U.S. patent application number 17/826924 was filed with the patent office on 2022-09-08 for method for preparation of liquid mixtures.
The applicant listed for this patent is Cytiva Sweden AB. Invention is credited to Enrique Napoleon Carredano, Elenor Strandberg.
Application Number | 20220283062 17/826924 |
Document ID | / |
Family ID | 1000006362229 |
Filed Date | 2022-09-08 |
United States Patent
Application |
20220283062 |
Kind Code |
A1 |
Carredano; Enrique Napoleon ;
et al. |
September 8, 2022 |
Method for Preparation of Liquid Mixtures
Abstract
The present invention relates to the preparation of liquid
mixtures, and more particularly to the preparation of a liquid
mixture, such as a buffer, wherein the conductivity of the liquid
mixture is measured and the pH indirectly determined if the buffer
concentration is known. Another object of the present invention is
to provide a method of preparing a liquid mixture with a
predetermined pH value by using conductivity as feedback control
parameter.
Inventors: |
Carredano; Enrique Napoleon;
(Uppsala, SE) ; Strandberg; Elenor; (Sigtuna,
SE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cytiva Sweden AB |
Uppsala |
|
SE |
|
|
Family ID: |
1000006362229 |
Appl. No.: |
17/826924 |
Filed: |
May 27, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16804440 |
Feb 28, 2020 |
11371919 |
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17826924 |
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15022501 |
Mar 16, 2016 |
10605710 |
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PCT/SE2014/051109 |
Sep 26, 2014 |
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16804440 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 27/06 20130101;
G01N 1/38 20130101; G01N 21/3577 20130101; G05D 21/02 20130101;
G01N 21/41 20130101; B01J 47/15 20170101; G01N 21/33 20130101 |
International
Class: |
G01N 1/38 20060101
G01N001/38; G05D 21/02 20060101 G05D021/02; B01J 47/15 20060101
B01J047/15; G01N 27/06 20060101 G01N027/06; G01N 21/33 20060101
G01N021/33; G01N 21/3577 20060101 G01N021/3577; G01N 21/41 20060101
G01N021/41 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 30, 2013 |
SE |
1351142-3 |
Claims
1. A buffer system comprising, a. a conductivity sensor for
measuring conductivity of a buffer diluted with a salt solution to
a specific buffer concentration; b. a conductivity sensor for
measuring conductivity of the buffer before dilution with the salt
solution; c. a concentration sensor for measuring or calculating
concentration of the buffer before and after the salt dilution; and
d. means for adding controlled amounts of buffer components to said
buffer based on the measured conductivity and the measured or
calculated concentration of the buffer to maintain buffer
concentration constant until a specific conductivity value is
obtained, wherein said specific conductivity value is correlated to
a predetermined pH value and the buffer concentration by a surface
model trained using a training data set with known concentrations,
specific conductivities and pHs obtained from a training set of
buffer systems and based at least in part on measuring the pH for
the solutions belonging to the training data set and using a
numerical regression method to obtain the surface model equation as
a regression model from the training data.
2. The system of claim 1, wherein the buffer is an acetate buffer,
a citrate buffer, a phosphate buffer, or a Tris buffer.
3. The system of claim 1, wherein the surface model equation is of
the form: pH=A+B*concentration of the buffer+C*concentration of the
buffer{circumflex over ( )}2+D* specific conductivity; and wherein
A, B, C, and D are buffer specific constants determined through the
regression modelling of the training data.
4. The system of claim 1, wherein the surface model equation is of
the form; pH=A+B*concentration of the buffer+C*concentration of the
buffer{circumflex over ( )}2+D* specific conductivity+E* specific
conductivity{circumflex over ( )}2; and wherein A, B, C, D, and E
are buffer specific constants determined through the regression
modelling of the training data.
5. The system of claim 1, wherein the concentration of the buffer
is measured by any of Index of Refraction (IoR), Infrared Red (IR),
and Ultraviolet (UV) absorbance at several wavelengths.
6. The system of claim 1, wherein the concentration of the buffer
is calculated from known flows from stock solutions of known
concentrations and flows of other components like water and
non-buffering salts or additives to the point of mixture of the
buffer.
7. The system of claim 1, wherein the concentration of the buffer
is calculated from the measured conductivities, IR values, or IoR
values and known flows from stock solutions and the flows of other
components like water and non-buffering salts or additives to the
point of mixture of the buffer.
