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An areal method for calibrating microburettes
ANALYTICA
130
AN
AREAL
JOHN
METHOD
I3. BUCK.
Labomtmy
CIUBfICA
ACTA
FOR CALIBRATING
MARGARET
o/ Physuai Bdiuda
L. KEISTER,
VOL.
4 (x950)
MICROBURETI-ES
AND
Biology, Naticnd ZnstAdas x4 Maryknd (U.S.A.)
M.
R. ZELLE
o/ Hdth.
IN-I-RODUCXION
Technical difficulties~~ 2~a. 4 have precluded direct gravimetm: calibratirr ; microburettes and microinjection pipettes in the range o. I to 1.0 ,vI, a&’ BW + limited anaiyses of delivery variation to volume far larger tba.n the ins.%& sl#’ claimed lower limit& 4. &a. Since delivery error is not constant +$ l8, Lm volumes’. and since u nsuspected orifice crro~s may wcuf. and experimental variation between repeated deliverws --tfuk. &xal over an instrument’s entire range. The following simple determinations on volumes as small as 0.x ~1. METRODS
Tba method iu based on the fact that if different volanxs of hquuI LT~ srpreu4
Four gravimetric and four areal calibrations are presented in Tables x and 2, and these, with four additional areal calibrations done on a diRerent day, are Ra~-#s
p. 134.
16 t4*7t
13.22
18
out
in
7.63
18
6.41
10
out
in
3.98
tt
1.3
2.2
3.0
215
47
7.3
3.02
in
t9
out
8.9
10.6
%
VU.
COd.
Mesa
2.21
1.36
mg
wt.
Mean
Run I
13.96
7.02
3.50
1.78
PI
vol.
corr.
m-
water
16
18
22
22
22
at
22
21
ObS.
No.
IYPIILLIR
14.81
13.19
7.6o
6.39
3-W
I.2
1.3
4.3
312
4.7
5.8
8.0
1.16 2.98
8.7
I.38
%
Var.
con.
-
14.00
7.0
3.48
t-77
Irl --
t9
18
21
21
20
21
21
20
Ob.
No.
--
II.13
to.94
5.60
543
1.83
2.70
I.44
I.31
=‘8
wt.
blean
2.3
1.8
5.3
3-4
7.4
4.2
10.4
11.4
%
VU.
4lM Coeff,
Run L
14.07
7.03
3.52
1.76
ccl
vol.
cm.
lI.I4
ro.95
22 21
5.63
514:
II 21
2.83
2.73
I.43
1.30
me
Htan wt.
1.8
2.5
4.0
310
6.7
3.t
8.5
to.2
%
Wt.
bhn cd.
Run 2
t9
t9
18
23
ObS.
No.
I-
KaOSCtte
‘4.07
7.03
3.34
I.73
P’
vol.
corr.
BY FRACtIONAL BIIVOLUTIONS D? A 32 TItRKAD PBlt INCli
t ml TUBBRCDLIN ~YRINGB’
bfcan Mean CO&. wt. vol.
Run 2
W
I
OR WITHDRAWN (“IN”)
USED WITH
BXPXLLBD (“OUT:‘)
TABLE
’ Both water experiments done on one day. both kerosene experimenta on another. The difference between “in” and “oat” wel&ts was shown, ltt other erpuimenb, to be exactly twice the wc&ht of liquid which evapratd in the pod required to make one measurement (citbn in or out). Hence the “comFted volume” was computed 81 the tneatt of “in” and ’ out” weighta divided by the specific gravity of the liquid (0.785 for tho kerosene wed). Impeller turned at rate of I revolutlot~ pa minute. Delivery tip withdrawn during wei@ing.
1.0
o-5
0.25
22
21
Ob.
No,
in
out
Fraction of one rev.
0.125
? * -8 _--
WIIGHTS 01 WATER AND XBROSINl
H
P
J. B. BUCK,
132
M. L. KEISTER, TABLE
AREAS
OF
SPOTS
PRODUCED
TRACTIONS
I Approx. 1
Fraction of one rev.
