G. M.
Gaucher
University of Co!gary
Calgary, Alberto, Cenada
An lntrodu<tion to Chromatog raphy
(1early for the practicing chemist and
particularly for the practicing biochemist an u nder-
standing of basic chromatographic principies along
with a working knowledge of the varíous types of
chromatography are indispensable. Thus the object of
this presentation is to serve as a preliminary practica!
introduction and guide to chromatography .
Historical Development ( J )
The separation technique known as chromatography
was extensively used and described for the first time by
Tswett (2, 3), a Russian chemist-botanist in 1903.
His interest in the green and yellow pigments of
chloroplasts resulted in one of the first descriptions of
what is now known as aclsorption column chromatog-
raphy.
When a petroleum ether solution is filtered through a colum n
of adsorbent (1 use mainly calcium carbonate which is tamped
firmly into a narrow glass tube), the pigments are resolved, ac-
cording to the adsorption sequence, from top to bottom into
various colored zones, since the more stronly adsorbed pigments
displace the more weakly adsorbed ones and force them farther
downward. This separation becomes practically complete when,
af ter the pigment solution has flowed through, one passes a stream
of pure solvent through the adsorbent colurn n. Like light rays
in the spectrum, the diff erent components of a pigment mixture,
obeying a law, separate on the calcium carbonate column and
can thus be qualitatively and quantitatively determ.ined. I call
such a preparation a chromatogram, and the corresponding
method the chromatographic method.
. . .It is self-evident that the adsorption phenomena described
are not restricted to the chlorophyll pigrnents and one must as-
sume that ali kinds of colored and colorless chernical compounds
are subject to the same laws.
Perhaps becau se the need was not yet great
enough, this powerful new separation technique went
virtually unused until 1931 when Kuhn, Winterstein,
and Lederer (4) separated the isomers of such polyene
pigments as carotene on a "Tswett" column. The
chromatographic separation of many natural pigment s
followed. While such colored hydrophobic compounds
were admirably separated by this techrúque, the nee.d
to separate colorless, hydrophilic compounds, such as
the constituents of the proteins, the polysaccharides,
and the nucleic acids, was increasingly evident . Thus
th next chromatographic advances utilized the phe-
nomena of ion exchange and partition. While ion
exchange was first recognized as a property of alumínum
silicates in soils, the widespread use of ion-exchange
chromatogra phy awaited the synthesis of the first ion-
exchange resins by Adams and Holmes (5) in 1935.
Then in 1941, Martin and Synge (6) developed pa rti-
tion column chromatography. They found that a mix-
ture of amino acids applied to the top of a sílíca gel
column containing definite amounts of water could be
separated by passi1.1g a suitable organic solvent through
the column. Soon after this in 1944 Consden, Gordon,
and Martin (7) replaced the silica gel support by strips
of paper and so initiated the wide-spread use of pa per
chrmnatagraphy. In the same year, Craig (8) estab-
lished the usefulness of the non-chromatographic
multiple partition separation technique of counter
current clistribution by developing the first practical
apparatus. A subsequent design by Craig and co-
workers (9, 10) is still in use today. A further advance
in partition chromatography occurred in 1952 when
James and Martin (11 ) published the first account of
gas-liquicl chromatography in which the mobile phase is
a gas rather than a liquid as in paper chromatography.
Unnoticed during this period of partition chromatog-
raphy's rapid development was the beginning of a
dramatic improvement in adsorption chromatography
which involved the utilization of an adsorbent in the
form of a thin !ayer on a glass plate. Thus the descrip-
tion by Stahl (12) in 1958 of an ingenious device for
preparing these "open columns" initiated the rapid
adoption of thin-layer chromatogra phy. Finally the
discovery by Porath and Flodín (13) in 1959 that gels of
crossliI1ked dextran (Sephadex) possessed the prop-
erties of an almo t perfect "molecular sieve" led to the
development of the unique chromatographic procedure
called gel filtration which fractionates molecules such
as proteins by virtue of diff erences in their size. Stim-
ulated particu1arly by the biochemist's continuing
interest in separating the complex molecular com-
ponents of the cell, advances in chromatographic meth-
ods will undoub tedly continue. The extraordinary
advances in our understanding of the biological world
at the molecular level which have resulted from the use
of chromatographic methods are innumerable and
unparnlleled.
