V O L U M E 24, NO. 7, J U L Y 1 9 5 2
1143
methylcyclohexane and toluene mixtures. This method of analysis is based on the autoniatic recording of the change in refractive index of the percolate during an adsorption analysis and integrating the area under the curve.
=
21
= displacement of phototube in inches = distance from refractometer cell to phototube
T LY
=
d
I
I
I
I
angle of incidence of light as diagonal boundary
= chart divisions = over-all sensitivity
of refractometer expressed m An per chart division, d df = refractomil-i.e., optical sensitivity expressed as An per 1-mil movement of Dhototube: for this instrument m = 1.24 x 10-4 ~n s = recorder sensitivity expressed as chart divisions equivalent to 1refractomil = volume of component (solute) volume of solution containing component nl = refractive index of pure solvent n2 = refractive index of pure component (solute) n, = refractive index of solution A = area under curve in refractogram in chart squares R = rate of flow, chart divisions per milliliter Q = refractom = volume equivalent to 1 refractom v, = volume of component eluted
D I
volume of percolate
2‘
v,
v =
L
I
I
I
I
0
IO
20
30
40
I
EFFLUENT VOLUME IN MILLILITERS
Figure 9. Hefractogram on Silica Gel of a Neutral Oil from Coal Hydrogenation B.p. 165-178O C.
,
Furthermore, the method is applicable to any adsorption analysis where the components are eluted singly. Another practical application of the instrument is in the adsorption analysis of complex mixtures in which the components are not eluted singly. The automatically recorded refractogram not only permits the taking of fractions during adsorption analysis according to the development of bands, but also permits rapid comparison of the relative amounts of saturates and aromatics present in hydrocarbon mixtures. Thus, the use of this instrument promises to be of value in adsorption analysis. ACKNOWLEDGMENT
The authors are indebted to A. A. Orning and George Waechter for their advice and technical assistance in this work. NOMENCLATURE
a
= amount of component
c
=
concentration of component
v,
LlTERATURE CITED
(1) Claesson,
S.,Ann. S. Y . A c a d . Sci., 49, 183-203 (1948). (2) Claesson, S.,Arlziv Kemi Mineral. Geol., 20A, No. 3 (1945).
(3) Dinneen, G. U., Bailey, C. W., Smith, J. R., and Ball, J. S., ANAL.CHEM.,19,992-8 (1947). (4) Hagdahl, L., and Holman, R. T., J . A m . Chem. Soc., 72, 701 (1950). (5) Hurd, C. D., Thomas, G. R., and Frost, A. A., I M . , 72, 3733 (1950). (6) Jones, H., Ashman, L.. and Stahly, E., AXAL.CHEM.,21, 1471 (1949). (7) Kegeles, G., and Sober, H., Division High Polymer Chemistry, Symposium on Physicochemical Methods in Study of High Mo!ecular Weight Xatural Products, 119th Meeting Am. Chem. Sac., Boston, Mass. (8) Mair, B. J., I n d . E m . Chent., 42, 1235 (1950). (9) O’Connor, Ruell, I b i d . , 40, 2102 (1948). (10) Thomas, G. R., O’Konski, C . T., and Hurd, C. D., AXAL.CHEM., 22,1221 (1950). (11) Tiselius, A . , and Claesson, S., Arkiv Kemi Mineral. Geol., 15B, No. 18 (1942). (12) Zaukelies, D., and Frost. -4.A , ANAL.CHEM.,21, 743 (1949). RECEIVEDfor review March 21, 1951. Accepted M a y 5 , 1952. Presented in part before t h e Division of Gas s n d Fuel Chemiqtry a t the 120th M e e t ing of the AMERICAN CHEmcaL SOCIETY, New York, N. Y .
Apparatus for Precise Volumetric Calibration with Adapter to Simulate Natural Drainage WILLIAM K. THOMPSON Dicision of Laboratories and Research, A-ew York State Department of Health, .41bany, 2V. Y .