ON OP
0.25%
Run
al-Ctl
0.25 0.50
178 278 485 884 I 272 1484 2180 3772
1.00
G10g
0.02 0.04
0.08 0.125 0.16
ONE
I’APOR
01’
mcthylcnc
I
Run
l-
cocff
>f var. % IO.4
8.5 ;:: 3.4 3.9 3.9 2.9 29
No. >bs.
Iucnr1 arca
40 3s 26
775 279 495 873 ‘377
20
DYE A
SOLUTXONS
Saturated
-I-
2 COCfl.
Jf var %
Run
29
IO
1422
42
r5
IL
ZZG.}
2.9
IO
J2
3850 6330
3.8
IO
4.1
10
Run
2
cocff.
cocff.
,of var
311s.
6.0
VARIOUS
Sudan IV in kcrosenc
I
NO.
25 25 23 ‘7
BY
IblI’ELLI3R*
--
I
blue
13.3 I I.6 7.8
EXPELLEI3
MICROSYRINGE
I.5 fG
I 4
4 (1950)
II BY
REVOLUTION
aqueous
hlcnn
0.01
PII.TER
VOL.
M. R. ZELLE
nf vnr
% 577 1010 I 876 3295 4867 5904 8578 15199 26473
% 21
2:; 4.6 5.6 4.0 5.3 3.4 3.1 I.3
14 17 16 13 17 II IO IO
565 10x6 1829 3210 4692 5938 8200 14852 2G213
s.9 4.4 6.3 3.9 4.1 2.3 4.1 3.6 x.8
* “Arcas” in arbitrary units (multrply by 0.024 to convert to mma). Whatman No. I paper. All runs done on same clay, and all spots in a single cxperimcnt made on same p~cce of paper. Impeller turnctl at rate of 1 rcv./mrnutc. Weqht(mg)
as 100
200
400
700~000 2000
Area
5000 loo00 2000030000 APQO (‘r I 5)
Relation bctwccn fractional ravolulrons of rmpclling screw, the weights of alrquots of liquid cxpcllcd, and tho arcas of spots produced on filter popcr by such ahquots. Crosses, gravimctric dctcrminations from Table I (the duphcatc cxpcrimcnts csactly supcrrmpose). Open circles, arcal data from Tnblc II. Closed circles, arcal calibrations made on a second day. Fig.
I.
plotted in Fig. I on log axes. The results show that fractional turns of the impeller screw are directly and strictly proportional to the corresponding weights of liquid Re/eyanccs 9. 134.
VOL.
4 (1950)
CALIBRATION
OF
MICROBURETTES
133
expelled, over the range of 1/8to I revolution; that fractional turns are regularly related to the areas of the spots made by corresponding volumes of liquid, over the range 1/,w to I revolution; and that duplicate calibrations on the same day agree very closely. Assuming that screw revolutions and quantities of liquid delivered were linearly related, not only over the range accessible by gravimetric calibration but down to 0.09 ccl, the areal method reflected accurately this linearity of delivery. An analysis of variance of spot area showed that 85% of the overall variation was due to actual variation in delivery. (Variation between spot sizes on different samples of Whatman no. I paper, between measurements of the same spots by different people, and between repeated measurements of the same spots by one person were negligible). Similarly, the variations in the gravimetric analyses were mainly ascribable to variation in delivery, balance error being significant only for the smallest volume (ca. 1.75 ~11).Accordingly, since the coefficients of variation are comparable over the range covered by both calibrations, it can be concluded that the methods are of equivalent accuracy, and, further, that the area1 method gives a valid estimate of delivery variation over the full range of the instrument. Though both dyes give usable results, the relationship between methylene blue spot sizes is somewhat less regular than with Sudan, probably because the former spots have slightly serrated margins. However, Sudan spots fade somewhat over several months and hence are less suitable for permanent records. It is of interest that whereas volumes derived gravimetrically (Tab. I) are in the expected I :2: 4: 8 ratio, the same volumes estimated by the area1 method are related by an exponent