Apparatus for Precision Calibration of Pipets, Volumetric Flasks, and Burets WILLIAM R. THOMPSON Division of Laboratories and Research, New York State Department of Health, Albany, N. Y.
length with stopcock B about 4 or 5 cm. above the Y-joint. The top of this branch is a butt at G like that above A * Each of these is fitted with an inverted No. 8 stop er bored with a slightly smaller hole for tight fitting around tge 7-mm. glass tubing, on which it is mounted with the glass butts halfway through in each case (& and G). The other halves of the borings SUPPlY joints to apparatus placed vertically above. The stopper at G is surrounded by a short glass cylinder, about 4 em. high and 4 cm. in outside diameter, to form a well. The stopper a t Q is surrounded by a moat with a drain to a waste bottle. The moat may be made by use of a rubber ring with chamfered inner edge and the drain supplied by a boring fitted with a small glass tube. The apparatus is mounted in clamps on a firm upright rode. g., a ring stand clamped to the bench With a C-clamP- Between clamp jaws and apparatus are placed rubber stoppers suitably bored and slit on one side, one between c and the yjoint and another 3 or 4 cm. above B. MERCURY CHARGEAND RESERVOIR.To the butt beyond F is attached rubber tubing, leading to reservoir R a t suitable height, with a Hoffman clamp, H (or another stopcock), just beyond the butt near F. Either through this lead, or a temporary substitute, vacuuII1is applied at F and also at B and A as required to fill the apparatus with mercury from the tip be!ow D to and through stopcocks A , B, and F with careful exclusion of air. Then reservoir 8 is filled with mercury and placed in position, and air is expelled from the connections to F by slightly whirling part of the rubber tube. STANDARD PIPET. A and D are left closed and C and E open (partly, a t least) while mercury is allowed to run through F and B partly to fill well G. Through this mercury, the surface of which is cleaned by aid of a small glass vacuum attachment, if necessary, a standard pipet, S , is inserted and joined (glass to glass) through the rubber sto per a t G. The pipet is provided with capillary arms above anjbelow its bulb, adapted, if necessary, at the lower end to make a good fit (about 7 mm. in outside diameter). For convenience of reading, the arm capillary volume has been made about 0.005 ml. per cm. for a 0.5-ml. standard pipet and 0.03 ml. per cm. for 3- or 5-ml. standards. The volume of mercury that is delivered from an initial mark, 0, above the bulb to a point below is read on a scale, V , calibrated by weight of mercury delivered from the tip through open E, C, and D with A and F closed and controlled at B. The tip is touched to a level mercury surface a t the beginning, the dischar ed mercury caught in a tared vessel, and the tip touched to the?evel surface of mercury in it at the end of delivery. At the top of pipet S may be attached an overflow safety vessel, Z (of well-known type), leading to a catch bottle. HoFever, careful adjustment of clamp H and the level of R make this unnecessary. Sis clamped lightly a t the bulb to the main support rod. WATERCHAMBER.A vessel, W , on the main vertical stem above A is attached through the rubber stopper at &, previously flooded with a few drops of water to cover the hole in the stopper so as to exclude air from the joint. W is adapted to fit this joint below, about 7 or 8 mm. in outside diameter at the base, to allow formation of a mercury-water interface in a large-diameter tube (25 mm. or more in inside diameter); and to admit attachment of pipet P through a rubber stopper a t the top of W.. W is provided with a surrounding moat, T , at the top, conveniently made all of glass with a drain, X, leading t o a waste bottle. Alternatively the moat and drain might be made with a rubber ring as at Q, or with a rubber stopper around W and a glass attachment like that at Z but without the dome top. ATTACHMENTS FOR PIPETTO BE CALIBRATED. The tip of pipet P must be above the mercury-water interface throughout all calibration operations on it. Bny parts required to be visible for reading or observation of flow should be well above or below the rubber stopper at T . The bulb of a small pipet, if near the tip, may be placed below T . Air is carefully excluded by flood1ng.W with water just before insertion of stopper T , the overflow passing away through X. In calibration between marks, P will have an upper mark, U,and a lower mark, L. L may be placed, as convenient, above or below T . In calibration to drain and touch, L may be a temporary mark-. g., a fine rubber ring cut from tub-
T
HE apparatus described here was designed for rapid calibration of pipets and flasks well within usual talerances-e* g.9 one part per thousand for volumes of lo ml. or more, and within 0.01 ml. for those less than 10 ml. I n practice i t appears to be subject to errors usually less than one tenth as great. A pipet of 0.5-ml. capacity may be measured to about 0.001-ml. approximation of calibration by other accepted means (3).
