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Qualitative and quantitative analysis of mixtures of sulfonamides. i
ANALYTICA
QUALITATIVE
AND
MIXTURES
CHIMICA
QUANTITATIVE OF
VOL.
ACTA
SULFONAMIDES.
ANALYSIS
7
(1952)
OF
I
This scrics of p;Lpcrs is part of an investigation, to which in November rggr a prize was awarded from the Blombcrg-funds of the State University at Lcyden. It contains a few methods concerning the qualitative and quantitative analysis of mixtures of a number of therapeutically used sulfonamides. At present, the clinical importance of these mixtures is steadily increasing. Mention must be made of the following restrictions: The investigation was set up for the purpose of finding one or more methods, I. by which it would bc possible to analyse every mixture of the sulfonamides which were talccn into account. This means that a general scheme had to be elaborated. M’c rcstrictcd ourselves to mixtures that did not contain more than 3 or 2. 4 sulfonamides, so fairly simple techniques could be used. 3. Further it was assumed that the mixtures did not contain any other comIn the last article, however, a few techniques pounds besides sulfonamides. concerning extraction of sulfas from tablets and ointments will be briefly reviewed. The sulfonamides which were taken into account are summarized in Table I. The trade names given thcrc will bc used throughout the present work. A list of synonyms may be found in NORTHEY~. A-number of important properties can be derived from the structure. WC give a brief survey: Sulfonamides with a. free aromatic amino group can form salts with acids I. and are consequently readily soluble in dilute hydrochloric acid. A few hydrochlorides arc sparingly soluble, however (irgafen, irgamid) ; hence they can easily be separated from the others. The salts of a number of organic and inorganic acids (picric acid and silicotungstic acid, for cxamplc) arc sparingly soluble in water and can be identified microchemically by their charnctcristic crystalline forms. We cannot use these salts, however, in the first part of our general scheme for qualitative analysis (separation of the components of the mixture), since they are only suitable for identification and confirmatory tests of the individual compounds. The same holds for many Rcferenccs
p.
56.
VOL.
7 (r95z)
ANALYSIS
OF
SULFONAMIDES
43
I
and coupling, reacother properties of the aromatic amino group: cliazotization tions with furfurol, #-dimethyl-aminobcnzaldehyde, sodium hypochloritc, phenol, lead dioxide, formaline, etc. Colours and precipitations, which occur during these reactions, are reviewed by many authors (SW, for example, LAPIBIU?), but, in neither for the sulfonamides general, they did not prove to be characteristic, as a group, nor for the individual compounds.
____-___--__ _.___ -Narnc
_. _-.-_-___ ---
H,S
Sulfacllazlllc Sod
_ -_. _-
l~orlllulo _-. .-
(‘-\-_ >
-
--.____
._ -_--
_
--____ _-.___
*\ -. -5O,SI-I
-
\
>
CCI,
--’
,s
-- /-\_
..- _.-- -._
s -/
1u111 sulf,lcll‘lzlllc
I-t,S
-.
-cs _>
-so,st-I
Na-salt
c L-1, ‘_*,,&_/--\__So _ ‘\.__/
Sotlium Sill
fa111czatl11
llc!
Sj-L
2
_P- -/
\s-_,) ‘CH,
n-it-LZIlt.
c t-i 3
IrgclfCIl
Sodium irgdfcti
/
H,S
-(->.-
H-CO
-
<
1-1,s.
Socliu tn irgnmicl
Su-L;llt
/,_I> so.,s
coI-1,-c1-1--c&--c
I SO.,X\;;I
I-I---co--c1-t
/I-i- \
1-1---s kJO,Si,
$-H,N-_(_>-S0,61-1-c Sctl1um sulfatlllazcrlc l?cjcr*c?~crs
p.
5G.
-->
-Cl-l,
Sil-\iLlt
Trgarnirl
S~~lu~cptazlllc
- so,s
SZI-Sillt
s
-J
=C(CI-I,),
- -so,is;i-1, __ >
.__.
P.
-_ _ _
L.
_-____ -
DIt: REEDER
-_-.--
-- _
Frzrnuln --
_
___--. _ _
.. ,~
/CH,
_- -
. _...
_.._I
scptazinc and succinylsulfntliia;zolc contain a secondary Soluscptazinc, aromatic nn~ino group. The last two coml>ounds arc sparingly soluble in &ICI, however. In succinyls~~Ifatl~i~zo1 this is clue to the n~i~hbourin~ -CO- group and the bcnzenc-nucleus (both arc electron-attracting). Since the I-I-atoms of the -S0,NH2 and -S02NI-I groups in-e more or less 3* acidic, the formation of salts is possilAc. The sulfonamides arc soluble in dilute as it forms a sodium hydroxycle solution, thcrcforc. Irgafcn is an exception, sodium salt which is but sparingly soluble in dilute NaOH. Sulfaguanidinc is insoluble in NaOl-I, since the SO,=N-group does not contain ionic I-i-atoms. Many salts of heavy mctnls arc insoluble in tsater and can be used for microchemicnl iclcntification. 4. Sulfonamides with the -SO,NH-12 groull cztn be hydrolysccl by boiling arc not with concentrated acids; in gcncml, however, the compounds R-NH, very useful for iclcntifi~tiot~ 13urposcs, in view of the similarity of many of their yhysical and ciicmicd propertics (see hPskxE~, STAINIER AND hP1&RE3, 3_.
-ikAVAGLIQ).
In addition to thcsc well-known prollcrtics, WC shall make USC of the different solubilities of the sulfonnmidcs in a nnmbcr of organic solvents. Many data on this subject have been published, but, since many are still lacking (or, sometimes, Rcf:lcrcwcs
p.
56.
ANALYSIS OF SULFONAnfIDES
VOL. 7 (1952)
I
45
unreliable), we ourselves estimated another number of solubilities. using the technique of SCHULECK AND ROSZA 5. Table II covers both data of the literature and of our own determinations. The solubilities of the sulfonamides in chloroform, petroleum ether, diethylethcr and the higher alcohols are so slight, that WC did not use these solvents in the various separations. Many schemes have been published. by means of which the identity of an unknown, pure, sulfonamide can be established0-z3, These schemes arc mainly based on more or less specific colour- and precipitation-reactions. However, not a single one is suitable for identifying the components of a mixture, since in this case previous separations are necessary. Hitherto, a few investigations concerning the qualitative and quantitative analysis of various mixtures of sulfonamides have been published ‘0.22-2a. They all are dealing with particular cases, involving chiefly mixtures of two or three of sulfathiazole, sulfadiazinc, sulfamerazine and sulfamezathine. the following: Their results will be discussed subsequently, together with those of our own investigations.