A
SYSTEhf of calibration has been described (16) that is
based upon equal-volume displacement in a standard pipet or buret, S, and in the apparatus, P , being calibrated. I t differs from others previously developed (1, d, 11) in that measurement in the standard is made with a secondary liquid (mercury) that does not wet its walls; thus a source of variation with a trend toward increasing bias ia avoided. The primary liquid, in t h e vessel under calibration, may be any that forms a layer upon the secondary; as a rule, it may be considered to be water over mercury. Reilly and Rae described the original apparatus (10); some subsequent modifications ( l a , 16, 18) and an adaptation to microvolumetric measurement ( 1 7 ) have been discussed (2.2). The prasent purpose is to describe the modifications of apparatus and technique in current use, especially with regard to use of an alternative form of the vessel, W , which contains the biliquid
interface, so that naturtil uniestiicted drainage of pipets may be closely simulated in the calibration process. This modification of W , shown in Figure 1 approximately to scale diagrammatically with the rest of the calibration apparatus, differs from the usual vessel W in having a largebore side arm and stopcock, Y ( a t least 10 mm. in internal diameter), through which drainage from the pipet, P , is allowed to an overflow tube above Y , whence it drains from a trough, T’,through X’ to waste. As needed, water from reservoir R’may be introduced through stopcocks E’ and E and the side arm to W . E’ has e two-way branching system below (not shown in the figure): one to R‘,the other joining a lead from X’that terminates in the neck of a filter flask somewhat above its side arm, which is provided with a tube leading to a sink. The filter flask serves as a trap without blocking drainage from 5’” through X’; if mercury is allowed to flow into the side arm to E, it may be drained through E‘ to this trap. Flexible transparent tubing (Tygon) joins E’ to E and to R’; it is used also to join F’ to the mercury reservoir, R. F’ and F
ANALYTICAL CHEMISTRY
1144
are part of a glass lead attached by a semiball joint a t the hottom of an H-shaped tube; the H cross-connection has a 45’ slope. The present stopcock E should not be confused with one that was SO designated on the original apparatus ( 1 5 ) but promptly eliminated (16),and its function taken over by additional stopcocks, A ‘ and B . A , B, C, and F are used as open or shut gates (mounted with handles toward the front). All four should never be left shut longer than necessary to permit deliberate operation. B‘ is left open either to S , o r to S’ (handle mounted toward the right, as shown), and A , D,and F’ are used as required to control flow rates (always partly open Tyith handles toward the rear, so that they are less likely to he turned hy mistake j. Stopcocks A , A ‘ , B , B‘, C, D , F , and F’ have a 2-mm. bore and are provided with springs to hold the plug in place against a pressure of 50 pounds per square inch. They are obtainable from the H. S. Martin Co., Evanston, Ill. These stopcocks and most of the semiball joints are lubricated with a mixture, made a t room temperature, of equal parts of the regular and of the high-vacuum type silicone stopcock grease (Don--Corning). Standard-taper joints (10/30) above semiball joints or the latter alone are used to attach the standard pipets to the apparatue a t G and G ‘ , and a semiball joint to attach W at &; thus a former minor source of error is eliminated (15). A grease that is easier to remove from glass should be used for these joints and stopcocks Y , E , and E‘; the commercial Nevastane XX (Keystone Lubricating Co., Philadelphia, Pa.) or an aluminum stearate-iilboline mixture, 35 grams to 100 ml., described by Hamilton and Van Slykc ( 3 , p. 233, footnote 3) is suitable. Size (18/7) semihall joints are used throughout, held together by spring pinchclamps; their use decreases the risk of breakage or of change of internal capacity of the apparatus under stress of operation, and facilitates adaptation of the apparatus to varioue purposes by substitution of appropriately modified parts. Cleaning is easier also. Troughs, T and T’,are made from rubber cushions for 500-mI. centrifuge bottles. They are about 7 5 mm. in diameter, and should be bored to fit snugly at 7’ and T’, respectively, and to take a small glass drain-tube inset (X and X ’ ) . X drains to Z”, and X ’ to the trap flask and sink. The stopcock pair C and D and delivery tip vertically below B , and the lead from F attached vertically below A , have interchanged their former positions to save over-all height a t the expense of a little more difficulti in filling the apparatus. Aside from these modifications, the form and operation of the apparatus are about as before (15, 16). Mercury Charge. The mercury is introduced SO that it always rises as it advances with a vacuum above its surface a t a pressure less than that of 3 cm. of mercury, and joints and stopcocks a r e tapped lightly as the mercury rises above them; the object is to minimize entrapment of air. Reservoir R is fillecj with F‘ closed; th: F-F connection piece is detached a t the semiball joint, and inverted, and vacuum is applied. Mercury is allowed to advance slowly through F , the piece being rotated as needed, so that the surface rises almost to the joint. Then F is closed, the vacuum is relieved, and the piece is attached to the H-tube as in Figure 1. Then, with D closed, B’ turned to S, and with A , A’, B, B’, and C wide open, vacuum is used in W and S, and the tip below D is submerged in mercury, which is gradu a l l y allowed t o rise through D almost to the Figure 1. Modified Calibracross connection of the tion Apparatus H-tube. Then C i s closed and mercury is slowly adVessel W with side a r m for simumitted through F. When lation of unrestricted drainage
The apparatus, previousl? designed for equal-volume displacement calibration, eliminated one source of error by use of a nonwetting liquid (mercury) in the standard; but small errors were still attributable to lack of rigidity in certain joints and of facilities for close simulation of natural unrestricted drainage of a pipet. The first is met by use of ground joints; the second by drainage from the pipet to an overflow ria a side arm and stopcock of large bore, which is closed during the measured refilling. A demonstration of single-observation settings of new marks on nine 10-ml. transfer pipets shows all to be within National Bureau of Standards tolerances, using scarcely more than the middle third of the bull’seye. Better precision, lower costs in manufacture, or both are made possible. Other modifications facilitate use and adaptation to many purposes.
it reaches beyond Q into W as in Figure 1, stopcock A la closed, and when it reaches the V-scale in S , B’ is closed, the vacuum, is relieved in TV and S , F is closed, and B’ is opened to S Mercury is run out through C till the level falls between B’ and B. Then C is closed, vacuum is applied to the top of S’, and mercury is admitted through F till the level in S’has reached,the V’-scale, whereupon B is closed and the vacuum is relieved in S . Water Charge. Vessel TY is filled with water, allowed to run down the inside wall so as to avoid bubble formation (15); but if the modification with side arm and reservoir, R’, is used, water is introduced from R’ through E‘ and E with Y open till the standpipe above it overflows, whereupon it is closed; and when water overflows W into trough T stopcock E is closed.