of about 1.2 (calculated from the Sudan results in Tab. 2). This means that spot area does not increase as rapidly as does volume. A reasonable explanation of this is the fact that the ratio of circumference to area falls with increasing spot size, so that proportionately more solvent evaporates from the spot without carrying dye out into the paper. This possibility is supported by the fact that the largest Sudan spots are slightly, and the largest methylene blue spots markedly, darker than the smallest ones. Supplementary tests showed that methylene blue spots are less regular in outline and 20% larger on the S & S paper than on the Whatman; that the length-width ratio of the spots is about 1.1 on the Whatman paper and 1.3 on the S & S; and that both papers are suitable for area1 calibrations. Drying the paper at 60” instead of 25” C. was found not to affect methylene blue spot size, but to decrease Sudan spot size a significant 7 %. Overnight standing of the filter paper in saturated water vapour increased methylene blue spot size zg%_ Accordingly, day-to-day variations in laboratory temperature and humidity are considered the cause of the small but significant differences between area1 calibrations made on different days (e.g., the Sudan lines in Fig. I), and it is recommended that if calibrations are required on more than one day they be carried out in constant temperature and humidity as with other chromatographic techniques. Reierences
p. r34.
134
J. B. BUCK,
M. L. KEISTER,
M. R. ZELLE
VOL.
4 (1950)
Grateful acknowledgement for valuable suggestions is made to JEROME CORNof the Division of Public Health Methods, U.S. Public Health Service.
FIELD
SUMMARY At a given tompcrature and humidity the areas of spotz produced on filter paper by minute droplets of dyo solution are related exponentially to the corresponding volumes. Arcal calibration can bo used to checlc tho linearity of dehvcry of microburettes and microinjection syringe systems, and to estimate the variation between rcpeatcd deliveries of volumes as small as 0.x ~1. Rl%UMl? Pour une temp6rature et unc humiditeS d&ermindcs, les surfaces dcs taches, produitcs sur papier filtro, par dcs t&s pctites gouttcs d’une solution dc colorant, sont propox-tionnelles, expononticllement, aux volumes correspondants. Ce pro&d& d’btalonnagc peut &re utilid pour v6rifier In regularity d’houlcment des microburettes et dcs scringues do micro-injection, et pour estimer les variations de volumes jusqu’h 0.1 ~1. ZUSAMMENFASSUNG Bei einor bostimmtcn Tompcratur und Feuchtigkeit sind d~c von sehr klcinen Tropfen ciner Farbelbsung auf Filterpapicr hinterlassencn Flccke, den cntsprechenden Volumina exponenticll proportional. Diese Kalibriormethode kann beniitzt werden, urn die RegelmUsigkeit des Ausflusses von Mikroburetten und Mikroinjcktionsspritzen zu kontrolhcren und die Volumtlnderungen bis zu 0.1 ,~l zu vcrfolgen. REFERENCES 1 J. W. TREVAN, Lancct, 202 (1922) 786. Technique” * R. CEZAMBERS AND M. J. KOPAC in MCCLUNG’S “Hawdbook of Microscopical (1937) Hober. s M. J. KOPAC in REYNIZR’S “Micrurgical and Germ-Free Methods” (1943) Thomas. 4 P. F. SCHOLANDER, G. A. EDWARDS AND L. IRVING, J. Biol. Ghem., 148 (x943) 495. 6 E. M. P. WXDMARK AND S. L. C)RSKOV, Biochem. Z., 201 (x928) 15. a K. LINDERSTR&U-LANG AND H. HOLTER, Compr. rend. Iraw lab. Carlsberg, xg (x931) Ig pp. 7 L. YOUNG, Can. J. Research E, 25 (x947) 137. e J. B. BUCK, Anal. Record, gg (1947) 71, and Rev. Sci. Inslrumsnls, 20 (x949) 676.