_ -.._
\vntcr ____ --.
__--_-.
Soluscptazille Sodium irgamid Sodium irgafcn Sodium sulfacliazinc
~-
-.
ZI-l,OH --.---_-
--
:z: z-x
___ ---.
-
7 y&HCI 5 y:,NaOH -- _____ -t- i-
+
+++
Sodxum sulfnmerazlnc Sodium sulfamczathlne Sodium sulfacetamide Sodium sulfapyridlnc Sodium sulfathiazolc Sulfacetarnidc Sulfanilamidc Sulfadiazinc Sulfamerazinc Sulfamezathinc Elkosin Sulfathlazol Sulfametl~ylthiazol Sulfapyritllnc Irgafen Irgamicl Septazinc Succinylsulfathiazolc Sulfaguanidinc
(
.----
+
- ._--_
((:l-I,),co -/-
-t
-
-
---
++-I+++ +++
+_+t ii= -------------_--------
__.
i-l+
+-t-s+-t--t -----t+/+--++ -I- + -‘T-
-
+ f +++ +++
i-
+
: -: 1- 5 -t++ +++ ff-t ----+++
-: -t
i- -t -t + + -t +++
-I--t +++ -t -I- -I-
::$Z -
rtz= -t/:z: z-: +-t-t ++ --t--+ + -t”+‘-+ -I- +
---
+-I-
-!-
+
+L
The investigations to be reported were divided into a number of separate general studies, These can be briefly summarized as follows: References
p.
56.
I'. L. DE
REEDER
VOL.
7 (1952)
I. Qualitative analysis. a. Preliminary examinations. b. Separations and identity tests. c. Chromatography. II. Quantitative analysis. a. Quantitative estimations without previous separation of the components of the mixture b. Quantitative estimations after previous separation of the components of the mixture. III. A few additional investigations. The solubilitics indicated hold at zoo C. Solubility-ranges arc given instead of accurutc values, since literature-data seldom agree mutually. The vast amount of literature, which is included in this table, is not further specified. -t_ + -t_ Solubility 10 y0 or more - Solubility less than 0.x o/o -Solubility 0.5-0.1 y. + -t Solubility 10-z %, Solubility r-o.5 “/0 Solubility 2-r o/ iI.
Q~~nlitativc
nnalysis
Previous to separation of the mixture into well-defined groups and finally into its components, a number of preliminary examinations arc made. The positive and negative evidences, thus obtained, arc particularly useful as an aid in subsequent operations. The material to be tested must be available in relatively large quantities, howcvcr. Thercforc, these methods can never become of clinical importance. Moreover. they take up a lot of time. Chromatographic methods supply this deficiencies, and for that reason will be separately and amply discussed. Though spcctrometric methods as well are suitable for analysing small quantities of material, they have but little value in our case, since the spectra (in visible and ultraviolet regions) of the different sulfonamides arc very like. A brief discussion about these spectra will bc given in II. a.
concerning I. The outward aspect of the mixture rarely can give information the prcscnce or abscncc of certain sulfonamides. Most of these are colourless or sometimes faintly yellow. Only a few sulfas are colourcd, for csample prontosil soluble (blood red) and OH H,SO,S
-
prontos~l
other azo-sulfonamides. Rrfcrcwccs
$.
56.
They
are of little
soluble importance
at present,
however,
and
ANAL’t’SIS
OF
SULFC?N.A3IIDES
1
47
Fzg:, ra. Mixture of r;ulfadiaeinc, s~iifamezathine and ~ulf~niian~iclc. Two dlffercnt crystallme forms (a and b); may be also a thwd one ? Fig. I b. The mixture of Fig. I a, after previous lsolatmn from a tablet and rccryhtall~atton from much alcohol. 3Fig. xc. Sulfathiazolc and sulfaclmzine. I*!g. zti. Sulfathiazolc and sulfadiazlne, rccrystallwxl from acctonc after isolation. Fig. xc. Sulfaguanldmc and sulfadiazlnc. F1g. If. Irgamicl and irgafcn.
48
P. L. DE
REEDER
VOL.
7 (1932)
no mixtures which contain them have been put on the market. Consequently, WC did not implicate them in our investigations. If the sample that is to be analysed is an aqueous solution, it can contain only water-soluble sulfonamides. In practice, this comes to the presence of one or more of the sodium salts of Table I. Only one of the azo-compounds mentioned is soluble in water (prontosil soluble) ; it colours the solution deeply red. All other compounds give colourlcss solutions. Next, a little quantity of the solid mixture is microscopically examined. By this examination it is sometimes possible to establish the presence of two or three distinct compounds, which can be distinguished by differences in the crystals present 5 commercial mixtures, the presence of characteristic and specific crystalline forms can hardly be expected; moreover, milling and mixing often destroy the initial forms. Consequently, by simple microscopical examination no conclusions can be drawn about the presence of some particular sulfonamide. On the other hand, if emphasis is placed not upon the crystal forms as a criterion for identification, but upon several geometric and optical properties of the crystal fragments, the findings may be conclusive as regards the mere number of components present. This knowledge may of course bc very helpful in subsequent analysis. Drawings of microscopic images of a few mixtures arc given in Fig. I. In this figure, crystal fragments that are clearly different from each other have been indicated by u, band c. It is thus highly probable that they correspond with different components of the mixture. Microscopical examination may be of greater value if it is attended with crystaloptical measurements (isotropy or anisotropy, sign of double refraction, angles of extinction, values of the different refraction indexes). A number of optical data has been given by KEENAN w. However, the performance of crystal-optical investigations is rather unfamiliar to the average pharmacist or chemist-analyst, so we have not included this part of the physical e.xamination of solid mixtures in our work. In aqueous solutions a test for the presence of sodium ions may be carried 2. out. The following procedure was found to bc adequate. To one drop of the solution to be tested sufficient acetic acid (3 %) is added to make it slightly acid (litmus paper). If a precipitation is formed, the solution is filtered. To one drop of the clear filtrate one drop of zinc-uranyl-acetate reagent (rather concentrated solution of zinc-uranyl-acctatc, strongly acidified with acetic acid) is added. If the crystals of the triple-acetate do not separate, one drop of the original solution is evaporated to dryness and one drop of the reagent is added to the solid residue. Viewed under the microscope by transmitted light the characteristic monoclinic twins of tetrahedras possess an octahedral aspect; they appear to be colourless, but are in reality faintly yellow (Fig. 2). Rcfcre9rces p. 5G.
