Delivery Precision of Micropipets Carl F. Ernanuel San Juan Research Laboratory, Bellevue, Wash. 98007
Chromatographic methods so widely used today have the decided advantage of utilizing extremely small test samples for separation. One microliter or even 0.1 microliter will suffice in most cases for a complete qualitative and quantitative analysis. Competent analysts are well aware of the importance of calibrating the contained volume of these micropipets and, in any case, detailed procedures for this can be found in several textbooks on microchemistry. Excellent procedural and theoretical discussions concerning accuracy and precision of micromeasuring devices are given by Conway ( I ) , Kirk ( 2 ) , Steyermark ( 3 ) ,Benedette-Pichler ( 4 ) ,and many others. It has been my experience that the delivery precision of several commonly used micropipets is often a greater source of error than the uncertainty of the stated nominal volume or even that of the combined errors of the chromatographic process itself. This uncertainty, in connection with the many types of micropipets which are commercially available, convinced me that a need existed to determine the delivery precision of several of the more commonly used types. Delivery precision can be influenced by a number of variables: (1) The type of pipet, (self-filling, piston controlled, micrometer controlled, etc.). (2) Material of construction (glass, metal, plastic). (3) Errors associated with adjustment of a meniscus to a marked volume line. (4) Type of pipet tip (geometry, material of construction). ( 5 ) Method of application of pipet tip to a surface (angle of application, motion). (6) Method of withdrawing or extruding the sample (absorption on substrate, pressure). ( 7 ) Inner surface effects (hydrophobicity, inadvertent presence of grease). (8) Particulate contaminants. (9) Calibration errors between different pipets of the same type and nominal volume. In the accompanying table, the results from an experimental analysis of several of these variables are presented. A standardized titration procedure was used in each case. Before use in this study, each pipet was carefully cleaned in sulfuric acid-dichromate solution (or in detergent if made of stainless steel) and then thoroughly rinsed with water. The inner surfaces were not allowed to dry prior to use but were well rinsed with a standard sample solution (5N HzS04). In one case, (Class I pipet, Exp. 3, Table I), the inner surfaces were siliconized before use. The pipet was filled to a certain volume either by capillarity (if self-filling) or by use of a manually operated plunger or micrometer screw. The results were then examined with a hand lens to be certain of the result. The filled pipet was applied to a 5-mm circle of well washed and dried filter paper and emptied either by the absorptive power of the paper, or by “blow-out,” or by use of the pipet plunger as the case may be. Next, the wetted paper was placed in a tiny porcelain dish, and this was followed by adding 0.20 ml of water containing methyl red. the latter being delivered with a 1.0-cm3 syringe equipped with a (1) E Conway, “Micro-Diffusion Analysis and Volume(ric Error,” D Van Nostrand Co., Inc., New York, N.Y., 1959, Chapters 5 and 6. (2) P. Kirk, “Quantitative Ultramicroananalysis,” John Wiley and Sons, Inc., New York, N.Y., 1950, Chapter 2. ( 3 ) A. Steyermark. “Quantitative Organic Analysis,” Academic Press, New York, N.Y., 1961, p 116 etseq. (4) A. Benedette-Pichler, “Essentials of Quantitative Analysis,” The Ronald Press Co . New York, N Y , 1956, p 113 et se9
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Chaney stop adjusted to 0.20 ml. A fine glass fiber attached to a vibration unit (Table I, footnote 1) provided vigorous stirring while the delivered acid was being titrated with 0.02N sodium hydroxide. The latter was dispensed with a Lazarow microburet provided with a No. 27 platinum tip immersed below the liquid surface. By using a very strong sulfuric acid solution (5N) dispensed from the pipet and a dilute base (0.02N) solution for the microburet titration, the volumetric errors in measuring the titrant were minimized in a ratio of 250 to l while still retaining a sharp end point. The titration and end point uncertainties were estimated to have a standard deviation as a per cent of the mean of 0.186 (see control experiment No. 18, Table I). Two hundred and twenty-one titrations were carried out by one person using the same technique, solutions, and titration equipment. One can draw several conclusions from an examination of the results in Table I. (1) A self-filling pipet is capable of about the same delivery precision as a similar volume pipet which has been carefully adjusted (manually) to a marked volume (Compare Class I pipets with those of Class V in Table I). (2) An inexpensive and simple glass capillary pipet is capable of the best delivery precision from among those tested. This is especially true if the pipet is internally coated with a silicone preparation. [A properly siliconetreated pipet ( 5 ) labeled “to contain” a certain volume delivers a larger volume (or the entire contained volume) than an uncoated pipet which never directly delivers what it contains. However, the difference determined in this study for 1-microliter pipets was less than the standard error of delivery and thus can be ignored.] Careful “blowout” of pipet volume did not result in appreciable loss in delivery precision (Classes I and V of Table). (3) Delivery precision decreases as pipet volume decreases. Proper design can probably overcome this problem to some extent. (4) The most commonly used pipets, those which contain a reservoir from which only a fraction of the contained liquid is to be voided by use of a plunger, suffer from poor reproducibility of delivery. *Probably this is due to variable absorptive effects (surface tension) of the receiving surface. The best results with this type pipet are obtained with a slanting-tip metal needle using a 30” attack. Results are improved if the entire volume to be delivered is first formed as a tiny drop on the slanted needle tip before touching to the substrate (Class I1 pipet, Exp. 4). (5) The “plunger within a needle” pipet, a rather expensive instrument, is somewhat more precise in delivery than the “plunger within a barrel” pipet which has a separate delivery tip (Class IV). (6) Material used to construct the delivery tip has little relationship to delivery precision. (7) Best results are obtained by using one individual pipet for all samples delivered in a given analysis operation. The errors of deliverv are amroximatelv doubled ._ when a new pipet is used for each sample to be delivered; even so, the error is small (Class V pipet, Exp. 16 and 1’7). It is ironic that the most expensive ultramicro pipet ($100) gave the poorest precision- at the I-microliter level (5) E Duggan, and K Smith. Science. 116, 305-6 (1952)
-
Table I. Delivery Precision of Micropipets :xp. IO.