8. The system of claim 1, which is computer implemented.
9. A method, comprising: controlling a buffer formulation using the
buffer system of claim 1.
10. A method, comprising: controlling an in-line dilution system
using the buffer system of claim 1.
11. A method, comprising: screening experiments using the buffer
system of claim 1, wherein pH and the concentration of a buffer is
used as a design of experiment (DoE) parameter.
12. A device comprising a computer and memory storing a computer
program product comprising instructions that, when executed by the
computer, causes the computer to perform a method of preparing a
buffer having a predetermined pH value, the method comprising, a.
measuring conductivity of a buffer using a conductivity sensor; b.
measuring or calculating concentration of the buffer; and c. adding
controlled amounts of buffer components to said buffer based on the
measured conductivity and the measured or calculated concentration
of the buffer to maintain buffer concentration constant until a
specific conductivity value is obtained, wherein said specific
conductivity value is correlated to a predetermined pH value and
the buffer concentration by a surface model, comprising using a
training data set with known concentrations, specific
conductivities and pHs obtained from a training set of buffer
systems; and measuring the pH for the solutions belonging to the
training data set and using a numerical regression method to obtain
the surface model equation as a regression model from the training
data.
13. The device of claim 12, further comprising a conductivity
sensor and a concentration sensor and the computer for calculating
pH from measured conductivity.
14. The device of claim 12, further comprising a conductivity
sensor and allowing input of buffer concentration and the computer
for calculating pH from measured conductivity.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 16/804,440 filed on Feb. 28, 2020, which claims the priority
benefit of U.S. application Ser. No. 15/022,501 filed on Mar. 16,
2016 which claims the priority benefit of PCT/SE2014/051109 filed
on Sep. 26, 2014, which claims priority benefit of Swedish Patent
Application No. 1351142-3 filed on Sep. 30, 2013, the entire
contents of which are hereby incorporated by reference herein.
FIELD OF THE INVENTION
[0002] The present invention relates to a method for the
preparation of liquid mixtures, and more particularly to the
preparation of a liquid mixture, such as a buffer, wherein the
conductivity of the liquid mixture is measured and the pH
indirectly determined.
BACKGROUND OF THE INVENTION
[0003] A buffer solution is typically an aqueous solution
consisting of a mixture of a weak acid and its conjugate base, or a
weak base and its conjugate acid, and has the property that the pH
of the solution changes very little when a small amount of strong
acid or base is added to it. Buffer solutions are therefore used as
a means of keeping pH at a nearly constant value in a wide variety
of chemical applications, including e.g. chromatography,
filtration, etc. In general, a buffer solution may be made up of
more than one weak acid and its conjugate base. For instance, a
wider buffer region may be created by mixing two buffering agents
with overlapping individual buffer regions.
[0004] A buffer solution having a desired pH, and optionally, with
a desired ionic strength, may be prepared by calculating the
necessary amounts of the ingredients of the buffer and mixing them.
While it is often necessary to solve several different equations
for calculating the pH of a relatively simple mixture of a weak
acid (or base) and a weak base (or acid) depending on their
relative concentrations, there is commercial software available for
performing such calculations.
[0005] Another possibility is to use feedback control as in
WO/2011/162666 where the accuracy of the pH measurement limits the
accuracy of the pH of the buffer. However calibration of pH meters
is both time consuming and often troublesome. The pH meters may not
be very accurate, have slow responses and may be easily
contaminated by salts for example. The pH measurement is also
dependent on temperature, with different buffers having a variety
of temperature constants. Usually the reading of the pH meters
cannot be corrected to a standard temperature.
[0006] Thus, there is still a need within the art to improve
pH-measurements and to provide reliable instruments for this
purpose.
SUMMARY OF THE INVENTION
[0007] The present invention provides a reliable method and means
for measurement of pH which avoids the drawbacks of prior art.
[0008] The present invention relates to a method to indirectly
determine the pH of a buffer solution if its conductivity and
concentration are known by using a surface model equation of the
form
pH=f(conductivity,concentration)
or
pH=f(conductivity, buffer concentration, salt concentration)
[0009] where the surface model equation has been previously
obtained by fitting such equation to previously measured pH values
for a training data set. The training data set comprises values for
the measured or controlled concentration and the measured
conductivity of solutions containing the buffer at a plurality of
different concentrations and conductivities, and optionally
different temperatures, and the surface model equation is obtained
by fitting the resulting data to the formula described.
