- Email: [email protected]
A multipurpose micropipette
MICROCHEMICAL JOURNAL
9, 46-51 (1965)
A Multipurpose Micropipette! BENJAMIN
W.
GRUNBAUM
Environmental Physiology Laboratory, Department of Physiology, University of California, Berkeley, California Received December 14, 1964
Quantitative microchemical analyses require accurate measurement of the original sample to be analyzed. Since most microanalytical work is carried out with liquids, the capillary micropipette becomes the basic parameter of measurement. In aliquoting microliter quantities of reagent or unknown, speed of transer of repetitive samples is a major factor in the successful analysis. Ten years ago, Grunbaum and Kirk (2) described a self-adjusting and self-dispensing all-glass micropipette. The design was based on a pycnometer-type tube; both ends of the measuring tube terminated in a capillary fine enough to make unnecessary any adjustment of volume after filling from either end. Even though this design had all the requisites of a multipurpose micropipette, it was not easy to use for the following reasons: (a) the flow of liquid had to be controlled by a syringe or by mouth suction, thus slowing the operation considerably; (b) the tapered opening to the reservoir bulb was too narrow to permit easy filling and emptying of reagent solutions; quick cleaning was difficult; and (c) no provision was made for keeping the pipette in a secure holding position when it was not in the hand of the operator. The present paper describes a modification of the original pipette of Grunbaum and Kirk. The micropipette is a complete unit designed for onehand operation. The pipettes are uniform in outer dimensions regardless of the quantity of liquid they will measure and can be used for storage of reagent for reasonable periods of time. A specially designed rack serves as a holder from which the pipettes can be moved easily in and out while in use. When not in use the pipettes are held securely in an immobile position without the danger of breakage in 1
This study was supported by U.S. Public Health Service grant GM-09265. 46
A MULTIPURPOSE MICROPIPETTE
47
storage or transportation. The rack (Fig. 2) consists of a polypropylene stand and a clear Plexiglas cover. Six slotted sockets are attached to each side of the stand to hold a total of twelve pipettes. When the cover is in position the pipettes are securely locked in place. This is accomplished by the use of a clamp on top of the cover which exerts a slight pressure on the rubber bulbs on the pipettes and holds them firmly. OPERATION
Dispensing. The main structural difference between the original pipette (2) and the modifications described is in the incorporation of a rubber bulb (Fig. la). The inside lip of the bulb is forced across the upper circular projection. In this position the bulb cannot slip back and forth, but
I
L',
I
~~~~_-_E)J
a
b
e FIG. 1. The multipurpose micropipette (see text).
48
BENJAMIN W. GRUNBAUM
becomes an integral part of the pipette. To place reagent or solvent into the reservoir, the pipette is held in a vertical position, and the rubber bulb is removed; a disposable-type transfer pipette is used, as shown in Fig. Ib, to fill liquid to below the inner capillary end. The rubber bulb is then inserted between the two circular projections and the pipette is ready for use as a dispensing pipette.
Fro. 2. The rack, with cover in position.
When the pipette is held in a horizontal position with the inner tip submerged (Fig. Ic), the liquid flows automatically into the tube and stops when the tube is full. For quantities larger than 25 or 50 III the filling can be speeded up by placing the index finger on the perforation at the apex of the rubber bulb and applying slight pressure. The pipette is then rotated 180 (Fig. Id ) and the aliquot is slowly emptied into a dish. Alternately, the pipette can be held in a vertical position (Fig. Ie) and the aliquot 0
A MULTIPURPOSE MICROPIPETTE
49
transferred into a test tube. A good pipette must fill readily when submerged in a liquid. Also, when it is filled with liquid from tip to tip and held vertically, it must not empty, regardless of the hydrostatic head, unless slight pressure is applied to the rubber bulb . This criterion can always be applied with aqueous solutions but not always with organic solvents. Transfer of organic solvents can then be accomplished by holding the pipette in a horizontal or slightly diagonal position when emptying. Successive aliquots can be drawn rather rapidly and in a reproducible quantity. In a test of reproducibility, each of 10 aliquots was weighed in pipettes ranging in size from 10 to 100 ul. The reproducibility of the 10-~1 pipette was ± 0.1 mg (the latter being the sensitivity of the balance). This would introduce a possible error of 1%. The reproducibility of the l00-~1 pipettes was ± 0.2 mg or a possible error of 0.2%. Generally, the multipurpose pipettes can be used conveniently in sizes from 1 to 500 ul. Quantities of 5 ul or less should be emptied into existing liquid droplets, otherwise the surface tension (of aqueous solutions only) is too great and it is difficult to separate the pipette from the discharged droplet. The pipetting error in this procedure is minimized because a single pipette is used for aliquoting successive measurements. Sampling. For the sampling of unknowns the outer tip of the multipurpose micropipette should be wiped dry with tissue. The tip is then inserted into the "unknown" while negative pressure is applied to the rubber bulb. The " unknown" liquid then flows into the capillary pipette and is allowed to overflow into the reservoir bulb. While the pipette tip is still in the "unknown" liquid , the index finger is removed from the rubber bulb perforation and the pipette is withdrawn . The " unknown" aliquot is then automatically contained between the two tips of the pipette. The "unknown" is then transferred slowly and uniformly to the proper reaction vessel. The pipette tip is wiped dry with tissue and is ready for duplicate sampling or for aliquoting a second " unknown" in a similar manner. The "washing" of the inside of capillary pipettes in the fashion described is most effective when sampling liquids of the same kind. Any residual liquid adhering to the inner walls of the capillary is carried upward and disposed of by the second unknown. Since the pipettes are calibrated to deliver, any remaining residue is not a part of the aliquot of the "unknown." When the glass reservoir bulb fills to the inner capillary tip and the pipette is held vertically, the liquid is then emptied. This is best done by inserting a suction tube made of polyethylene or some other flexible plastic material.
