T I I K A M K K I C A N .IOUUNAI, OF C L I N I C A L
PATHOLOGY
Vol. 39, No. 3, p p . 235-241
Mureli, 1%3
C o p y r i g h t © 1003 b y T h e Williams & Wilkins Co.
1'rinled in
U.S.A.
FALLING DROP METHOD FOR DETERMINATION OF SPECIFIC
GRAVITY OF URINE
M A R T H A W I N S T E A D , A.B., M . T . (ASCP), N . D . SISSON, M . D . , AND 11. L. M A R K E Y
South Bend Medical Fovndalion,
Inc., 531 North Main Street, South Bend 1,
Indiana
Many efforts have been made to establish
a satisfactory method for determination of
the specific gravity of urine on a small
specimen. These began in 1926 when Kirkpatrick and Kling7 adjusted the relative
proportions of xylene and carbon tetrachloride in a hydrometer cylinder while
they simultaneously dropped the sample
drop. The specific gravity of the exact
mixture that would make the drop hover
Avas then measured by means of a hydrometer, and this specific gravity was taken
as representing that of the sample. Since
Aaron's' article in 1945, the accuracy of
measuring the specific gravity of urine by
means of the usual hydrometer has been
questioned, and numerous methods that
circumvent the hydrometer have appeared
in the literature. The gradient column, 4 ' 8
a series of tubes containing solutions with
known specific gravities into which drops
are allowed to fall,5, n and the specific
gravity balance methods 9 seem to be cumbersome for use in routine measurement
of the specific gravity of urine in the busy
hospital laboratory.
The falling drop method herein described,
which has been in use in our laboratory
since .1.956, is basically similar to that of
Barbour and Hamilton. 2 It is simply a
measure of the time required for a uniform
drop of immiscible fluid to traverse a 12-in.
distance in a solution of fixed specific
gravity. The specific gravity of urine is
found on a time-wersMS-specific gravity
standard curve established with sucrose
solutions of known specific gravity.* '
Received, March 15, 1902; revision received,
September 21; accepted for publication, November
14.
Miss Winstead is Chief of Quality Control, D r .
Sisson is Pathologist., and Mr. Markey is Technical
Advisor.
* Sodium chloride solutions may also be used
for standardization. 0
235
F I G . 1. Falling drop column
236
WINSTEAD BT
The accuracy of falling drop methods
(if corrections for temperature and proper
standardization are considered) has been
well established in relation to the gravimetric and refractometric methods. 6,12 Comparison is made between the values for the
specific gravity when a simple American
Optical Company refractometer (A-0 TS
Meter)f and 4 urine hydrometers from 3
different manufacturers are used.
METHOD
1. Draw the urine into a 1-ml. serologic
pipet and immerse the tip approximately
Ys in. into the fluid column.
2. Permit a drop (0.05 ml.) to form on
the tip of the pipet, and remove the pipet
from the fluid in order to release the drop
from the tip.
3. With a stopwatch, time the fall of the
drop from the upper to the lower line on
the column.
4. Record the time and find the value for
the specific gravity on the calibration chart.
MATERIALS AND EQUIPMENT
1. Falling drop column. The column
(Fig. 1) is made of heavy gage glass and
has a uniform internal diameter of Yi hi.
It is scribed with 2 lines, an upper and a
lower, which are 12 in. apart. J The fall rate
is the time needed for the drop to fall from
the upper to the lower of these 2 lines. The
bottom of the column is fitted with a Teflon
stopcock that allows the urine drops that
are collecting to be drawn off without
disturbing the mixture in the column. In use,
the column is held exactly vertical on a
buret stand. (Note—This column should
be siliconed with G. E. Drifilm #SC-87;§
aqueous silicones have proved to be not
suitable.) Gentle rinsing with detergent
cleans the column, but resiliconing must be
performed occasionally. Old silicone is
| Manufactured by American Optical Company,
10 S. Wabash Avenue, Silversmith Bldg., Box 804,
Chicago 90, Illinois.
J Available from Research Apparatus Co., Inc.,
Ill W. Mill St., Wauconda, Illinois.
§ Available from General Electric Chemical
Dcpt., PiUsfield, Massachusetts.
Vol. S&
AL.
TABLE 1
W E I G H T S FOR SUCROSE STANDARDS
Specific Gravity*
Sucrose Standards
Cm./100 ml.