.
Standard Pipet or Buret. All standardization and calibration values are given in the usual way as estimates of volume that would be obtained under like conditions but with the temperature at 20” C. The standard S or S’ pipets (or burets) are modified only in having an outer standard-taper or semiball joint for connection to the apparatus a t G or G’; if they are to be used inverted, a like joint is required on the other end, but the alternative of having a V-scale on both arms of the S-pipet or separate pipets for each of these uaes is preferable. Because B’ is used only for a choice between standards a t G or G’, it is assumed that it is turned wide open in the re uired direction; and, for simplicity, unprimed letters are usediere by preference for designation of a standard pipet or buret, its parts, and point of attachment. S may be standardized as previously (1.5), attached at G with F and A closed, C and D wide open, and flow controlled a t B. The mercury delivered from the tip below D is collected as the level in S passes from 0 to a stipulated point on the V-scale; its temperature and weight are observed and the volume delivered as of 20” C. is calculated as usual. However, the apparatus in Figure 2 is simpler and less subject to errors that may be worth consideration in such standardizations, though negligible in ordinary calibrations. It is used like an automatic pipet or buret with the upper stopcock partly closed to control flow rates. With this apparatus a nominally 10-ml. S-pipet gave the estimated volume delivery values of 9.9967 and 9.9966 ml. ( a t 20” C. j, and hence the correction of -0.0034 ml. was taken to apply to the nominal value in use. By weight of mercury delivered between this point and others on the V-scale, a set of corrections for the scale waa prepared in tabular form by use of linear interpolations. As a preliminary to experiments described below, the same Spipet was checked on the complete apparatus in the original manner; this gave the corresponding value, 9.9972 ml., a difference of only 6 parts per hundred thousand, that was considered negligible. Pipets to Deliver with Unrestricted Drainage. Suppose that a pipet, P, is to be calibrated under conditions simulating unrestricted vertical drainage of water into a weighing bottle (the time SO required, until the meniscus apparently comes to rest, being designated by t ) with the tip held against the wet wall of the
4
1145
V O L U M E 24, NO. 7, J U L Y 1 9 5 2 vessel a t that time and removed by a horizontal motion a t a stipulated time thereafter, which may be as soon as practicable or after a prescribed n-aiting period. Five seconds x-ere so allowed in the experimental observations here reported; but, either 1 or 2 seconds might be preferable. However, the present purpose is to show comparability of the equal-volume displacement and the gravimetric calibrations under similar conditionp, rather than to judge the merit of minor differences in such stipulat'ione. The level of apparent rept of the meniscus is called L , and it,s distance above the tip is measured (to about the nearest 0.1 mm.), usually taken as the mean of several observations; but it is not necessary to diaw water into P much above L at each trial. The time of unrestricted iiow may be used, a t leart occasionally, as a c l d < on thr h i u i a t i o n to be macle.
y n
r
\
I", ' ',
P is inserted in a rubber stopper to fit into the top of TV, but either L is etched on P or a rubber ring is set a t L carefully; water is flooded over the top of W and the stopper is inserted without entrapment of bubbles and with the tip of P set between 0 and 1 mm. above the flood level on the overflow above stopcock Y . The setting should be checked by actually opening Y bvide and running xvater through E' and E to overflow into 7" (about as when the pipet is draining when Figure 2. Standhalf full of water, but this need ardization Of S-Pipet h e estimated only roughly). Flooding may serve also to remove some impurities that tend to concent,rate a t the surface ( 7 ) . The S-pipet is filled to the 0 mark with mercury, ready for use; F , C, anci B are closed, but A is opened. Y is closed, and water is admitted through E to 11- till the level in P is a t U,the mark to be calibrated. The ]eye1 at C or later a t L may be adjusted by use of a tambour effect ( 1 5 ) on the stopper a t T by pressure exerted by a half-clamp, Jl (shown in Figure 1); but it is better t o avoid such pressure, and not to clamp P except to restrict its lateral motion. Level adjustment, if needed, a t U before drainape or a t L :ifterward may be effected readily by use of E and B ' , instead: To lower the level E is closed, E' is opened to drain momenLarily and then closed, and E is reopened; to raise the level in P stopcock E' is closed, E is opened, and light pressure is exerted upon the flexible tubing between them as required to raise the level to the desired point, whereupon E is closed. K i t h the water level set a t U ,the starting position for a calibration is reached (as shown in Figure 1); the setting a t 0 in S should be checked. Drainage of P is started by quickly opening Y wide; then E' is closed and E is opened to be ready for any adjustment that may be needed. The meniscus should come to apparent rest Tvithin 1 mm. of L; the time elapsed in seconds is called the simulation value oft, and during the prescribed waiting period Y is closed and the level in P i s brought to L; a t the end E is closed and B opened to allow the level in P to return to