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A linear-gradient mixing device for viscous solutions
ANALYTICAL
BIOCHEMISTRY
A Linear-Gradient
3,
236-243
(1962)
Mixing
Device
G. LEW From the McCollum-Pratt
Institute, Baltimore,
Received
for Viscous
Solutionsl”
CHOULES The Johns Maryland
September
Hopkins
University,
7, 1961
INTRODUCTION
In the course of work in this laboratory with density gradient electrophoresis (I), the need was encountered for a dependable gradient mixing device that was neither unduly complex nor time consuming in its operation. Two qualities were required in addition to those above: the gradients formed should be linear, to reduce lengthy calibration procedures; and viscous solutions containing up to 50% sucrose should be effectively mixed. The apparatus presented here produces reproducible, linear gradients with liquids as widely varying in viscosity as saturat,ed sucrose solution and water at room temperature. The device operates according to the principle developed by Bock and Ling (2) and is essentially a modification of the syringe gradient mixing device constructed by Bock and described by de Duve, Berthet, and Beaufay (3) in their review of gradient devices. Bock’s device produces linear gradients without the use of differential exponential drives, which are difficult to construct. Another modification of Bock’s device that has been used successfully in this laboratory is the simple mixer of Britten and Roberts (4), which has a gravity feed instead of syringes or pistons. It was found, however, that a gravity feed does not give entirely reproducible results with dilute solutions and fails completely with viscous, concentrated sucrose solutions. APPARATUS
Essentially, the apparatus (Figs. 1 and 2) consists of two cylindrical chambers containing solutions of different concentrations. Liquid is forced out of both chambers by two pistons, which are coupled to a common drive and move at a constant speed. Solution is forced from ’ This work u-as supported in part by Contract AT(30-l)-1822 Energy Commission and The Johns Hopkins University. ‘This is contribution No. 348 from the McCollum-Pratt 236
between Institute.
the
Atomic
LINEAR
FIG.
:lssemhly mounted.
1. Photograph are at the
of the starting
gradient position
GRADIER’T
237
MIXER
mixing apparatus. for a run. Note
The pistons and drive how the plastic block is
one chamber into the other, where it is mixed with a second solution. Mixing is accomplished with a screw turbine that circulates liquid through the mixing chamber and back through a special return channel as pictured. The spiral portion of the channel is an open groove in the mixing chamber wall so that fluid may flow through the chamber at all times during the forward passage of the piston. After mixing, liquid from the second chamber flows out into a receiving tube, the concentration varying linearly with volume of flow according to the equation of Bock and Ling (2) : c = C%- (cz - Cl) (1 - 2))%‘% where cI and cz are the initial solution concentrations and a, and a2 are the cross-sectional areas of t,he two chambers. Since a, and a2 are equal in our device, the exponent drops out and the resulting gradients are linear. The Z-cc dead space in the return channel reduces the slope of the gradient but does not alter the linearity. The initial step in construction of the apparatus (Fig. 2) was to make an aluminum mold 5 in. square and 1 in. deep. Two aluminum tubes 0.5 in. in diamet’er were prepared, open at one end and closed at the other. These were pushed through positioning holes in one side of the mold so that the closed ends projected 4 in. into the mold in oositions correspond-
238
CHOULES
FIG. 2. The gradient mixing apparatus. A and A’ are cylindrical sample chambers in a cast block of Ward’s Bio-Plastic. B is a circulation channel for screw pump C. The spiral portion of the channel is a groove in the wall of chamber A. D is a plunger valve. E, E’, and E” are inlet-outlet tubes for filling and emptying the sample chambers. The aluminum guard plate L seats two “0” rings, which prevent leakage around pistons F. G is a threaded drive shaft that pushes aluminum bar H as it turns in the threaded hole of the bar. I is a slip clutch. Stabilizing rods J slide freely through stationary mounting block K. An expanded view of the screw pump is shown in the insert. The Y&-in. shaft is machined down to I/s in. to minimize friction with the rubber “0” ring M. The larger “0” ring R seals the outer circumference of the a/s-in. aluminum washer 0. Another washer P and a mounting bracket Q complet,e the assembly.