VOL. 7 (1952)
ANALYSIS
OF
SULFONAMIDES
I
49
If the mixture to be tested is solid, it may be extracted with water and the aqueous solution so obtained tested for sodium. A positive sodium reaction indicates the presence of one or more sodium sulfonamides. It must be borne in mind, however, that in addition to these salts, other sulfonamides may bc present which are not sodium salts. decompose upon heating above the 3. All sulfonamides Fig. 2. Sodium zinc melting-point. At first, some form deeply coloured prouranyl ncetotc ducts (sulfanilamide : violet, sulfadiazine: red, sulfapyridine : (60 x). yellow, then violet, etc.), next, they all decompose entirely into gaseous and liquid products (aniline, ammonia, sulphur dioxide) and a black residue. As a preliminary examination the expulsion of hydrogen sulfide and the appearance of a sublimate is of much importance. Hydrogen sulfide is expelled upon heating of one or more of the following: 3a. Na-irgafen septazine soluseptazine Na-irgamid Na-sulfadiazine
sulfamerazine Na-sulfamerazine sulfamethylthiazole elkosin sulfamezathine
Na-sulfamezathine sulfathiazole Na-sulfathiazole succinylsulfathiazole
Noteworthy is the distinction between irgafen, irgamid and sulfadiazine expulsion of H,S) and their sodium salts, which do expel H,S. However, apparently strange phenomenon was established beyond all doubt. TABLE
Compound
Sublrmate
Elkosin
G-amino-2,4-clin1ctl1ylyyrlmiclinc
Irgafcn
3,q-dimethylbenzoiiitril
Sulfadiazine and Na-sulfacliazine
2-ammopyrini1d1ne
Sulfamerazine and 4-methyl-2-aminosodium salt pyrlmidlnc Sulfamezathme and sodium salt Sulfaguanicline Ammonium sulfite, together with a little ammonium sulfate. Rc/cre?tccs
p. 56.
(no this
III hIeltlll~-
point
181-183~ GGO 127-1~8~ I 5g-rGo0 X52-153” Sublimates
Outward appearance of the sublimate Yellow liquid, congeals to a nearly colourless, Glassy mass. Yellow, sometimes colour!ess liqwd, congeals after bomc t1mc to a soft mass. Colourless solid ; somctimch yellow parts, caused by aniline. Colourless solid, somctnncs yellow parts. Colourlcss solld, somctlmes yellow parts. Colourlcrs :ol~cl.
P. L. DE
50
REEDER
VOL.
7
(1952)
The performance of the test is described below. 3b. Only a few sulfonamides give a sublimate on heating; therefore, its formation is the more characteristic. Moreover, a number of these few compounds are of great importance at present (sulfadiazine, sulfamcrazine and snlfamezathine), and may be found in nearly every mixtur e of sulfas. Good distinctive reactions for the compounds present in the sublimate are at hand, so the appearance of a sublimate is of great value as a preliminary test. The compounds which may bc found in the sublimate are included in Table III. It is remarkable that succinylsulfathiazole docsnot give a sublimate of succinic anhydride, and also that Na-irgafen does not give one like irgafen. On macro-scale the heat test may be performed in the following way. A little quantity of the solicl, previously dried * mixture is slowly heated in a little test-tube of hard glass. Only the extreme end of the tube is heated, while it is held nearly horizontally to prevent running back of condensing liquids. The substance melts and decomposition soon starts. Finally the tube is heated until dark-red-hot, while it is slowly rotated. During heating a test-paper soaked in lead-acetate solution is placed in the upper end of the tube. Evolving of hydrogen sulphidc is indicated by a brown
or blaclc colour. When the decomposition has been sublimate is tested for the compounds
completed, mentioned
the tube is cooled and the in Table III (column 2).
The erformancc of the test on a micro-scale is clcscribcd below. The formation of a su &llmatc on heating of clkosin, irgafen and sulfaguanidine and of the various soclium salts is not clcscrlbcd m the literature. \Ve lclcntlfied the sublimating compounds by rccrystalllsation (climetl~ylbcnzonitril : alcohol/water I : I) or rcsublimation the other compounds) and dcterminatlon of the melting point and microchcmica I reactions ( (NI-14)aS0,, (NH,),SOJ. The compounds may be detected by means of the following reactions: a. Raybin-reaction to prove the presence of z-aminopyrimidine. b. Saponification with cont. HCl, extraction with ether and sublimation of the residue after evaporation of the ether. Dimcthylbcnzoic acid is then detected in this sublimate. c. Microchemical test for NH, with H,PtCl,. d. Microchemical test for SOam with calcium acetate. 4-methyl-2-aminopyrimidinc and 4,G-dimethyl-z-aminopyrimidine may be detectedby means of the reaction of HUCKNALL AND TURFITT~~ but this reaction gives better results when it is carried out with the original mixture. No reaction is available to prove the presence of 6-amino-z,4-dimethylpyrimidine (from elkosin). A brief discussion of the reactions mentioned may follow. * The mixture must bc well clricd, since the sublimating compounds (except ~NH,),SOJ are all very soluble in wntcr. If a solution has to bc tested, rt is cvaporated to clryness on the water-bath; the residue is powdered and used In the heat test. 1?cfcrc91ccs
p.
56.
VOL.
