chromatography seem obvious. Au-
tomatic monitoring of column effluents
by available methods are necessarily
coniplicatcd by the prcsence of the
eluting solute, usually present in much
higher concentration than the substance
being separated. Successful water elu-
tion chromatography might make pos-
sible the detection and measurement
of eluted peaks by a simple physical
means such as a refinement of the con-
ductivity mcthod demonstratd here,
ultraviolet absorbance, p H measure-
ment, or possibly others. -4s pointed
out by Thompson and Morris ( I S ) , the
chemical mildness to which the separat-
ing substances arc subjected might be
advantageous with labile compounds.
I n prcparative work the ease with which
the purified material c a n be recovered
cannot bc escet,ded by any ot,her
method of chromat,ogrLipliy. In large
scale separations, such as those that
might be carried out indust~rially. the
elimination of reagcnt cost \vould bc a n
important factor.
X disadvantage of this typt, of chro-
matography as dewloped so far, lies
in its lack of versatility. From avail-
able data it ~vouldbe diffiidt to devise a
uwful schcme for tlie scyaration of all
the protclin amino acids on a single
column or even by an orderly succcssioii
of diffc,rent columns. If the isolation
of ccvtniii individual amino acids is
intendcd, a successful mctliod (mild cer-
t a i n l ~ i. i o be
~ drvelopcd.
P u t of tlir difficulty ill pcrforniing
prcy:trntivc, c-hromatography n i t h these
techiliqrics lit's in the e f h t of column
loatling t i n tht' iiosition of peaks.
'The ~licwdslin~icof clution p d s that
oceurs xhenever a relatively abundant
solute, is present a n d wliich is h ~ l dhack
to some degree by tlw resin is ade-
quately t~sp1:iiiii~d by a tlit~orc~tical
c o i i d i w t i o n of self-sl~ar~~t~riirig
ant1
self-(liffiising boinidariw ( 2 1 ) . Froni
Stable Diazo Salts for Chromatographic
Spray Reagents
IRWIN A. PEARL and PATRICIA F. McCOY
The Institute of Paper Chemistry, Appleton, Wis.
b Commercially produced stable diazo
salts Of a number of aromatic amines
provide readily available spray rea-
gents for locating and identifying
phenolic compounds and aromatic
amines on paper chromatograms,
Twenty phenolic compounds were
spotted on paper and sprayed with
water solutions of 30 different stabi-
the data presented here i t seenis prob-
able that the position of a n y skewed
peaks would advance if more solute were
present and retard if less material were
put on the column. This uncertainty
in the posit'ion of a peak Tould give
rise to inconstant results with different
mixtures and would cause the most
trouble when separations are incomplete.
This type of difficulty is encountered in
other types of preparative chromatog-
raphy but in some, such as carrier
displacement chromatography ( I ) , it
is less conspicuous.
Many of the resins tested seemed to
have acidic or basic properties in the
desirablt, range but behaved sluggishly,
perhaps because of high cross linkage.
Xmberlite IRC-50 is such a material.
.A carboxylic resin of lower cross linkage,
rlnibcrlite SI<-89. cschanged more rap-
idly but, perhaps bccause of a less
dense structure, is somrivhat gelatinous
in tcsturc and n.Iit11 vc'ry finely ground
allows so litth flo\v of n-atc'r as to be
nearly usc~lcs~.I t is possiblc that r&ns
might, be prqiarecl \vith tlie desired
nciditicxs or bxsicities and yet possess
wtisfactorj- pliysit>al p i m p c ~ t i t ~Es-
. ( 4 ) Fisher, 5 . F.,Kunin, R., J . Pliys.
periment,s dcsc:ribetl here intlicut,c, that
only a few s w h resins would hr~nec~led;
columns of an>. rcquisitcs acidity or
basirity could be prq)Lirid b>- mising.