[0010] In a first aspect the invention relates to a method of
preparing a liquid mixture with a predetermined pH value without
using a pH meter by adding and reducing controlled amounts of
buffer components, preferably in liquid form, to said liquid
mixture; and stop adding and reducing buffer components when a
specific conductivity value is obtained, wherein the reduction and
addition of buffer components is done in such a way as to maintain
the buffer concentration constant and wherein said specific
conductivity value corresponds to the predetermined pH value of
said liquid, and wherein said specific conductivity value is
correlated to said predetermined pH value and the buffer
concentration by a mathematical model, such as a surface model
equation performed on a specific buffer system.
[0011] The invention also relates to a method to indirectly
determine the pH of a buffer solution by measuring its conductivity
and measuring or controlling the buffer concentration, and wherein
said specific conductivity value is correlated to said
predetermined pH value by a mathematical model.
[0012] When using a surface model equation it may be of the
form
pH=A+B*concentration+C*concentration{circumflex over (
)}2+D*conductivity
wherein A, B and C and D are buffer system specific constants.
[0013] Alternatively the surface model equation may be of the
form
pH=A+B*concentration+C*concentration{circumflex over (
)}2+D*conductivity+E* conductivity{circumflex over ( )}2
wherein A, B and C and D and E are buffer system specific
constants
[0014] In an alternative embodiment the surface function giving the
predicted pH as function of the conductivity and concentrations is
obtained through regression modelling of training set of mixtures
with known concentration, conductivity and pH.
[0015] The concentration of the buffer may be measured by any of
the following IoR, IR, and UV absorbance at several
wavelengths.
[0016] An alternative way of measuring or controlling the buffer
concentration is to prepare stock solutions of acid and base
respectively to a known concentration for instance by diluting
weighted amounts of buffer salts to measured volumes and then to
control or measure the flow from the stock solutions and of water
to the common point of mixture of the buffer. Yet another way to
control or measure the buffer concentration is to measure the
conductivity or another property like IR light absorbance or other
absorbance or the IoR signal on the stock solutions, then use those
signals to back calculate the concentration of the stock solutions
using beforehand determined conductivity (or IR or IoR) versus
concentration curves. Using the information obtained in this way on
the concentration of the stock solutions and the information on the
measured or controlled flows to the mixing point it is then
possible to calculate the concentration of the mixed buffer.
[0017] With the knowledge of the buffer concentration and the
conductivity of the buffer it is possible according to the present
invention to determine the pH using a surface model equation of the
pH as function of the conductivity and the concentration of the
buffer solution. For every type of buffer solution such a surface
model equation can be pre-determined by (i) using a training data
set belonging to the relevant area of concentration and
conductivity considered. (ii) Measuring the pH for the solutions
belonging to the training data set and (iii) using a numerical
regression method like for instance multi-linear regression (MLR)
or partial least squares (PLS) method to obtain the surface model
equation as a regression model from the data.
[0018] Preferably the method according to the invention is
computer-implemented.
[0019] In a second aspect, the invention relates to use of the
above described method for controlling a buffer formulation system
or an in-line dilution system. The method may also be used in
screening experiments wherein pH and concentration is used as a
design of experiment (DoE) parameter.
[0020] In a third aspect, the invention relates to a device
comprising a computer program product comprising instructions for
causing a computer to perform the method as described above.
[0021] The device may comprise a conductivity sensor and
concentration sensor and means for calculating pH from measured
conductivity using the above described method.
[0022] The device may also allow input of buffer concentration and
include means for calculating pH from measured conductivity using
the steps of the above described method.
[0023] In the following, the invention will be described in more
detail, by way of example only, reference being made to the
accompanying drawing.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 shows the results from scaled and centred
coefficients for pH according to the experimental details in
Example 1 below.
[0025] FIG. 2 shows a graph of the observed versus predicted values
of pH according to the experimental details in Example 1 below.
[0026] FIG. 3 shows a graph showing the resulting measured pH
values from buffers prepared in two different ways. By recipes and
by "pH by conductivity-feedback". Both values are compared to
target value, see Example 2 below.
[0027] In order to facilitate an understanding of the disclosed
invention, a number of terms will be defined below.
