Separation Methods in Organic Chemistry and Biochemistry

238 Pages·1969·3.939 MB·English

by  Frank J. Wolf (Auth.)

Separation Methods in
Organic Chemistry and
Biochemistry
FRANK J. WOLF
Merck Sharp & Dohme Research Laboratories
Rahway, New Jersey
ACADEMIC PRESS New York and London 1969
COPYRIGHT © 1969, BY ACADEMIC PRESS, INC.
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Preface
The isolation of pure substance is frequently the major laboratory effort
required for the solution of a chemical or biochemical problem. This may
require considerable time and effort. At any point in the solution of an
isolation problem more than one method or combination of methods can
usually be employed. The choice of the most rapid and convenient procedure
requires careful evaluation of many factors which include the available
equipment, the time and assay facilities required for a particular step, use
of previous knowledge or experience, and the scale of operation.
A major objective of this book is to provide perspectives for the commonly
used methods and indications for their use. The determination of molecular
properties useful in separation based on micro test methods, paper chromato-
graphy, thin-layer chromatography, and electrophoresis is described. Illustra-
tive examples of each method are included.
Separations are classed generally as group or fractionation methods
depending on the selectivity or fractionation needed. The theoretical principles
of group-separation procedures, liquid-liquid partition, ion-exchange
selectivity, gel permeation, and adsorption are included. Methods of in-
fluencing the selectivity coefficients are discussed.
The basic theory of fractionation methods is developed and the principles
of application are discussed in terms useful to practicing chemists whose major
interest is the use of separation methods to accomplish a given objective and
not separation per se.
It is hoped that the book will serve as a useful guide to the solution of many
practical problems encountered in the laboratory practice of organic chemistry
and biochemistry.
I am indebted to many co-workers in the Merck Sharp & Dohme Research
Laboratories who have offered many helpful suggestions during the prepara-
tion of the manuscript, especially L. Chaiet, R. G. Denkewalter, T. E. Jacob,
E. A. Kaczka, A. J. Kempf, T. W. Miller, H. Shafer, and R. Weston. The
V
VI Preface
cooperation of M. Tishler, L. H. Sarett, and H. B. Woodruff, through whose
efforts many facilities of the Merck Sharp & Dohme Research Laboratories
were made available, is gratefully acknowledged. I also wish to recognize the
invaluable assistance of Mrs. M. M. Tatro for secretarial help in preparing the
manuscript.
Rahway, New Jersey
May, 1969 FRANK J. WOLF
Introduction
I. TYPES OF SEPARATION CONSIDERED
Separation processes are based on mechanical and chemical methods. No
separation can be achieved without some mechanical means. For the purpose
of this book only separations in which the substances being separated are
present in the orginal mixture in forms which cannot be physically separated
are considered. Methods based on the degradation or decomposition of
unwanted substances are likewise not included.
Consequently the separation methods of most interest are those which can
be applied to solutions of two or more substances. During the separation
process at least two phases are present and the separation is based on the
unequal distribution of the substances between these phases. In solvent
extraction and partition chromatography methods the phases are liquid. In
other types of chromatography one of the phases is a solid.
II. DISTRIBUTION COEFFICIENT
A. Group Separation
The relative affinity of a substance for two phases is the distribution coeffi-
cient. This is frequently expressed as the ratio of concentration in the two
phases. The ratio of the distribution coefficients for two substances is the
separation factor, /?, for the substances in the system under consideration.
This is customarily expressed as a value greater than 1 rather than a fraction.
If the ß value is very large the separation is a "group" or type separation. If ß
is small a fractionation method is required. Thus, a group separation of acetic
and propionic acid from glucose can be carried out by extraction of an
aqueous solution with an immiscible solvent such as ethyl acetate. The further
separation of acetic and propionic acids requires a fractionation method.
In the above example the group separation is that of a "polar" from a
1
2 Introduction
"nonpolar" substance. Separations may also be used to group materials
based on their ionic properties. To a lesser extent molecular size can be used
in grouping materials. Most separations of complex mixtures are carried out
by using a group method at an early stage. Since the group separation step
results in enrichment of related substances, one or more fractionation steps
for the final separation of pure substances are usually required.