Hon-ever, tlie mising of ver!. -trong and
w r y w : t k a ( d resins in iiixiiy propor-
tions would wcni g e n t ~ t l ltlisadvan-
~~
tagcous i i i that the rrcalc resin xould
act niainlj, as a nicchaniixl rlilucnt or
filler and little of its t~wliangr~ c.al)ac.ity
v-ould bc utilized. -1 scrics of acidic
(cation) csc~huiigi~rcyitis with suitnble
physical propCrtiw :ind app:iwnt pK
v ~ l u o s bitn.c,cm 1 antl 6, pcrlixps a t
int(n:ils uf :ipprosim:itc~ly 1 to 2 p K
units. antl a similar 5erit.r of basic
(anion) psclimigc resins with a n up-
parent pI< range bctwccw 8 and 12
n-oultl prolxihly allow useful chromnto-
lized diazo salts. The colors produced
were noted before and after sub-
sequent spraying with saturated sodium
so,utions. A great variety
of colors were noted for the various
combin~tions- These cOmmercial sa'ts
appear to be stable for chromate-
graphic spray use for extended periods.
graphs of nearly all organic electrolytes.
These resins should have relatively low
cross linkages to allow rapid exchange of
dissolved solutes, and high mechanical
rigidities to allow useful flow rates
even when finely divided. If all the
resins now available had this combina-
tion of properties, both series would be
nearly complete.
ACKNOWLEDGMENT
The authors are indebted to the
Rohm & Hans Co.for generous gifts of
the Amberlite resins XE-89, XE-168,
IR-45. XE-112, and XE-58. The Ka-
tional Aluminate Corp. kindly donated a
sample of Xalcite X-219 and the
Chemical Procws Co. furnished the
Duolite rwins C-62. C-63, and A-4.
LITERATURE CITED
(1) Biic:hanan. 11. L . , A s . 4 ~ .CHEar. 31,
833 (1959).
(2) Buchanan, 1). I,, J . Biol. Cheni. 229,
211 (195ij.
(3) Davies, C. l V . j Biochem. .I. 45, 38
(1949).
Chem. 60, 1030 (1956).
( 5 ) Hamilton. P. B..ANAL.Cr-mar.30, :314
(1958).
( 6 ) IToore, S., Stein, IT. H., J . R i d .
Chem. 176,367 (1948).
( 7 ) Ibid., 211, 803 (1954).
18) Pai.tridw. S. 11.. Brimlev. 11. C..
\ -
Biociiem.2.'49, 153 11931). "
(9;) Ihid., 51, 628 (1952).
(11.)) Partridge, S. M., Kestall, I!. G.,
' Ibid., 44, &l8(1949).'
(11) Iteichcnberg, D., "Ion Exchangers in
Organic :ind Biochemistry," C. Calmon
and T. R . E. Krrssman, eds., p. 90, In-
terscience, S c w York, 1957.
(12) Spies, J . R., Charnliers, D. C., J . B i d .
Cherri. 191,79ti !1951).
(13) Thompson, J. F., Iloi-ris, C:. ,J.>
Arch. Biochern. Biophys. 8 2 , 380 (1959).
RECEIVEDfor review Ilarch 24, 1960.
hccepted .July 22, 1960. LVork was slip-
ported i n part by the V.s. Pulilic Hralth
Pcrvice.
I 5 A i'(wnt pwliminary coniniuiiit~a-
t'ion (9) the use of Fast Red Salt
GG, a comnic,i,rial!y avai1aI)le staldizcd
diazo salt of p-nitroaniline, as a spray
reagc>nt for plienolic compounds related
t o wood cheniistqr, was reported. The
advantages noted for its use over the
classical procedure of diazotization of
the p-nitroaniline immediately hefore
VOL. 32, NO. 1 1 , OCTOBER 1960 1407
use led to an investigation of other
commercially available stabilized diazo
salts as spray reagents for phenolic
compounds, especially those related
to our studies in xvood chemistry.
This paper reports thp iesults of these
investigations.
Individual diazotized aromatic amines
often give spots of varying color when
applied to various phenolic materials,
and such materials have been excep-
tionally useful in the past foi, detection
of specific phenolic compounds. For
example. diazotized p-nitroaniline gives
spots of entirely different color when
applied to chromatograms of vanillic,
syringic, p-hydroxybenzoic, ferulic, and
p-coumaric acids, a mixture commonly
encountered in hydrolyzates of woods
( 2 ) . In many instanceq a different or
more pronounced coloi is produced
upon subsequent spraying with an
alkaline solution such as sodium car-
bonate. On the other hand, many
phenolic compounds of similar structure
give essentially identical colors with
individual diazotized aromatic amines,
and differentiation or identification
becomes possible only by the use of
many different diazotized amines. I n
accordance with classical procedures
the necessity for keeping unstable stock
solutions of these amines on hand pre-
cluded the use of a variety of diazotized
amines for routine spraying of paper
chromatograms. The stability, avail-
ability, and ease of application of the
commercial stabilized diazo salt suggest
its use for differentiation and identifi-
cation of individual phenolic com-
pounds. In this connection, Neu ( 1 )
has suggested the use of stabilized
diazonium salts of 4-amino-3,6-
dimethoxy - 4' - nitroazobenzene, 4-
benzamido - 2,s - diethoxyaniline, and
di-o-anisidine for the detection of indi-
vidual flavones.