Adsorption and Partition
Chromatography may be defined as an analytical
technique (14) for separating compou nds on the basis of
diff erences in affinity for a stationary and mobile phase.
These diff erences in affinity involve the processes of
either adsorption or partitíon. Adsorption involves
the binding of a compound to the surface of a solid
phase. For example, the purification of a compound
by solution in an organic solvent followed by treatment
with activated charcoal is dependent upon the. impmity
being adsorbed preferentially onto the charcoal. Par ti-
tioQ. on the other hand involves the relative solubility of
a compound in two phases resulting in the "partition"
of that compound between the two phase . For exam-
ple, the extraction of an organic compound from an
aqueous solutíon with ether is dependent upon the
organic compound being dissolved preferentia lly in the
Volume 46, Number 1 1 , November 1 96 9 / 729
 ether phase. Thus, as indicated in the table, the vari-
ous types of chromatography may be cla sified as
either partition chromatography or adsorption chro-
matography depending upon whether the stationary
pha. e is a liquid or a solid, respectively. As indicated
the mobile phase may be either a liquid or a ga. .
Counter Current Distribution
As previously indicated, the possibility of develop-
ing a practical separation technique based on the parti-
tion of a solute between two immiscible solvents led in
turn to the development of partition chromatography
by Martin and Synge (6) and counter current distribu-
tion by Craig (8). In each case the respective authors
provided a theoretical treatment of their technique.
Thus Martin and Synge (6), in analogy to the "theoreti-
cal plate" description of fractional distillation, presented
one of the first theoret ical treatments of column
chromatography and Williamson and Craig (15)
derived a mathematica l treatment of counter current
distribution capable of accurately predicting their
experimental results. The theoretical plate treatment
of chromatography and the mathematical basis of
counter current distribution have been aptly sum-
marized by Johnson (16) and Clark (17), respectively.
In addition the more fundamental physico -chemical
basis of chromatography has been discussed by Giddings
(18).
Because of its theoretical simplicity and mathemat -
ical predictability, counter cunent distribution rep-
resents an ideal model of partition chromatography .
Thus an understand ing of this tech nique leads most
readily to an appreciation of basic chromatographic
principies. The usual counter current distribution ap-
paratus (19) consists of a series of interconnected glass
tubes (from 10-1000) so designed that i n each tube an
upper (mobile) phase can be shaken with a lower
(stationary) phase, allowed to separate from this lower
phase, and then automatically transferred to the next
tube leaving the lower phase behind. A counter cur-
rent distribution experiment begins with a solu te parti-
tioned between two phases in the first tube of a series.
A measure of the relative affinity of a solute for these
two phases is given by that solute's partition coefficient
K greater the number of transfers. In addition as an
K = C,./ C1
where e,, and e, are the concentrations of solute in the
upper and lower phases respectively. If the volumes
of the two phases are equal, the partition coefficient
may be redefined as
K = x/y
where x is the fraction of the-solu te in the upper phase
and y is the fraction of the solute in the lower phase
such that x + y = l. Thu. as illustrated in Figure 1,
·with zero transfers having taken place, the solute is
distributed in tube O according to this partition co-
efficient. One "transfer" then consists of
moving this upper phase(s) to the next tube(s) containing fresh
lower phase and adding fresh upper pbase to the first tube,
af ter which
all contacting pbases are mixed and allowed to separate
Af ter one such transfer the fraction of solute remaining
in tube O (i.e., y ) is partitioned so that y of this fraction
73Ó / Journal of Chemical Education
NUMBE R OF
TRANSFERS
T UBE
NUMBER
BINOMIAL
ET
o
o
l 11 + y )2
Figure 1. A two tra nsfer counter current distribution scheme.