U (or, if a new mark is to be set on P , the flow is stopped when the mercury level in S has reached the required point on the V-scale). During this refilling of P, flow may be controlled by partly closing either A' or A or both. A slight temporary pressure by hand on the stopper a t T toward the end of the refilling may be used to set the shape of the meniscus properly; but the stopper must merely flex and return to its previous position, else the observation is iiivalidated. Ilternativelg, water may be allowed to rise somen-hat above the required point, and then return with the aid of vacuum applizd to the top of S; this sets the water meniscus escellently. However, there should never be a vacuum in S n-hen the final reading is taken. l l i n u t e amounts of gas that remain tr:ipped in the apparatus a t various points expand and contract with change in pressure. The plan is to have them under nearly the same pressure a t the initial and final resdings; that is why stop-
cock B is used as the gate-when closed, B supports the pressure of mercury above it, and pressure variations on its other side are relatively small by design of W . However, the magnit,ude of such variations should be checked, as is readily done; indeed, i t is often convenient to make minor adjustments in level (as in setting the level a t 0) by alternately rotating B and either F or C according as the level in S is to be raised or lowered. Likewise the required level in refilling P may finally be approached in small increments by alternate rotation of B and A (with or without vacuum applied a t the top of S ) I t is advisable to have a sensitive thermometer wedged with a suitably cut piece of rubber stopper entirely within TV (the rubber kept clear of the mercury to avoid errors due to it.s compressibility) and another t'hermometer alongside the apparatus. The influence of temperature changes, unimportant except during the actual volumetric displacement ( 1 5 ) , may be gaged by a rough estimate of the total volumes of water and mercury involved and their coefficients of thermal volume expansion. Such influences should be kept negligibly small with respect to the volume and kind of apparatus being calibrated.
Cleaning. For preliminary cleaning of glassware if the use of chromium is not objectionable, the writer formulated a ternary mixture, chromium trioxide-sulfur trioxide-water, in weight proportions about 2: 68: 30. A simple method of preparation has been given (PSI. All apparatus prior to calibration was allowed to stand overnight in this cleaning fluid and afterward estensively rinsed with tap and diFtilled Ivater.
X
I -51
00
'
01
02
03
04
05
06
07
08
CUMULATIVE PROBABILITY ESTIMATE
09
fp,
.O
Figure 3. For n random observations, x k , arranged in order of ascending magnitude, the expectation that a future x under like conditions will be less than x k is p k = k/(n+l); x denotes discrepancy between gravimetric and volumetric calibration (in parts per 10,000). Open circles are for observations made on a 10-ml. transfer pipet; black circles are for new settings on labels on different pipets; squares are for means of four observations each way after etching a mark (for six of these that survived an accident)
EXPERNEST1. -4 nominally 10-ml. transfer pipet with inner diameter about 3.2 mm. a t the etched mark, U,was alternately calibrated gravimetrically, V ' , and then volumetrically, V , with a rubber ring a t level L , where the meniscus finally rested after drainage in the immediately preceding gravimetric observation. Tht: discrepancies in parts per 10,000, x = lo3 (V' - V ) , for nine pairs of observations form the graph with points represented by open circles in Figure 3. The n points, respectively, have x k in order of ascending magnitude as ordinate and P, = k/(n 1) as abscissa (which are simply successive tenths, since
+
ANALYTICAL CHEMISTRY
1146 n = 9); points are jointed successively to guide the eye and aid linear interpolation.
modified vessel W of the type shown in Figure 19 of (2f)but with a semiball joint for attachment a t Q may be wed.
-4s shon-n elsewhere (12, 1 4 ) , such a graph of n random observations under stipulated experimental conditions furnishes an estimate, p , of the probability that a future observation under the stipulated conditions will yield a value of z less than that of the point ( p , x) on the graph. For points corresponding to the actual observed values of z the estimate was shown ( l a )to be unbiased.
Burets and Extraction Chambers. The previously described apparatus for calibration of burets, etc., is modified as shown in Figure 4,having a semiball joint for attachment at Q and another on the delivery tube to ease tension; it also is provided with a trough, T , .crith drain, X. A Van Slyke-Seill-Folch extraction chamber (19, 20) is shown ready for calibration, filled with water run partly into the cup above its stopcock, b, but with the delivery tube left dry. The apparatus not shown in Figure 4 beyond Q is a6 in Figure 1, except that the S-pipet is of the inverted form with the mercur) level at 0 on the lower arm and the V-scale on the upper arm. The chamber stopcock, b, is turned to the dry tube at the last practicable moment; then B is opened with flow controlled by A or A ’ or both, so as to meet stipulated conditions for drainage. The high mercury level in W causes flow into S and out of the extraction chamber, and flow is stopped \vhen the meniscus is a t the required mark in the chamber. The calibration value is then read on the V-scale.