DEFINITIONS
[0028] Buffer
[0029] As used herein, a buffer solution is an aqueous solution
typically consisting of a mixture of a weak acid and its conjugate
base or a weak base and its conjugate acid. It has the property
that the pH of the solution changes very little when a small amount
of strong acid or base is added to it. Buffer solutions are used as
a means of keeping pH at a nearly constant value in a wide variety
of chemical applications
[0030] Conductivity (Electrolytic)
[0031] The conductivity (or specific conductance) of an electrolyte
solution is a measure of its ability to conduct electricity. The SI
unit of conductivity is "siemens" per meter (S/m).
[0032] Ionic Strength
[0033] The ionic strength of a solution is a function of the
concentration of all ions in the solution (half the sum of
concentration multiplied by the square of ionic charge for all
ions). The ionic strength is typically given in the unit
mol/dm.sup.3.
[0034] Surface Model Equation
[0035] A surface model equation as used here is a function of the
form f(x,y) where x and y are two independent variables which are
properties of a buffer solution. More specifically in this context
x (or y) refers to the conductivity of the buffer solution and y
(or x) refers to the buffer concentration.
DETAILED DESCRIPTION OF THE INVENTION
[0036] As mentioned above, the present invention relates to the
preparation of liquid mixtures, and more particularly to the
preparation of a liquid mixture, such as a buffer, wherein the
conductivity of the liquid mixture is measured and the pH
indirectly determined if the buffer concentration is known.
Furthermore the invention relates to the provision of a method of
preparing a liquid mixture with a predetermined pH value by using
conductivity as feedback control parameter.
Below the methods used for predicting pH, determination of surface
model equation and calculation of pH are described,
respectively.
Predicting the pH
[0037] One object of the present invention is thus is indirectly
determine the pH of a buffer solution if its conductivity and
concentration are known. As compared to pH measurements,
conductivity measurements are fast and reliable and can be
temperature corrected to for instance room temperature 25 degrees
C. as standard. Furthermore, usually calibration on a yearly basis
is enough for conductivity meters. Thus it should be advantageous
to measure the conductivity instead of the pH.
[0038] The present invention provides also a method of preparing a
buffer with a given buffer concentration to a predetermined pH
value without using a pH meter. There are other methods where this
is possible to do for instance a buffer solution with a desired pH,
and optionally, with a desired ionic strength, may be accurately
prepared by calculating the necessary amounts of buffer ingredients
and mixing them as previously described in EP 2269055 B 1. However
in contrast to those methods, the method described in the present
invention does not use recipes and instead uses measured
conductivity as feedback control parameter. The use of feedback
control to obtain a buffer with desired properties is not new. For
instance one such method is described in WO/2011/162666.
Nevertheless, there is a large difference and advantage as compared
to that method where measured pH is necessary as control parameter.
In the present case, the correct buffer with correct pH and buffer
concentration is obtained by combining conductivity measurement
with control of buffer concentration by some means.
[0039] The measuring or controlling of the buffer concentration may
be achieved in different ways. For instance the measuring or
controlling of the buffer concentration may be achieved by using
Index of Refraction (IoR) techniques or Infra Red (IR)
spectroscopy, UV absorbance or other methods.
[0040] An alternative way of measuring or controlling the buffer
concentration is to prepare stock solutions of acid and base
respectively to a known concentration for instance by diluting
weighted amounts of buffer salts to measured volumes and then to
control or measure the flow from the stock solutions and of water
to the common point of mixture of the buffer. Yet another way to
control or measure the buffer concentration is to measure the
conductivity or another property like IR light absorbance or other
absorbance or the IoR signal on the stock solutions, then use those
signals to back calculate the concentration of the stock solutions
using beforehand determined conductivity (or IR or IoR) versus
concentration curves. Using the information obtained in this way on
the concentration of the stock solutions and the information on the
measured or controlled flows to the mixing point it is then
possible to calculate and control the concentration of the mixed
buffer.
[0041] Thus as it is possible according to this invention to
calculate the pH by monitoring the conductivity of the buffer as
long as the buffer concentration is calculated or controlled it is
also possible to determine the amount of ingredients, acid and base
with conductivity feedback control.
