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Modern Methods of Organic Analysis.

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through a layer of barium stearate about 200 8, thick or
across several solvent molecules without radiation to a
receiver substance which emits fluorescent light. Similar
energy transfer phenomena are known to occur in systems
such as anthracene/tetracene, and in the ‘plastic scintillators’
used in nuclear technology.
Fiber Damage due to Anthraquinonoid Dyes
F. DBrr, Miinchen (Germany)
Yellow anthraquinone dyes dehydrogenate their substrate
on exposure to light. The known dyes can be divided into
two groups according to the electronic structure of their
lowest excited state. In the first group, which included most
yellow dyes, the lowest excited state is a triplet n-rr* state in
which a non-bonding, localized n-electron from the oxygen
of the keto group passes into a delocalized n*-orbital of the
carbon skeleton. The free-radical electron remaining on the
oxygen reacts with the surroundings. Almost all the dyes
which cause active damage belong to this group. In the
second groii:), the lowest triplet level of the delocalized xelectrons T(z:-;s*)
;s depxssed below the T(n-n“) level by the
action o f substituents. This group includes the inactive dyes.
Tnis hypothesis is supported by the detection of n-x* absorption bands in djes which cause damaze, by the flashspectroscopic detection of ketyl radicals in active benzophenone derivatives, and of semiquinone radicals in active
anthraquinone derivatives. These radicals react further by
second-order kinetics. They are absent from all the inactive
compounds investigated. Flavanthrone, which belongs to the
second group, is progressively reduced on cellophane to the
semiquinone radical ion, when irradiated with light of 365 m:*
wavelength in its second, weak absorption band. It is assumed that a T(i1-n”) state, which lies above the T(n-n*)
level, can also rcact in the casc I f hindered deactivati3n.
[VB 837/161 IE]
German version: Angew. Chem. 76, XI9 (1964)
Translated by Express Translation Service, London
Modern Methods of Organic Analysis
A symposium under the above title organized by the Analytical Chemistry Sections of the Royal Dutch Chemical Society
and the German Chemical Society was held in Eindhoven
(Holland) from May 20th to 23rd, 1964.
From the lectures:
Determination of Acetoacetic Acid i n 0.1 ml of Blood
F. Bu/zner, Heidelberg (Germany)
Acetone is liberated from the acetoacetic acid in the blood
sample and isolated by microdiffusion. Disadvantages of the
Conway microdiffusion apparatus are that the diffusion cell
is not gas-tight at 30--40°C, and a temperature gradient
cannot be maintained between the inner and outer chambers.
In a new apparatus described, a temperature difference can
be maintained between the two reacting solutions. Acetone
reacts in alkaline solution with salicylaldehyde to form red
1,3-disalicylideneacetone.The reaction is influenced by 1. the
reactant concentrations; 2. oxygen; 3. daylight; and
4. slow thermal dissociation of the disalicylideneacetone in
alkaline solution; this last factor makes the final color dependent on temperature. Under appropriate conditions,
10-8 mole of acetone can be separated and determined.
Improvement and Automation of the Determination
of Creatinine
K . Beyermann, Mainz (Germany)
Creatinine can be separated rapidly and selectively by precipitation as its tetraphenyl borate complex in the presence of
butylamine as carrier. The optimum conditions for precipitation (pH 3 i 1, I04-fold excess of tetraphenyl borate and
103-fold excess of butylamine, room temperature) were determined by studies with [14C]creatinine [I]. For 10-2-103 [*g
of creatinine per ml, the efficiency of isolation is 97-100 %.
About 300 substances representing either normal constituents
of biological materials or pharmaceutical preparations did
not interfere. The few compounds which are coprecipitated
d o not interfere in practice with the subsequent determination. The precipitate is dissolved in dimethylformarnide, and
the red color produced by reaction between creatinine and
[I] K . Beyermann, Z . analyt. Chem., in the press.