Apparatus Figure 1 gives a schematic diagram of the essential elements of the calibration apparatus. Convenient dimensions are given below. CONTROL VESSEL. A double Y-tube is constructed from Six Standard-taPer stopcocks (Preferably Pyrex) of about 2-mm. bore, with connecting tubes of approximately 4-mm. bore and 7mm. outside diameter. The Y-angles are about 60". Vertically ascending from a 7-cm. delivery tip are stopcocks DJ c, and A with intervals of 6 and 10 cm.7 respectively. The stem above A extends 5 cm. to a neatly fire-polished butt a t &. Midway between A and C is joined the branch ascending at 30" from horisontal. About 6 cm. along this is stopcock E and 8 cm. farther is F with another butt end after about 3 or 4 cm. Midway between E and F is a vertically ascending branch about 14 cm. in
R
FIGURE 1. APPARATUS FOR PIPETCALIBRATION BY
REFILLINGOR MERCURY WEIGHING)
268
,
ANALYTICAL EDITION
March 15, 1942
ing-placed where the meniscus stands after completing drainage with the tip against the wet wall of a receiving vessel (S), or touching the tip of the drained pipet to a level water surface and then slowly withdrawing it, as prescribed by convention. The former is usually preferred, but the latter was used in experiments described below. Vessel W is clamped firmly to the main support rod (with care not to interfere with readings). It is convenient to place a rubber stopper at the top of pipet P to be calibrated, and adjust a clamp from the main support so that its movable jaw presses gently upon the top. This furnishes a micrometer screw type of adjustment, M , acting on the remote stopper a t T as a tambour to effect minor adjustment of the air-water interface in the pipet a t the upper mark, U, and later at the lower mark, L, or at the actual ti as required in blow-out or to-contain types of calibration. SPight adjustments a t the lower points are usually admissible in calibrations, if the pipet is simply refilled with a definite volume of water after drainage, approximately as in use.
269
Operate F to fill pipet P up to U (approximately). Close F , open D, and adjust C to give the required outflow rate. Repeat the filling and drainage till the adjustment is satisfactory. If air has been admitted to W , remove the pipet and stopper a t T and replace, after flooding W as previously. A small amount of air in W might not interfere appreciably with preliminary adjustment of C but might affect later operations considerably. d o s e A , o n B, and fill standard pipet S to 0. To facilitate this, E may set in advance so as to restrict subsequent downward flow and then mercury run from F into S till above 0, F closed, and D opened to run mercury slowly down to 0. Close B , then D, and open A . Adjust water level in P to U (by F , D, and M as necessary). Open D wide, and drain pipet P to the lower mark as required. Adjust by M , if necessary. Open B , control flow rate by E , and run back to U (or down to the prescribed V in the standard, or both in succession) using B as shut-off. Read V on S, or set new mark U on P if the previous mark is found in repeated trial to yield a delivered volume outside tolerable limits. If required as a final check calibration, the weight and temperature of mercury delivered from the tip below D may be used, provided that the drainage is made by control a t D alone, with M not used except at U. In calibration of volumetric flasks or tubes, the procedure is essentially the same except that an inverted U-delivery tube with a fine tip (illustrated in Figure 2) is used a t T instead of P; and, after delivery, water is added to the system, as required, by dipping the tip of this tube in water and running mercury out through D. If air bubbles form in the delivery tube, they may be removed by running more air into it (0 ening D) to contact the entrapped air and following by reversafof flow (closing D, opening F ) to run the air out. Just before the start and at the finish of the measured delivery, the tip is touched to a water surface, first in the beaker used for filling and, finally, within the vessel being calibrated.
