Mixtures With Ether
The use of diethyl ether, known simply as “ether”, requires special care by the user be- cause of its very low b.p. and flash point. Before use, the ether should be tested for ab- sence of peroxides; also, any solvent residues left over after the chromatographic pro- cess must be disposed of separately from other solvents. The reason why the DAB (1993, Appendix 2) prescribed the solvent system “ether + methanol” (90 + 10 ml) for the identity test for stinging nettle root is difficult to understand, as, in summer months, the composition of this solvent system after charging it into the TLC chamber would be the same as that of the starting mixture only if the work was performed in a climatic laboratory. Use of this solvent system will in fact kill two birds with one stone, as the detection of scopoletine (Fig. 53a3) under 365-nm UV is performedbeforethe derivatization with vanillin-phosphoric acid and the determination of -sitosterol (Fig. 53a1). However, depending on the quality of the starting drug, the scopoletine content can be very low, so that a clear identification becomes more like a guessing 82 4 Solvent Systems, Developing Chambers and Development
game. Separate preparations and TLC systems for these two substances have been per- formed as follows. For -sitosterol an identity test is performed on a sample taken from every container of a batch (Fig. 53b), and for scopoletine the purity test is per- formed on a mixed sample, i.e. a homogeneous mixture of the samples taken from the containers of one batch, after concentrating the substance, using a solvent mixture suitable for coumarins (Fig. 53c) [51]. For -sitosterol, the solvent system “toluene + ethyl acetate” (50 + 20 ml) is quite satisfactory, not only for qualitative detection but also for assay after appropriate validation (Fig. 53d). All the experimental conditions for the chromatography of the dry extract of stinging nettle root are given in Table 14.
The chromatogram obtained using the DAB solvent system is also given for compari- son. Here, the development was performed at a temperature of 5 °C. The result is shown in Fig. 53a2.
Figure 53:see Photograph Section.
Table 14: Dry extract of nettle root
DAB 10 Test for -sitosterol Test for scopoletine Sample solutions Reflux with methanol +
ethyl acetate + toluene, filter, concentrate
With methanol ultrasonic bath, centrifuge:
100 mg DE/ml
Dissolve in water, extract with HCl- acidified CHCl3, org. phase = 0, dissolve R in CHCl3: 500 mg DE/ml
Sorbent Prescribed:
Silica gel G Used:
TLC silica gel 60 F254
20 × 20 cm (Merck 1.05715)
TLC silica gel 60 F254 20 × 20 cm
(Merck 1.05715)
TLC silica gel 60 F254 10 × 20 cm
(Merck 1.05729) Used transversely Solvent system Ether+ methanol
(90 + 10)
Toluene + ethyl acetate (50 + 20)
Acetone + water + ammonia solution conc. (45 + 3,5 + 1,5) Reference
substances
Scopoletine + cholesterol (1 + 30 mg/20 ml)
-Sitosterol 1 mg/ml
Scopoletine 0,1 mg/ml
Applied sample 20 l 20 l 10 l
volume 20 × 3 mm 10 mm 10 mm
Chamber saturation Yes Yes Yes, not less than
30 min
Migration distance 10 cm 10 cm 7 cm
Development time 16 min/5 °C 16 min 12 min
11 min/22 °C Derivatization
reagent
Vanillin-phosphoric acid
Vanillin-phoshoric acid
Detection at UV 365 nm
hRf-values:
-Sitosterol 67–73 39–43 91–96
Scopoletine 51–58 11–16 38–42
org. phase = 0: organic phase is evaporated to dryness, R: residue
83 4.1 Solvent Systems
Chlorinated Hydrocarbons (CHCs)
Following a decision of the Conference of the German Ministers of Culture, Education and Church Affairs, CHCs must no longer be used in school chemistry lessons, and must be excluded from the laboratory as soon as possible. Unfortunately, this will not happen very quickly, as these substances are often prescribed in the DAB monographs and in pharmacopoeias of other countries, e.g. as solvents for ibuprofen and haloperi- dol or in the preparation of samples of drugs and drug preparations, e.g. chloroform for greater celandine and dichloromethane for liquid extract of thyme. They are also often used for the subsequent chromaphotography, e.g. chloroform and its mixtures with toluene for steroid hormones and dichloromethane for liquid extract of thyme.
Some modern precoated layers have been developed with the object of avoiding CHCs in solvent systems [52]. For example, separations have been performed on RP- 18 layers using solvent systems without any CHCs, which in contrast are used for chro- matography on silica gel layers. A comparison of CHC-containing and CHC-free TLC systems is given in two examples below. Figure 54a,b shows chromatograms of theoph- ylline, theobromine and caffeine, and Fig. 55a,b the scanned chromatograms of furose- mide and spironolactone. Tables 15 and 16 give the experimental conditions.