LINEAR
GRADIENT
MIXER
239
ing to the sample chambers (A and A’) of the finished device. To form channel B, a single length of l/&in. aluminum wire was wound one turn around tube A to form a 3-in. spiral with one free end connecting to the closed end of tube A’, forming a bridge between tubes A and A’. The other free end was bent back parallel with tube A and cut off slightly below the end of tube A. The aluminum wire is wound tightly enough around tube A to hold the wire in place without further support. The mold was then filled with Ward’s Bio-Plastic,3 and treated according to directions sent with the plastic. After casting, the aluminum parts in the interior of the block were removed by dissolving in 6 N HCl in preparation for the subsequent machining operations. The first step in machining was to fasten an aluminum guard plate L over the top end of the plastic block by means of screws. This plate has two 0.5-in. holes to match the holes in the block. With the guard plate firmly in position, the 0.5-in. holes were reamed with a ?&-in. precision reamer. This reaming operation cut away approximately half of the inner diameter of the spiral portion of channel B so that this spiral portion is now a groove in the wall of tube A. The guard plate was then removed and a groove was machined in the wall of each of the plate’s reamed holes to seat t.wo rubber “0” rings.4 These “0” rings make leakproof seals around the pistons F without requiring the precision necessary in syringe construction. The guard plate was finally glued back onto the plastic block with epoxy resin to prevent leaks between the plate and the block. Two cylindrical rods F were machined from Plexiglas to fit the reamed holes with 0.002-in. clearance. The housing for the screw turbine C was formed by drilling a 1/4-in. hole in the side of the block, so as to connect the free end of channel B with the bottom of the piston chamber A. About one-half the length of the l/!-in. hole was expanded with a s/s-in, drill to accommodate the “0” rings and washers that seal the turbine housing against leakage. The screw turbine, made from stainless steel or aluminum rod, had 8 threads/in. and is driven at 1600 rpm? It is important that the threads be roughly square in cross section to minimize friction with the liquid being pumped. In the finished apparatus the screw turbine C! pumps liquid from channel B into chamber A. This liquid circulates through the ‘The Bio-Plastic was obtained from Ward’s Natural Science Establishment Inc., P. 0. Box 1712, Rochester, New York. 4“O” rings type MS9021-16, 0.614 in. inside diameter, made by Precision Rubber Products Corporation, General Office, 3110 Oakridge Drive, Dayton 17, Ohio. ‘We recommend a l/60-HP, 1725-rpm motor for this purpose, such as Bodine type NS I-13, No. 2224 E.
240
CHOULES
solution contained in chamber A and out again into channel B at the intersection with the end of piston F. If it is recalled that the spiral portion of channel B is a groove in the wall of tube A, it can be seen that the entire liquid content of tube A is circulated by the screw turbine no matter what position piston F is in. The machining of the block was completed by inst,alling valve D on the channel connecting the two sample chambers and by drilling 3&-in. holes for the three inlet-outlet tubes (E, E’ and I?). The shaft of D is threaded so that by turning it the Teflon insert at its tip is forced either in, to close channel B, or out to open the channel, depending on the direction the knob is turned. E, E’, and E”’ are 2-in. lengths of 18 gage stainless steel hypodermic needle tubing, to which suitable lengths of Tygon tubing are attached. The Tygon portion of the tubes make it possible to clamp off the inlet-outlet, tubes by means of pinch clamps. The inlet E to the arm of the spiral channel is used for flushing air bubbles out of the system during filling operations. This is important because the mixing pump is not self-priming. The assembly is mounted on an aluminum plate, the plastic block being supported by two aluminum bars at a suitable level to couple with the drive motors. The drive shaft G is threaded with 20 threads/in. so that the aluminum bar H, which is also threaded, is pushed forward or backward as the drive shaft turns. The drive shaft is coupled to the drive motor6 through the slip clutch assembly I.7 The stabilizing rods ,J are attached to the drive bar H and slide freely back and forth through the mounting block K. The slip clutch is employed as a safety device to prevent the drive mechanism from exerting too much force on the pistons when they come to the limit of their travel. The drive motor is reversible so that the drive mechanism can be returned to the starting position by running the motor backward at high speed. METHODS
AND
RESULTS
To prepare for filling the mixing device, 10 to 20 cc of water is injected with a syringe into the arm of the spiral channel B through the inlet E with both of the other inlets (E’ and E?“) open. This clears the system of bubbles. Next, the valve D is closed separating the two sample chambers, and the mixing chamber A is flushed with the dense solution (con“The Gerald ‘R33 ponents,
drive motor is a l/100 reduction gear motor, model 2T60-100, obtained K. Heller Co., Las Vegas, Nevada. adjustable slip clutch assembly obtained from Precision Instrument 477 Atlantic Avenue, East Rockaway, New York.