Received August Igth, 1949
130
AN
AREAL
JOHN
METHOD
I3. BUCK.
Labomtmy
CIUBfICA
ACTA
FOR CALIBRATING
MARGARET
o/ Physuai Bdiuda
L. KEISTER,
VOL.
4 (x950)
MICROBURETI-ES
AND
Biology, Naticnd ZnstAdas x4 Maryknd (U.S.A.)
M.
R. ZELLE
o/ Hdth.
IN-I-RODUCXION
Technical difficulties~~ 2~a. 4 have precluded direct gravimetm: calibratirr ; microburettes and microinjection pipettes in the range o. I to 1.0 ,vI, a&’ BW + limited anaiyses of delivery variation to volume far larger tba.n the ins.%& sl#’ claimed lower limit& 4. &a. Since delivery error is not constant +$ l8, Lm volumes’. and since u nsuspected orifice crro~s may wcuf. and experimental variation between repeated deliverws --tfuk. &xal over an instrument’s entire range. The following simple determinations on volumes as small as 0.x ~1. METRODS
Tba method iu based on the fact that if different volanxs of hquuI LT~ srpreu4
Four gravimetric and four areal calibrations are presented in Tables x and 2, and these, with four additional areal calibrations done on a diRerent day, are Ra~-#s
p. 134.
16 t4*7t
13.22
18
out
in
7.63
18
6.41
10
out
in
3.98
tt
1.3
2.2
3.0
215
47
7.3
3.02
in
t9
out
8.9
10.6
%
VU.
COd.
Mesa
2.21
1.36
mg
wt.
Mean
Run I
13.96
7.02
3.50
1.78
PI
vol.
corr.
m-
water
16
18
22
22
22
at
22
21
ObS.
No.
IYPIILLIR
14.81
13.19
7.6o
6.39
3-W
I.2
1.3
4.3
312
4.7
5.8
8.0
1.16 2.98
8.7
I.38
%
Var.
con.
-
14.00
7.0
3.48
t-77
Irl --
t9
18
21
21
20
21
21
20
Ob.
No.
--
II.13
to.94
5.60
543
1.83
2.70
I.44
I.31
=‘8
wt.
blean
2.3
1.8
5.3
3-4
7.4
4.2
10.4
11.4
%
VU.
4lM Coeff,
Run L
14.07
7.03
3.52
1.76
ccl
vol.
cm.
lI.I4
ro.95
22 21
5.63
514:
II 21
2.83
2.73
I.43
1.30
me
Htan wt.
1.8
2.5
4.0
310
6.7
3.t
8.5
to.2
%
Wt.
bhn cd.
Run 2
t9
t9
18
23
ObS.
No.
I-
KaOSCtte
‘4.07
7.03
3.34
I.73
P’
vol.
corr.
BY FRACtIONAL BIIVOLUTIONS D? A 32 TItRKAD PBlt INCli
t ml TUBBRCDLIN ~YRINGB’
bfcan Mean CO&. wt. vol.
Run 2
W
I
OR WITHDRAWN (“IN”)
USED WITH
BXPXLLBD (“OUT:‘)
TABLE
’ Both water experiments done on one day. both kerosene experimenta on another. The difference between “in” and “oat” wel&ts was shown, ltt other erpuimenb, to be exactly twice the wc&ht of liquid which evapratd in the pod required to make one measurement (citbn in or out). Hence the “comFted volume” was computed 81 the tneatt of “in” and ’ out” weighta divided by the specific gravity of the liquid (0.785 for tho kerosene wed). Impeller turned at rate of I revolutlot~ pa minute. Delivery tip withdrawn during wei@ing.
1.0
o-5
0.25
22
21
Ob.
No,
in
out
Fraction of one rev.
0.125
? * -8 _--
WIIGHTS 01 WATER AND XBROSINl
H
P
J. B. BUCK,
132
M. L. KEISTER, TABLE
AREAS
OF
SPOTS
PRODUCED
TRACTIONS
I Approx. 1
Fraction of one rev.