MATERIALS
Stabilized diazo salts were obtained
from three commercial suppliers. These
materials are identified in Table I.
The twenty phenolic compounds and
their R, values related to wood chem-
istry are listed in Table 11.
PROCEDURE
Whatman No. 1 paper was spotted
with a 4% solution of the phenolic
compound in acetone or some other
suitable organic solvent. From 50 to
100 pg, of phenolic compound were
employed. The solvent was allowed
to evaporate, and the spot was exposed
t o ammonia vapor and sprayed immedi-
ately with a 0.057, solution of the stable
diazo salt in water, the paper was
allowed to air-dry, and the color, if any,
was recorded. After 30 minutes the
paper was sprayed with a saturated
solution of sodium carbonate in water
and allowed to dry. The color ITas
again recorded.
I n some instances the acetone solution
of the phenolic compound appeared to
size the paper and cause the spray
reagent to form a ring around the com-
pound spot. I n most cases this could
be obviated by preliminary development
of the spot in one of the commonly used
phenolic compound developers such as
10 :3 :3 butanol-pyridine-water.

RESULTS A N D DISCUSSION
Table I. Commercial Stable Diazo Salts"
YO. Commercial Nameb Stabilized Diazo Salt of The colors of all 1200 possible com-
binations were noted and recorded in
1 Fast Red AL Salt a-Aminoanthraquinone
Naphthanil Diazo Red AL accordance with Ridgway's color stand-
2 Fast Red 3GL Salt 4Chloro-2-nitroaniline ards and nomenclature (4). However,
Naphthanil Diazo Red 3G only representative data are given here,
3 Fast Scarlet GB Salt 2,5-Dichloroaniline and only common names for colors are
Xaphthanil Diazo Scarlet 2G employed. Several of the compounds
4 Fast Scarlet R Salt 2-Amino-4-nitroanisole
Naphthanil Diazo Scarlet R tested gave essentially the same color
5 Fast Blue B Salt o-Dianisidine with all diazo reagents, indicating that
Xaphthanil Diazo Blue B
-
6 Fast Orange GG Salt
Fast Ponceau L Salt
m-Dichloroaniline
2-Amino-5-nitrotoluene
in these cases, the nature of the phenolic
compound had little influence on the
;i Fast Red GL Salt 4-.4mino-3-nitrotoluene resulting azo dye. Thus, acetosyringone
9 2-.4mino-5-chlorotoluene gave a yellow-brown to brown color
10 4-Amino4 '-methoxydiphenylamine with every diazo reagent. Similarly! 2,5-
11 2-Amino-4-chlorotaluene
12 2-Amino-3-nitroanisole dihydroxyacetophenone yielded a green,
13 4-Amino-2,5-dimethoxy-4 '-nitroazobenzene and vanillil gave a yellow to gold color
14 Fast Blue Salt BBX 4-Benzamido-2,5-diethoxyaniline with every diazo reagent test'ed. For
15 Fast Bordeaux Salt BD 4-Amino-2,5-dimethoxybenzonitrile the most part, however, each phenolic
16 Fast Brown Salt Y 2-Amino-2'-chloro-4-methoxy-5-methyl-4'-
nitroazobenzene compound gave a variety of colors with
17 Fast Corinth Salt V 4-Amino-2,4'-dimethvl-5-methoxy-2'-nitro- the different diazo reagents. I n no
azobenzene instance did a diazo reagent give essen-
18 Fast Garnet Salt GBC 4-Amino-2',3-dimethylazobenzene t'ially the same color with every phenolic
19 Fast Orange Salt GR o-Xitroaniline
20 Fast Red Salt GG p-Nitroaniline compound, although some reagents did
21 Fast Red Salt ITRN N,N-diethyl. 3-amino-4-methoxybenzene- give much less variation in colors than
sulfonamide most.