from Com-
LINEAR
GRADIEN’I’
MIXER
‘241
centrated sucrose) by injecting it into the spiral channel B through inlet E, as is the water used in clearing out the bubbles. The bubble formed by connecting the syringe to the inlet is pulled back into the syringe before injecting the sucrose solution. That procedure is used in all subsequent injections. After flushin,, 0 the bottom outlet E’ is closed and 10 cc of the concentrated sucrose solution is injected and the inlet E is clamped off. This forces the piston out to its starting position without risking bubble formation from minute leaks that might develop if the piston were put under tension while drawing in the sample. The other sample chamber A’ is flushed by injecting a dilute solution through the hole that serves as both an inlet and outlet for this chamber. Finally, 10 cc of the dilute solution is injected into A’ and the inlet-outlet B clamped off. The above filling procedure is performed with the apparatus horizontal, to aid in expelling bubbles. After filling, the apparatus is mounted vertically to make full use of convection in stirring the mixing chamber. When this is done the motor driving the screw turbine is started, the chamber-connecting valve D is opened, the mixing chamber outlet E’ is opened, and finally the drive motor is turned on t.o turn the drive shaft G at 5-10 rpm. Six trial runs were made with the completed apparatus making gradients between 5070 sucrose solution and distilled water. A small amount of acid fuchsin was added to the water so that a visible dye gradient was formed in addition to the sucrose gradient. This dye gradient then permitted the determination of the linearity and reproducibility of the gradients spectrophotometrically. The range 0 to 50% sucrose was chosen because it fits the needs of this laboratory; however, a preliminary run with saturated sucrose and water indicates that the device is equally good for broader ranges of sucrose concentration. All trial runs were performed at room temperature, but present operation of the device in a cold room is satisfactory. Samples were collected by changing collecting tubes once each minute to give approximately l-cc samples at a drive shaft speed of 5 rpm. The optical densities of the samples were then read at 543 rnp. Figure 3 shows a set of three runs made in one afternoon. The curves would normally fall one on top of the other, but for clarity they have been shifted 0.1 optical density unit apart. Small differences in slope are due to differences in the drive shaft speed between the runs, since the motor was not regulated. This altered the sample size slightly from run to run but not the shape of the actual gradient formed. For purposes of comparison, the data from all six curves were normal-
I
3
5
7
9
TUBE
II
13
15
17
I9
NUMBER
FICZ. 3. Gradients formed between a 50% sucrose solution and a dilute acid fuchsin solution. The points on the curves represent the optical densities at 543 my, the maximum for acid fuchsin, of samples taken from the gradient device at I-min intervals. The curves show three consecutive runs with the same solutions. For clarity the curves are displaced 0.1 optical density unit apart; otherwise they would fall on top of each other.
ized and a regression was calculated* to the general equation for a straight
for the combined line:
data, according
y=n+bx *We wish to thank Mr. Robert Burow of the computations center of The Johns Hopkins University for doing the machine computations for the statistical analysis presented here.
LINEAR
GRADIENT
MIXER
243
where y is the dependent variable, in this case optical density, and x is the independent variable or sample number. The standard deviation from the regression line was 3.2% of the mean value of y, and the 99% fiducial limits of the slope, b, were 1.035 k 0.015. Examination of the standard deviations of the individual values of y from the regression line showed no trends that might be interpreted as departure from linearity. It is concluded from the statistical analysis that the described apparatus fulfills the objectives set for it. SUMMARY
A gradient mixing device is described that forms linear concentration gradients and is suitable for mixing viscous liquids. Construction details are given and data are presented for gradients formed between 50% sucrose solution and water at room temperature. Statistical analysis shows that the slope of the regression line calculated from the data is constant within k1.576 for the 99% fiducial limits. REFERENCES G. L., AND BALLENTINE, R., Anal. Biochem. 2, 59 (1961). 2. BOCK, R. M., AND LING, N. S., Anal. Chem. 26, 1543 (1954). 3. DE DTJVE, C., BERTHET, J., AND BEAUFAY, H., Prog. in Biophys. and Biophys. 9, 325 (1959). 4. BRITTEN, R. J., AND ROBERTS, R. B., Science 131, 32 (1960). 1. CHOULES,
Chem.