Experimental Results
FIGURE 2. AD.4PTATION WITH DELIVERY TUBEFOR CALIBRATION OF VOLUMETRIC FLASKS OR OTHER VESSELS
Stopcock C is adjusted in preliminary trial to control flow rate conventionally, so as to approximate conditions of drainage of pipet P expected in actual use. E is used chiefly to control flow rate from S to P. The other stopcocks, F , B , A , and D are usually either closed or wide open in use. Accordingly, it is convenient t o distinguish C and E from the others by a red rubber cover, made from tubing with a little hole cut in the side and slipped over the stopcock handle.
Procedure Have in place the required standard pipet, S , and suitable vessel, W . Take care to eliminate any entrapped air. Usually keep C and E partly open, and never have A , B , F , and D all closed longer than necessary to deliberate operation. Have H adjusted to give steady flow so as not to entrap air in the standard pipet when refilling (from V to 0) with B and F wide open and A and D closed. Then, with B closed and A open, adjust mercury in W through F or D , so that it will extend up to the wider cylindrical portion but not high enough to reach pipet P in later operations-i. e., the water in W between the top of the mercury, level and the pipet bottom should have a volume slightly exceeding that to be contained in the pipet. The rubber stopper for use with M may be attached at the pipet top before inserting the pipet into W . If the ipet has a stopcock, keep it open throughout. Ffood W with water. Immediately insert P and the stopper at T ; or insert the stopper only, run water up to flood the hole, and insert the pipet tip through the drop of water and the stopper, if only the tip is to enter W . The latter is convenient when several pipets may be calibrated without removal of the stopper a t T ; the former is required when the bulb is to be placed within W or when a temporary marker (rubber ring) is used near the tip as in drain and touch calibration. Adjust the clamp a t M to give a slight tension.
A standard 0.5-ml. pipet was made with marks in close approximation on the V-scale t o 0.5 ml. and 0.005 and 0.010 ml. above and below. Recalibration at the 0.5-ml. mark by weight of mercury delivered (as described above) gave the V estimates, 0.49924, 0.49974, 0.49921; mean V E 0.4994. The difference of 0.01 ml. in volume delivered corresponded approximately t o 20 mm. on the V-scale. As a n illustrative application, a 0.5-ml. Ostwald pipet was calibrated in various ways. Its natural outflow time was between 6 and 7 seconds. In drain and touch delivery (touching as previously described after 5 seconds more) calibration from the original mark by weight of water delivered gave the volume estimates, 0.4915, 0.4913, 0.4917, 0.4903; and % 0.4912. The position of the meniscus in the tip after touching was noted (about 29 m m from the end) and a fine red rubber ring set as lower mark L in that position. I n the volunietric apparatus against the standard pipet with the indicated outflow times in seconds (D. T.), the following values of V were obtained: T.' = 0.4915, 0.4915, 0.4915, D. 7'. = 5.8 to 6.6; V = 0.4930, 0.4930, 0.4935, D. T . = 21.0 to 21.4; V = 0.4940, 0.4940, 0.4940, D. 2". = 5 5 4 to 55.8. Thus, at natural drainage rate T' 0.4915 volumetrically and V 0.4912 gravimetrically, a difference of 0.0003 ml. This is remarkable in view of the rapid outflow rate. Volumetric readings were made to the estimated nearest 0.0005 ml. (tenth of smallest scale division on 8-i. e., to thenearest millimeter). Thus, it would seem that even greater precision might be attainable with closer reading, but the precision was already far greater than sought for the purpose. The same pipet was calibrated also to deliver from another initial mark, U , to the tip, simulating blowout and to-contain calibration according as the drainage time was approximately that of free delivery or relatively very great. The results are given in Table I. For drainage time (D. T.)of 134 seconds or more, there appears close approximation in the 13 observations to their mean value, 0.49885 ml. Recalibration of the dry pipet with mercury, using the meniscus correction (S), gave the volume estimates, 0.4983, 0.4987, and 0.4993 ml. with a mean V of 0.49877 ml. S o t only are the volumetric
v
270
Vol. 14, No. 3
INDUSTRIAL AND ENGINEERING CHEMISTRY
TABLEI. YOLUMETRICMEASUREOF DELIVERYTO GIVENDR.4INAGE TIMES Drainage Time Range, Seconds 7 1-8 0 22-23 65-GG 134-138 420-412
1 6-1 S
MI. 4885 4895
Mean
4890
TABLE11.