7 (xg52)
ANALYSIS
OF
SULFONAnllDES
X
5I
3i*3z. A few mg of the sublimate is dissolved in I ml a. The Raybin-reaction of a solution of resorcinol (5 %) in ethyl alcohol (96 */$); I ml of concentrated sulfuric acid is then added. The temperature may rise to 80” C and a blood-red colour develops if z-aminopyrimidine is present. Upon dilution with zs ml icewater and neutralisation with ammonia, the colour turns blue with a reddish tint. If sufficient aminopyrimidine is present, a red precipitation may be obtained by pouring the solution on ice and adding sodium bisulfite. This precipitation yields a blue colour when alkali or ammonia is added. Especially the first blood-red colour is very sensitive. When carried out on a micro-scale, 0.0~ rng aminopyrimidine may be detected. In addition to this discussion, a few notes may follow. It is essentially that the sublimate is added to the resorcinol-solution before I. addition of the sulfuric acid. The other sequence yields a faint colour or no colour at all. The colour is most intensive after about I minute. 2. 3. The reaction is completely specific for z-aminopyrimidinc. Sublimates of mixtures which did not contain sulfadiazine always gave a negative reaction. A few aldehydes and ketones also give a colour reaction with resorcinoi, but there is little chance, if any, of finding these in a mixture of sulfonamides. 4. Aminop~~idine may also be obtained by acid hydrolysis of sulfadiazine and of sodium sulfadiazine (c/. structure). The Raybin-reaction may thus be performed with the original mixture itself. In this case, the solution is gently heated after addition of the sulfuric acid. The reaction is again completely specific for sulfadiazine and sodium sulfadiazine. 5. The other colour reactions, mentioned by RAYBIN~~ and performed with the blood-red solution, do not give conclusive evidence of the presence or absence of suifadiazine or sodium sulfadiazine. 6. The presence of ammonium sulfite in the sublimate (from s~fa~a~id~e) causes evolution of sulfur dioxide on addition of sulfuric acid. This does not inhibit the reaction of aminop~~idine. 7. The reaction may be carried out on a micro-scale by beating a sample of the mixture in a capillary that is placed in a metal block. The capillary must stick out about x.5 cm over the block. 0.5-x mg of the mixture is placed in the capillary and the inside of the upper part is cleaned by wiping with a piece of filter paper. The block is then heated to 300” C and held at this temperature for rg minutes. The sublimate appears as a yellow or colourlcss, liquid or solid mass nearly 5 mm under the end of the capillary. After cooling, the upper part of the capillary is cut off and broken to pieces. The Raybin-reaction is now best performed in a white porcelain crucible with a few drops of resorcinol-solution and sulfuric acid. may be detected by the method of DE WOLFP~. z ml b. Dimethylbenzonitril concentrated WC1 are placed in a test tube and IO-SO mg of the sublimate added. Rcferc~tcss
p. 56.
P.
L.
DE
REEDER
VOL.
7 (rg52)
The solution is boiled for x5 .&nutes; evaporated water is but sparingly supplied, so that after boiling has been completed only a small volume of the solution is left. After cooling, the solution is extracted with 2 ml of ether. The ethereous extract is dried with sodium sulfate for half an hour, and then evaporated to dryness in a small watch glass (diam. I-Z cm). Next, the watch glass is covered with an object slide, which may be cooled by means of a piece of moistened cottonwool, and the whole is placed on a metal block, which previously has been heated to 120-150~ C. After about ten minutes the object slide is covered with a sublimate of 3,+dimethylbenzoic acid. This substance can be further identified in the following ways: D&YC’sd The sublimate forms thin, colourless transparent, ; O==_~~G&+ xo six-sided plates and prisms, which are often long Y J r/ and narrow (Fig. 3). *;q u%L] II 2, The melting point is 164~ C. 0 /J PQ~,J@\O 3. To one drop of NaOH (x “/u) the ~ethylbenzoic acid is added until the solution is completely u 22%2~0:0 saturated. A few pieces of solid calcium acetate are Prg. 3. Sublimate of added to the clear solution, The calcium salt of 3,4-dimetltylbcriaoic acid the benzoic acid is obtained in form of rosettes of (Go x). long, very slender needles, which cover the pieces of calcium acetate (Fig. 4)” c. The ammonium ion (from sulf~~a~idine) is detected in an aqueous solution of the sublimate by the well-known reaction with HePtCI,. To one drop of the dilute solution to be tested, acidified with HCl, one drop of a concentrated solution of H&X.II~ is added. Brilliant, highly refractive, deep-yellow octahedra separate at once {Fig. 5).
I;‘ig. 4. Calcium salt of 3,4-climethylbcn~aic acid (60 x ).
Fig.
5. (NH,),PtCl, (60 x).
The presence of other compounds in the sublimate (mentioned in Table III) did not interfere with the reaction. Care must be taken, however, of the presence of potassium ions from the glass powder, which is present when the sublimation is performed on a micro-scale (cf. a, 7). In this instance, ammonia may be separated prior to identification by gently warming the solution to be tested after &7fcvcnces
$3.
545.
var.. 7
(1952)
AEJALYSSS
OF
SULFONAMIDES
I
53
a drop of NaOH has been added, and fixation of expelled Nl-la in a drop of HCl. d. The sulfite ion is detected in an aqueous solution of the sublimate by means of the microchemical reaction with calcium acetate. A drop of aqueous calcium acetate solution (which must not be too concentrated) is added to a small drop of an aqueous solution of the sublimate. Strongly birefringent, tiny discs and spherulites are formed. The separation is slow and the discs always remain small. If the solution of the sublimate is caused to flow into the drop of the reagent, however, the small discs and spherulites increase rapidly in size, and after a few minutes prisms separate, which may be seen at fairly moderate magnification (Fig. 6). If the drop has been almost evaporated to dryness, the clusters of needles of CaSO, appear (Fig. 7). @
0 x;
&;Q
0x. Q 8p
~OQJc? LG& G. CaSO$j (Go x ).
P1g* 7. cnso,
(Go x) *
The reaction proved to be completely specific for the sulfite ion. Dimethylbenzoic acid does not interfere, as the reaction is performed with an aqueous solution of the original sublimate, in which only dimcthylbenzonitril is present. Another reaction of great value is the vanilline-reaction of I~UCKNALL AND T:;FITT I* which indicates the presence of the mcthylpyrimidine-dcrivates sulfamerazine and sulfamezathine. To one drop of sulfuric acid a few crystals of vanilline are added; after mixing, a little of the solid mixture to be t&cd is added. The drop is then gently heated above a micro-flame. The changes of colour are examined on a white background. Most sulfonamides yield a yellowish or slight green colour, just like a blank test. The following exceptions are therefore of importance: sulfamerazine and sodium salt sulfamezathine and sodium salt irgamid and sodium salt
colour : intense red ; ,, intense red ; very dark green or black; the edges ,, of the drop are usually dark red.
It is highly remarkable that the third methylpyrimidine-derivate, elkosin, does not give the characteristic red colour. The reaction proved to be specific for the six compounds mentioned. However, if the mixture contains irgarnid together with one or more of the methylpyrimidines mentioned, the dark green colour of irgamid masks completely the red one of the methylp~imidines. We tried to avoid this difficulty by performing the Rc~i.W11casp.
