MODIFICATIONS OF RAST’S MICRO METHOD FOR

MOLECULAR WEIGHT DETERMINATIONS.

BY JAMES H. C. SMITH AND WILLIAM G. YOUNG.
(From the Coastal Laboratory of the Carnegie Institution of Washington,
Carmel-by-the Sea, California.)

(Received for public&ion, August 5,1927.)

In the course of investigations of photosynthesis in plants it

has become clearly evident that more precise knowledge of the
cell constituents is a prerequisite to an understanding of the
mechanisms concerned. Because of the fact that a great deal
of labor and time are involved in obtaining even small quantities
of many of these compounds in a high state of purity, micro
methods must be considered for obtaining the desired information.
This applies also to the pigments of the chloroplasts. Although
considerable work has been done on these pigments, investiga-
tions in this laboratory have revealed the fact that several funda-
mental properties as, e.g. the molecular weights of such com-
pounds as carotin and xanthophyll, have not been established with
certainty and that their exact determination offers a number of
difficulties. The method to be described has proved to be very
satisfactory for a variety of substances and requires only very
small amounts of material and simple apparatus.
Rast (1) proposed a simple micro method for the determination
of molecular weights which has been applied by Houben (2),
Wittka (3), Sadikov and Michailov (4), Mason (5), Pastak (6),
and Carlsohn (7) to a variety of compounds. Rast’s method is,
in short, the adaptation of the ordinary method used by organic
chemists for the determination of the melting point of organic
oompounds to the determination of the lowering of the melting
point of camphor by a solute. From this depression the molecu-
lar weight of the solute is calculated. Camphor has a molecular
lowering of 39.7” and has the advantages of ease of purification
and great solvent powers, It is very fluid in the liquid state and
crystallizes readily without supercooling.
289

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290 Molecular Weight Determinations

The object of this communication is to describe certain modi-

fications of the original method which further increase its useful-
ness and accuracy and to add data to those already existent to
show the dependability of the procedure and its limitations. In
Rast’s method, the substance, the molecular weight of which was
to be determined, was melted with a definite amount of camphor
and the mixture allowed to solidify; a portion of this was then
used for the determination of the melting point of the mixture.
This is one of the steps in the procedure which has been changed,
because thereby smaller amounts of the substance can be used

FIQ. 1.

and because in some instances it was observed that Rast’s pro-

cedure has decided disadvantages when waxy mixtures were
obtained or readily oxidizable solutes were being investigated
(carotin or xanthophyll). The present procedure also obviates
any possible loss of camphor through sublimation.
Apparatus.-The apparatus which was finally found to be the
most useful in our laboratory is diagrammed in Fig. 1. A tall
300 cc. beaker (E) was covered with an asbestos sheath to elimi-
nate the large heat loss and almost Ned with Parawax or glycerol.
Two windows (F), 2.5 X 4.5 cm. were cut in the asbestos cover-
 J. H. C. Smith and W. G. Young 291
ing so that the melting point tube (G) could easily be seen by the
illumination from the electric light placed directly behind the
bath. The melting point tube was held completely submerged
by the special holder (C), a frame made from copper wire and
suspended from clamp (B), so that the melt in the capillary tube
was close to the mercury bulb of the thermometer (A). The
thermometer used in the earlier determinations was an ordinary
grade of laboratory thermometer graduated in whole degrees.
Splendid results were obtained with it but the accuracy and ease
of making the temperature readings were much increased by
resorting to a thermometer, graduated in 0.1” divisions, and with
a working range from lOO-200°C. The bath liquid was stirred
by hand with a stirrer (0). The source of heat was a small
Bunsen burner (I). A sheet of paper (J), hung just back of the
bath so that its lower edge was level with the top of the rear
window, served to cut off the glare from the light, and made the
manipulation more accurate. A second thermometer hung so
that the bulb came at the middle point of the thermometer (A)
recorded the average temperature of the exposed stem. This
average temperature was used in making the correction for the
exposed stem.
Manipulation.-It is essential that the melting point tubes
have very thin walls so that there will be no temperature lag
between the bath and the substance in the tube. Tubes 2 mm.
X 6 cm. were found to give the best results. The tubes were
made by drawing out thin walled test-tubes, cutting to the de-
sired lengths, and sealing one end with as little accumulation of
glass as possible. The tubes were stored in a desiccator to mini-
mize the amount of absorbed moisture.
When a molecular weight was to be determined, a tube was
taken from the desiccator, wiped with a clean dry cloth, and
weighed on a good analytical balance to the nearest 0.01 mg. by
the method of swings. From 1 to 3 mg. of the substance, the
molecular weight of which was to be determined, was then intro-
duced into the tube. The tube was wiped again and reweighed
with the same accuracy. Now the desired amount of camphor
(about 10 times the weight of the unknown) was put on top of
the sample and roughly weighed so that it was known that approxi-
mately the right amount had been added.
 Molecular Weight Determinations