500-553
10-4
4950 4965 4960 4955 4960
4975 4970 4980
4990
4985 4985 4985 4985 4985 4990 4990
4985 4985 4990 4990
5000 4995
495s
4975
4900
1986
40SS
1998
G R ~ V I M E TCALIBRATIOXS RIC O F 3ML. PIPET
VOLUMETRIC AND
Observer W. R T
r
Mean
x
T I P IN
V'
11I
v1
3.003 3,002
3.003
3.0027
Obsen CI C E , F V
v
Obaeiver 1' 1f1
P hI L. V' .lfl
M1
M1.
3.0010 3.0014 3.0019
3.001 3.003 3.002
3.0000 3.0020 3.0017
3.002 3.002
3.002
3.0014 3,0018 3.0014
3.0014
3.0020
3.0012
3.0020
3.0015
and gravimetric means in close approximation to each other, but only 2 of 13 individual observed volumetric values differ from 0.4988 ml. by more than 0.0005 ml. and the greatest deviation is only 0.0012 ml. Gravimetric calibration to blow-out after free drainage gave the volume estimates, 0.4928, 0.4932, 0.4918, 0.4930, with a mean of 0.4927. This differs by 0.0031 ml. from the volumetric mean for D. T. between 7.4 and 8.0 seconds (Table I). Obviously, with such short drainage time, considerably more variation in delivery is to be expected than in the cases approximating complete drainage more closely, and such an influence is evident by comparison with the first column of Table I where D. T. is approximately 2 seconds. Although the same size of capillary tubing could be used for larger standard pipets, actually 0.03 ml. per cm. was the volume difference on the scales of the 3- and 5-ml. S-pipets used. Accordingly, a comparison of results by different observers was made to indicate reproducibility of calibration of a stopcock pipet for delivery of 3 ml. betreen marks. As a control on the volumetric estimates ( V ) , the mercury delivered each time was caught and weighed in a tared vessel to give a corresponding gravimetric estimate (V'). The results are given in Table 11. The apparatus may readily be adapted, as indicated in Figure 3, by use of a modified vessel, W , so t h a t flow from the vessel to be calibrated goes directly to the standard pipet, S. This is useful in calibration of instruments such as burets and Van Slyke-Neil1 chambers for manometric gas analysis. c-4LIBRATION OF
MICROBURET.
As illustrated in Figure 3, a modified vessel, W , was made as follows: From a 7-mm. outside diameter adapted tip to fit Q, a 15-mm. outside diameter tube rose t o 34 cm. above, joined t o a bulb about 50-mm. outside diamter and Pcm. length with tapered ends about 3 or 4 cm. above and below, Ioining an 8-mm. outside diameter delivery tube, arched
FIGURE 3. MODIFIED VESSELW FOR CALIBRATIONBY DIRECTFLOW, ALL ONE PIECE FROM Q TO T
at the top and turned down leading to a level approximating that of stopcock D, then turned upward again and adapted t o a cup, T , similar to that in the vessel described above although a trough was not actually used. The modified vessel W , was joined at Q as reviously described, clamped just below the bulb and arouna the cup below T about 15 cm. t o the left of C. Mercury was run up almost t o its top. Then water was added at cup T, and run into the bulb by withdrawing mercury through D. At fist some air was trapped in the bulb and top of the delivery tube, but this was removed by repeating the raising and lowering of the mercury level, and supplying water a t T as re uired. In the manner previously employed, the tip of a 10-A.microburet was inserted through a stopper at T , and the buret clamped near the t o in a vertical position. Using a standard pipet, S, but with 0 be!& and V-scale above, successive withdrawals were made from the microburet t o readings as iven in Table I11 with the estimated cumulative volumes (sum o f successive readings on the standard). Just before each withdrawal, the buret setting was carefully checked t o guard against any drift, such as might result from atemperaturechangeduringanintervalofdelay. However, there was seldom any need for resetting. The delivery speed was about 1ml. in 40 seconds, controlled b E. A set of observations at about 0.5-ml. intervals was ma& and a t about 5-ml. intervals by each of two observers. The results are given in Table 111,with gravimetric data subsequently obtained by weight of mercury delivered in the manner described for check calibrations in the case of the 3-ml. pipet ( V ' ) .