56.
QUALITATIVE
AND
MIXTURES
CHIMICA
QUANTITATIVE OF
VOL.
ACTA
SULFONAMIDES.
ANALYSIS
7
(1952)
OF
I
This scrics of p;Lpcrs is part of an investigation, to which in November rggr a prize was awarded from the Blombcrg-funds of the State University at Lcyden. It contains a few methods concerning the qualitative and quantitative analysis of mixtures of a number of therapeutically used sulfonamides. At present, the clinical importance of these mixtures is steadily increasing. Mention must be made of the following restrictions: The investigation was set up for the purpose of finding one or more methods, I. by which it would bc possible to analyse every mixture of the sulfonamides which were talccn into account. This means that a general scheme had to be elaborated. M’c rcstrictcd ourselves to mixtures that did not contain more than 3 or 2. 4 sulfonamides, so fairly simple techniques could be used. 3. Further it was assumed that the mixtures did not contain any other comIn the last article, however, a few techniques pounds besides sulfonamides. concerning extraction of sulfas from tablets and ointments will be briefly reviewed. The sulfonamides which were taken into account are summarized in Table I. The trade names given thcrc will bc used throughout the present work. A list of synonyms may be found in NORTHEY~. A-number of important properties can be derived from the structure. WC give a brief survey: Sulfonamides with a. free aromatic amino group can form salts with acids I. and are consequently readily soluble in dilute hydrochloric acid. A few hydrochlorides arc sparingly soluble, however (irgafen, irgamid) ; hence they can easily be separated from the others. The salts of a number of organic and inorganic acids (picric acid and silicotungstic acid, for cxamplc) arc sparingly soluble in water and can be identified microchemically by their charnctcristic crystalline forms. We cannot use these salts, however, in the first part of our general scheme for qualitative analysis (separation of the components of the mixture), since they are only suitable for identification and confirmatory tests of the individual compounds. The same holds for many Rcferenccs
p.
56.
VOL.
7 (r95z)
ANALYSIS
OF
SULFONAMIDES
43
I
and coupling, reacother properties of the aromatic amino group: cliazotization tions with furfurol, #-dimethyl-aminobcnzaldehyde, sodium hypochloritc, phenol, lead dioxide, formaline, etc. Colours and precipitations, which occur during these reactions, are reviewed by many authors (SW, for example, LAPIBIU?), but, in neither for the sulfonamides general, they did not prove to be characteristic, as a group, nor for the individual compounds.
____-___--__ _.___ -Narnc
_. _-.-_-___ ---
H,S
Sulfacllazlllc Sod
_ -_. _-
l~orlllulo _-. .-
(‘-\-_ >
-
--.____
._ -_--
_
--____ _-.___
*\ -. -5O,SI-I
-
\
>
CCI,
--’
,s
-- /-\_
..- _.-- -._
s -/
1u111 sulf,lcll‘lzlllc
I-t,S
-.
-cs _>
-so,st-I
Na-salt
c L-1, ‘_*,,&_/--\__So _ ‘\.__/
Sotlium Sill
fa111czatl11
llc!
Sj-L
2
_P- -/
\s-_,) ‘CH,
n-it-LZIlt.
c t-i 3
IrgclfCIl
Sodium irgdfcti
/
H,S
-(->.-
H-CO
-
<
1-1,s.
Socliu tn irgnmicl
Su-L;llt
/,_I> so.,s
coI-1,-c1-1--c&--c
I SO.,X\;;I
I-I---co--c1-t
/I-i- \
1-1---s kJO,Si,
$-H,N-_(_>-S0,61-1-c Sctl1um sulfatlllazcrlc l?cjcr*c?~crs
p.
5G.
-->
-Cl-l,
Sil-\iLlt
Trgarnirl
S~~lu~cptazlllc
- so,s
SZI-Sillt
s
-J
=C(CI-I,),
- -so,is;i-1, __ >
.__.
P.
-_ _ _
L.
_-____ -
DIt: REEDER
-_-.--
-- _
Frzrnuln --
_
___--. _ _
.. ,~
/CH,
_- -
. _...
_.._I
scptazinc and succinylsulfntliia;zolc contain a secondary Soluscptazinc, aromatic nn~ino group. The last two coml>ounds arc sparingly soluble in &ICI, however. In succinyls~~Ifatl~i~zo1 this is clue to the n~i~hbourin~ -CO- group and the bcnzenc-nucleus (both arc electron-attracting). Since the I-I-atoms of the -S0,NH2 and -S02NI-I groups in-e more or less 3* acidic, the formation of salts is possilAc. The sulfonamides arc soluble in dilute as it forms a sodium hydroxycle solution, thcrcforc. Irgafcn is an exception, sodium salt which is but sparingly soluble in dilute NaOH. Sulfaguanidinc is insoluble in NaOl-I, since the SO,=N-group does not contain ionic I-i-atoms. Many salts of heavy mctnls arc insoluble in tsater and can be used for microchemicnl iclcntification. 4. Sulfonamides with the -SO,NH-12 groull cztn be hydrolysccl by boiling arc not with concentrated acids; in gcncml, however, the compounds R-NH, very useful for iclcntifi~tiot~ 13urposcs, in view of the similarity of many of their yhysical and ciicmicd propertics (see hPskxE~, STAINIER AND hP1&RE3, 3_.
-ikAVAGLIQ).
In addition to thcsc well-known prollcrtics, WC shall make USC of the different solubilities of the sulfonnmidcs in a nnmbcr of organic solvents. Many data on this subject have been published, but, since many are still lacking (or, sometimes, Rcf:lcrcwcs
p.
56.
ANALYSIS OF SULFONAnfIDES
VOL. 7 (1952)
I
45
unreliable), we ourselves estimated another number of solubilities. using the technique of SCHULECK AND ROSZA 5. Table II covers both data of the literature and of our own determinations. The solubilities of the sulfonamides in chloroform, petroleum ether, diethylethcr and the higher alcohols are so slight, that WC did not use these solvents in the various separations. Many schemes have been published. by means of which the identity of an unknown, pure, sulfonamide can be established0-z3, These schemes arc mainly based on more or less specific colour- and precipitation-reactions. However, not a single one is suitable for identifying the components of a mixture, since in this case previous separations are necessary. Hitherto, a few investigations concerning the qualitative and quantitative analysis of various mixtures of sulfonamides have been published ‘0.22-2a. They all are dealing with particular cases, involving chiefly mixtures of two or three of sulfathiazole, sulfadiazinc, sulfamerazine and sulfamezathine. the following: Their results will be discussed subsequently, together with those of our own investigations.