The open end of the tube was then sealed by heating slowly
from about 1 cm. back of the opening to the open end. This
drove out the adhering camphor along with some of the air.
When the fumes of the camphor ceased to escape the end was
melted together without difficulty. The tube was then cooled,
wiped with a dry cloth, and reweighed accurately. It is a great
advantage to use a closed tube, because the tube can be completely
submerged, thus avoiding condensation of camphor on the cooler
exposed walls and as many readings as desired can be taken with-
out any danger of changing the composition of the solution due
to a continuous loss of camphor from an open tube.
During these operations the bath was brought to a temperature
slightly above the melting point of the pure camphor. The end
of the sealed tube containing the mixture was immersed in the
bath and the contents melted. By quickly removing the tube
from the bath, inverting, and giving it a violent shake the con-
tents were completely distributed over the walls of the tube.
By again submerging the tube the mixture was melted and run
into the bottom of the tube. This process was repeated as often
as necessary to insure complete mixing. The tube was then half
immersed in the bath. The camphor sublimed onto the upper
part of the tube and when the tube was again completely sub-
merged the camphor melted, washing any solute adhering to the
side walls into the main body of the solution. By alternate freez-
ing and melting the solution was further mixed.
The actual observations of the freezing and melting points were
then taken. The melting point was taken as the temperature at
which the last crystal disappeared when the temperature was
being raised. The bath temperature was then lowered until the
first crystal of camphor reappeared. This temperature was
recorded as the freezing point. Freezing points and melting points
were taken alternately until a series of concordant and reliable read-
ings was obtained. By so doing a good degree of accuracy was
obtained. The lag between the two points usually amounted to
u-2.0”. The reproducibility of the average of the two tempera-
tures was about 0.1-0.2” however. Since the lowering of the
melting point was in the neighborhood of 20” this gave an accuracy
of about 1 per cent. The melting point of the camphor was taken
in the same manner as that of the solution. Better results were
 J. H. C. Smith and W. G. Young 293
obtained by taking the average of the melting and freezing points,
than by using the temperature at which the last crystal disap-
peared as in the technique of Rast. It is reasonable that such
should be the case too, for the mixing in the melting point tube
is not good. This will make the observed melting point too high,
because the layer of the solution in the immediate vicinity of
the melting camphor will be richer in camphor than the thoroughly
mixed solution would be. Carlsohn (7) has also pointed out
errors arising when substances are used which are soluble in
camphor with difficulty. The freezing point observed, on the
other hand, will be too low due to supercooling. The average
however will be nearer the true melting point of the camphor in
the solution than either of the actual observations. For this
reason we have introduced this modification and shown its worth
by experimental observations. One precaution needs special
emphasis; viz., the melting point tube during the observations must
be completely submerged.
Calculation.-The molecular weight was then calculated from
the accumulated data. The simplest way to explain the use of
these data is actually to record one set of data and one calculation.
Freezing Point Constant of Camphor.-The molecular weights
were calculated by the usual formula
looow
m=K-
W A Ti

where m is the molecular weight of the solute, w is the weight of

of the solute, W is the weight of the solvent, ATt is the lowering
of the freezing point of camphor observed, and K is the lowering of
the freezing point of camphor when 1 mol of solute is dissolved in
1000 gm. of camphor, or the cryoscopic constant for camphor.
It is essential that we have the correct value for K before we
can calculate molecular weights by this method. There seems
to be some question as to the correct value for this constant,
however, since different investigators have published widely
differing results. Rast (1) gives the value as 39.7, and Houben
(2) uses the closely agreeing value 40.0. M. Jouniaux (S), how-
ever, has obtained the average value, 49.5, from a large number of
determinations which are not consistent among themselves.
Efremov (9) has found 48.5 as the average of his determinations.
294 Molecular Weight Determinations