CUMUL.4TIVE VOLUME TABLE111. EST~VATED READING TO GIVENREADING
a
FROM ZERO
Reading
VI (W. R. T.)
Vz (P.hI. L.)
MI.
M1.
M1.
VdVo Ml.
0.5 1.0 1.5 2.0 2.5
0.5020 0.9990 1.5000 1.9985 2.5055
0.5015 1.0025 1.5035 2.0015 2.5065
1.0010 0.9965 0.9977 0.9985 0.9996
3.0 3.5 4.0 4.5 5.0
3.0115 3.5120 4.0135 4.5200 5.0250
3.0110 3.5135 4.0105 4.5155 5.0185
1.0002 0.9996 1.0007 1.0010 1.0013
5.5 6.0 6.5 7.0 7.5
5.5250 6.0280 6.5245 7.0300 7.5335
5.5195 6.0180 6.5150 7.0190 7.5190
1.0010 1.0017 1.0015 1.0016 1.0019
8.0 8.5 9.0 9.5 10.0
8.0345 8.5410 9.0435 9.5465 10.0490
8.0235 8.5270 9.0290 9.5325 10.0350
1 0014 1:0b16 1.0016 1,0015 1.0014
5.0 10.0
5.019 10.040
5.017 10.033
1.0004 1.0007
5.0 10.0
5.015' 10.0320
5.016'' 10.034"
Gravimetric observations, V', made subsequently by W. R.
T.
Discussion The present system of calibration by equal volume displacement differs primarily from others previously developed (1, 6,4) in that the measurement is made in the standard pipet with a liquid (mercury) which does not wet the glass. Thus is avoided a source of drainage variation which otherwise may become progressively worse with use. Obviously, another liquid forming a similar layer above mercury may be used if required instead of water. The volume range employed thus far has been from 0.5 to 10 ml. Great variations in pressure on the glass by the mercury might interfere with precision in large volume calibration; and, perhaps, use of volumes exceeding 100 ml. might give unsatisfactory results. I n the case of the apparatus constructed by the author, addition of a gas pressure equivalent to about 48 cm. of mercury in the standard pipet, with B closed, caused a depression of about 0.002 ml. on the V-scale. Slight influences of this nature are compensated automatically by the suggested
ANALYTICAL EDITION
March 15, 1942
method of calibration of the standard pipet system, and a check upon variability of such influence is covered by replicate observations. Only those variations in temperature that occur during the usually short period required for actual measured volume displacements affect the precision. If the volume coefficient of thermal expansion is the same for the glass of the standard pipet as for that of the apparatus being calibrated, and their temperatures are equal, correction t o a prescribed standard temperature is automatically made. Otherwise, a correction for a difference in thermal expansion coefficient may be introduced in the usual manner. The system should be most useful where many pipets, small flasks, or tubes are to he calibrated. Manufacturers of
271
such apparatus should thus be aided in meeting tolerances prescribed by the National Bureau of Standards for certification, with a consequent saving by a lower frequency of rejection. Furthermore, improvement in precision of setting initial marks on apparatus may permit actual reduction of some such tolerances without increase in cost.