EXPERIMEKT 2. To test the precision with which new marks may be set on transfer pipets, nine pipets of nominally 10-ml. volume were prepared. A single volumetric observation (taking V = 10.000)provided a new mark, set on a strip of label pasted on the upper arm. The maker’s marks were so far off (at least 5 mm.) that they did not bias the observer. Each pipet, immediately after removal from the volumetric apparatus, was recalibrated by a single gravimetric observation, V’. For ex erimental purposes it seemed &est thus toavoid any possible observer bias that might occur in replicate observations. The corresponding times, t , respectively, under the simulation conditions and in the unrestricted flow during the subsequent gravimetric observation were as follows: 12.4, 12.4; 23.0, 23.3; 16.3, 16.0; 30.0, 31.4; 20.2, 20.8; 20.0, 20.6; 16.4, 17.0; 16.7, 17.0; and 23.4, 24.0. The respective gravimetric estimates of volume gave V’ = 4-05 9 . 9 9 8 5 , 10.0036, 10.0012, 9.9977, 9.9987, 9.9956, 9.9998. 10.0068, and 10.0031.
!
As before, z = 103(v’
- V)
is taken as discrepancy in parts per 10,000 to form a cumulative probability graph as black circles also shown in Fiaure - 3: again n = 9 and hence the abscissal values are successive tenths. If we consider that we are aiming to meet Bureau of Standards tolerances ( r ) , we fail with four of the nine pipets
i,
At the 50-ml. mark, where a mercury meniscus is set in actual use of the chamber, the difference in volume between a water and mercury menidcus in a tube of the given inner diameter should be taken into account in the usual way ( 9 ) . Corrections, usually small but not negligible, are thus established for each mark. Occasionally, large errors arr found; thus two chambers in succession gave 0.475 and 0.600 ml., approximately, at the nominal 0.5-ml. marks in recalibration. Syringes and Syringe Pipets. Apparatus for ejection of given amounts of solution, such as glass syringes or syringe pipets, may be calibrated (18, 2 1 ) by use of another modification of vessel W , as shown in Figure 5. It is like that in Figure 4 except that, instead of the delivery tube, there is a cup a t the top, joined to the bulb by a short narrow tube on which is an etched level ring, li. The procedure is like that for buret or extraction-chamber calibration; the water level is a t U initially and the mercury level a t 0 on the inverted-form S-pipet. ?;early all the nominal valueis run into the S-pipet, then the syringe is discharged as much as practicable directly into the bulb of W(the need e being inserted through the constricted neck), the last is discharged into the bottom of the cup; then flow is allowed throu h B until the tvater menisccls is again a t L‘. $he calibration value is read oh the Vscale of S .
2
.--
Figure 4. Rlodified )‘essel rp’ for Calibration by Direct Chamber Ready for Calibration
with regard to outflon time t , which should be 20 to 60 seconds; likea ise the inner diameter a t the capacity mark should not exceed 4 mm. but is between 4.4 and 4.9 in all nine. However, this may be considered only as a slight handicap in the present experimental attempts t o set a mark within the volume tolerance interval (10 & 0.02 ml.); and all nine shots appear to have landed in the middle 34% of the bull’s-eye.
A ring was etched on each pipet as near as practicable to the newly set mark, U , and likewise a t the estimated rest point, L. After recleaning, volumetric recalibration gave as the mean of 10) .X 108 ,= four observations, respectively, the values ( V 6.5, 8.5, 4.!, 3.6, 0.8, 0.8, 0.9, 5.0, and 0.1. .4fter cleaning again, the following means of four gravimetric observation8 were ob4.0, -, 8.5, tained: (V’ - 10) X lo3 = 7.1, 9.1, 7.5, 4.0, and -. The blanks are due to accidental breakage of three of the nine pipets in the last cleaning; fortunately, as the broken pipets had appeared about the least in error, it seems safe to say that all nine of the newly etched marks had been set within the middle half of the bull’s-eye. A cumulative probability graph of discrepancies (parts per 10,000) with squares to denote the points for the remaining six pipets is given also in Figure 3; in this case, since n = 6, the successive intervals on the abscissa] scale are sevenths. Pipets to Deliver between Marks, to Blow Out, or to Contain. The technique for other types of pipet calibration is in accord with previous directions, except that the level a t U and L may be adjusted in the improved manner given above. Volumetric Flasks. The previous technique (15) ma be used with a delivery tube a t the top of W instead of pipet but a
-)
J;
t)ISCUSSION
Figure 5. R I o d i fi e d Vessel W f o r C a 1 i b r ation of Syringes According to Thomp s o n and Murdick (18)
The apparatus should be mounted on stands firmly clamped or bolted to the work bench, the upright rods cross-braced in several directions so as to provide triangulation to oppose tendencies to sway with shifts in the loads. However, the semihall joints minimize the influence of such sway on the internal capacity of the essential parts during the mercury-water displacement measurement. Indeed, rigid clamping is not attempted; instead, rubber stoppers are bored and cut ( 1 5 )tomake thickcushions between theglass apparatus and any clamp. C‘se of glass joints facilitates changes of parts to avoid unduly large capacities in calibration of small volumes; this decreases the influence of temperature variation (16).