BIOCHEMISTRY
A Linear-Gradient
3,
236-243
(1962)
Mixing
Device
G. LEW From the McCollum-Pratt
Institute, Baltimore,
Received
for Viscous
Solutionsl”
CHOULES The Johns Maryland
September
Hopkins
University,
7, 1961
INTRODUCTION
In the course of work in this laboratory with density gradient electrophoresis (I), the need was encountered for a dependable gradient mixing device that was neither unduly complex nor time consuming in its operation. Two qualities were required in addition to those above: the gradients formed should be linear, to reduce lengthy calibration procedures; and viscous solutions containing up to 50% sucrose should be effectively mixed. The apparatus presented here produces reproducible, linear gradients with liquids as widely varying in viscosity as saturat,ed sucrose solution and water at room temperature. The device operates according to the principle developed by Bock and Ling (2) and is essentially a modification of the syringe gradient mixing device constructed by Bock and described by de Duve, Berthet, and Beaufay (3) in their review of gradient devices. Bock’s device produces linear gradients without the use of differential exponential drives, which are difficult to construct. Another modification of Bock’s device that has been used successfully in this laboratory is the simple mixer of Britten and Roberts (4), which has a gravity feed instead of syringes or pistons. It was found, however, that a gravity feed does not give entirely reproducible results with dilute solutions and fails completely with viscous, concentrated sucrose solutions. APPARATUS
Essentially, the apparatus (Figs. 1 and 2) consists of two cylindrical chambers containing solutions of different concentrations. Liquid is forced out of both chambers by two pistons, which are coupled to a common drive and move at a constant speed. Solution is forced from ’ This work u-as supported in part by Contract AT(30-l)-1822 Energy Commission and The Johns Hopkins University. ‘This is contribution No. 348 from the McCollum-Pratt 236
between Institute.
the
Atomic
LINEAR
FIG.
:lssemhly mounted.
1. Photograph are at the
of the starting
gradient position
GRADIER’T
237
MIXER
mixing apparatus. for a run. Note
The pistons and drive how the plastic block is
one chamber into the other, where it is mixed with a second solution. Mixing is accomplished with a screw turbine that circulates liquid through the mixing chamber and back through a special return channel as pictured. The spiral portion of the channel is an open groove in the mixing chamber wall so that fluid may flow through the chamber at all times during the forward passage of the piston. After mixing, liquid from the second chamber flows out into a receiving tube, the concentration varying linearly with volume of flow according to the equation of Bock and Ling (2) : c = C%- (cz - Cl) (1 - 2))%‘% where cI and cz are the initial solution concentrations and a, and a2 are the cross-sectional areas of t,he two chambers. Since a, and a2 are equal in our device, the exponent drops out and the resulting gradients are linear. The Z-cc dead space in the return channel reduces the slope of the gradient but does not alter the linearity. The initial step in construction of the apparatus (Fig. 2) was to make an aluminum mold 5 in. square and 1 in. deep. Two aluminum tubes 0.5 in. in diamet’er were prepared, open at one end and closed at the other. These were pushed through positioning holes in one side of the mold so that the closed ends projected 4 in. into the mold in oositions correspond-
238
CHOULES
FIG. 2. The gradient mixing apparatus. A and A’ are cylindrical sample chambers in a cast block of Ward’s Bio-Plastic. B is a circulation channel for screw pump C. The spiral portion of the channel is a groove in the wall of chamber A. D is a plunger valve. E, E’, and E” are inlet-outlet tubes for filling and emptying the sample chambers. The aluminum guard plate L seats two “0” rings, which prevent leakage around pistons F. G is a threaded drive shaft that pushes aluminum bar H as it turns in the threaded hole of the bar. I is a slip clutch. Stabilizing rods J slide freely through stationary mounting block K. An expanded view of the screw pump is shown in the insert. The Y&-in. shaft is machined down to I/s in. to minimize friction with the rubber “0” ring M. The larger “0” ring R seals the outer circumference of the a/s-in. aluminum washer 0. Another washer P and a mounting bracket Q complet,e the assembly.