Literature Cited
. .
(4)
English, S., J. SOC.Glass Tech., 2, 216-19 (1918). Findlay, Alexander, p. 35, London, Longmans, Green and Co., “Practical Physical Chemistry”, 3rd ed., 1919. Peters, J. P., and Van Slyke, D. D., “Quantitative Clinical Chemistry”, Vol. 2 (Methods), pp. 3-23, Baltimore, Williams & Wilkins, 1932. Stott, Verney, J. SOC.Glass Tech., 8, 38-43 (1924).
Riboflavin Analvsis of Cereals d
Application of the Microbiological Method JOHN S. ANDREWS, HAROLD M. BOYD, AND DAVID E. TERRY General Mills, Inc., Research Laboratories, Minneapolis, Minn.
A
MOTU’G the rapid methods proposed for the estimation
of riboflavin, the microbiological and fluorometric appear to be the most promising (8,7). Both have been shown to give comparable results on a wide variety of biological materials (6) and to agree satisfactorily with values obtained by animal assays (1, 3, 8, 10, 11). Neither method, however, is universally applicable without modifications designed to remove interfering impurities. The microbiological procedure may be adversely affected by extraneous solids (8) or other nonflavin growth-influencing factors (4), while the fluorometric procedure is accurate only if pigments and nonflavin fluorescent substances are compensated for or removed (6,6,Q). For the past two years the authors have used the microbiological method for the examination of cereals and cereal products, and more recently have applied the fluorometric technique to this same study. Numerous instances have been found where the results from the two methods did not agree and these findings have led to a study of the factors which are responsible for this lack of agreement.
Microbiological Studies While in the microbiological method the direct assay of the solid, finely ground cereals has an advantage in the possibility that incomplete extraction may thereby be eliminated, the presence of the suspended solids can lead to unreliable results (8). When different levels are used in the microbiological assay there is frequently a marked tendency toward appreciably lower values as the weight of the sample is increased. This introduces uncertainties as to the accuracy of the average of the several levels (Table I). I n extracts prepared by autoclaving with water and centrifuging, this tendency is decreased although not entirely removed. If uniformity is taken as a criterion of reliability, the values for the aqueous extracts would be more acceptable. However, the assays thus obtained are materially lower than those from the direct assay and a t once the question of extraction efficiency is introduced. Either extraction is incomplete or some nonflavin growth factor is removed. But if the latter
is present and is responsible for the higher values in the direct assay, why is there a falling off in the assay values as the weight of the sample is increased? If one attempts to improve on the extraction conditions by treating the sample with takadiastase prior to removing the undissolved solids, there is a further change in the assay results. The absence of uniformity a t the different levels is almost entirely removed, but the riboflavin values are still lower! For this reason it is believed that completeness of extraction is a minor problem compared to the behavior of the organism under the different assay conditions. I n order to determine whether the residue remaining after extraction contained any appreciable quantity of riboflavin, it was subjected to assay. The solid had to be used, since further extraction yields extracts very dilute in riboflavin. The results indicated that refined cereal residues contained 6 to 8 per cent of their original flavin, while cereal grains contained up to 20 per cent. However, these assays were as erratic as the direct assays on the original solid samples and no decision could be made as to whether the results were due to riboflavin in the residues or to the presence of extraneous solids in the assay medium. The latter can cause fictitiously high values which could readily account for the “apparent” riboflavin content of the residues. A partial answer to these problems was found in the examination of cereals to which pure riboflavin had been added.
TABLEI. EFFECTOF WEIGHT OF SAMPLE MICROBIOLOGICAL ASSAY Cereal Product
ON
DIRECT
Weight of Sample
Riboflavin
Gram
IrLl./O.
Whole wheat flour
0.053 0.068 0.0S3 0.09s
Patent flour
0.21 0.27 0.33 0.39
.Lv.
Av.
2.33 2.20 2.13 1.80 2.13 0.40 0.41 0.30 0.32 0.37