[0042] Whether the method described in here is used for predicting
the pH of a buffer or for using conductivity feedback control to
produce a buffer with correct pH and buffer concentration a central
feature of this invention is the determination of a surface model
equation for the pH as function of the conductivity and the buffer
concentration in other words a function of the form f(x,y) where x
(or y) refers to the conductivity of the buffer solution and y (or
x) refers to the buffer concentration. It will be now described how
this equation can be obtained.
Determination of Surface Model Equation
[0043] The surface model equation of the form
pH=f(conductivity, concentration)
or
pH=f(conductivity, buffer concentration, salt concentration)
for the buffer can be produced by fitting obtained pH data from a
finite number of points in the (conductivity, concentration) space
to the equation above. Design of Experiments (DoE) can be used with
advantage but is not necessary. This step is not difficult to do
for anyone skilled in the art of fitting an equation to the data
measured for a training set. The obtained equation is valid within
certain accuracy in the area covered by the discrete number of
points in the training set.
Calculation of pH
[0044] Finally, the pH is calculated as predicted pH of the desired
buffer solution by using the above described surface model equation
and measured values of conductivity together with corresponding
measured or controlled values of the buffer concentration.
[0045] The method of the invention may be implemented by software
run on an electrical data processing device, such as a computer.
Such software may be provided to the computer on any suitable
computer-readable medium, including a record medium, a read-only
memory, or an electrical or optical signal which may be conveyed
via electrical or optical cable or by radio or other means.
[0046] Predicting the pH as described above has not been described
before somewhere else. In previous occasions, the prediction of the
pH or the formulations of buffers at a given pH have been
described. For instance in US20110039712 A1 and EP 2269055 B1, but
those methods or similar methods do not use conductivity and
require recipes. On the other hand, another method describes the
prediction of conductivity from it's constituents PCT SE2011/051513
but that method does not describe how the pH could be retrieved
from conductivity measurement without needing to calculate for each
ionic species of said plurality of species the molar conductivity
by the formula:
=.sub.0-K.times.Sqrt(c)
wherein is the molar conductivity, .sub.0 is the molar conductivity
at infinite dilution, c is the concentration of the ionic species,
and K is the Kohlrausch coefficient, and wherein K and .sub.0are
obtained from a data set comprising predetermined values for K and
.sub.0 for each ionic species.
[0047] Predicting the pH as described here requires only the
calibration of a surface equation of the form
pH=f(conductivity, buffer concentration)
or
pH=f(conductivity, buffer concentration, salt concentration)
with a valid range at a given accuracy and simply the measurement
of the conductivity and the control or measurement of the buffer
concentration and if necessary the salt concentration. Predicting
the pH as described here does not require to calculate for each
ionic species the molar conductivity, or .sub.0 is the molar
conductivity at infinite dilution or Kohlrausch coefficients.
[0048] Predicting the pH as described here may be used for several
purposes. Exemplary applications include the use for controlling a
buffer formulation system or an in-line dilution system. Such
prediction of pH may also be used in screening experiments wherein
pH and concentration (but not conductivity) are used as design of
experiment (DoE) parameters. Still another application is for
determining the exact concentration of a stock solution or a buffer
by measuring its pH and conductivity.
[0049] The method described above may be used to obtain the
specific surface model for a specific buffer system in conjunction
with a specific concentration of non-buffer salt (for instance
NaCl). One can also generalize the surface equation to include
concentration of salt, i.e. f(x,y,z) where z is concentration of
salt. Alternatively keep the concentration of NaCl constant and
obtain one model for each concentration of NaCl.
[0050] The invention will now be described in more detail, by way
of example only, by the following non-limiting Examples.
EXAMPLES
Example 1: Surface Model Equation for the Ph as Function of the
Concentration and the Conductivity
[0051] In this experiment, the conductivity and the buffer
concentration has been used to indirectly determine the pH. Modde
version 9.0 was used to obtain the surface model equation as a
regression model including the coefficients of the model.
[0052] Three different concentration levels and six different
conductivity values for each concentration of citrate buffer were
included in the training set (table). The buffers were mixed using
a lab scale Akta explorer system (GE Healthcare Biosciences AB)
with five pumps with Unicorn 5 control software (GE Healthcare
Biosciences AB) where the concentration was controlled by setting
the flow manually from stock solutions of acid (0.5 M citric acid)
and base (0.5 M Na3 citrate) and from water. The conductivity was
measured off-line using linear temperature correction with constant
2.1 and the pH was measured off-line using a calibrated HANNA pH
meter.