760
picric acid is determined spectrophotometrically. Protein must
first be separated off by precipitation with trichloroacetic
acid; this results in a creatinine loss of less than 1 %. Other
separation procedures (ion exchange, adsorption on Fuller’s
earth) gave lower recoveries. A fully automatic, programmed
apparatus makes it possible to complete 10 creatinine determinations per hour.
Thin-Layer Chromatography on Silica Gel
Impregnated with Silver Nitrate
F. C . den Boer, Vlaardingen (Holland)
Substances with C = C double bonds form complexes with
silver ions. Hence olefinic substances can be separated on
thin layers of silica gel previously impregnated with silver
nitrate. The thin layers can be prepared quickly, and 0.1 to
100 mg of mixtures can be separated quickly, readily identified, and isolated quantitatively.
Typical analyses include the separation of the methyl esters
of the fatty acids in soya oil having the same chain length
but different numbers of double bonds, analysis of a mixture
of the triglycerides of linoleic, oleic, and stearic acid, and
the analqsis o f a mixture of acetylated methylsterols.
Separation of Steryl Acetates b y Thin-Layer
Chromatography
1. W. Copius-Peereboom, Leiden (Holland)
Mixtures of animal and vegetable fats were analysed by
identifying the sterol types present by means of a thin-layer
chromatography reversed-phase technique on kieselguhr G
impregnated with undecane (b.p. 190-220 “ C ) . On development o f the plates with acetic acid/acetonitrile (25:75),
various steryl acetates, e.g. the acetates o f @-sitosterol,stigmasterol, cholesterol, desmosterol, and Ag(l’)-dehydroergosterol, are separated into small semi-circular bands.
The rates of migration of sterols are characterized by their
carbon numbers (Nc values), i.e. the number of carbon
atoms minus the number of double bonds.
Unfortunately, in this system, certain sterol pairs have equal
RF values, e.g. cholesterol and brassicasterol ; ergosterol and
7-dehydrocholesterol. Such “critical pairs” of sterols were
separated by adding 0.5 P: bromine to the mobile phase according to the procedure of Kauftztrnn.
Angew. Chem. internat. Edit. ] VoI. 3 (1964) 1 No. I I
Critical pairs of sterols can also be separated on silica gelG layers impregnated with silver nitrate. The acetates of
various sterols are separated with chloroform/light petroleum/
acetic acid (25:75:0.5) as solvent, e.g. the pairs: dihydrocholesterol/choleslerol, A7-~holesterol/cholesterol,AT-cholesterol/5-dihydroergosterol,
lanosterol/agnosterol. Sterols with
a greater number of ethylenic linkages display lower RF values
in this system.
Colorimetric Sub-micro Determination of Bromine
in Organic C o m p o u n d s
T. R. F. W. Fennel1 and J. R . Webb, Farnborough, Hants
(England)
Sulfophthalein dyes in strongly acidic solution exhibit high
intensity absorption in the 500-550 mF spectral region. It
was found that on treating bromide with bromate in the acidic
dye solutions, a decrease of the absorption intensity occurred
which was proportional to the amount of bromide present.
Detailed investigations with several dyes showed that ocresolsulfophthalein (cresol red) was most suitable for the
determination of bromide.
By varying the initial concentration of dye, 2-10, 5-20, and
10-40 pg amounts of bromide were determined with standard deviations of 0.08,0.12, and 0.16 pg respectively. Within
these ranges, straight-line calibration curves were obtained.
Apart from chloride and iodide, ions in the amounts that
occur in sub-micro analyses d o not interfere provided that
oxidizing and reducing agents are absent.