(i.e., y 2) remains in the lower phase while x of this frac-
tion (i.e., :1-, y) goes i nto the upper phase. A similar
partitioning of the fraction of solute transferred to tube
1yields the distribution x 2 and yx in the upper and lower
phases, re -pectively. Finally the fraction of the total
solute found in each tube (both phases !) is given below
the tubes (i.e., y and x ) while the sum of these fractions
is given to the right as i ts binomial equivalent (i.e.,
(x + y) 1). Upon following the results of the second
transfer the pattern which emerges is such that the
distribution of solute in each tube af ter n transfers is
seen to be given by the terms of the binomial expansion
of (x + y ) 11
1' 1
(x + y )n = Í: r!(n r)! xryn - r
T = 0
Thus given the partition coefficient for two compou nds
and the number of transfers eff ected, the distribution of
each compound in each tube can be accurately cal-
culated. Such calculations readily demonstrate that
for two solutes originally mixed in tube O, their degree
of separation increases the greater the difference be-
tween theiT respective partition coefficients and the
increased number of transfers tends to increase the
resolution between these solute peaks, each peak be-
comes shorter and wider.
Partition chromatography can then be looked upon
as ati extension of this technique in which the contin-
uous motion of a mobile phase over a stationary phase
adhering to a solid support replaces the step-wise
transfer of an upper phase to succeeding individual
lower phases. Chromatography is superior to counter
current distribution in many instances, since with much
simpler equipment a much smaller arriount of material
may be separated into its components with better
resolution. For example, chromatography on a small
20-cm long silica gel column can yield a resolution
equivalent to approxi mately 1000 transfers . Such
chromatography columns are common and in gas-
liquid chromatography special capillary columns equiv-
alent to greater than 500,000 transfers may be pre-
pared. A greater equivalent number of transfers and
therefore better separations are commonly obtained by
 increasing the length of the column
using slower fiow rates, and thus operating closer to equilibrium
conditions
increasing the porosity or decreasing the size of the particles mak-
ing up the solid phase or the solid support for the liquid phase
The Basic Types of Chromatography
Having examined the partition of a solute between a
stationary and mobile phase, a brief description of the
major types of chromatography is possible. In addi-
tion to consulting the literature cited for practica !de-
tials of theory and procedure , a number of jomna ls and
general texts are of interest (20-28) . Inaddition to the
above mentioned references, a great wealth of informa-
tion on biochemical applications of chromatography is
to be found in the many multi-volume works which
deal with biochemical techniques ( e.g., Colowick and
Kaplan (29)).
In partüion column chrornatography , a column is
packed with a porous solid of high surface area (e.g.,
silica gel, cellulose) which has been coated with the
liquid stationary phase (usually water). The compo-
nents of a mixtme are then separated by passing a
mobile phase (usually an organic solvent mixture)
through the column. Thus depending on the relative
solubility of the components in the stationary phase,
they are partitioned between the two phases and move
clown the column at diff erent rates (e.g ., components
which are more soluble in the stationary phase move
morn slowly down the column). A variety of both
hydrophobic and hydrophilic compounds (from fatty
acids to proteins) have been separated on such columns.
In paper chromatography (30), a paper sheet takes
the place of a packed column, and the components of a
mixture are partitioned between cellulose-bound water
(stationary phase) and an organic solvent mixture
(mobile pbase) whjch moves through the paper by
. capillary action. This method has been extensively
employed in virtually all fields and has the specific
advantages of simplicity in equipment and technique
along with the remarkable ability to resolve very small
amounts of material particularly when two dimensions
are used. It is less useful for preparative or quantita-
tive separations than is partition column chromatog-
raphy.
In zone electrop horesis (31, 32) , a solid support such
as paper (paper electrophoresis) or cellulose acetate
strips, or starch and agar gels (gel electrop11oresis)
contains an electrolyte (a buff er solution) in which the
components of a mixture move by virtue of their charge
and the electric current passed through the electrolyte .