Because of the discrepancy in these values it is necessary to

evaluate the constant from other data. There are two methods
available for the calculation of this constant: first from cryo-
scopic systems which behave normally throughout, and second
from the thermodynamic equation involving the value for the
latent heat of fusion. The latter method is perhaps the more
accurate and generally useful, because the constant calculated
by the use of the latent heat of fusion is not dependent on the
specific properties of individual solutions. There are three ways
in which the latent heat of fusion may be obtained, exclusive of
the freezing point method: first, calorimetrically; second, from
vapor pressure data for solid and liquid camphor; and third, from
the rate of change of freezing point with pressure. The first of
these methods has never been used and the second method is not
practicable because of the inaccuracy of the vapor pressure data.
This leaves only the third which can be employed. By this
method, Hulett (10) found the latent heat of fusion to be 9.4
calories per gm. While this value is not as accurate as we need
for this purpose it will serve as a check on the values for K given
in the literature.
In searching for cryoscopic systems which behave normally
throughout, we found only one system which served our purpose,
viz. the system camphor-menthol. By using the data for this
system given in Landoldt-B&stein (11) it is possible to verify
Hulett’s value for the latent heat of fusion from the thermody-
namic equation
Rln 1-
2
Lf =

assuming Lr, the molal heat of fusion, to be independent of the

temperature. 2 is the mol fraction of solvent in the solution,
Tt is the observed freezing point, and Tro is the freezing point of
pure camphor. The other symbols have their usual significance.
The results of this calculation are shown in Table I. This value
is in excellent agreement with Hulett’s observation.
If Hulett’s value is used and substituted in the thermodynamic
equation already given, it will now be possible to calculate the
 J. H. C. Smith and W. G. Young 295

cryoscopic constant. This can be done by calculating the freez-

ing point lowering for a given mol fraction and then dividing the
calculated lowering by the molality. These values are tabulated
below.
1
Molality. K
z Ti
0.1 1.015 4.7 47.0
0.5 1.076 20.0 40.0
1.0 1.153 37.6 37.6

While the numbers in the last column should be corrected for the
change of Lr with temperature, this is impossible because the
data are not available for the heat capacities of liquid and solid
camphor.
From the foregoing calculation it is readily seen that when the
lowering is about 20”, the cryoscopic constant is very nearly 40.0,

TABLE I.

z 1
log J
z 3 & (k-k, L* 4

0.9264 0.03342 0.002336 0.002231 0. ooO105 1458 9.58

0.8854 0.05269 0.002407 0.002231 0.000176 1362 9.02
0.7673 0.12156 0.002625 0.002231 0.000394 1414 9.29
0.7035 0.15290 0.002709 0.002231 0.000478 1465 9.63

Average...................................................... 9.38

the value given by Rast (1) and Houben (2). For this reason we
will continue to use the value published by Rast, viz. 39.7, a value
which has been shown to give reliable results by the method in
use.
Care must be exercised in applying this constant to all samples of
camphor, however, for Datin (12) has shown that camphor from
various sources has different vapor pressures. There is great
need of a critical investigation of the physicalpropertiesof camphor.

Melting Point of Pure Camphor.

In order that the melting point of the camphor would be com-
parable with that of the solution, the same method of taking
and recording the observations was used.
296 Molecular Weight Determinations
Tem,per$ure Readings.. Auxiliary Data.
Meltiq pant. Freez~$! pant.
YY.
177.4 176: 6 Average temperature of exposed
lT7.3 176.5 column.......................... 37.5
177.4 176.4 Scale reading at point of immersion. 97.0
177.36 176.50 Stem correction*. . . . . . . . . . . . . . . . . 1.72
Average, 176.93”. Melting point correction.. . . . . . . . .178.65
Melting Point of Solution.
Material.
Thymol. Camphor.

Weight of tube and substance.. . . . . . . . . . . . . O.g642 0.1%

‘I “ “ . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.12684 0.13042
“ “ substance . . . . . . . . . . . . . . . . . . . . . . . 0.00358 0.03626
Temperature Readings. Auxiliary Data.
Melti~~point. Free@g point.
*c.
152.6 148.7 Average temperature of exposed
152.2 148.7 column........................ 43.0
152 .O 149 .o Scale reading at point of im-
152.06 148.80 mersion.. . . . . . . . . . . . . . . . . . . . . . 23.0
Average, 150.43”. Stem correction*. . . . . . . . . . . . . . . . 2.02
Melting point correction.. . . . . . .152.45
Lowering of melting point.. . . . . . 26.20
1000 39.7 X 0.00358
Molecular weight = x = 149.6.
0.03626 X 26.20
Theory = 150.16, per cent error = 0.37.
* Stem correction = (t - i) (t - a) (0.000154), where t is the observed
temperature; i is the scale reading at the point of immersion of the ther-
mometer in the bath; a is the average temperature of the exposed column;
0.000154 is the coefficient of expansion of mercury in glass (13).