The writer believes that it is more convenient to have standard-taper joints (as in Figure 1)on the 8-pipets than to use semiball joints directly, except to save over-all length with large 8pipets; but this is a close question. Whether it is worth while to have the ’oint shown just above C is doubtful; a previous satisfactory model lacked this and those just below A and B. How-
V O L U M E 2 4 , NO. 7, J U L Y 1 9 5 2
1147
ever, no difficulty has been encountered in use of many joints and stopcocks with the recommended lubricants, though additional clamps may be required with added joints. By release of stress the semiball joints appsrently tend to decrease error rather than contribute to it. There is practically no influence of temperature upon the Calibration by equal-volume displacement except for temperature changes during the actual displacement from unknown to standard or vice versa (15, 16, 2f). In the latter case, as in Experiments 1 and 2, the refilling may be done rapidly without risk of drainage errors, say in 15 seconds or less. The volume of water and mercury involved in producing such errors in the apparatus was about 140 ml.; about half was mercury. At 20" C. the coefficient of thermal expansion of water is roughly 0.00020, a t 25" it is about 0.00026; that of mercury is about 0,00018 throughout this range. Accordingly, the error induced through thermal expansion should not exceed 0.031 ml. per degree change in tempxature in that range; expansion of the glass would be a counter influence. If the temperature increased a t t,he rate of 0.1" C. per minute during refilling of the nominally 10-ml. pipet in Experiment 1, which may be supposed t'o take 15 seconds, the error induced would obviously be less than 8 parts per 100,000. The risk of such errors shoulJ always be considered and t,heir probable magnitude a t leafit roughly estimated. The apparatus should be shielded from sudden changes in temperature; customarily the operator should avoid exhalation directly toward the parts of the apparatus that would induce such errors. However, a technician with mouth within 15 cm. of vessel W , deliberately exhaling through the mouth toward the lower part of W containing mercury and the thermometer, was never able to increase the temperature more than 0.6" C. in 10 minutes in several experiments. I n one of these a fine-bore pipet as P was used to indicate actual volumetric error that would have been induced, readings being made on this and on thermometers inside and outside W a t the end of successive 10minute intervals. The apparatus was previously a t rest,, the technician breathed on it as indicated for 20 minutes, then retired and ho person came near it except for the readings for the next, 20 minutes. The results were as follows, roughly in accord tyith the estimated expansion per degree: Time, AI1r1,
0 10 20 30 40
Volume Reading, 111. 0.0000 0.0095 0.0146 0 0105 0 0065
Temperature, C. Outside 24.4 24.5 24.8 24.9 24.9 25.0
I n TV
24.8 24 6
24.5 24 5
For biologic fluids many chemists follow Van Slyke (9) in preferrihg pip& calibrated for blow-out or for between-mark delivery. The latter may be useful in precise measurement of small volumes. Precision of calibration by equal-volume displacement is shoxn by data (15, 8 1 ) obtained with a previous model. Thus, new upper marks were set on six 0.2-ml. 0stwald\'an Slylre pipets. Mean recalibration volumes in 10-5 ml. were: 20,085, 20,026, 20,032, 20,004, 20,018, and 20,001, res!)ectivelg, for right observations (half by each of two observem) on each pipet. S o observation deviated from the corresponding mean by more than 0.00042 ml. and the .rtandard deviation estimate for individual observations (48 with 42 degrees of freedom) was 0.0001ti ml. The total effective volume of the apparatus was about 70 nil. Similarly, ten 0.5-ml. Ostwald pipets for blowout delivery were given new marks and recalibrated ( 2 1 ) . Two observations by each of tivo persons on each pipet gave the mean recalibration volumes in ml. : 5003, 5030, 5008, 4984, 4989, 4998, 4983, 5008, 4996, and 4999, respectively, from which no observation deviated by more than 0.0008 ml., and the standard deviation estimate (for 40 observations with 30 degrees of freedom) was 0.00039 ml. Both the accuracy and precision of similar calibration observations by different persons have been indicated (15)by corresponding objective gravimetric checks. The equal-volume displacement may likewise be made rapidly without danger of drainage errors in calibration of flasks (15) or syringes (18, 2 1 ) ; but in calibration of burets, etc., a8 illustrated in Figure 4, the measurement is made during displacement of liquid from the vessel under calibration to the standard. This must be done in a stipulated way, usually with longer time required for the measurement with consequently greater risks of serious thermal expansion errors. It is usually (16) important t,o have the total effective volume of fluid as small as practicable, as well as to guard against important temperature fluctuations. Studies of delivery of burets or measuring pipets a t different rates of drainage may be used to indicate errors resultant from different usage of these instruments, or to supply factors for correction (21). Thus a slow drainage might be used in calibra-