Analysis of D r u g s in Biological Fluids b y the
Tropaolin M e t h o d
A . Huussler and P . Hujdu, Frankfurt/Main-Hoechst
(Germany)
The formation of chloroform-soluble complexes with Tropaolin-00 (4-Phenylaminoazobenzene-4'-sulfonic acid) by
many basic pharmaceuticals can be used for both qualitative
and quantitative analysis [2]. Using a n Autoanalyzer [3], a
continuous record of reaction kinetics can be obtained by
this method, e.g. of the ester hydrolysis of local anesthetics
at low concentrations (10-50 pg/ml) in serum. The calculated reaction constants show that the rate of hydrolysis
is strongly species-dependent. This finding is in agreement
with the literature [4].Complex formation can also be used
to extract basic drugs selectively from body fluids. By fluorimetry of the extract, less than 10-6 g of drug material can
be detected.
Automatic Combination of Thin-Layer
C h r o m a t o g r a p h y and a Temperature-Programmed
Gas-Chromatograph
R . Kaiser, Ludwigshafen/Rhein (Germany)
With increasing molecular weight, the uncertainty in the
qualitative identification of compounds by gas chromatography increases sharply. Here automatic combination of a
gas chromatograph and a thin-layer chromatograph can
help. A thin-layer plate is attached to the adapted, strongly
heated outlet of a temperature-programmed gas-chromatograph and is moved from one end to the other, the movement
being controlled either by time or by recorder output. The
~~
~-
[2] A . Haussler and P. Hajdi, Arzneimittel-Forsch. 12, 41 1
(1962).
[3] A . Huussler and P. Hajda: Wandel in der chemischen Technik. Hanser, Miinchen 1963, p. 112.
[41 R . Muschaweck, Lokalanasthesie und Lokalanasthetika.
Thieme, Stuttgart 1959, p. 103.
Angew. Chem. internat. Edit. / Yo/.3 (1964) / No. I1
material from each small gas-chromatographic peak, and
the impurities between peaks, are deposited as individual
spots or as a line by local cooling. Large peaks (relatively
large quantities of material) are deposited in several spots on
the start line. The thin-layer plate is then developed with a
suitable mobile phase. In this way, the substances contained
in the gas-chromatographic peaks are rechromatographed,
but on the basis of different principles, so that better separations can be achieved. The developed thin-layer plates are
treated with specific chemical reagents; the finished thinlayer chromatograms provide, in association with the gas
chromatogram, a n abundance of frequently surprising
additional information.
The Reaction of Nucleotides and Nucleic Acids
with Diazonium Salts
H . Kossel, Munich (Germany)
As Zillig and Verwoed [5] showed by the example of the reaction of hydroxylamine with s-RNA, chemical methods are
useful for the base-specific degradation or base-specific modification of nucleic acids for sequence analysis. The reaction
of diazonium salts with nucleotides and nucleic acids was
examined from this aspect.
The diazonium salts used were diazotized p-sulfanilic acid,
diazotized 2,4-dichloroaniline, diazotized p-nitroaniline,
and diazotized 4-nitroaniline-3-sulfonic acid, all in 3- to
10-fold excess at 2°C. The reactions with the nucleotides
guanylic acid (Gp), adenylic acid (Ap), and cytidylic acid
(Cp) were readily followed by extinction measurements in the
370-440 m p region and by high-voltage paper electrophoresis.
Gp, Ap, and Cp all reacted with diazotized p-sulfanilic acid,
G p quickly and quantitatively, but C p only to the extent
of about 20 %. Uridylic and thymidylic acids did not react.
The p H optima were 10.2 (Gp) and 10.7 (Ap, Cp). The dyestuffs produced were not, or were only in small part, true azo
compounds, but rather diazoamino derivatives with the
structure Rl-N=N-NH-RZ;
this was deduced from the
following observations:
1. The dyes decompose in weakly acidic medium to reform
the starting materials.
2. They show spectroscopic pKB values of 11.9 (Gp) and 9.0
(AP, CP).
3. Their formation is inhibited by formaldehyde, which reacts with amino groups.
The dye formed with A p is the one most readily reconverted
into the starting materials. Decomposition of the product
from G p gives some xanthidylic acid, the deamination product of guanylic acid.