DISCUSSION.

In Table II, a summary of the results obtained by this method

is recorded. The determinations marked with an asterisk were
not done in the micro way but followed the original method of
Rast ; aiz., by transferring a small portion of a previously prepared
solution to the melting point tube. In some instances it was
observed that this procedure had decided disadvantages. The
manipulation described here yielded much better results. The
work on the molecular weights of carotin and xanthophyll will
be described in another place. Duplicate determinations check
excellently. There are a few facts which should be noted. From
Table II it will be seen that acids show considerable abnormality.
 J. H. C. Smith and W. G. Young 297

This behavior is being investigated and already interesting results

have been obtained. Alkaloids and osazones caused blackening
of the melt which showed that reaction had set in. It is interest-
ing that Sadikov and Michailov (4) got reasonably good results
with these classes of compounds. Further work needs to be done
with these types.
TABLE II.

Results of Determinations on Molecular Weights by the M~CTO Method.

Substance. we3ht
aample.
Uolecula
weight
)bwrved
Molecula
weight
theory.

Nephthalene.*. .....
-I-r--
0.0101 0.1390 22.3 129.3 128.1
p&r c‘mt

0.94
cY-Naphthol.*. ........ 0.0150 0.2072 19.8 145.0 144.1 0.62
“ 141.5 144.1 1.80
......... 0.00291 0.03025 27.01
Hydroquinone.*. ..... 0.0108 0.2008 19.5 109.6 110.1 0.46
Benzoic acid.*. ...... 0.0114 0.1120 30.2 133.8 122.0 9.7
Succinic “ ....... 0.00233 0.03683 15.6 161.0 118.07 35.5
Salicylic “ ....... 0.00304 0.02307 36.13 144.80 138.08 4.87
Diphenylamine ....... 0.00357 0.02874 29.4 167.8 169.19 0.83
Bensidine ............ 0.00347 0.03285 22.35 187.5 184.2 1.8
Thymol.. ............ 0.00358 0.03626 26.20 149.6 150.16 0.37
Acetanilide ........... 0.00183 0.02175 24.3 137.4 135.12 1.69
Urethane ............. 0.00177 0.03102 23.69 95.6 89.06 7.30
“ ........... 0.00227 0.03580 26.28 95.8 89.06 7.57
Hexachlorobenzene ... 0.00312 0.02695 16.4 280.2 284.73 1.58
p-Nitrotoluene ....... 0.00286 0.03069 27.0 137.1 137.1 0.00
Azobenzene ........... 0.00255 0.03242 16.87 185.1 182.16 1.65
Dibromobenzene ...... 0.00402 0.04170 15.9 240.5 235.98 1.92
Brucine ..............
Phenylglucosazone ....

* Original method of Rast.

The greatest need in this field of investigation, however, is to

find other solvents which have properties similar to those of cam-
phor, so that when abnormal results are obtained in one solvent
other solvents may be used. The properties which are essential
are large freezing point depression, ease of purification, sharp
melting and freezing points, little supercooling, low viscosity in
the liquid state, wide solvent powers when melted and none when
solid. Other solvents are being investigated in this laboratory
298 Molecular Weight Determinations

but no substance has been found which is nearly so useful as

camphor.
We wish to express our appreciation for the many helpful sug-
gestions made by Dr. H. A. Spoehr.
BIBLIOGRAPHY.

1. Rast, K., Ber. them. Ges., 1922, IvB, 1051,3727.

2. Houben, J., J. prakt. Chem., 1922, cv, 27.
3. Wittka, F., Chem. Abst., 1924, xviii, 3732.
4. Sadikov, V., and Michailov, A., Biochem. Z., 1924, cl, 363.
5. Mason, F. A., J. Sot. Chem. Ind., 1926, XIV, 199.
6. Pastak, I., Bull. Sot. chim., 1926, xxxix, 82.
7. Carlsohn, H., Ber. them. Ges., 1927, lx B, 473.
8. Jouniaux, M., in Tables Annuelles Internationales de Constants et
Donnees Numeriques, 1914, iii, 305.
9. Efremov, N., in Tables Annuelles Internationales de Constants et
Don&es Numeriques, 1913, ii, 853.
10. Hulett, C. A., Z. physik. Chem., 1898, xxviii, 629.
11. Landoldt, H., Bornstein, R., and Roth, W. A., Physikalisch-chemische
Tabellen, Berlin, 4th edition, 1912,543.
12. Datin, P., Ann. Physik., 1916, v, 218.
13. Kamm, O., Qualitative organic analysis, New York, 1923, 116.