1.002
2.012
3.030
4.056
5.0S9
S.240
9.306
11.470
1.003
1.007
1.011
1.015
1.019
1.031
1.035
1.043
* At room temperature of 23 C.
removed by means of allowing a dichromatesulfuric acid cleaning solution to stand in
the column for 1 hr.
Instructions for siliconing. Silicone isapplied in the following manner:
A. Close the small end of the column with
a rubber stopper and pour in a small amount
of Drifilm.
B. Plug the large end of the column and
rotate so that the entire surface is covered.
C. Pour out excess Drifilm, unstopper,
and leave the column in an upright position
overnight to dry.
D. Rinse with tap water and dry by
rinsing with acetone.
2. Thermometer hung beside the column.
Although corrections are ordinarily unnecessary, they must be made for drastic
changes in room temperature. The usual
<j'f»e
in
scconas
FIG. 2. Calibration curve for specific gravity
March 1963
SPECIFIC
GRAVITY
OF
237
URINE
TABLE 2
SPECIFIC GRAVITY OF SUCROSE STANDARDS
Hydrometers*
Standard Calculation of
Sucrose by Weight
Refractometer
Falling Drop
No. 1
No. 2
No. 3
No. 4
1.007
1.011
1.015
1.017
1.021
1.032
1.035
1.042
1.007
1.009
1.013
1.018
1.021
1.033
1.039
1.043
1.002
1.009
1.015
1.018
1.022
1.034
1.037
1.04G
1.008
1.012
1.014
1.010
1.021
1.031
1.039
1.045
sec.
1.003
1.007
1.011
1.015
1.019
1.031
1.035
1.043
1.003
1.0005
1.0105
1.0145
1.0185
1.0305
1.0345
1.043f
48,
23,
17,
13,
11,
8,
50
23
17
13
11
8
7M, 7K
VA, <$A
* Values are corrected to 1.000 with distilled water and are also corrected for temperature.
f Solution was diluted 1:2 because the refractometer read only to 1.035.
TABLE 3
SPECIFIC GRAVITY OF U R I N E BY M E A N S
REFRACTOMETER, F A L L I N G D R O P , AND
HYDROMETER AT 23 C.
Urine
No.
Refractometer
1
2
3
4
5
0
7
S
9
10
11
12
13
14
15
10
17
18
19
20
1.008
1.008
1.0075
1.015
1.014
1.013
1.0145
1.010
1.019
1.019
1.023
1.025
1.024
1.023
1.0235
1.0215
1.025
1.0255
1.027
1.032
Hydrometers*t
Falling
Drop
1.008
1.008
1.010
1.013
1.0135
1.013
1.015
1.010
1.019
1.019
1.020
1.023
1.0225
1.021
1.023
1.020
1.025
1.027
1.025
1.031
OF
No. 1
No. 2
No. 3
No. 4
1.012
1.011
1.012
1.015
1.018
1.019
1.020
1.021
1.024
1.023
1.025
1.030
1.029
1.020
1.029
1.024
1.029
1.011
1.011
1.012
1.015
1.018
1.020
1.021
1.023
1.024
1.026
1.030
1.028
1.026
1.031
1.025
1.030
1.014
1.010
1.011
1.014
1.017
1.017
1.018
1.018
1.023
1.024
1.025
1.029
1.028
1.020
1.029
1.023
1.028
1.011
1.010
1.012
1.015
1.018
1.018
1.021
1.022
1.024
1.024
1.027
1.030
1.028
1.028
1.029
1.025
1.029
—
—
—
—
—
1.030 1.031 1.030 1.030
1.035 1.037 1.030 1.037
* Values arc corrected to 1.000 with distilled
water and arc also corrected for t e m p e r a t u r e .
f —indicates t h a t there was not sufficient
urine for determining specific gravity.
correction of 0.001 specific gravity for
each 3 C. variation is suitable for this
method 2 ' 7 and should be applied for room
temperature outside the 22 to 24 C. range.
3. Standard sucrose solutions. For use in
selecting the appropriate points on the
time-versMS-specific gravity graph, the required solutions of sucrose and their specific
gravities are listed in Table 1. Although
several dilutions of a concentrated solution
could have been used, individual sucrose
weighings were made in order to establish
independent points on the graph.
4. Solution for the column. This material
is prepared in the following manner:
A. Add 28 ml. of bromobenzene* to 72
ml. of kerosene in a graduated cylinder.
Mix well and fill the column with the
mixture to approximately 2 in. from the top.