50
BENJAMIN W. GRUNBAUM
Other uses. For viscous solutions and for highly volatile solutions such as standard iodine or standard fatty acid made up in an organic solvent, the multipurpose micropipette can be used in two ways: (a) the standard solution is placed in the reservoir bulb and dispensed very slowly to prevent erratic drainage; (b) the standard solution is drawn into the pipette very carefully so that it just fills to the end of the inner tip. It is then expelled like a regular capillary pipette. Pure solvent is then introduced through the pipette from the reservoir bulb, which can be charged with solvent at any time. This rinsing solvent can be measured by passing it through the pipette any number of times. The second method is, of course, more accurate because the solute has been completely transferred. If it is desirable to have an accurate aliquot without additional dilution, a multipurpose pipette can be fitted with a special adapter and the aliquot can be centrifuged into a test tube. The multipurpose micropipette can also be treated with a nonwetting agent such as Desicote (Beckman Instruments Inc., Fullerton, California) and recalibrated to contain rather than to deliver. In this case aqueous solutions will drain dry. Storage oj reagents. For the sake of accuracy, the amount of reagent used in microchemical determinations is generally made up in much larger quantities than can be used. Reagents should not be kept in the micropipette for more than a day or two, depending on the type of reagent. If the reagent in the pipette is not to be used every day, the pipette and reservoir should be emptied, cleaned, and stored dry. However, if the micropipette is used routinely every day the perforated rubber bulb may be exchanged for a solid one at the end of a series of determinations. The reservoir bulb would then essentially be stoppered. Before dry storage the pipette can be cleaned in routine fashion like any other pipette. DISCUSSIO:-l
The extensive adoption of microchemical methodology by the routine clinical laboratories has created a need for some kind of universal pipette. In 1955 Grunbaum and Kirk described their all-glass, self-adjusting and self-dispensing micropipette, and in 1957 Sanz proposed a self-adjusting polyethylene pipette (3) which is essentially an overflow pipette attached to a plastic squeeze bottle. Compared to all-glass pipettes it has the advantage of being unbreakable and, of course, is naturally hydrophobic. The
A MULTIPURPOSE MICROPIPETTE
51
plastic pipette part was changed to a glass capillary in a modification of the all-plastic pipette by the Coleman Company (1). The Sanz overflow pipettes require a high degree of manual dexterity to handle properly, and the drainage in small capillaries is poor. Also, the reservoir (the plastic squeeze bottle) is much too large for safe keeping of reagents. Reagents are best kept in a properly stoppered container which is opened only occasionally to remove a sufficient quantity for a day's work or a series of determinations. For the transfer of reagent a disposable clean plastic or glass pipette is used. This prevents any contamination and concentration changes. In summary, a convenient multipurpose micropipette is described as an aid in most phases of microchemistry. ACKNOWLEDGMENT The author acknowledges with gratitude the help received from Microchemical Specialties Co., 1825 Eastshore Highway, Berkeley, California in the construction of the multipurpose pipettes and rack. REFERENCES 1. CAMPBELL, D. ]., COMFORT, D., AND BELL, R. D., An evaluation of two ultramicro systems for clinical chemistry. Am. J. Clin. Pathol. 38, 323-329 (1962). 2. GRUNBAUM, B. W., AND KIRK, P. L., Self-adjusting and dispensing micropipet. Anal. Chem, 27, 333 (l955). 3. SANZ, M. C., Ultramicro methods and standardization of equipment. Clin. Chem. 3,406·419 (1957).