tion of a buret in small subintervals, supplemented by calibrations over-all a t various drainage rates used for prorating. Similar studies (15) led to a method of to contain calibration of a pipet in excellent accord with the usual gravimetric method with mercury and meniscus correction: Thus, a 0.5-ml. Ostwald pipet was drained in between 134 and 552 seconds and the mean of 13 refill volumes was 0.49885 ml., whereas the mean of three gravimetric observations WAS 0.49877 ml. Only 2 of the 13 volumetric values differed from 0.4988 ml. by more than 0.0005 ml.; the greatest deviation was 0.0012 ml. Like comparisons between volumetric and gravimetric calibrations were made with this pipet bot,h in blow-out delivery and in a simulation of natural drainage (15). Of course, an independent type of measurement of any quantity is always valuable a t least as an occasional check, especially if simpler assumptions are involved; and reference to recommendat,ions of authorities on use or calibration of given types of apparat'us (7-9)should not be neglected. Intended use should be the paramount consideration in choice of conditions of calibration. Rapid delivery may be needed for some purposes. However, it should not be supposed that the volume delivered by a buret in calibration with slower flow may be read on the instrument aft,er rapid delivery followed by a waiting period, so that the total elapsed time is the same as in the calibration. A buret system free from drainage errors even in rapid delivery and reliable to within 0.0002 ml. in such delivery has been based on the equal-volume displacement apparatus (17, 2 1 ) ; holl-etrer, semiball joints should be used at G and Q a s for the calibration apparatus. Vessels for use with reagent in contact \vit,h mercury or separated by an intervening fluid have been described ( 2 1 ) ; they are readily attached at Q or detached without disturbing the reagent or entrapment of air. Alany may be kept ready for use, simply hung over a horizontal rod or pipe; and a simplified stopcock system may be used, eliminat'ing A ' , B ' , F ' , and U but preferably retaining C and a tip below for removal of mercurj-, so reservoir R need not be lowered for the purpose. In unrestricted drainage of a pipet with final touching of the tip to the wet wall of the receiving vessel and horizontal removal from contact', there may be considerable variation in the level, L , of the meniscus of the remaining liquid. I t should be measured promptly with the pipet still vertical. Variation in L may result from variation in the character of the surface touched and the angle of inclination of the pipet to the surface. However, the comparisons in Experiment 1 were sensibly free from such influences, as the same level, L, found in a gravimetric calibration observation was used in the immediately succeeding volumetric observation. Moreover, the nine distances of L from the tip were all in the range from 12.5 to 13.0 mm. In this case, observation of L was expedited by having rings etched 12.0 and 14.0 mm. from the tip, the level being estimated to the nearest 0.1 mm. The volume between the tL5-o marks was about 0.004 nil. Accordingly, an error of 0.25 mm. in setting a level belon- 15.0 should induce a volume error less than 0.0005 ml. S o w , consider the waiting period, w , in seconds after t,he meniscus comes to apparent rest. If it is stipulated that w be approximately zero, there is obvious danger of premature removal of the ipet, perhaps with considerable consequent error. However, ?more than negligible variation in delivered volume, V , is induced by variation of w between 1 and 5, perhaps the pipet design or mode of delivery should be considered unsuited to the pur ose. Alternat'e gravimetric observations with w < 1 and wit{ w = 5 gave the following results, expressed as increase in delivery in parts per 10,000: 9.0, 0.0, 0.3, -0.9, and 1.1; the same pipet as in Experiment 1 was used. I t was subsequently set in the calibration apparatus of Figure 1 carefully so that the level, L , at the end of fiow was between 12.5 and 1Y.O mm. from the tip. The consequent saving in time of adjustment permitted calibrations with waiting periods, w , less than 2 seconds. The mean of four values of V with w 5 2 was 9.9877 ml., the mean of four with 2 < w 5 5 was 9.9978 ml., and the mean of three with w = 15 was 9.9963 ml. The (intraclass) estimate of standard deviation was 0.0018 ml.; obviously, the differences are not statisticallr significant. The outflow times, 1, all were in the interval from 31.5 to 32.2 seconds as determined by an assistant who also timed the immediately following waiting period, w . illthough values of w = 1.2, 1.8, 2.0, and 1.2 u-ere scored in successive attempts to operate with w = 2 seconds or less, such hurried operation seems hardly to be recommended.
It is not suggested that the equal-volume displacement technique entirely replace the established standard technique of calibration (7-9); these should be used a t least with each type of apparatus as a check. Obviously, differences in the technique may mean some relative bias, though it' appears negligible in most
A N A L Y T I C A L CHEMISTRY
1148 respects, so far as has been investigated. Experiment 2 indicates that, even under somen-hat unfavorable circumstances, the volumetric technique may be practicable n ith some economic advantage. Note. The methods of statistical inference applied to the data of Experiments 1 and 2 are part of a development that is not only more firmly founded but often (as a t present) simpler to apply than theory based on the assumption of the conventional normal distribution. These methods are sometimes called “nonparametric” but preferably “distribution-free” methods of statistical inference; the latter term v-as proposed by Wilks (2.4) in an extensive review of the subject, which is treated in many recent texts (4-6‘,21).
(10) (11) (12) (13)
Chemistry,” Vol. 2, “Methods,” Baltimore, Williams and Wilkins Co., 1932. Reilly, J., and Rae, W. iV.,“Physicochemical Methods,” 4th ed., Vol. 1, pp. 47-8, New York, D. Van Nostrand Co., 1943. Stott, Verney, J . SOC.Glass Technol., 8 , 38-43 (1924). Thompson, IT. R., Ann. Math. Statistics, 7, 122-8 (1936). Thompson, W.R., Annual Report of Division of Laboratories and Research, K. Y. State Department of Health, p. 18, 1940; pp. 26-7, 1942; pp. 20-2, 1943; p. 20, 1945; pp. 31-5,
1948. (14) Thompson,
W.R., “Assumption Economy in Scientific Statistical Analysis,” presented at a panel discussion on Application of Statistical Methods to the Evaluation of Biological Data a t meeting of SOC.Am. Bact., Cincinnati, May 17, 1949. (15) Thompson, 1%‘. R., ISD. EXG.CHEN., AXIL. ED., 14, 268-71 (1942).
