
The newHPTLC Premium Purity Plate(Merck) is especially designed for demanding pharmacopoeia applications. It is carefully wrapped in a special plastic coated alumi- num foil to prevent any plastic deposits from the wrapping material that could appear as an unknown extra zone when using medium-polar solvent systems such as toluene/
ethylacetate (95/5). These plates have no GLP-laser code.
Figure 10 shows three commercial labels for precoated layers with explanatory notes. The concepts explained in this Chapter are again clearly represented for the user, so that taking a plate out of the “wrong” box should be a thing of the past!
Figure 10. Examples of labels used to identify precoated layers.
(a) Silica gel 60 with normal separation efficiency (b) Water-tolerant high-performance plate
(c) Silica gel high-performance plate suitable for AMD (Automated Multiple Development).
25 2.3 What Types of Precoated Layers are there?
Sorbent layer with
normal separation efficiency Fluorescence indicator with excitation wavelength
GoodLaboratoryPractice (coded plate with individual plate number)
Dimensions and quantity of the plates
Sorbent material Silica gel 60 Batch number
Merck article no.
Macherey-Nagel article no. AutomatedMultipleDevelopement
Very thin sorbent layer
“HighPerformanceThin LayerChromatography"
ReversedPhase material Completely water-tolerant
Dimensions of the concentrating zone Acid-stable fluorescence indicator
(a)
(b)
(c)
Figure 10(legend see page 25) 26 2 Precoated Layers