Between a simple screw-top jar and an automated multidevelopment system (AMD) there is a very wide range of TLC developing chambers, differing from each other in materials of construction, but especially in price, and finally also in the chromato- graphic result obtained in the various fields of application. The chambers for “classi- cal” ascending chromatography are of glass and have glass or stainless steel lids, while the bodies of the horizontal chambers are of PTFE (“Teflon”) and have glass lids, as can be seen in Fig. 56, which shows a selection of developing chambers. All the auto- matic chambers commonly used today in analytical equipment are built into a metal housing packed with electronic circuitry.
87 4.2 TLC Developing Chambers
Figure 56. Selection of developing chambers for TLC Back row from left to right:
Screw top jar (brown glass) for maximum plate size 5 × 8 cm Double-trough 10 × 10 cm chamber with stainless steel lid (CAMAG) Double-trough light-weight 20 × 20 cm chamber with glass lid (CAMAG) Flat-bottomed 20 × 20 cm chamber placed on its side for drying
Chamber, 20 × 10 cm, with V-shaped bottom and stainless steel lid (DESAGA) Cylindrical 10 × 20 cm developing chamber with glass lid (DESAGA)
Front row from left to right:
Horizontal 5 × 5 cm chamber (DESAGA), common name: “baby chamber”
Horizontal 10 × 10 cm chamber (DESAGA)
Screw top jar (clear glass) for maximum foil size 4 × 8 cm (Macherey-Nagel)
4.2.1.1 TLC Chambers for Vertical Development
For teaching in schools, the principle of TLC can be demonstrated using equipment never intended to be used for this analytical method and in the lowest price bracket (up to 50 DM). Hellendahl’s staining trough recommended by Surborg in 1981, like the screw-top glass jars in the TLC “micro-set” of Macherey-Nagel, also falls into this category. Both these types of chambers are suitable for precoated layers on foils up to 4 × 8 cm in size. Necessity is the mother of invention, and even preserving jars from mother’s pantry have been used with great success in teaching [54].
The circular development chambers now commonly used for the TLC plate formats 5 × 20 and 10 × 20 cm (DESAGA) stem from the era of paper chromatography.
These containers may often be misappropriated for keeping flowers fresh, but in general their preferred use is for in-process control. They are not particularly heavy, and are still in the lower price bracket (up to 180 DM). Also in this price range are the flat-bottomed “light-weight chambers” for plate sizes of 20 × 20 and 20 × 10 cm obtainable since 1996 and the flat-bottomed chambers for 10 × 10 cm plates 88 4 Solvent Systems, Developing Chambers and Development
(CAMAG). According to the manufacturer’s information, the latter are not avail- able in light-weight material, as chambers of this size do not weigh very much even in the normal material.
Most classical TLC developing chambers (also known as “tanks”) are in the me- dium price bracket (up to 500 DM). For the plate sizes 10 × 10 cm, 20 × 10 cm and 20 × 20 cm, these are available in rectangular form and as double-trough chambers with V-shaped bottoms or with side grooves for simultaneous blank chromatogra- phy for prewashing. One gets the impression that the heavier the chamber the dea- rer it is. The only exceptions to this are the “light-weight chambers” in the double- trough form for plate sizes 20 × 10 and 20 × 20 cm. These weigh only ca. 25 % of the usual tanks, and even the larger chambers can be carried comfortably in one hand.
Breakages caused by dropping heavy tanks when cleaning them in the sink should now be a thing of the past!
New development chambers for small UTLC plates (60 × 36 mm) are available:
CAMAG markets a small double-trough chamber. Baron has modified a staining trough for microscope slides with a tailor-made cap and a plate holder as a small development chamber.
If they are used correctly, all the TLC developing chambers described after this point in the book fulfill the requirements for identity testing, purity determinations and assays.