Figure 54:see Photograph Section.
The replacement of CHCs prescribed in official publications will take some years, as past experience shows. The question therefore arises why every user should not be free to use alternative environmentally friendly solvents after appropriate validation, with- out the necessity for audits or discussions with FDA inspectors or other authorities, bearing in mind the fact that the pharmacopoeia permits the use of alternative testing procedures provided that these lead to the same result as that obtained by the pre- scribed procedure (DAB, Chapter IV, General Instructions).
Practical Tipsfor the replacement of CHCs:
In many cases, it is possible to replace CHCs by other solvents. Here, it can happen that a larger volume of the new solvent is required to dissolve a given amount of substance. The volume of solution applied must be correspondingly larger.
If there is not enough time available to devise a CHC-free chromatographic system, an attempt should at least be made to replace the chloroform in the solvent system by dichloromethane. However, the method must then be revalidated.
84 4 Solvent Systems, Developing Chambers and Development
Figure 55. Direct optical evaluation of spironolactone and furosemide from the contents of a capsule measured by the TLC Scanner II (CAMAG) at 254 nm
(a) Chromatography on TLC silica gel 60 F254with CHC-containing SS according to the DAB (b) Reversed-phase chromatography on RP-18 TLC foil with CHC-free SS
1 1.6 g furosemide/spot, 2 4.0 g spironolactone/spot
Table 15: Spironolactone and furosemide
DAB 10 Alternative (free of CHC)
Sample solution 2 mg each of spironolactone CRS and furosemide/ml methanol Sorbent TLC silica gel 60 F254 TLC alusheet RP-18 F254s
20 × 20 cm (Merck 1.05715) 20 × 20 cm (Merck 1.05559) Solvent system Chloroform + methanol Acetone + water (90+10)
+ acetic acid (70+30+1)
Applied sample 10 l 10 l
volume 10 mm bandwise 10 mm bandwise
Chamber saturation No Yes No Yes
Migration distance 10 cm 10 cm 10 cm 10 cm
Development time 50 min 24 min 33 min 16 min
hRf-values:
Spironolactone Front 83–87 56–58 68–72
Furosemide 93–95 53–56 70–73 82–85
85 4.1 Solvent Systems
Table 16: Theophylline, theobromine, caffeine
DAB 10 Renger et. al. [144] Alternative (free of CHC)a)
Sample solutions 200 mg/4 ml MeOH 100, 120, 140 g/ml 50 mg/10 ml methanol + 6 ml CHCl3 Methanol + water
(1+1v/v)
Sorbent Prescribed:
Silica gel G Used:
TLC silica gel 60 F254
20 × 20 cm (Merck 1.05715)
HPTLC silica gel 60 F254GLP
20 × 10 cm (Merck 1.05613)
TLC alusheet RP-18 F254s
20 × 20 cm (Merck 1.05559)
Solvent system 1-Butanol
+ chloroform + acetone + ammonia solution conc.
(40 + 30 + 30 + 10)
Toluene+ 2-propanol + acetic acid (16 + 2 + 1)
Methanol + water (60 +40)
Applied sample Purity test: Assay: Purity test:
volume 10 l = 200 g
20 × 3 mm Limit test:
1 l = 120 ng (100 %-value) 4 mm bandwise
40 l = 200 g 10 mm bandwise Identity test:
1 g = 0,5 % 10 g/10 mm
Chamber saturation Yes No No
Migration distance 10 cm 3 cm 10 cm
Development time 40 min 15 min 63 min
hRf-values:
Theophylline 10 g 15–19 Approx. 27b) 49–53
Theobromine 10 g 43–47 56–59
Caffeine 10 g 73–78 36–39
a) The chromatographic system is very environmentally friendly, therefore recommended for schools.
b) Acc. to a private communication of the authors.
”Critical” Solvent Compositions
The following two examples are not quite so problematical with respect to handling of the solvents, but are critical with respect to the reproducibility:
It is not recommended to store solvent systems that contain alcohols and acids, as these components tend to esterify, and this eventually leads to an altered chromato- gram. An example of this is the frequently used mixture “1-butanol + glacial acetic acid + water”.
Saponification in ester-containing solvent systems should also be regarded as a problem, as the free acids and alcohols cause a shift in the polarity, which again strongly influences the chromatographic results.
86 4 Solvent Systems, Developing Chambers and Development
Solvent systems in whose phase diagram there is in any danger of a miscibility gap are also critical. Even with small changes in temperature, these can lead to emulsion formation, i.e. separation of the mixture (example: the DAB solvent system for the sugars fructose, glucose, lactose and sucrose).