_ -.._
\vntcr ____ --.
__--_-.
Soluscptazille Sodium irgamid Sodium irgafcn Sodium sulfacliazinc
~-
-.
ZI-l,OH --.---_-
--
:z: z-x
___ ---.
-
7 y&HCI 5 y:,NaOH -- _____ -t- i-
+
+++
Sodxum sulfnmerazlnc Sodium sulfamczathlne Sodium sulfacetamide Sodium sulfapyridlnc Sodium sulfathiazolc Sulfacetarnidc Sulfanilamidc Sulfadiazinc Sulfamerazinc Sulfamezathinc Elkosin Sulfathlazol Sulfametl~ylthiazol Sulfapyritllnc Irgafen Irgamicl Septazinc Succinylsulfathiazolc Sulfaguanidinc
(
.----
+
- ._--_
((:l-I,),co -/-
-t
-
-
---
++-I+++ +++
+_+t ii= -------------_--------
__.
i-l+
+-t-s+-t--t -----t+/+--++ -I- + -‘T-
-
+ f +++ +++
i-
+
: -: 1- 5 -t++ +++ ff-t ----+++
-: -t
i- -t -t + + -t +++
-I--t +++ -t -I- -I-
::$Z -
rtz= -t/:z: z-: +-t-t ++ --t--+ + -t”+‘-+ -I- +
---
+-I-
-!-
+
+L
The investigations to be reported were divided into a number of separate general studies, These can be briefly summarized as follows: References
p.
56.
I'. L. DE
REEDER
VOL.
7 (1952)
I. Qualitative analysis. a. Preliminary examinations. b. Separations and identity tests. c. Chromatography. II. Quantitative analysis. a. Quantitative estimations without previous separation of the components of the mixture b. Quantitative estimations after previous separation of the components of the mixture. III. A few additional investigations. The solubilitics indicated hold at zoo C. Solubility-ranges arc given instead of accurutc values, since literature-data seldom agree mutually. The vast amount of literature, which is included in this table, is not further specified. -t_ + -t_ Solubility 10 y0 or more - Solubility less than 0.x o/o -Solubility 0.5-0.1 y. + -t Solubility 10-z %, Solubility r-o.5 “/0 Solubility 2-r o/ iI.
Q~~nlitativc
nnalysis
Previous to separation of the mixture into well-defined groups and finally into its components, a number of preliminary examinations arc made. The positive and negative evidences, thus obtained, arc particularly useful as an aid in subsequent operations. The material to be tested must be available in relatively large quantities, howcvcr. Thercforc, these methods can never become of clinical importance. Moreover. they take up a lot of time. Chromatographic methods supply this deficiencies, and for that reason will be separately and amply discussed. Though spcctrometric methods as well are suitable for analysing small quantities of material, they have but little value in our case, since the spectra (in visible and ultraviolet regions) of the different sulfonamides arc very like. A brief discussion about these spectra will bc given in II. a.
concerning I. The outward aspect of the mixture rarely can give information the prcscnce or abscncc of certain sulfonamides. Most of these are colourless or sometimes faintly yellow. Only a few sulfas are colourcd, for csample prontosil soluble (blood red) and OH H,SO,S
-
prontos~l
other azo-sulfonamides. Rrfcrcwccs
$.
56.
They
are of little
soluble importance
at present,
however,
and
ANAL’t’SIS
OF
SULFC?N.A3IIDES
1
47
Fzg:, ra. Mixture of r;ulfadiaeinc, s~iifamezathine and ~ulf~niian~iclc. Two dlffercnt crystallme forms (a and b); may be also a thwd one ? Fig. I b. The mixture of Fig. I a, after previous lsolatmn from a tablet and rccryhtall~atton from much alcohol. 3Fig. xc. Sulfathiazolc and sulfaclmzine. I*!g. zti. Sulfathiazolc and sulfadiazlne, rccrystallwxl from acctonc after isolation. Fig. xc. Sulfaguanldmc and sulfadiazlnc. F1g. If. Irgamicl and irgafcn.
48
P. L. DE
REEDER
VOL.
7 (1932)
no mixtures which contain them have been put on the market. Consequently, WC did not implicate them in our investigations. If the sample that is to be analysed is an aqueous solution, it can contain only water-soluble sulfonamides. In practice, this comes to the presence of one or more of the sodium salts of Table I. Only one of the azo-compounds mentioned is soluble in water (prontosil soluble) ; it colours the solution deeply red. All other compounds give colourlcss solutions. Next, a little quantity of the solid mixture is microscopically examined. By this examination it is sometimes possible to establish the presence of two or three distinct compounds, which can be distinguished by differences in the crystals present 5 commercial mixtures, the presence of characteristic and specific crystalline forms can hardly be expected; moreover, milling and mixing often destroy the initial forms. Consequently, by simple microscopical examination no conclusions can be drawn about the presence of some particular sulfonamide. On the other hand, if emphasis is placed not upon the crystal forms as a criterion for identification, but upon several geometric and optical properties of the crystal fragments, the findings may be conclusive as regards the mere number of components present. This knowledge may of course bc very helpful in subsequent analysis. Drawings of microscopic images of a few mixtures arc given in Fig. I. In this figure, crystal fragments that are clearly different from each other have been indicated by u, band c. It is thus highly probable that they correspond with different components of the mixture. Microscopical examination may be of greater value if it is attended with crystaloptical measurements (isotropy or anisotropy, sign of double refraction, angles of extinction, values of the different refraction indexes). A number of optical data has been given by KEENAN w. However, the performance of crystal-optical investigations is rather unfamiliar to the average pharmacist or chemist-analyst, so we have not included this part of the physical e.xamination of solid mixtures in our work. In aqueous solutions a test for the presence of sodium ions may be carried 2. out. The following procedure was found to bc adequate. To one drop of the solution to be tested sufficient acetic acid (3 %) is added to make it slightly acid (litmus paper). If a precipitation is formed, the solution is filtered. To one drop of the clear filtrate one drop of zinc-uranyl-acetate reagent (rather concentrated solution of zinc-uranyl-acctatc, strongly acidified with acetic acid) is added. If the crystals of the triple-acetate do not separate, one drop of the original solution is evaporated to dryness and one drop of the reagent is added to the solid residue. Viewed under the microscope by transmitted light the characteristic monoclinic twins of tetrahedras possess an octahedral aspect; they appear to be colourless, but are in reality faintly yellow (Fig. 2). Rcfcre9rces p. 5G.