9, 46-51 (1965)
A Multipurpose Micropipette! BENJAMIN
W.
GRUNBAUM
Environmental Physiology Laboratory, Department of Physiology, University of California, Berkeley, California Received December 14, 1964
Quantitative microchemical analyses require accurate measurement of the original sample to be analyzed. Since most microanalytical work is carried out with liquids, the capillary micropipette becomes the basic parameter of measurement. In aliquoting microliter quantities of reagent or unknown, speed of transer of repetitive samples is a major factor in the successful analysis. Ten years ago, Grunbaum and Kirk (2) described a self-adjusting and self-dispensing all-glass micropipette. The design was based on a pycnometer-type tube; both ends of the measuring tube terminated in a capillary fine enough to make unnecessary any adjustment of volume after filling from either end. Even though this design had all the requisites of a multipurpose micropipette, it was not easy to use for the following reasons: (a) the flow of liquid had to be controlled by a syringe or by mouth suction, thus slowing the operation considerably; (b) the tapered opening to the reservoir bulb was too narrow to permit easy filling and emptying of reagent solutions; quick cleaning was difficult; and (c) no provision was made for keeping the pipette in a secure holding position when it was not in the hand of the operator. The present paper describes a modification of the original pipette of Grunbaum and Kirk. The micropipette is a complete unit designed for onehand operation. The pipettes are uniform in outer dimensions regardless of the quantity of liquid they will measure and can be used for storage of reagent for reasonable periods of time. A specially designed rack serves as a holder from which the pipettes can be moved easily in and out while in use. When not in use the pipettes are held securely in an immobile position without the danger of breakage in 1
This study was supported by U.S. Public Health Service grant GM-09265. 46
A MULTIPURPOSE MICROPIPETTE
47
storage or transportation. The rack (Fig. 2) consists of a polypropylene stand and a clear Plexiglas cover. Six slotted sockets are attached to each side of the stand to hold a total of twelve pipettes. When the cover is in position the pipettes are securely locked in place. This is accomplished by the use of a clamp on top of the cover which exerts a slight pressure on the rubber bulbs on the pipettes and holds them firmly. OPERATION
Dispensing. The main structural difference between the original pipette (2) and the modifications described is in the incorporation of a rubber bulb (Fig. la). The inside lip of the bulb is forced across the upper circular projection. In this position the bulb cannot slip back and forth, but
I
L',
I
~~~~_-_E)J
a
b
e FIG. 1. The multipurpose micropipette (see text).
48
BENJAMIN W. GRUNBAUM
becomes an integral part of the pipette. To place reagent or solvent into the reservoir, the pipette is held in a vertical position, and the rubber bulb is removed; a disposable-type transfer pipette is used, as shown in Fig. Ib, to fill liquid to below the inner capillary end. The rubber bulb is then inserted between the two circular projections and the pipette is ready for use as a dispensing pipette.
Fro. 2. The rack, with cover in position.
When the pipette is held in a horizontal position with the inner tip submerged (Fig. Ic), the liquid flows automatically into the tube and stops when the tube is full. For quantities larger than 25 or 50 III the filling can be speeded up by placing the index finger on the perforation at the apex of the rubber bulb and applying slight pressure. The pipette is then rotated 180 (Fig. Id ) and the aliquot is slowly emptied into a dish. Alternately, the pipette can be held in a vertical position (Fig. Ie) and the aliquot 0
A MULTIPURPOSE MICROPIPETTE
49
transferred into a test tube. A good pipette must fill readily when submerged in a liquid. Also, when it is filled with liquid from tip to tip and held vertically, it must not empty, regardless of the hydrostatic head, unless slight pressure is applied to the rubber bulb . This criterion can always be applied with aqueous solutions but not always with organic solvents. Transfer of organic solvents can then be accomplished by holding the pipette in a horizontal or slightly diagonal position when emptying. Successive aliquots can be drawn rather rapidly and in a reproducible quantity. In a test of reproducibility, each of 10 aliquots was weighed in pipettes ranging in size from 10 to 100 ul. The reproducibility of the 10-~1 pipette was ± 0.1 mg (the latter being the sensitivity of the balance). This would introduce a possible error of 1%. The reproducibility of the l00-~1 pipettes was ± 0.2 mg or a possible error of 0.2%. Generally, the multipurpose pipettes can be used conveniently in sizes from 1 to 500 ul. Quantities of 5 ul or less should be emptied into existing liquid droplets, otherwise the surface tension (of aqueous solutions only) is too great and it is difficult to separate the pipette from the discharged droplet. The pipetting error in this procedure is minimized because a single pipette is used for aliquoting successive measurements. Sampling. For the sampling of unknowns the outer tip of the multipurpose micropipette should be wiped dry with tissue. The tip is then inserted into the "unknown" while negative pressure is applied to the rubber bulb. The " unknown" liquid then flows into the capillary pipette and is allowed to overflow into the reservoir bulb. While the pipette tip is still in the "unknown" liquid , the index finger is removed from the rubber bulb perforation and the pipette is withdrawn . The " unknown" aliquot is then automatically contained between the two tips of the pipette. The "unknown" is then transferred slowly and uniformly to the proper reaction vessel. The pipette tip is wiped dry with tissue and is ready for duplicate sampling or for aliquoting a second " unknown" in a similar manner. The "washing" of the inside of capillary pipettes in the fashion described is most effective when sampling liquids of the same kind. Any residual liquid adhering to the inner walls of the capillary is carried upward and disposed of by the second unknown. Since the pipettes are calibrated to deliver, any remaining residue is not a part of the aliquot of the "unknown." When the glass reservoir bulb fills to the inner capillary tip and the pipette is held vertically, the liquid is then emptied. This is best done by inserting a suction tube made of polyethylene or some other flexible plastic material.