B. Fractionation Separations—Reasons for Using Them
Any separation method which produces a product of high purity in good
yield requires either countercurrent contact of two phases or very high
separation factors.
Suppose that two substances are present in equal amounts in a solution. A
batch separation step is applied by introducing a second phase, either liquid
or solid, which removes 91 % of A and 50% of B. Since the distribution coeffi-
cients of A and B are 10 and 1, respectively, the separation factor, /?, is 10.
After the phases are separated one phase will contain 50% of B in 85%
purity and the second phase will contain 91 % of A in 64% purity. If the
distribution coefficients do not change with the concentrations of A and B, it
can be seen that there is no method of utilizing successive contacting of
phases by which both good yields and purity can be obtained. This is true
even though the starting material is 50% pure and a system having a ß of 10
is used. Thus, for batch operation, even in successive stages, a much larger
separation factor is needed if both high yield and purity are to be obtained.
If the second phase used in the above example is an immiscible liquid, the
use of another solvent is not likely to markedly change the separation factor
unless one of the compounds is affected by one of the solvents. Thus, methyl
stéarate and methyl oleate were observed to have a ß of 1.6 (K= 17.6 and
10.8, respectively) in a system of 95% methanol-water-isooctane and a ß of
2.6 (K =19 and 7.3, respectively) in hexane-acetonitrile. The change in
separation factor with the two systems is not of any great usefulness for group
separation but may be significant for fractionation processes. Since the two
solvent systems are rather different, the change in separation factor is about as
large as can be obtained in the absence of some additional factor. It has
observed that certain heavy metal salts, especially mercuric and silver salts,
form association complexes with olefinic bonds. If mercuric acetate is added
to the first solvent pair, the resulting complex with methyl oleate has increased
polarity and the distribution coefficient is reduced from 10.8 to 0.28. Since
methyl stéarate is unaffected, a ß of 63, suitable for group separation, is
obtained. Other similar effects may be due to strong hydrogen bonding of one
component in one system and not in another. A change in pH which influences
the ionic state of one of the compounds also markedly affects the separation
III. Use of Micro Methods 3
factor. If the distribution coefficient is not independent of concentration the
separation factor of two compounds differs depending on the concentration
of each and/or the amount of second phase employed. No predictions can be
made for such systems in the absence of considerable experimental data.
C. Methods of Fractionation
If the separation requires substantial enrichment it is usually necessary that
at least one step employ countercurrent contacting of two phases. Most
laboratory countercurrent contacting methods are carried out by maintaining
one phase stationary and moving the other phase past the stationary phase.
In chromatography the stationary phase is solid. In countercurrent extraction
techniques both phases of the system are liquids.
III. USE OF MICRO METHODS
The evolution of the rapid and simple techniques of paper and thin-layer
chromatography and zone electrophoresis has been of great value in seeking
methods or systems having adequate separation factors. Although these
methods are frequently used as identification tools or in studies of purity, they
can be also used to determine the chemical and physical properties of an
unknown substance. The determination of differential migration rates of
various substances under a variety of conditions can be carried out rapidly.
The data obtained may be directly applicable in the design of a separation
process, such as solvent extraction, ion exchange, or chromatography, and
its large-scale application. In addition, these methods provide procedures for
determining the behavior of the molecule in a complex environment. This
study is usually necessary, even for known compounds which might be part
of a synthesis reaction mixture, since the actual prediction of a "best" system
based only on the structural features of the compound being isolated is
uncommon.
These tests are useful in determining methods of carrying out both group
and fractionation separations and in predicting the behavior of the desired
compound in the separation step.
IV. EVALUATION OF SEPARATION PROCESSES
A separation method or process can be evaluated using four criteria.