22 Fast Red Salt PI)C S-n-butyl-3-amino-4-methoxybenzene- Although it is not a phenolic com-
sulfonamide pound. quinic acid wm included in this
23 Fast Red Salt R C S 2-Amino-4-chloroanisole
24 Fast Red Salt R L 2-Amino-3-nitrotoluene study because of its occurrence in so
25 Fast Scarlet Salt G 2-Amino-initrotoluene many natural products and because of
2ti Fast Violet Salt B S 4-Benzamido-2-methoxy-5-met hylaniline its close relationship to many of the
27 Fast Yellow Salt GC o-Chloroaniline phenolic acids encountered in our
28 Variamine Blue Salt RT 4-Aminodiphen ylamine
29 Variamine Blue Salt FG 4-Amino-3-methoxydiphenylamine studies. Furthermore, i t w m thought
30 Fast Orange Salt RDN 2-Chloro-5-trifluoromethylaniline that, quinic acid might give colors
similar to those given by syringic acid
a Sources of diazo salts: Kos. 1-12, Koppers Co., Inc.; Nos. 1-5, E. I. du Pont and the like which do not have ring
de Nemours & Co., Inc.: Nos. 13-30, Antara Chemicals Division of General Aniline and positions available for diazo coupling.
Film Corp.
b The h a hthanil" names for diazo salts 1-5 are names employed by E. I. du Pont de However, results obtained with quinic
Xemouriii Zo., Inc. acid indicated that no coupling or color
formation took place with any of the

1408 ANALYTlCAL CHEMISTRY

 diazo reagents. Instead, the quinic
acid usually acted as a sizing agent for
the paper and gave a rejection spot.
Many of the phenolic compounds with
ring positions available for diazo cou-
pling also gave such rejection spots with
some of the diazo reagents. 2-Hydroxy-
5-methoxybenzoic acid displayed this
activity more than any of the phenols
tested.
Syringic acid, having no free ring po-
sitions either ortho or para to the pheno-
lic hydroxyl group, gives bright' colors
with almost every diazo reagent em-
ployed. The bright blue obt'ained
with Fast Red Salt GG (diazotized p-
nitroaniline) is one of our most charac-
teristic color tests. iilthough the m ~ c h -
anism of color formation in this instance
is not knon-n, it is suspected that coup-
ling takes place a t one of t'he vacant ring
positions. The additive activation of
both methoxyl groups to each of these
positions apparently is sufficient, for the
coupling reaction.
Illustrative data are given in Table 11,
which records the colors given by se-
lected diazo salts of Table I with all of
the phenolic compounds. The colors
recorded are only those obtained after
spraying with saturated sodium car-
bonate solution. The colors reported
in Table I1 are those obtained with
phenolic spots obtained by evaporation
of an acetone solution applied t o the
paper. The colors obtained in this man-
ner mag be somewhat different in hue
from the color obtained with the same
compound which has been chi,omato-
graphed and absorbed by the fibers of
the paper, Therefore, it is possible that
colors obtained from these same com-
binations of phenolic compounds and
diazo salts after chromatography in
other laboratories might differ somewhat
from the reported colors. Accordingly,
care should be exercised in using these
reported colors for absolute identifica-
tions.
The effect of concentration of pheno-
lic compounds on the color produced by
the diazo salt's was checked. Light,
(25 fig.), medium (50 fig.), and heayy
(100 fig.) spots of vanillic, syringic, and
p-hydroxybenzoic acids were applied to
Whatman Xo. 1 paper and processed
\vith Fast Scarlet R Salt (diazo salt of
2-amino-4-nitroanisole) and with Fast
Red Salt GG (diazo salt of p-nitroani-
line) as described. The colors obtained
were analyzed on the General Elect'ric
recording spectrophotometer. The
curves obtained indicated that the same
color was produced by the same com-
bination of phenolic material and diazo
salt a t all three concentrations.
It has come t o our attent,ion recently
( 5 ) that, for dyestuff use, many of the
commercial stabilized diazo salts are not
completely stabilized and are refrig-
erated to prolong their useful life.