50
BENJAMIN W. GRUNBAUM
Other uses. For viscous solutions and for highly volatile solutions such as standard iodine or standard fatty acid made up in an organic solvent, the multipurpose micropipette can be used in two ways: (a) the standard solution is placed in the reservoir bulb and dispensed very slowly to prevent erratic drainage; (b) the standard solution is drawn into the pipette very carefully so that it just fills to the end of the inner tip. It is then expelled like a regular capillary pipette. Pure solvent is then introduced through the pipette from the reservoir bulb, which can be charged with solvent at any time. This rinsing solvent can be measured by passing it through the pipette any number of times. The second method is, of course, more accurate because the solute has been completely transferred. If it is desirable to have an accurate aliquot without additional dilution, a multipurpose pipette can be fitted with a special adapter and the aliquot can be centrifuged into a test tube. The multipurpose micropipette can also be treated with a nonwetting agent such as Desicote (Beckman Instruments Inc., Fullerton, California) and recalibrated to contain rather than to deliver. In this case aqueous solutions will drain dry. Storage oj reagents. For the sake of accuracy, the amount of reagent used in microchemical determinations is generally made up in much larger quantities than can be used. Reagents should not be kept in the micropipette for more than a day or two, depending on the type of reagent. If the reagent in the pipette is not to be used every day, the pipette and reservoir should be emptied, cleaned, and stored dry. However, if the micropipette is used routinely every day the perforated rubber bulb may be exchanged for a solid one at the end of a series of determinations. The reservoir bulb would then essentially be stoppered. Before dry storage the pipette can be cleaned in routine fashion like any other pipette. DISCUSSIO:-l
The extensive adoption of microchemical methodology by the routine clinical laboratories has created a need for some kind of universal pipette. In 1955 Grunbaum and Kirk described their all-glass, self-adjusting and self-dispensing micropipette, and in 1957 Sanz proposed a self-adjusting polyethylene pipette (3) which is essentially an overflow pipette attached to a plastic squeeze bottle. Compared to all-glass pipettes it has the advantage of being unbreakable and, of course, is naturally hydrophobic. The
A MULTIPURPOSE MICROPIPETTE
51
plastic pipette part was changed to a glass capillary in a modification of the all-plastic pipette by the Coleman Company (1). The Sanz overflow pipettes require a high degree of manual dexterity to handle properly, and the drainage in small capillaries is poor. Also, the reservoir (the plastic squeeze bottle) is much too large for safe keeping of reagents. Reagents are best kept in a properly stoppered container which is opened only occasionally to remove a sufficient quantity for a day's work or a series of determinations. For the transfer of reagent a disposable clean plastic or glass pipette is used. This prevents any contamination and concentration changes. In summary, a convenient multipurpose micropipette is described as an aid in most phases of microchemistry. ACKNOWLEDGMENT The author acknowledges with gratitude the help received from Microchemical Specialties Co., 1825 Eastshore Highway, Berkeley, California in the construction of the multipurpose pipettes and rack. REFERENCES 1. CAMPBELL, D. ]., COMFORT, D., AND BELL, R. D., An evaluation of two ultramicro systems for clinical chemistry. Am. J. Clin. Pathol. 38, 323-329 (1962). 2. GRUNBAUM, B. W., AND KIRK, P. L., Self-adjusting and dispensing micropipet. Anal. Chem, 27, 333 (l955). 3. SANZ, M. C., Ultramicro methods and standardization of equipment. Clin. Chem. 3,406·419 (1957).