TABLE-US-00001 TABLE 1 No Concentration (mM) Conductivity (mS/cm)
pH (measured off-line) 1 20 1.511 3.89 2 20 1.997 4.34 3 20 2.51
4.73 4 20 3.01 5.15 5 20 3.49 5.58 6 20 4.01 6.06 7 50 2.51 3.33 8
50 2.99 3.6 9 50 3.47 3.83 10 50 3.97 4.04 11 50 4.47 4.23 12 50
4.95 4.4 13 80 2.48 2.81 14 80 2.97 3.03 15 80 3.49 3.22 16 80 3.97
3.39 17 80 4.45 3.57 18 80 4.99 3.72
Partial least square regression analysis was used to obtain a
surface model equation as a relationship among concentration,
conductivity and pH (FIG. 1). A test set with seven citrate buffers
was done to test the prediction capability of the model (Table
2).
TABLE-US-00002 TABLE 2 Test set for prediction capability test of
the model. pH Concentration Conductivity (measured Lower Upper (mM)
(mS/cm) off-line) Predicted pH value value 30 3.95 4.92 5.01 4.82
5.20 30 2.45 4.08 4.20 4.01 4.38 40 2.48 3.63 3.73 3.50 3.96 40
4.45 4.59 4.80 4.58 5.02 70 2.96 3.18 3.00 2.80 3.19 70 3.97 3.57
3.54 3.39 3.70 70 4.95 3.91 4.08 3.86 4.29
The measured pH values for all of the runs were in the predicted
interval so that the model has been proved to be capable of
allowing indirect determination of the pH (FIG. 2).
Example 2: Making a Buffer of a Given pH Using Conductivity
Feedback Control
[0053] It is an object of the present invention to provide a method
of preparing a liquid mixture with a predetermined pH value by
using conductivity as feedback control parameter. No recipes and no
in-line pH meters are necessary in this case. A total of 21
different buffers were used to compare buffer preparation in this
way (Table 3) and compared to a second case where the flows of the
incoming solutions were controlled and the buffers were prepared
using recipes. The target conductivity values for the first method
which is the method described in the present invention were the
conductivity values obtained by measuring the temperature corrected
conductivity from the result files of the second method. According
to the results, preparation of buffers through conductivity
feedback showed no difference in pH compared to buffers prepared by
recipes and pH meters (FIG. 3).
TABLE-US-00003 TABLE 3 Buffers prepared in two different ways, by
recipes and by conductivity feedback. Stock Stock Stock 1 Buffer
system solution 1 solution 2 solution 3 1 20 mM acetate pH 5.0 0.5M
1.0M n/a 2 20 mM acetate pH 5.0 HAc NAc*3H20 3M NaCl 1M NaCl 3 100
mM acetate pH 5.0 n/a 4 100 mM acetate pH 5.0 3M NaCl 1M NaCl 5 100
mM acetate pH 5.0 3M NaCl 2M NaCl 6 20 mM citrate pH 3.5 0.5M 0.5M
n/a 7 20 mM citrate pH 3.5 Citric acid * Na3 Citrate 3M NaCl 1M
NaCl 1H20 *2H2O 8 100 mM citrate pH 3.5 n/a 9 100 mM citrate pH 3.5
3M NaCl 1M NaCl 10 25 mM citrate pH 5.0 1M NaCl 200 mM NaCl 11 25
mM citrate pH 5.6 1M NaCl 225 mM NaCl 12 20 mM phosphate pH 7 0.4M
0.4M n/a 13 20 mM phosphate pH 7 NaH2PO4 * Na2HPO4* 3M NaCl 1M NaCl
1H2O 2H2O n/a 14 50 mM phosphate pH 7 15 50 mM phosphate pH 7 3M
NaCl 1M NaCl 16 50 mM phosphate pH 7 3M NaCl 2M NaCl 17 30 mM
phosphate pH 6.5 n/a 18 30 mM phosphate pH 6.5 1M NaCl 50 mM NaCl
19 30 mM phosphate pH 6.5 3M NaCl 1M NaCl 20 20 mM Tris pH 8 0.3M
0.3M n/a 21 50 mM Tris pH 8 Tris base Tris HCl 3M NaCl 1M NaCl
* * * * *