LITERATURE CITED
English, S.,J . SOC.Glass Technol.. 2, 216-19 (1918). Findlay, Alexander, “Practical Physical Chemistry,” 3rd ed., London, Longmans, Green and Co., 1919. Hamilton, P. B., and Van Slyke, D. D., J . Biol. Cheni., 150, 231-50 (1943).
Johnson, P. O., “Statistical RIethods in Research,” pp. 117-19, 169-83, Sew York. Prentice-Hall. 1949. Kendall. M. G., “Advanced Theory of Statistics,” 2nd ed., Vol. 2, pp. 83-4, London, Charles Griffin and Co., 1948. Mood, A. ll.,“Introduction to the Theory of Statistics,” pp. 385-418. New York, McGraw-Hill Book Co., 1950. Osborne, N. S., and Veasey, B. H., B d l . Bur. Standards, 4, 553601 (1908).
Peffer, E. L.. and Mulligan, G. C . , \ h t . Bur. Standards, Circ. C434 (1941).
Peters, J. P., and Van Slyke, D. D., “Quantitative Clinical
(16) Ihid., 15, 118 (1943).
(17) Thompson, W.R., J . Bact., 47,582 (1944). 118) Thompson, W.R.. and hfurdick. P. P.. Annual Report. Division- of Laboratories and Research, S . P.State Department of Health, p. 23, 1943. (19) Van Slyke, D. D., and Folch, J., J . Biol. Chem., 136, 509-41 (1940). (201 Van Slyke, D. D., and Neiil, J., Ibid., 61, 523-84 (1924). (21) Wadsworth, A. B., “Standard RIethods of the Division of Laboratories and Research of the Sew York State Department of Health,” 3rd ed., Baltimore, Williams & Wilkins Co., 1947. (22) Ibid., chapter on “Volumetric Calibration and Microvolumetric
Measurement. ” (23) Ibzd., 2nd and 3rd eds., hlethod F 78 B. (24) Wilks, S.S..Bull. A m . Math. SOC.,54, 6-50 (1948) R E C E I V Efor D review November 21, 1951.
Accepted May 22, 1952.
Simple Fractionating Device for Chromatographic Analysis Application to the Study of Carbohydrates L. A. BOGGS, L. S. CUENDET, MICHEL DUBOIS, AND FRED SRIITH Division of Agricultural Biochemistry, University of Minnesota, S t . Paul, Minn. A simple automatic apparatus was needed for the quantitative chromatographic separation of mixtures of sugars and their methyl derivatives on a scale large enough to provide sufficient material for making crystalline derivatives. The construction and operation of the apparatus developed are described. By the use of phenol saturated with water and a column of cellulose powder, a number of simple sugars have been separated. Similarly with methyl ethyl ketone-water azeotrope as the solvent, mixtures of methylated sugars may be readily re-
I
SVESTIGATIOKS into complex polysaccharides have been largely concerned with the separation and identification of
partial and complete hydrolytic products of the polysaccharides themselves and their methyl derivatives, and as a result the main structural features of a number of these important naturally occurring substances have been established (31). There are still many unanswered questions concerning the structure of these substances, and it has become increasingly apparent that much more attention should be paid to the finer structural details of even such polysaccharides as cellulose, starch, and glycogen, which hitherto have been regarded as relatively simple. Already there is evidence that some structural revisions may be necessary (3,7 , 18). Such investigations into the fine structure of polysaccharides clearly require a procedure for the quantitative separation of small amounts of sugars of their derivatives in a pure form from mixtures in which the required compound map be a minor constituent. Some success has attended fractional distillation procedures in
solved into their components. In this manner, it has been possible to determine the composition of methylated and unmethylated polysaccharides. The apparatus provides a simple and very useful tool for the quantitative separation of sugars and their derivatives from mixtures in which some of the components constitute only a small fraction of the whole. It thus becomes possible to determine the finer structural details of polysaccharides. The apparatus is of general use for fractionating eluates from chromatographic columns.
the past (16, 25), but the present prohlems require the handling of much smaller amounts of material and also a greater precision. This can be achieved to a limited extent by partition chromatography on large sheets of filter paper, but until thicker papers become available this does not constitute a practical solution t o the problem. Consequently, it has been found more effective to replace the sheets of filter paper by tubes packed Lvith powdered cellulose (21, 63),starch (28),silica (4), alumina (24, 2 7 ) , and various silicates (14). Only in this wa.v has it been posRible to obtain enough pure material for complete identification of some of the minor but perhaps highly important constituents of complex polysaccharides; such a task was regarded until very recently as Kell nigh impossible. Flolving chromatography on columns of absorbents with or without the added partitioning effect ( I O , SO) can be a tedious operation, but it can be greatly facilitated if it is used in conjunction with an automatic fractionating device (11, 15, 21, 32, 34, 38). A device (21, 38) that has been extensively used for