The direction and distance moved depends on the
charge on the particle (pH of buff er) and the applied
field (voltage and· current). Low-voltage electrophore-
sis (5-20 V/cm) is of general value for protein separa-
tions, while high-voltage electrophoresis (50-200 V/cm)
is of particular value for separating amino acids and
peptides . This technique is not chromatography but is
closely related and is particularly useful when used in
conju nction with chromatography.
In gel filtration (33) , or molecular sieving a hydro-
philic gel (e.g ., Sephadex, a cross-linked dextran; Bio-
Gel P, a polyacrylamide ; or Agarose, a non-ionic galac-
tan from agar) in the form of porous beads contains the
aqueous stationary phase which is only distinguished
from the rnobile phase by its immobilization within the
bead . The ability of a particular molecule to penetrate
the gel particle is determined solely by the molecule's
size and shape and the porosity of the particle. Thus
the components of a mixture are separated accordi ng to
their size by virtue of a diff erential distribution between
easily displaceable water present in the inter-bead space
(mobile phase) and water immobilized inside the beads
(stationary phase) . The value of gel filtration in tbe
desalting (an alternative to dialysis), separation, purifi-
cation, and molecular weight determination of bio-
polymers, (particularly proteins and enzymes) has
resulted in its extensive U§e in biochem istry.
In gas-liquid chromatography (34, 35), a colu mn is
packed with a porous inert solid (e.g., Celite or
crushed firebrick) coated wi th a thin layer of an
involatile liquid as the stationary phase. Components
of a mixture are separated by being partitione d be-
tween this stationary phase and a gaseous mobile phase
which is usually helium. Of greatest value in separat-
ing organic compounds, glc (vpc) is limited to those
compounds which are at least somewhat volatile. Thus
biopolymers such as proteins which are not readily
volatilized cannot be gas-chromatographed. How-
ever, many essentially non-volatile compounds such
as sugars can be gas-chromatographed by converting
them to suitably volatile derivatives. The enhanced re-
solving power of gas chromatography results from the
ability to maintain fast flow rates in long columns due
to the mobile gas phase's low viscosity and ability to
rapidly attain equilibrium. Gas chromatography is
also favored by its ease of automation.
In adsorption column chromatography, the separation
of a mixture's componen ts is determined by the differ-
er'i.tial adsorption of these components on an "active"
solid such as alumina, silica gel, or charcoal (the sta-
tionary phase) as an organic solvent (the mobile phase)
containing them passes over it. " Thu:s weakly ad-
sorbed components will travel down the column more
rapidly than ·strongly adsorbed ones. The tendency
of components to "tail," the diffi.culty in preparjng
adsorbents of uniform activity, and the tendency of
these adsorbents to catalyze chemical changes often
result in partition chromatography being preferred
over adsorption chromatography.
In thin layer chromatography (36, 37 ), rather than
packing the adsorbent into a column, the adsorbent
(stationary phase) is spread over a glass plate in a
thin film of even thickness. The chromatographic
separation then takes place by allowing a solvent
(mobile phase) to move up the plate by capillary ac-
tion as i n paper chromatography. TLC combines the
capability of adsorption chromatography in the separa-
tion of non-polar compounds with the practical sim-
plicity and sensitivity of paper chromatography . In
addition tlc is much faster (30 min/ 10 cm versus 4
hr/10 cm) than paper chromatography and usually
yields superior resolution (spot sizes remain approxi-
mately the same as those applied to the origin as op-
posed to the substantial enlargement found in paper
chromatography) . The variety of diff erent adsor-
bents available and the ease with which adsorbed water
may be retained or elíminated (by drying) makes both
partition and adsorption thin layer chromatography
possible. Hence tlc is rapidly replacing many forms of
cbromatography including paper chromatography as
Volume 46, Number 1 1 , N ovember 1 969 / 731
 Types of Chromatography Based on the Stationary Phase
-· ---Phases--
Chromatography Type l\fobile Stationary Examples
Partition column chromatography
liqtúd liqwd• Paper chromatography
Partition chromatographyb Thin layer chromatography (tlc)
Gel filtration (Sephadex chromatog-
raphy)
gas liqtúd Gas-liquid chromatography (glc)
or Vapor phase chromatography
(vpc) .