These are (1) yield, (2) separation, (3) capacity, and (4) efficiency. The advan-
tages of having a high yield are obvious but if this is achieved with little
separation the method is not satisfactory. Likewise if the separated product is
obtained in low yield the method may be of little value. Some separation
4 Introduction
methods are readily applied on large scale and with large amounts of material;
others can be applied on small scale only and hence would be less satisfactory
on a capacity basis. Various criteria in the determination of efficiency are
possible. Thus, excessive time, equipment, reagent, or labor cost may render
a separation method impractical. An illustration of this is the analysis of
amino acids by ion exchange chromatography. Although pure amino acids
in a buffer solution are obtained from the chromatography column the method
is not nearly efficient enough to be considered useful in the preparation of
amino acids. The separation, although complete, is inefficient when equipment,
time, and reagents are considered.
In summary, the overall separation of a pure substance from a complex
mixture is a stepwise process employing a group separation and a fractionat-
ing separation. The group separation may be a batch process. The fractiona-
tion separation is a countercurrent process.
I General Principles
I. STABILITY
Before attempting to determine which separation method is most applic-
able to a particular substance it is necessary to know conditions under which
the compound is stable. A preliminary investigation should be undertaken to
assess the effect of pH, solvent, temperature, light, and oxygen on the
substance. Although some commercial separation methods can be carried out
using a condition of great instability for the desired substance, these are based
on detailed knowledge of the substance. Benzylpenicillin is rapidly decom-
posed at acidic pH. The rate of decomposition is influenced by both tempera-
ture and pH. However, a commercial process relies on the successful extraction
of the free acid into solvent and reextraction into water as an alkali metal salt.
High yields are obtained over these steps by using continuously flowing
streams and centrifugal separators in such a way that the entire extraction and
reextraction is complete in about 1 min. Such rapid transfers are usually not
possible in ordinary laboratory equipment; hence it is desirable to avoid
unstable conditions if possible.
Stability information is especially desirable in dealing with substances of
biological origin. Frequently these materials are found to be unstable to
drastic changes in pH or to some other laboratory stress. Fractionation steps
may require prolonged contact and if the material is unstable may even result
in lower, rather than higher, purity.
Some type of assay method is usually known for the desired substance.
Useful stability information can be obtained using methods which do not
require precise assay information if extended time or drastic conditions are
used. Thus it may be more meaningful to determine the effect of pH on
stability by using brief exposures to drastic pH changes than to attempt to
determine rates of decomposition at close pH intervals. Similarly, temperature
increases can be substituted for time provided conditions are chosen
judiciously.
5
6 /. General Principles
Although such stability studies require material, time, and assays and may
appear to detract from the objective of obtaining pure material, the latter
may be substantially aided by such information.
II. GROUP SEPARATIONS
Substances can be grouped based on ionic properties, polar properties,
or molecular size. Each of these will be discussed in greater detail under the
method employed. A general outline of the groups possible and the methods
of obtaining these groups is given in this section.
A. Molecular Size
1. Dialysis and Ultrafiltration
The separation mechanism can be simply visualized as based on molecular
size and shape. A membrane is used which has pores of sufficient size to allow
smaller molecules to pass but which inhibits the passage of larger molecules.
Thus in a batch process, two fractions are obtained. The exclusion properties
of membranes vary drastically. Cellophane membranes can be specially
treated to permit the passage of molecules as large as 20,000 m. wt. but the
cellophane types commonly available are useful for separating substances of
about 5000 m. wt. or lower from higher molecular weights. Special mem-
branes have recently become available which are applicable to the separation
of much lower molecular weight substances, using filtration. Reverse osmosis
(high pressure filtration) can be used to separate salt from water using cellulose
acetate films. Certain types of ion exchange membranes permit the passage
of substances having molecular weights of less than 1000 but exclude higher
molecular weight compounds.
2. Gel Filtration
The procedure consists of applying a solution containing substances of
different molecular weights to a column containing a swollen gel in the desired
solvent and developing the column with the solvent. The column can be
considered as containing two types of solvent, that within the gel particle
and that outside the gel particle. Large molecules, which cannot permeate the
gel, appear in the column effluent after a volume equivalent to the solvent
outside the gel has emerged from the column. Small molecules, which per-
meate the gel matrix, appear in the effluent after a volume equivalent to the
total liquid volume within the column has emerged. A typical separation is
depicted in Fig. 1.1.
As a group separation method, the procedure can be used to separate
substances which do not permeate the stationary phase from those which do.