ON OP
0.25%
Run
al-Ctl
0.25 0.50
178 278 485 884 I 272 1484 2180 3772
1.00
G10g
0.02 0.04
0.08 0.125 0.16
ONE
I’APOR
01’
mcthylcnc
I
Run
l-
cocff
>f var. % IO.4
8.5 ;:: 3.4 3.9 3.9 2.9 29
No. >bs.
Iucnr1 arca
40 3s 26
775 279 495 873 ‘377
20
DYE A
SOLUTXONS
Saturated
-I-
2 COCfl.
Jf var %
Run
29
IO
1422
42
r5
IL
ZZG.}
2.9
IO
J2
3850 6330
3.8
IO
4.1
10
Run
2
cocff.
cocff.
,of var
311s.
6.0
VARIOUS
Sudan IV in kcrosenc
I
NO.
25 25 23 ‘7
BY
IblI’ELLI3R*
--
I
blue
13.3 I I.6 7.8
EXPELLEI3
MICROSYRINGE
I.5 fG
I 4
4 (1950)
II BY
REVOLUTION
aqueous
hlcnn
0.01
PII.TER
VOL.
M. R. ZELLE
nf vnr
% 577 1010 I 876 3295 4867 5904 8578 15199 26473
% 21
2:; 4.6 5.6 4.0 5.3 3.4 3.1 I.3
14 17 16 13 17 II IO IO
565 10x6 1829 3210 4692 5938 8200 14852 2G213
s.9 4.4 6.3 3.9 4.1 2.3 4.1 3.6 x.8
* “Arcas” in arbitrary units (multrply by 0.024 to convert to mma). Whatman No. I paper. All runs done on same clay, and all spots in a single cxperimcnt made on same p~cce of paper. Impeller turnctl at rate of 1 rcv./mrnutc. Weqht(mg)
as 100
200
400
700~000 2000
Area
5000 loo00 2000030000 APQO (‘r I 5)
Relation bctwccn fractional ravolulrons of rmpclling screw, the weights of alrquots of liquid cxpcllcd, and tho arcas of spots produced on filter popcr by such ahquots. Crosses, gravimctric dctcrminations from Table I (the duphcatc cxpcrimcnts csactly supcrrmpose). Open circles, arcal data from Tnblc II. Closed circles, arcal calibrations made on a second day. Fig.
I.
plotted in Fig. I on log axes. The results show that fractional turns of the impeller screw are directly and strictly proportional to the corresponding weights of liquid Re/eyanccs 9. 134.
VOL.
4 (1950)
CALIBRATION
OF
MICROBURETTES
133
expelled, over the range of 1/8to I revolution; that fractional turns are regularly related to the areas of the spots made by corresponding volumes of liquid, over the range 1/,w to I revolution; and that duplicate calibrations on the same day agree very closely. Assuming that screw revolutions and quantities of liquid delivered were linearly related, not only over the range accessible by gravimetric calibration but down to 0.09 ccl, the areal method reflected accurately this linearity of delivery. An analysis of variance of spot area showed that 85% of the overall variation was due to actual variation in delivery. (Variation between spot sizes on different samples of Whatman no. I paper, between measurements of the same spots by different people, and between repeated measurements of the same spots by one person were negligible). Similarly, the variations in the gravimetric analyses were mainly ascribable to variation in delivery, balance error being significant only for the smallest volume (ca. 1.75 ~11).Accordingly, since the coefficients of variation are comparable over the range covered by both calibrations, it can be concluded that the methods are of equivalent accuracy, and, further, that the area1 method gives a valid estimate of delivery variation over the full range of the instrument. Though both dyes give usable results, the relationship between methylene blue spot sizes is somewhat less regular than with Sudan, probably because the former spots have slightly serrated margins. However, Sudan spots fade somewhat over several months and hence are less suitable for permanent records. It is of interest that whereas volumes derived gravimetrically (Tab. I) are in the expected I :2: 4: 8 ratio, the same volumes estimated by the area1 method are related by an exponent of about 1.2 (calculated from the Sudan results in Tab. 2). This means that spot area does not increase as rapidly as does volume. A reasonable explanation of this is the fact that the ratio of circumference to area falls with increasing spot size, so that proportionately more solvent evaporates from the spot without carrying dye out into the paper. This possibility is supported by the fact that the largest Sudan spots are slightly, and the largest methylene blue spots markedly, darker than the smallest ones. Supplementary tests showed that methylene blue spots are less regular in outline and 20% larger on the S & S paper than on the Whatman; that the length-width ratio of the spots is about 1.1 on the Whatman paper and 1.3 on the S & S; and that both papers are suitable for area1 calibrations. Drying the paper at 60” instead of 25” C. was found not to affect methylene blue spot size, but to decrease Sudan spot size a significant 7 %. Overnight standing of the filter paper in saturated water vapour increased methylene blue spot size zg%_ Accordingly, day-to-day variations in laboratory temperature and humidity are considered the cause of the small but significant differences between area1 calibrations made on different days (e.g., the Sudan lines in Fig. I), and it is recommended that if calibrations are required on more than one day they be carried out in constant temperature and humidity as with other chromatographic techniques. Reierences
p. r34.
134
J. B. BUCK,
M. L. KEISTER,
M. R. ZELLE
VOL.
4 (1950)
Grateful acknowledgement for valuable suggestions is made to JEROME CORNof the Division of Public Health Methods, U.S. Public Health Service.
FIELD
SUMMARY At a given tompcrature and humidity the areas of spotz produced on filter paper by minute droplets of dyo solution are related exponentially to the corresponding volumes. Arcal calibration can bo used to checlc tho linearity of dehvcry of microburettes and microinjection syringe systems, and to estimate the variation between rcpeatcd deliveries of volumes as small as 0.x ~1. Rl%UMl? Pour une temp6rature et unc humiditeS d&ermindcs, les surfaces dcs taches, produitcs sur papier filtro, par dcs t&s pctites gouttcs d’une solution dc colorant, sont propox-tionnelles, expononticllement, aux volumes correspondants. Ce pro&d& d’btalonnagc peut &re utilid pour v6rifier In regularity d’houlcment des microburettes et dcs scringues do micro-injection, et pour estimer les variations de volumes jusqu’h 0.1 ~1. ZUSAMMENFASSUNG Bei einor bostimmtcn Tompcratur und Feuchtigkeit sind d~c von sehr klcinen Tropfen ciner Farbelbsung auf Filterpapicr hinterlassencn Flccke, den cntsprechenden Volumina exponenticll proportional. Diese Kalibriormethode kann beniitzt werden, urn die RegelmUsigkeit des Ausflusses von Mikroburetten und Mikroinjcktionsspritzen zu kontrolhcren und die Volumtlnderungen bis zu 0.1 ,~l zu vcrfolgen. REFERENCES 1 J. W. TREVAN, Lancct, 202 (1922) 786. Technique” * R. CEZAMBERS AND M. J. KOPAC in MCCLUNG’S “Hawdbook of Microscopical (1937) Hober. s M. J. KOPAC in REYNIZR’S “Micrurgical and Germ-Free Methods” (1943) Thomas. 4 P. F. SCHOLANDER, G. A. EDWARDS AND L. IRVING, J. Biol. Ghem., 148 (x943) 495. 6 E. M. P. WXDMARK AND S. L. C)RSKOV, Biochem. Z., 201 (x928) 15. a K. LINDERSTR&U-LANG AND H. HOLTER, Compr. rend. Iraw lab. Carlsberg, xg (x931) Ig pp. 7 L. YOUNG, Can. J. Research E, 25 (x947) 137. e J. B. BUCK, Anal. Record, gg (1947) 71, and Rev. Sci. Inslrumsnls, 20 (x949) 676.
Received August Igth, 1949