Automatic developing chambers were developed especially for work in accordance with the GMP/GLP guidelines, and with their aid the user is relieved of all supervi- sory functions (and can go to lunch with a clear conscience!). At ca. 5500 DM for the simple version of Baron (produced by DESAGA) and ca. 10 000 DM per set of ADC (automatic development chamber) equipment manufactured by CAMAG, these are in the upper price bracket and are therefore usually found only in the lab- oratories of the pharmaceutical industry, where they are used for high-qualityquan- titative TLC. However, it should not go unremarked at this point that ADC offers not only comfort at the workplace but also certainty with respect to reproducibility, which is today regarded as standard in pharmaceutical quality control. Without au- tomatic operation, the daily volume of work could not be coped with.
The AMD method of Burger is also based on vertical TLC. This system for auto- mated multiple development of thin-layer chromatograms is discussed in Chapter 11 “Special Methods in TLC”. This equipment falls into the top price bracket and is included here only for completeness.
89 4.2 TLC Developing Chambers
4.2.1.2 TLC Developing Chambers for Horizontal Development
With developing chambers for horizontal development, the chamber type again deter- mines the price.
The 5 × 5 cm horizontal chamber of Kraus is in the bottom price bracket [55]. To ob- tain good thin-layer chromatograms with this “mini” apparatus, sometimes also known as a “baby chamber”, the user first needs to gain some experience with it.
With this proviso, the Kraus chamber can be used in teaching and for identity test- ing in pharmacies with good results. Kraus recommends the 1/16 TLC plates with normal silica gel 60, but the pre-scored 10 × 10 cm HPTLC silica gel 60 plates (Merck Article No. 1.05644), which can readily be broken into four plates of the 5 × 5 cm format, are more suitable. With these, all work, including assays, can be performed, provided the appropriate fittings are available and the work is per- formed correctly [56, 57].
The “baby chamber” has now been adopted by the DAC, and the World Health Or- ganization (WHO) has accepted it for monitoring medicinal plants and natural products [58].
The manufacturer of the Kraus chamber (DESAGA) also markets a horizontal chamber of similar type for the plate size 10 × 10 cm. This also falls into the medium price range, and, after a suitable period of familiarization (as with the “baby cham- ber”), can be used for identity testing. For purity testing using the large amounts of substance often recommended, the suitability of this developing chamber should be tested. With these horizontal developing chambers, the chromatographic develop- ment has to be performed in one direction only.
A different manufacturer (CAMAG) produces two horizontal developing cham- bers for the plate sizes 10 × 10 cm and 20 × 10 cm, mainly for use with HPTLC plates. These types of chamber enable the samples to be developed from the two opposite sides towards the middle, giving an available migration distance of only 4.5 cm. With optimal operation, ca. 70 samples can be analyzed in parallel on the 20 × 10 cm plate. These developing chambers are mainly used where a large number of samples must be analyzed quantitatively in a short time. However, this type of chamber is not suitable for use at low temperatures, as in this case liquid can eas- ily condense out and cause problems. The price per set of equipment is over 100 DM.
The Vario-KS chamber supplied by Geiss and Schlitt [59] and marketed by CA- MAG is a special variation of the horizontal development technique. As well as five separate solvent troughs, there are also four different interchangeable conditioning trough inserts. This enables up to five solvent systems to be investigated simulta- neously under the same conditions on the same TLC plate. Moreover, this type of chamber enables the relative atmospheric humidity to be kept constant and differ- ent types of conditioning of the layer to be carried out. This type of chamber is rec- ommended for development work, as a great deal of time can be saved in the opti- mization of separating problems by skillful selection of the experimental conditions.
The cost of the Vario-KS chamber, including accessories, exceeds 3000 DM (1996).
90 4 Solvent Systems, Developing Chambers and Development
Developing chambers for horizontal development were originally conceived as sandwich chambers (S-chambers) in which the plate, with the layer underneath, is placed above the counter-plate at a distance of 0.5 mm. Without this counter-plate, a filter paper soaked in solvent can be placed at the bottom of the chamber, en- abling development to be performed in a saturated chamber. Chromatography can be performed with only a few ml solvent in horizontal chambers, which is, of course, economic and reduces disposal problems. However, the question arises how much of a four- or five-component solvent system is actually used if only 2 ml is consumed in the development process in the 5 × 5 cm horizontal chamber?