VOL. 7 (1952)
ANALYSIS
OF
SULFONAMIDES
I
49
If the mixture to be tested is solid, it may be extracted with water and the aqueous solution so obtained tested for sodium. A positive sodium reaction indicates the presence of one or more sodium sulfonamides. It must be borne in mind, however, that in addition to these salts, other sulfonamides may bc present which are not sodium salts. decompose upon heating above the 3. All sulfonamides Fig. 2. Sodium zinc melting-point. At first, some form deeply coloured prouranyl ncetotc ducts (sulfanilamide : violet, sulfadiazine: red, sulfapyridine : (60 x). yellow, then violet, etc.), next, they all decompose entirely into gaseous and liquid products (aniline, ammonia, sulphur dioxide) and a black residue. As a preliminary examination the expulsion of hydrogen sulfide and the appearance of a sublimate is of much importance. Hydrogen sulfide is expelled upon heating of one or more of the following: 3a. Na-irgafen septazine soluseptazine Na-irgamid Na-sulfadiazine
sulfamerazine Na-sulfamerazine sulfamethylthiazole elkosin sulfamezathine
Na-sulfamezathine sulfathiazole Na-sulfathiazole succinylsulfathiazole
Noteworthy is the distinction between irgafen, irgamid and sulfadiazine expulsion of H,S) and their sodium salts, which do expel H,S. However, apparently strange phenomenon was established beyond all doubt. TABLE
Compound
Sublrmate
Elkosin
G-amino-2,4-clin1ctl1ylyyrlmiclinc
Irgafcn
3,q-dimethylbenzoiiitril
Sulfadiazine and Na-sulfacliazine
2-ammopyrini1d1ne
Sulfamerazine and 4-methyl-2-aminosodium salt pyrlmidlnc Sulfamezathme and sodium salt Sulfaguanicline Ammonium sulfite, together with a little ammonium sulfate. Rc/cre?tccs
p. 56.
(no this
III hIeltlll~-
point
181-183~ GGO 127-1~8~ I 5g-rGo0 X52-153” Sublimates
Outward appearance of the sublimate Yellow liquid, congeals to a nearly colourless, Glassy mass. Yellow, sometimes colour!ess liqwd, congeals after bomc t1mc to a soft mass. Colourless solid ; somctimch yellow parts, caused by aniline. Colourless solid, somctnncs yellow parts. Colourlcss solld, somctlmes yellow parts. Colourlcrs :ol~cl.
P. L. DE
50
REEDER
VOL.
7
(1952)
The performance of the test is described below. 3b. Only a few sulfonamides give a sublimate on heating; therefore, its formation is the more characteristic. Moreover, a number of these few compounds are of great importance at present (sulfadiazine, sulfamcrazine and snlfamezathine), and may be found in nearly every mixtur e of sulfas. Good distinctive reactions for the compounds present in the sublimate are at hand, so the appearance of a sublimate is of great value as a preliminary test. The compounds which may bc found in the sublimate are included in Table III. It is remarkable that succinylsulfathiazole docsnot give a sublimate of succinic anhydride, and also that Na-irgafen does not give one like irgafen. On macro-scale the heat test may be performed in the following way. A little quantity of the solicl, previously dried * mixture is slowly heated in a little test-tube of hard glass. Only the extreme end of the tube is heated, while it is held nearly horizontally to prevent running back of condensing liquids. The substance melts and decomposition soon starts. Finally the tube is heated until dark-red-hot, while it is slowly rotated. During heating a test-paper soaked in lead-acetate solution is placed in the upper end of the tube. Evolving of hydrogen sulphidc is indicated by a brown
or blaclc colour. When the decomposition has been sublimate is tested for the compounds
completed, mentioned
the tube is cooled and the in Table III (column 2).
The erformancc of the test on a micro-scale is clcscribcd below. The formation of a su &llmatc on heating of clkosin, irgafen and sulfaguanidine and of the various soclium salts is not clcscrlbcd m the literature. \Ve lclcntlfied the sublimating compounds by rccrystalllsation (climetl~ylbcnzonitril : alcohol/water I : I) or rcsublimation the other compounds) and dcterminatlon of the melting point and microchcmica I reactions ( (NI-14)aS0,, (NH,),SOJ. The compounds may be detected by means of the following reactions: a. Raybin-reaction to prove the presence of z-aminopyrimidine. b. Saponification with cont. HCl, extraction with ether and sublimation of the residue after evaporation of the ether. Dimcthylbcnzoic acid is then detected in this sublimate. c. Microchemical test for NH, with H,PtCl,. d. Microchemical test for SOam with calcium acetate. 4-methyl-2-aminopyrimidinc and 4,G-dimethyl-z-aminopyrimidine may be detectedby means of the reaction of HUCKNALL AND TURFITT~~ but this reaction gives better results when it is carried out with the original mixture. No reaction is available to prove the presence of 6-amino-z,4-dimethylpyrimidine (from elkosin). A brief discussion of the reactions mentioned may follow. * The mixture must bc well clricd, since the sublimating compounds (except ~NH,),SOJ are all very soluble in wntcr. If a solution has to bc tested, rt is cvaporated to clryness on the water-bath; the residue is powdered and used In the heat test. 1?cfcrc91ccs
p.
56.
VOL.