This fact might be of great' concern to

VOL. 32, NO. 1 1 , OCTOBER 1960 1409

the dyestuff user who must reproduce
an exact shade. However, for use as
chromatographic spray reagent, the
instability is not so important. I n our
experience, the colors produced by the
first five stabilized diazo salts of Table I
after standing a t room temperature for
3 years were essentially identical with
those produced by fresh salts.
Surface Areas from Adsorption of Methyl Red
by Column Chromatography
HANS A. BENESI
Shell Development Co., Emeryville, Calif.
b The uses and limitations of a dye
adsorption method for the determina-
tion of the surface area of cracking
catalysts and allied solids have been
explored. The method is based on
the measurement of the length of the
colored zone produced by the chro-
matographic adsorption from benzene
solution of methyl red on a column of
powdered sample. The results ob-
tained indicate that this method can be
used to determine the accessible
surface area of alumina-silica and
silica gel with a probable error of 6%.
D YE ARSORPTION methods for the
determination of surface areas of
solids have been used for many years
(6-8). Their chief advantage is the
simplicity of the experimental technique.
Their disadvantage lies in the fact that
adsorption of dyes is highly specific;
consequently, no single dye can be used
to measure surface areas of all solids.
However, this type of method is well
suited for measurement of the surface
area of a given class of solids. Shapiro
and Kolthoff (IO) have already shown
that the area of silica gel surfaces can
be determined from measurements of
the adsorption of methyl red [o-(p-
dimethylaminophenylazo) benzoic acid i.
Apparently, methyl red is chemisorbed
on silica gel through an acid-base re-
action between the basic azo group in
the dye and the weakly acidic SiOH
groups on the silica gel surface. If this
is so, methyl red adsorption measure-
ments should also prove useful in the
determination of surface areas of crack-
ing catalysts, because such solids are
strongly acidic ( I , 11). The present
study explored this possibility.
Shapiro and Kolthoff measured
methyl red adsorption from benzene
1410 ANALYTICAL CHEMISTRY
ACKNOWLEDGMENT (2) Pearl, I. A,, Beyer, D. L., Laskoweki,
The authors thank the Antara Chemi- D., Ta pi42,779 (1959).
\ , Pearr I. A.. McCov.
(3) .., P. F.. ANAL.
cals Division of General Aniline and CHEM.32,132 11960).
Film Corp., the Organic Chemicals De- (4) Ridgway, R., ''Co!x Standards and
partment of E. I. du Pont de Nemours & Color Nomenclature, 1st ed., published
Co., Inc., and the Koppers Co., by author, Washington, D. C., 1912.
(5) Shand, E. W., Koppers Co:, Pitts-
Inc., for supplying the stabilized diazo burgh, Pa., private communication.
salts used in this study.
LITERATURE CITED
(1) Neu, R., 2. anal. ~Chem. 151, 321 RECEIVED for review April 21, 1960
(1956). Accepted July 20, 1960.
technique. I n the present study, this
difficulty was avoided by using a
chromatographic technique to measure
methyl red adsorption. Employing
such a technique, advantage was taken I
of the fact that the equilibrium methyl
red concentration behind a zone front
is essentially identical with the methyl
red concentration in the standard solu-
tion itself. Another advantage of the
treme simplicity.
Apparatus and Materials. T h e
chromatographic tube used is shown
2 l
in Figure 1.
Catalyst Composition, yo Manufacturer
MS-A-1 Aerocat 13 AlzOs, 87 s i o z American Cyanamid Co.
h1S-A-3 Aerocat 22 A1~01,78 SiOn iZmerican Cyanamid Co.
Filtrol SR 37 Alzo3, 63 si02 Filtrol Corp.
Alumina-silica 22 AlzOa, 78 SIOZ Sational lluminate Co.
Magnesia-silica 31 MgO, 69 Si02 Davison Chemical Co.
The wide range in surface area of the were obtained from Davison Chemical
samples shown in Figure 2 was ob- Co. The alumina and magnesia prep-
tained by sintering fresh catalysts. arations have been described else-
Sintering was brought about by calcina- where (1).
tion at 900" to 1000" C., by heating to Surface areas of all samples used were
565" C. in the presence of steam, or by determined from nitrogen adsorption
long term use in the refinery. measurements a t the boiling point of
Most of the silica gel samples used nitrogen using the Brunauer-Emmett-