Diazotized p-sulfanilic acid reacts correspondingly with R N A
and D N A ; cytidylic acid does not react. In contrast, Beer and
Mouclzriunukis [6] observed base-specific coupling of diazotized aminonaphthalenetrisulfonic acid with DNA.
Only the purine nucleotides react with the diazonium salts of
2,4-dichloroaniline, p-nitroaniline, and 4-nitroaniline, but
again G p reacts faster than Ap. The p H optimum for both
nucleotides is 8.5 for all three diazonium salts.
NMR-Spectroscopy for the Analysis of Organic
C o m p o u n d s with Known Functional G r o u p s
N . van M e w s , Amsterdam (Holland)
High-resolution N M R spectra can be used a) to distinguish
between primary, secondary, and tertiary monobasic acids
and for quantitative analysis of binary mixtures of these
[ 5 ] W. Zillig, D . W. Verwoed, and H . Kohlhage, Hoppe-Seylers
Z. Physiol. Chem. 332, 184 (1963).
[6] M. Beer and E. N. Mouchrianakis, Proc. nat. Acad. Sci.
U.S.A. 48, 409 (1962).
76 1
acids, and b) to differentiate between primary, secondary,
and tertiary monohydric alcohols and for the quantitative
analysis of binary and ternary mixtures of these alcohols.
The three types of monobasic acids can be distinguished by
the multiplet structure and intensity ratio of the a-CH2 and
a - C H proton resonance signals, provided that these d o not
overlap with the P-proton multiplets. The intensities of the
signals are determined relative to that of the carboxylic
proton resonance signal. For quantitative analysis of binary
mixtures, the intensity ratio can be used.
For the analysis of alcohols, the preparation of the sample
constitutes an important part of the procedure, for it must
ensure that any exchange of the hydroxyl protons comes to a
standstill [7]. The three types of alcohol then show differences
in the multiplicity of the hydroxyl signal and in its chemical
shift. This depends on the solvent and the temperature.
A second procedure, based o n comparison of peak heights
gives results of similar absolute accuracy (is %) in suitable
cases.
Relations between Structure and Rate Constants for the
Dissociation and Recombination of Carboxylic Acids
H . W. Niirnberg, Jiilich (Germany)
The rate constants for the dissociation and recombination
of 12 aliphatic and aromatic carboxylic acids in 1 M LiCl
have been determined for the first time by a new electrochemical method termed “High Level Faradaic Rectification” 181.
With the kinetic data now available for a greater number of
acids, the formulation of a correlation between the rate
constants and the structure of the acid molecules and their
anions is possible; previously such considerations had to be
based solely on equilibrium constants. Normally, recombination depends only upon diffusion, and a common value
~
1iter.mole-1.sec-1) is obtained for the rate
(k, m 3 . 5 1010
constant for recombination in 1 M LiCI, since the diffusion
coefficient of the hydrogen ion exerts the dominating
influence. Consequently there is a linear relationship between
the dissociation constant kd and the equilibrium constant.
Hence, for acids with n o r m a l b e h a v i o r , kd can be predicted from a knowledge of the equilibrium constant. The
influence of structure with its inductive and mesomeric effects
plays a part in determining the value of kd. Deviations from
normal behavior arise if recombination is retarded by steric
effects, e.g. particularly by the formation of intramolecular
hydrogen bridges in the anion (lactic acid, salicylic acid).
With these acids, only kinetic measurements give complete
information on the dissociation and recombination behavior. I n addition, the stability constant of the intramolecular hydrogen bridges follows from a knowledge of
kr. Finally, evidence of the salt effect o n the rate of dissociation and recombination can be obtained.
Conformational Analysis of Nitrosteroids
b y Circular Dichroism
G . Snatzke, Bonn (Germany)
Nitrosteroids show a Cotton effect at 280 mp, and sometimes
their absorption band at 320 my also is circular dichroitic.