B. Drop the 1.003 sucrose standard
twice and time its fall. This should require
50 sec. ± 3 sec. A time longer than 50 sec.
requires addition of kerosene; less time
requires addition of bromobenzene.
C. Adjust with the proper solution until
the sucrose 1.003 standard gives the correct
fall time. The adjusting solution is added
dropwise, and the column (if the lower
clamp is swung out of the way) can be
inverted by means of rocking it in the upper
clamp for easy mixing. (Note—Small droplets
of standard suspended in the mixture
affect the time. For the final check of time
after addition of the adjusting mixture,
allow the droplets to settle and retest the
1.003 sucrose standard.)
* Available from E a s t m a n - K o d a k
State St., Rochester 4, New York.
Co.,
343
238
WINSTEAD
ET
Vol. 39
AL.
TABLE 4
C O N F I D E N C E L I M I T S (3 S.D.)* FOR THE V A R I O U S M E T H O D S OF D E T E R M I N A T I O N OF SPECIFIC GRAVITY
Refractometer
No. 1
No. 2
No. 3
Falling Drop
Hydrometer No. 1
Avg. sp. gr.
3 S.D.
Avg. sp. gr.
3 S.D.
Avg. sp. gr.
3 S.D.
1.0050
1.0190
1.0310
No variation
No variation
No variation
1.0061
1.0199
1.0314
0.0006
0.0009
0.0005
1.0072
1.0217
1.0337
0.0025
0.0020
0.0020
S.D. = standard deviation.
5. Refractometer. An A-0 TS Meter is
used.
6. Hydrometers. Four hydrometers are
obtained from 3 different manufacturers.
CALIBRATION
Each of the standards should be dropped
at least twice and their fall times recorded.
A time-yersMS-specific gravity curve is constructed on a linear graph (Fig. 2), and a
chart for routine use may be prepared from
the graph.
RESULTS
The sucrose standards used for calibration of the falling drop method were checked
with the refractometer and with 4 different
urine hydrometers. Results are listed in
Table 2.
Twenty urines were tested for specific
gravity by means of the falling drop refractometer and 4 urine hydrometers. Results are summarized in Table 3.
In order to check reproducibility, 3
samples of urine with specific gravities of
1.005, 1.019, and 1.031 were tested 30 times
each by means of each method. The results
were then used to calculate 3 standard
deviations by the replicate formula:
I. S.
y N -1
These comparisons are listed in Table 4.
DISCUSSION
The specific gravity of the bromobenzenekerosene mixture may be established at any
point between and including 1.000 and 1.003,
but once it has been established, it obviously
must be kept the same, inasmuch as the
time of fall is directly related to the specific
gravity of the solution. The specific gravity
is, in turn, directly related to the temperature of the solution, and continual correction of temperature is required unless room
temperature is kept relatively constant.
The difference in specific gravity resulting
from a temperature change of 3 C. is 0.001;
therefore, if 23 C. is selected in the standardization, the room temperature may
range from 22 to 24 C. without correction.
The statement made by the manufacturer and others3 that the A-0 TS refractometer was compensated for temperatures
between 60 and 100 F. was viewed with
some suspicion until the sucrose standards
proved to yield values with this instrument
that were essentially identical to the standard values. On the basis of this finding, in
later portions of this work the refractometric value for specific gravity is accepted
as the true value.
The correction of the hydrometers used
in this work to 1.000 for distilled water
ranged from +0.004 to -0.001 (Aaron1
pointed out that the inherent calibration
error of hydrometers can be as great as
0.009). All hydrometers had been calibrated for 60 F. (15.5 C ) ; therefore, all
specific gravities they recorded required
the addition of 0.003 for our room temperature of 23.5 C. If, as we suspect, the average
laboratory seldom applies the required
correction for temperature, and if, as Miles10
states, the temperature error summates with
calibration error, it would seem reasonable
that hydrometer determination of specific
gravity frequently yields grossly inaccurate
values. Reference to Table 2 indicates that
even corrected, hydrometer values for specific
gravity are significantly in error.
March 1963
239
SPECIFIC GRAVITY OF URINE
The size of the sample drop may be judged
visually after one is accustomed to the
amount regularly released by delivery of
0.05 ml. from a 1-ml. serologic pipet. In
routine procedure, it is more rapid and
practical to drop the urine with a disposable
Pasteur pipet of uniform bore. The same
pipet may be used for a whole run of urines
if it is rinsed with sample between samples.