liquid solid Adsorption column chromatography
Adsorption chromatography Thin !ayer chromatography
electrolyte ionic polymer Ion exchange chromatography
solution
ª The technique of electrophoresis of ten uses the physical set-ups of liqwd-liquid partitiQ.n chromatography but is not chromatography
as such.
b Counter current distribution represents a mathematically predictable model of partition chromatography .

the method of choice far analytical separations and
sorne preparative separations.
In ion exchange chromatography (38, _ 39), ionized
compounds in aqueous solution (mobile phase) are
separated by virtue of their diff erences in aflinity far
ionized groups which are an integral part of an insoluble
salid phase (stationary phase). These ionic solids are
usually synthetic polystyrene resins in bead form which
contain either acidic groups (cation exchangers) or
basic groups (anion exchangers) . Thus a mixture of
cations in aqueous solution applied to the top of a
column packed with a cation exchange resin in the
hydrogen ion form will displace the hydrogen ions and
form a narrow adsorbed band at the top of the column.
Upan adding a solution of sorne other appropríate cat-
ion, the cations to be separated will be displaced, mov-
ing down the column at diff erent rates according to
their relative affinity far the resin. Because the ex-
change capacíty and therefore the eff ectiveness of
synthetic ion exchange resins is dependent upan
penetration of the resin bead by the ionic salutes to be
separated, the pare size of these beads determines the
size of the salute which can be eff ectively separated.
Thus while available ion exchange resins are of great
value in separating small molecules such as amino
acids, they exclude large macromolecules such as pro-
teins. However cellulose and dextran (Sephadex) ion
exchangers (i.e., DEAE (diethyl amino ethyl) cel-
lulose and DEAE Sephadex) are commonly used to
separate such macromolecules on the basis of íonic
affinities .
Having classified and described the various types of
chromatography ít is noteworthy that the simple diví-
COUNTER CURRE NT DISTRIBUTION
"0ATC H" i •cOMPLETC •
PROCE:DURE S ----;.. CHHY
sion of all types into either partition or adsorption
chromatography as in the table is not rigid. Virtually
all the types of chromatography mentioned exhibit a
combination of physical eff ects. Thus ion exchange
resins exhibit molecular sieve properties, while dextran
gels exhibit both ion exchange and adsorption proper-
ties. Both tlc and adsorption column chromatography
require sorne adsorbed water far optimum adsorption
chromatography. And paper chromatography separa-
tions can be achieved using water as both the mobile
and stationary phases! This situation exemplifies the
still very empirical natu.re of chromatography and the
fact that experience rather than theory is still of great-
est value.
Ch romatograph ic Methodology
It is evident from the various types of chromatog-
raphy described, that chromatographic methodology or
in other words the physical set-up used, may vary
considerably. This is indicated in arder of increasing
complexity in Figure 2. The resolution, simplicity,
and rapidity of any separation is often dependent upon
this physical set-up. Thus while "complete" chro-
matography is simplest and most rapid, "flowing" or
gradient elution chromatography is capable of greater
resolution . This is because flowing chromatography
eff ectively increases the length of a chromatographic
set-up, while gradient elution chromatography de-
creases peak broadeníng and therefore increases resolu-
tion.
While non-equilibrium conditions (caused far example
by high flow rates) may result in "tailing" and "front-
ing," it is the exístence of non-linear partition or adsorp-
tion isotherms which are the majar cause of such peak
" FLOWlNG " "GRAOl(NT ELUTION broadening eff ects. Thus the amount of a substance
CHR0MAT0GRAPHY CHROMATOGRAPHY

.. SJ..,Pl.( oll/11/NG OF -A lil OlllLl l'lt /I SC '*;t1C:H S -/1 MOtUL[ l'Jj/1$[ WHr C" - ...o.!11.[ PHASE
taken up by the stationary phase depends upon this
nro Plt•SCS IN ..