7 (xg52)
ANALYSIS
OF
SULFONAnllDES
X
5I
3i*3z. A few mg of the sublimate is dissolved in I ml a. The Raybin-reaction of a solution of resorcinol (5 %) in ethyl alcohol (96 */$); I ml of concentrated sulfuric acid is then added. The temperature may rise to 80” C and a blood-red colour develops if z-aminopyrimidine is present. Upon dilution with zs ml icewater and neutralisation with ammonia, the colour turns blue with a reddish tint. If sufficient aminopyrimidine is present, a red precipitation may be obtained by pouring the solution on ice and adding sodium bisulfite. This precipitation yields a blue colour when alkali or ammonia is added. Especially the first blood-red colour is very sensitive. When carried out on a micro-scale, 0.0~ rng aminopyrimidine may be detected. In addition to this discussion, a few notes may follow. It is essentially that the sublimate is added to the resorcinol-solution before I. addition of the sulfuric acid. The other sequence yields a faint colour or no colour at all. The colour is most intensive after about I minute. 2. 3. The reaction is completely specific for z-aminopyrimidinc. Sublimates of mixtures which did not contain sulfadiazine always gave a negative reaction. A few aldehydes and ketones also give a colour reaction with resorcinoi, but there is little chance, if any, of finding these in a mixture of sulfonamides. 4. Aminop~~idine may also be obtained by acid hydrolysis of sulfadiazine and of sodium sulfadiazine (c/. structure). The Raybin-reaction may thus be performed with the original mixture itself. In this case, the solution is gently heated after addition of the sulfuric acid. The reaction is again completely specific for sulfadiazine and sodium sulfadiazine. 5. The other colour reactions, mentioned by RAYBIN~~ and performed with the blood-red solution, do not give conclusive evidence of the presence or absence of suifadiazine or sodium sulfadiazine. 6. The presence of ammonium sulfite in the sublimate (from s~fa~a~id~e) causes evolution of sulfur dioxide on addition of sulfuric acid. This does not inhibit the reaction of aminop~~idine. 7. The reaction may be carried out on a micro-scale by beating a sample of the mixture in a capillary that is placed in a metal block. The capillary must stick out about x.5 cm over the block. 0.5-x mg of the mixture is placed in the capillary and the inside of the upper part is cleaned by wiping with a piece of filter paper. The block is then heated to 300” C and held at this temperature for rg minutes. The sublimate appears as a yellow or colourlcss, liquid or solid mass nearly 5 mm under the end of the capillary. After cooling, the upper part of the capillary is cut off and broken to pieces. The Raybin-reaction is now best performed in a white porcelain crucible with a few drops of resorcinol-solution and sulfuric acid. may be detected by the method of DE WOLFP~. z ml b. Dimethylbenzonitril concentrated WC1 are placed in a test tube and IO-SO mg of the sublimate added. Rcferc~tcss
p. 56.
P.
L.
DE
REEDER
VOL.
7 (rg52)
The solution is boiled for x5 .&nutes; evaporated water is but sparingly supplied, so that after boiling has been completed only a small volume of the solution is left. After cooling, the solution is extracted with 2 ml of ether. The ethereous extract is dried with sodium sulfate for half an hour, and then evaporated to dryness in a small watch glass (diam. I-Z cm). Next, the watch glass is covered with an object slide, which may be cooled by means of a piece of moistened cottonwool, and the whole is placed on a metal block, which previously has been heated to 120-150~ C. After about ten minutes the object slide is covered with a sublimate of 3,+dimethylbenzoic acid. This substance can be further identified in the following ways: D&YC’sd The sublimate forms thin, colourless transparent, ; O==_~~G&+ xo six-sided plates and prisms, which are often long Y J r/ and narrow (Fig. 3). *;q u%L] II 2, The melting point is 164~ C. 0 /J PQ~,J@\O 3. To one drop of NaOH (x “/u) the ~ethylbenzoic acid is added until the solution is completely u 22%2~0:0 saturated. A few pieces of solid calcium acetate are Prg. 3. Sublimate of added to the clear solution, The calcium salt of 3,4-dimetltylbcriaoic acid the benzoic acid is obtained in form of rosettes of (Go x). long, very slender needles, which cover the pieces of calcium acetate (Fig. 4)” c. The ammonium ion (from sulf~~a~idine) is detected in an aqueous solution of the sublimate by the well-known reaction with HePtCI,. To one drop of the dilute solution to be tested, acidified with HCl, one drop of a concentrated solution of H&X.II~ is added. Brilliant, highly refractive, deep-yellow octahedra separate at once {Fig. 5).
I;‘ig. 4. Calcium salt of 3,4-climethylbcn~aic acid (60 x ).
Fig.
5. (NH,),PtCl, (60 x).
The presence of other compounds in the sublimate (mentioned in Table III) did not interfere with the reaction. Care must be taken, however, of the presence of potassium ions from the glass powder, which is present when the sublimation is performed on a micro-scale (cf. a, 7). In this instance, ammonia may be separated prior to identification by gently warming the solution to be tested after &7fcvcnces
$3.
545.
var.. 7
(1952)
AEJALYSSS
OF
SULFONAMIDES
I
53
a drop of NaOH has been added, and fixation of expelled Nl-la in a drop of HCl. d. The sulfite ion is detected in an aqueous solution of the sublimate by means of the microchemical reaction with calcium acetate. A drop of aqueous calcium acetate solution (which must not be too concentrated) is added to a small drop of an aqueous solution of the sublimate. Strongly birefringent, tiny discs and spherulites are formed. The separation is slow and the discs always remain small. If the solution of the sublimate is caused to flow into the drop of the reagent, however, the small discs and spherulites increase rapidly in size, and after a few minutes prisms separate, which may be seen at fairly moderate magnification (Fig. 6). If the drop has been almost evaporated to dryness, the clusters of needles of CaSO, appear (Fig. 7). @
0 x;
&;Q
0x. Q 8p
~OQJc? LG& G. CaSO$j (Go x ).
P1g* 7. cnso,
(Go x) *
The reaction proved to be completely specific for the sulfite ion. Dimethylbenzoic acid does not interfere, as the reaction is performed with an aqueous solution of the original sublimate, in which only dimcthylbenzonitril is present. Another reaction of great value is the vanilline-reaction of I~UCKNALL AND T:;FITT I* which indicates the presence of the mcthylpyrimidine-dcrivates sulfamerazine and sulfamezathine. To one drop of sulfuric acid a few crystals of vanilline are added; after mixing, a little of the solid mixture to be t&cd is added. The drop is then gently heated above a micro-flame. The changes of colour are examined on a white background. Most sulfonamides yield a yellowish or slight green colour, just like a blank test. The following exceptions are therefore of importance: sulfamerazine and sodium salt sulfamezathine and sodium salt irgamid and sodium salt
colour : intense red ; ,, intense red ; very dark green or black; the edges ,, of the drop are usually dark red.
It is highly remarkable that the third methylpyrimidine-derivate, elkosin, does not give the characteristic red colour. The reaction proved to be specific for the six compounds mentioned. However, if the mixture contains irgarnid together with one or more of the methylpyrimidines mentioned, the dark green colour of irgamid masks completely the red one of the methylp~imidines. We tried to avoid this difficulty by performing the Rc~i.W11casp.
56.