I
s.lf-fulityl
2
'Fill t o mark"'
3
&1;~:%"2"
but
4
10 ul Hamllton l y r l r y (701N)urntry Hamilton Rmpatlng Dhp.n..r (PB600).
5
Dltto
6
Dltto
7
mtto
I
Dltto
'Ultramicro Syrltye' a micromotor con.rolled myrhgo).
10
Mtto
11
Dltto
12
Dltto
13
DlttO
MANNER
u
o
0.996
0.104
I . a7
2.44
11
3. 29
S a m am "5" but d i m p n s o r m d s y r k g o b l d vortical and munplo appllod to papor h l d horisontal.
14
I ,40
Each of throe mmparato drops wsro formod on needlo tlp. than oach appllod to p a p r a t 30' awl..
12
7. 50
Tan wltm d o l i n r o d .ubi* t i p warn h.ld vortically. touahlty horlaontal pap?.
I4
16.7
Samplo warn d o l l w r o d w h l h t i p t o u c h b a the p a p r . WAm i n d h o d at 30
ia
13. 0
12
14. 7
DlttO
wrt1c.l.
mtto
'
Glaaa, ground.
atto
.
Teflon ruodlo (0.022" ID) with mquarad-off
I . 77
11
12
No. 27 .tool needlo with mqurod-off tip.
19. 2
W
Slanting t a p r e d mu.1 tip
15
uicroc.p.5
Squared-off ondo. emptlod by capillarit y
Mtto
~
12 10
S a n u am "5" but doll-rod whllo d l o p l u o r and o y r l q o worm b l d horlsontJ. pap?
mtto
Hamilton Syringe. I ul (7001N), p l u n p x within needle t y p .
mtto
-
I3
am * 3 4 1hut 3 needlo w l t h 90' (mquarod-off) tip.
14
17
11 12
Dltto
-
16
Y
0.996
Sa-
-
DILIVIBY
Throo " a t o p " m d i s p n s o r woro uood; accumulated llquid on tlp t b n toushsd U pap.'.
Cementad mtool needlo with t a p r o d , b.vo1.d tip.
Sam. am "5" but rued 127 platinum. mquared-df tip.
or
Contontm c a r d u l l y blown-out
mtto
9
-
SHAPE O F DELIVERY T1P
TYPE
30'
approach, aa i n "12".
mtto
Ea& *ample doliverod with a
I6
5. 51
I2
0.640
I2
I . 17
I1
1.Ob
11
0.186
dlfferent Microcap. _ .
18
CONTROL
Umlng Chaney adaptor. 0.20 ml of dilutm acl$wam dopomiud In tiny porsolala d1.h M d titrated jumt am above with Lazaraw m i s r o m y r l ~ ounit and vihratien .tirrar.
I.
Microchemical Specialties C o . Berkeley. Calif.
3.
Hamilton Company Whittier, Calif. 50608
5.
Kkummond SclontUlc Cm. Broomall. Pa.
2.
Clay Adam., Inc. New York, N. Y. 10010
4.
R.G . l . , Ins.
6.
AIOS I d o n t U l s E4violoo St. Loulm. Mo.
VlnalMd. N. J.
while a simple capillary, costing about 5c, gave the best results at the same volume. This does not reflect upon the craftmanship of construction or the calibrated contained volume of the ultramicro pipet but rather upon the practical dynamics of usage. Authors describing analytical results and procedures almost never inform the reader concerning details of sample application or delivery, and yet it stretches the credulity of the reader when the total
analysis precision is so often reported to be in the f l to 5% range. As this paper attempts to demonstrate, the sample delivery errors are pften larger than 10 to 1570, but with care in usage and proper pipet selection, the deviation can be kept below 1%. Received for review November 3, 1972. Accepted February 6, 1973.
A N A L Y T I C A L C H E M I S T R Y , V O L . 4 5 , N O . 8, J U L Y 1973
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