8CUIC . FLASll,ETC
NOT P<lOC((O "AST THE [NO
OT THC SU1'0NARI' PHAS.C
OH .A SHECT Of' l'.APER , THlN
PROCEEOS 1'4ST THE ENO Of'
THE S TATi ONAlt,. PHA$[ OH ..
""P(lt QR COlVlllN
CON'TIN\IOVSL" CHU..GING
co...PCS1TrON ¡S f/4( C'Ol.IJ.VN
CLV•NT .
substance's concentration in the mobile phase. A plot
1..A CR l'l..U'C , PJ.CllCO TUH',
m
of "amount in stationary phase" (A,) versus "concentra-

t.
tion in the mobile phase" (Cm) at constant temperature
illustrates thís relationship. As illustrated in Figure 3

- 01 ¡ Dl ".... 1:1-J
y
¡iJhjjj
1ub• Hm!MI•-+
these adsorption or partition isotherms determine
whether a chromatography peak is symmetrical or
exhibits "tailing"
For example in or "fronting."
case B, as the band moves clown the
... 'ª · 'ª
-OCCOLOAIZ ATION •1Tt1
CH.UCO•L
-H PEll Ctd O!U'!'OGR.llP tn
-noN l.-/IYE R CMllOM4TOl;IUPMY
- OE SC[NOLNG P.llP[R
(MltOMATOGA/IPH
-PARf1110H , L flLTRUjQN
•DSORPTION • .llND IOH (X(ttAHlj.f_
column, the concentration of the compound in the
-51,.PL.[ [TH[lt -E TR USION CHlt0fOJ0GR.llF"IY -PAAT !TIC!ló.G CL flLTA.lltl°", GAS· COLU MN (HjlOlilJ.TOGA/IPM Y
EXTR.llCT!OM
-llCM OV&l.- OF Cn" .WITH
LIOUID , &OSOllPT !OH ANO ION
EC:H•HG[ C11fl01i1.11 TOGR.llPH r
mobile phase decreases at the back of the band while
A CUICIN DCHAHGf:
increasing at the front. Simuitaneously the amount of
compound held by the stationary phase increases at the
f ig ure 2. Types of ch romatogra phy based on the physical set-up. back and decreases at the front, reslilting in "tailing."

732 / Journa/ of Chemical Educa fion

 l-s.01herm Bcnd
··lL_ a . lI_
A
< S Jm e1r1 )
Cm
.!!.
!Toilil'IQI ··L_
··LL
f.
ffl'onlin9)
Cm
Figu re 3. ·The effect of odsorption or porlilion isotherms on peak shope
(28).
Tailing is almost inevitably found to some extent in
adsorption chromatography. Symmetrical peaks are
more common in partition chromatography . Fronting
is relatively rare.
Since the phenomenon of tailing is often the cause of
poor resolution (40) , an invaluable technique first
described by Alm, Williams, and Tiselius (41) is that of
gradient elution. This teclmique involves the use of
an eluant solution in which the concentration of the
most powerful eluting agent grad ually increases, thus
forming a concentration gradient clown the column.
The tail or rear portion of a peak is therefore always in
contact with a stronger eluant solution than is the
front of the peak. The tail therefore moves more
rapidly than the peak itself and is eliminated. A
variety of continuous elution gradients may be pro-
duced by using two eluant container of diff erent size
and/or shape (42, 43). As indicated in J<igure 4,
RE:LATIVE RE:LATIVE
CROSS -SE CTIQNAL
PPARATUS
W ARE.AS
Figure 4. A simple sel u p far producing elua nt grodients.