LINEAR
GRADIENT
MIXER
239
ing to the sample chambers (A and A’) of the finished device. To form channel B, a single length of l/&in. aluminum wire was wound one turn around tube A to form a 3-in. spiral with one free end connecting to the closed end of tube A’, forming a bridge between tubes A and A’. The other free end was bent back parallel with tube A and cut off slightly below the end of tube A. The aluminum wire is wound tightly enough around tube A to hold the wire in place without further support. The mold was then filled with Ward’s Bio-Plastic,3 and treated according to directions sent with the plastic. After casting, the aluminum parts in the interior of the block were removed by dissolving in 6 N HCl in preparation for the subsequent machining operations. The first step in machining was to fasten an aluminum guard plate L over the top end of the plastic block by means of screws. This plate has two 0.5-in. holes to match the holes in the block. With the guard plate firmly in position, the 0.5-in. holes were reamed with a ?&-in. precision reamer. This reaming operation cut away approximately half of the inner diameter of the spiral portion of channel B so that this spiral portion is now a groove in the wall of tube A. The guard plate was then removed and a groove was machined in the wall of each of the plate’s reamed holes to seat t.wo rubber “0” rings.4 These “0” rings make leakproof seals around the pistons F without requiring the precision necessary in syringe construction. The guard plate was finally glued back onto the plastic block with epoxy resin to prevent leaks between the plate and the block. Two cylindrical rods F were machined from Plexiglas to fit the reamed holes with 0.002-in. clearance. The housing for the screw turbine C was formed by drilling a 1/4-in. hole in the side of the block, so as to connect the free end of channel B with the bottom of the piston chamber A. About one-half the length of the l/!-in. hole was expanded with a s/s-in, drill to accommodate the “0” rings and washers that seal the turbine housing against leakage. The screw turbine, made from stainless steel or aluminum rod, had 8 threads/in. and is driven at 1600 rpm? It is important that the threads be roughly square in cross section to minimize friction with the liquid being pumped. In the finished apparatus the screw turbine C! pumps liquid from channel B into chamber A. This liquid circulates through the ‘The Bio-Plastic was obtained from Ward’s Natural Science Establishment Inc., P. 0. Box 1712, Rochester, New York. 4“O” rings type MS9021-16, 0.614 in. inside diameter, made by Precision Rubber Products Corporation, General Office, 3110 Oakridge Drive, Dayton 17, Ohio. ‘We recommend a l/60-HP, 1725-rpm motor for this purpose, such as Bodine type NS I-13, No. 2224 E.
240
CHOULES
solution contained in chamber A and out again into channel B at the intersection with the end of piston F. If it is recalled that the spiral portion of channel B is a groove in the wall of tube A, it can be seen that the entire liquid content of tube A is circulated by the screw turbine no matter what position piston F is in. The machining of the block was completed by inst,alling valve D on the channel connecting the two sample chambers and by drilling 3&-in. holes for the three inlet-outlet tubes (E, E’ and I?). The shaft of D is threaded so that by turning it the Teflon insert at its tip is forced either in, to close channel B, or out to open the channel, depending on the direction the knob is turned. E, E’, and E”’ are 2-in. lengths of 18 gage stainless steel hypodermic needle tubing, to which suitable lengths of Tygon tubing are attached. The Tygon portion of the tubes make it possible to clamp off the inlet-outlet, tubes by means of pinch clamps. The inlet E to the arm of the spiral channel is used for flushing air bubbles out of the system during filling operations. This is important because the mixing pump is not self-priming. The assembly is mounted on an aluminum plate, the plastic block being supported by two aluminum bars at a suitable level to couple with the drive motors. The drive shaft G is threaded with 20 threads/in. so that the aluminum bar H, which is also threaded, is pushed forward or backward as the drive shaft turns. The drive shaft is coupled to the drive motor6 through the slip clutch assembly I.7 The stabilizing rods ,J are attached to the drive bar H and slide freely back and forth through the mounting block K. The slip clutch is employed as a safety device to prevent the drive mechanism from exerting too much force on the pistons when they come to the limit of their travel. The drive motor is reversible so that the drive mechanism can be returned to the starting position by running the motor backward at high speed. METHODS
AND
RESULTS
To prepare for filling the mixing device, 10 to 20 cc of water is injected with a syringe into the arm of the spiral channel B through the inlet E with both of the other inlets (E’ and E?“) open. This clears the system of bubbles. Next, the valve D is closed separating the two sample chambers, and the mixing chamber A is flushed with the dense solution (con“The Gerald ‘R33 ponents,
drive motor is a l/100 reduction gear motor, model 2T60-100, obtained K. Heller Co., Las Vegas, Nevada. adjustable slip clutch assembly obtained from Precision Instrument 477 Atlantic Avenue, East Rockaway, New York.