Both bands involve n + TC* transitions, but only the first
transition exhibits the symmetrical properties of carbonyl
excitation. The rules found for ketones can thus be applied
to the 280 m y band with slight modifications [9].
For nitro compounds in which free rotation of the NO2
group is hindered, the relationship between the circidar dichroism and the axial or equatorial conformation of the NO2
[7] Proton exchange is suppressed by careful neutralization of
solvents and solutes.
[ 8 ] H . W. Nurnberg and G. C. Barker, Naturwissenschaften 51,
191 (1964).
[9] C. Djerassi: Optical Rotatory Dispersion. McGraw-Hill,
New York 1960.
762
group has been deduced; a modified octant rule is applicable
for nitro groups. The two planes of symmetry of the NO2
group and a third plane perpendicular to them lying between
the nitrogen atom and the two oxygen atoms divide the
space around the molecule as in ketones; the contributions
of the carbon atoms are, however, calculated with opposite
signs. Measurements of the circular dichroism of gem-nitrohalogenosteroids at -188 “ C support the rule given. From
the results it follows that in unhindered nitrocyclohexanes
the following conformations are energentically most favored :
H
H
Axial NOz g r o u p
equatorial NO2 group
Preparative Thin-Layer Chromatographic Separation of
Phosphatides and Thin-Layer Chromatographic
Identification of Their Hydrolysis Products
0.W. Thiele and W. Wober, Gottingen (Germany)
Phosphatides can be separated by thin-layer chromatography on silica gel-G with isobutyl ketone/formic acid/
water (40: 15: 2). Staining agents which have proved generally
applicable are: borate-buffered bromothymol blue solution
for lipids; ninhydrin for primary amines; and Dragendorff’s
reagent, followed by ninhydrin on the same plate, for quaternary amines.
Since column-chromatographic separation of phosphatides
seldom yields pure fractions, exclusive use of thin-layer
chromatography on the preparative scale is of great value.
The mixture is placed on the start line in a strip 18 cni long
(0.1 mg/cm). After development of the chromatogram and
staining of a narrow guide strip, the absorption material containing the desired fraction is scraped off the plate and
extracted.
After hydrolysis of the phosphatides, fatty acids, amines, and
phosphate esters (which alone are resistant to hydrolysis) are
obtained. The fatty acids are separated by gas chromatography of their methyl esters. The amines are separated by
thin-layer chromatography on silica gel-G with 96 ”/, v/v
ethanol/water (63 : 37), or n-butanol/glacial acetic acid/water
(3 : 1 : l), or n-propanol/34 % ammonia (67: 33). For the
identification of phosphate esters, a thin-layer chromatographic procedure on cellulose sheets is employed, using
either a n ammoniacal (e.g. 0.5 N ammonia/methanol, 2:3)
or an acidic system (e.g. glacial acetic acid/n-propanol/water
1 : 2: 1) as mobile phase. Compared with paper-chromatographic procedures, thin-layer chromatography on cellulose
has the advantages of improved zone sharpness, clean applicability of phosphate reagents (Hanes-Isherwood spray
reagent), and good resistance of the cellulose layers to
aggressive spray reagents.
[VB 819/147 IE]
German version: Angew. Chem. 76, 691 (1964)
Verdazyls and Related Nitrogen-Containing
Free Radicals
R. Kuhn, Heidelberg (Germany)
Verdazyls (1) [I] may be prepared from sugar phenylhydrazones; the solubility of such 1,5-diphenylverdazyls in
polar solvents is improved by hydrophilic substituents in the
3-position (T. Schwarz-Fischer). The following compounds
3-[~-gu/acto-,and 3 - [ ~ have been prepared: 3-[~-g/uco-,
[ I ] R. Kuhn and H.Trischmann, Mh. Chem. 95, 457 (1964); Angew. Chem. 75, 294 (1963); Angew. Chem. internat. Edit. 2, 155
(1963).
Angew. Chem. internat. Edit. / Vol. 3 (1964) No. I1
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