As may be seen in Table 3, the specific
gravity values of urine obtained by the
falling drop method compare well with those
obtained by the refractometric method.
The hydrometer values, however, vary
from the refractometric ones by a difference
of 0.002 to 0.007.
When the specific gravity of a single
specimen of urine was measured 30 times
with the refractometer, no variation in the
reading was obtained. Comparison of the
statistical data in Table 4 for 3 standard
deviations (S.D.) clearly indicates that
better reproducibility is obtained with the
falling drop method than with use of the
hydrometer.
In order to compare the falling drop and
hydrometer methods with regard to accuracy, the specific gravities for the 20
urines given in Table 3 were used to calculate " t " values. (This is a means of establishing whether or not differences between values
by different methods are significant.) The
refractometer value was regarded as the true
value.
For the falling drop method compared
with the refractometer method, the " t "
value of 1.9, calculated by means of the
equation:
d
is not a significant difference at the 5 per
cent level.
Comparison of the hydrometer with the
refractometer, however, resulted in a " t "
value of 11.6—a highly significant difference
at the 5 per cent level.
In order to maintain this accuracy in
routine use of the falling drop method, it is
suggested that the fall time for 2 sucrose
standards (1.003 and 1.035) be verified
daily. As stated in the discussion of calibra-
tion, the 1.003 standard should give 50 ± 3
sec. and the 1.035 standard, 7 ± Yi sec.
(or whatever time was obtained for this
standard calibration). Necessity for adjusting the column mixture may be detected
at once.
Standards should be kept in the refrigerator, but small aliquots for daily checking of
solutions may be left at room temperature
for periods as long as 2 weeks. Further precautions necessary are:
1. The column should be kept corked
when not in use, in order to avoid evaporation of the fluid.
2. The column should be kept away from
open flames and heat (Bunsen burners,
sunlight, and so on), in order to avoid fires
and alteration of the specific gravity of the
solution.
3. Tor better accuracy, times shorter
than 7 sec. should be rechecked on a 1:2
dilution of specimen with distilled water.
4. The drop of testing sample should be
kept free of air bubbles. The time is entirely
inaccurate if even a small bubble is present.
CONCLUSIONS
A falling drop method for determination
of the specific gravity of urine has been
described. It is accurate, extremely reproducible, and very simple for routine use.
Calibration is made with sucrose standards and is entirely independent of the urine
hydrometer. Specific gravity values are
identical to those read when an A-0 TS
Meter is used and thus furnish a means of
establishing true specific gravity values for
specimens of urine with which to compare
values obtained by other methods.
A simple check of the accuracy of daily
maintenance calibration is described.
There was no variation in the specific
gravity replicates made with the use of the
A-0 TS Meter. The 3 S.D. average value
for reproducibility of the falling drop method
was one-third that for the hydrometer
(0.0007 to 0.0022).
Judging from " t " test calculations, there
is no significant difference at the 5 per cent
level between the refractometer and the
falling drop values. There is, however, a
highly significant difference between the
refractometer and the hydrometer values.
240
Vol. 39
WINSTEAD ET AL.
SUMMARIO IN INTERLINGUA
• Es describite un methodo a gutta cadente
pro le determination del gravitate specific
de urina. Le methodo es accurate, reproducibile, e multo simple a executar in usos
routinari.
Le calibration es facite con standards de
sucrosa e es completemente independente ab
le hydrometro de urina. Le valores del
gravitate specific assi obtenite es identic con
illos legite in le uso del refractometro A-0 TS
e forni assi un medio pro establir ver valores
de gravitate specific de specimens de urina,
con le quales il es possibile comparar valores
obtenite per altere methodos.
Es describite un simple methodo pro le
controlo del accuratia del calibration de
mantenentia diurne.
Esseva constatate nulle variation in le
replicationes del determination de gravitates
specific effectuate con le uso del refractometro A-0 TS. Le valor medie de 3 D.S.
(deviation standard) pro le reproducibilitate
del methodo a gutta cadente esseva un
tertio de illo del methodo a hydrometro
(0.0007 a 0.0022).
A base de calculationes a test t il pote
esser asserite que il existe nulle significative
differentia al nivello de 5 pro cento inter le
valores a refractometro e le valores a gutta
cadente. Del altere latere, un differentia
altemente significative se notava inter le
valores a refractometro e le valores a hydrometro.
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H.
G.,
AND H A M I L T O N ,
W.
F.:
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