from Com-
LINEAR
GRADIEN’I’
MIXER
‘241
centrated sucrose) by injecting it into the spiral channel B through inlet E, as is the water used in clearing out the bubbles. The bubble formed by connecting the syringe to the inlet is pulled back into the syringe before injecting the sucrose solution. That procedure is used in all subsequent injections. After flushin,, 0 the bottom outlet E’ is closed and 10 cc of the concentrated sucrose solution is injected and the inlet E is clamped off. This forces the piston out to its starting position without risking bubble formation from minute leaks that might develop if the piston were put under tension while drawing in the sample. The other sample chamber A’ is flushed by injecting a dilute solution through the hole that serves as both an inlet and outlet for this chamber. Finally, 10 cc of the dilute solution is injected into A’ and the inlet-outlet B clamped off. The above filling procedure is performed with the apparatus horizontal, to aid in expelling bubbles. After filling, the apparatus is mounted vertically to make full use of convection in stirring the mixing chamber. When this is done the motor driving the screw turbine is started, the chamber-connecting valve D is opened, the mixing chamber outlet E’ is opened, and finally the drive motor is turned on t.o turn the drive shaft G at 5-10 rpm. Six trial runs were made with the completed apparatus making gradients between 5070 sucrose solution and distilled water. A small amount of acid fuchsin was added to the water so that a visible dye gradient was formed in addition to the sucrose gradient. This dye gradient then permitted the determination of the linearity and reproducibility of the gradients spectrophotometrically. The range 0 to 50% sucrose was chosen because it fits the needs of this laboratory; however, a preliminary run with saturated sucrose and water indicates that the device is equally good for broader ranges of sucrose concentration. All trial runs were performed at room temperature, but present operation of the device in a cold room is satisfactory. Samples were collected by changing collecting tubes once each minute to give approximately l-cc samples at a drive shaft speed of 5 rpm. The optical densities of the samples were then read at 543 rnp. Figure 3 shows a set of three runs made in one afternoon. The curves would normally fall one on top of the other, but for clarity they have been shifted 0.1 optical density unit apart. Small differences in slope are due to differences in the drive shaft speed between the runs, since the motor was not regulated. This altered the sample size slightly from run to run but not the shape of the actual gradient formed. For purposes of comparison, the data from all six curves were normal-
I
3
5
7
9
TUBE
II
13
15
17
I9
NUMBER
FICZ. 3. Gradients formed between a 50% sucrose solution and a dilute acid fuchsin solution. The points on the curves represent the optical densities at 543 my, the maximum for acid fuchsin, of samples taken from the gradient device at I-min intervals. The curves show three consecutive runs with the same solutions. For clarity the curves are displaced 0.1 optical density unit apart; otherwise they would fall on top of each other.
ized and a regression was calculated* to the general equation for a straight
for the combined line:
data, according
y=n+bx *We wish to thank Mr. Robert Burow of the computations center of The Johns Hopkins University for doing the machine computations for the statistical analysis presented here.
LINEAR
GRADIENT
MIXER
243
where y is the dependent variable, in this case optical density, and x is the independent variable or sample number. The standard deviation from the regression line was 3.2% of the mean value of y, and the 99% fiducial limits of the slope, b, were 1.035 k 0.015. Examination of the standard deviations of the individual values of y from the regression line showed no trends that might be interpreted as departure from linearity. It is concluded from the statistical analysis that the described apparatus fulfills the objectives set for it. SUMMARY
A gradient mixing device is described that forms linear concentration gradients and is suitable for mixing viscous liquids. Construction details are given and data are presented for gradients formed between 50% sucrose solution and water at room temperature. Statistical analysis shows that the slope of the regression line calculated from the data is constant within k1.576 for the 99% fiducial limits. REFERENCES G. L., AND BALLENTINE, R., Anal. Biochem. 2, 59 (1961). 2. BOCK, R. M., AND LING, N. S., Anal. Chem. 26, 1543 (1954). 3. DE DTJVE, C., BERTHET, J., AND BEAUFAY, H., Prog. in Biophys. and Biophys. 9, 325 (1959). 4. BRITTEN, R. J., AND ROBERTS, R. B., Science 131, 32 (1960). 1. CHOULES,
Chem.