variations in the size of cylindrical containers (43) is a
simple method of obtaini ng eluant gradients. Since the
two containers are open to thc atmosphere the liquid
levels in each drop at the same rate. Thus as indicated
in Figure 4 the rate of solution addition to the column
(R0 ) is equal to twice the rate of solution transfer from
container a to b (Ra) for a linear gradient. If the cross-
sectional areas (Aa, A 0), the initial concentrations
(Ca, C0) and the volumes (V = Va + V0) are known the
concentration of the solution produced (e) af ter any
given volume (v) has been added to the column, may
be readily calculated using the following equation
e = C. - (C. - C 0 )(1 -
In general linear and concave gradients have been
found to yield the best chromatographic separations
Appecronce
(42). 1ore complex gradients of virtually any shape
may be produced using the gradient device described
by Peterson and Saber (44) and marketed by Buchler
Tube N11mber
Instrument. , Inc., Fort Lee, New Jer ·ey. Finally it
¡¡:
o
2
jl_A_ should be noted that in the method described above the
·olutions in the reservoir and in the mixing chamber
w Tube N11mber ·h ould not d iff er in density by more than 53. How-
.
l Tube N11mber
ever a modified methocl of value for adsorption column
chromatography, where for example a methanol
(den ity 0.79) : chloroform (density 1.49)
grarlient is used, has been described by Wren (45) .
Literature Cited
(1) ZECHMEIBTER, L., "P.rogress in Chromatography 1938-1947," Chapman
and Hall, London, 1950.
(2) RomNSON, T., J. CHEM. Enuc., 36, 144 (1959).
(3) STRAIN, H. H., AND SnERMA, J., J. CHEM. Eouc., U, 235 (1967) .
(4) KUHN, R., WINTERSTEIN , A., AND LEDERER, E., Hoppe-Seyl .er's Z.
Physio! . Ghem., 197, 14 1 (1931).
(5) AoAMS, B. A., ANO HoLMEs , E. L., J . Soc. Chcm. lnd. (London), 54 ,
1 (1935).
(6) MARTIN, A. J. F., AND SYNGE , R. L. M., Biochem . J ., 35, 1358 (1941) .
(7) CoNSDEN, R., GonnoN , A . 1-l., AND MAHTIN , A. J. P., Biochem. J., 38,
224 (1944) .
(8) Cn.AIG, L. C., J. Biol. Chem., 155, 519 (1944) .
(9) CRAIG , L. c.. AND POST, o.. Ana!. Ghem., 21, 500 (1949).
(10) CRAIG , L . C., 1-IAUSMANN , \V., Aun ENS, P., A ND HAnFENIST , E. J.,
Anal . Ghe-m., 23 , 1326 (1951).
(11) JAMES, A. T., ANO MAllTIN, A . J. P., Biochem. J., 50, 679 (1952) .
(12) ST.rnL, E. Chemiker-Ztu. 82, 323 (1958).
(13) PonATH J., AND FLODt N, P.. Nature , 183, 1657 (1959) .
(14) CAssrnY, H. G., J. Cm:M. Eouc., 33, 482 (1956).
(15) W1LLIAMBON, B., AND CnAJG, L. C.. J . Biol.. Chem., 168, 687 (1947).
(16) JoUNBON, M. J., "Dcsign or Chrom atograph ic Proccdures" in UMDR EIT,
\'V . \V., BuRRJS, R. H., AND STAUFE R, J. ]1'. , "l\'Ianomet.ric Tech niq ues,"
('Hh Ed.), Bu rgess Publishing Co., Minneapol is, 1964 , p. 233.
(17) CLARK , J. M . Jn., ( Editor ) , "Experi mental Biochemistry ," W. H.
Freeman and Co., San Francisco, 1964., p . 190.
(18) GrnvrnGS, J. C., J. CHEM . En c., 44 , 704 (1967) .
(19) CRAJG, L. C., "Counter Current Distribu tion" in Ft..ORKIN, M., AND
STOTZ, E. J-1. (Editor s)i "Comprehensive Biochem istry," American
E l sevier , New York, 1962 , Vol. 4, p. l.
(20) ) Anal . Chem., American CbemicaI Society, Washington, various reviews
GRAOIENT
FLO PRODUCE O eacb April.
RATES
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(38) 11
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