Title of Invention

ELEMENTS FOR SEPARATING SUBSTANCES BY DISTRIBUTING BETWEEN A STATIONARY AND A MOBILE PHASE, AND METHOD FOR THE PRODUCTION OF A SEPARATING DEVICE

Abstract The invention relates to elements for separating substances by distributing between a stationary and a mobile phase. Said separating elements comprise any stationary phase and a support element. The separating elements are part of a set that in provided with at least two separating elements Ti (i ≥ 2) encompassing different stationary phase Si (i ≥ 2). The set comprises at least three pieces of each type of separating element Ti with a specific stationary phase Si. The separating elements can be coupled to each other or interconnected in another manner so as to form a separating device. The invention further relates to a method for producing an optimized device for separating substance mixtures.
Full Text Elements for separating substances by distributing
between a stationary and a mobile phase, and method for
the production of a separating device
The present invention relates to elements for
separating substances by distributing between a
stationary and a mobile phase, in particular for
chromatography, and a method of production of the
separating device comprising the stationary phase, in
particular a chromatographic column.
Chromatography is, a physicochemical method of
separation, in which the substances to be separated are
distributed between two phases, one of which, the
stationary phase, is fixed, whereas the other, mobile
phase moves in a defined direction.
The stationary phase is one of the two phases
comprising the chromatographic system. A solid phase
(sorbent), a liquid phase (solvent) or a gel can be
used as the stationary phase. In the case of the liquid
phase, this is applied to a solid (support), which may
also be involved in the separating process. In the case
of a bound phase, the layer with separating properties
is bound chemically to a support or to the inside
surface of a capillary.
Within the scope of the present invention, the
stationary phases also include the quasistationary
phases in liquid-liquid chromatography.
In order to protect the separating column against
contaminants during chromatographic separation, the
separating column is sometimes preceded by a precolumn.

In general the precolumn is filled with the same
stationary phase as the separating column, or a
material with lower retention is used.
In isocratic analysis the composition of the mobile
phase remains constant throughout the elution
operation.
In contrast, in gradient elution, the composition of
the mobile phase is varied continuously or
alternatively stepwise (step gradient).
In practice, substances of different polarities are
often separated chromatographically over a wide range
of polarity by gradient elution, as selective
separation can be achieved in this way. In this case, a
more polar mobile phase is added continuously or
stepwise to an initially usually nonpolar mobile phase
(normal-phase chromatography) or a less polar mobile
phase is added to one that is more polar (reversed-
phase chromatography).
For formation of the mobile phase by means of a
gradient, chromatographs are required for gradient
elution with at least two pumps for the different
solvents or at least one low-pressure mixer and control
software. The robustness of gradient separation is
always poorer than that of isocratic separation.
Another disadvantage of gradient elution is that after
elution with e.g. an increasingly more polar solvent
mixture, the chromatographic system must be brought
back again to the initial thermodynamic equilibrium
with respect to the initially nonpolar mobile phase in
the next elution, namely by extensive rinsing with the,
in this example, nonpolar phase, before the column can

be used for the next gradient elution. This
"backwashing" is generally carried out with fifteen to
twenty times the column volume and leads to a
prolongation of the total cycle time and a considerable
consumption of solvents.
Thus, separation of a large number of samples that are
to be verified and separated by gradient elution, for
example in quality control, has the disadvantage of the
prolonged total cycle time, whereas for routine
investigations of a large number of samples, the time
per analysis with good separation should of course be
as short as possible for economic reasons.
A further disadvantage of gradient elution is that the
varying composition of the mobile phase means that
certain detection principles cannot be employed. For
example, the use of detectors based on measurement of
refractive index is essentially ruled out when the
composition of the mobile phase varies.
Even the UV/VIS detectors that are generally used are
greatly restricted in their range of application in
gradient elution on account of many admixed solvents
and additives, e.g. methanol, THF, TFA, formic acid,
acetic acid etc., which are themselves strongly
absorbing in the UV range Even with mass spectrometers, which have been used as
universal detectors in recent times, secondary effects
occur in gradient elution, e.g. problems of ionization
in different chemical environments, making quantitative
analysis much more difficult.
There are also ecological disadvantages in separation
of substances by gradient elution: the mobile phase

contained in a mixture after elution can only be
recycled with difficulty, if at all, necessitating
expensive disposal of the solvent mixture and
additional costs for supply of the (fresh) solvents for
forming the gradient. Moreover, gradient elution
requires the use of far more expensive solvents, of
higher purity, than the isocratic technique.
Furthermore, backwashing of the separating column also
requires appreciable amounts of solvents, which cannot
be recycled.
When in practice, also within the scope of preliminary
tests, the user first develops, from columns with
different stationary phases, the column with the most
suitable stationary phase (such columns are supplied as
test columns), optimization of the separation technique
in practice is ultimately based on optimization of the
mobile phase, namely through selection of the type of
solvents, the pH value of the mobile phase, further
additives etc., which then lead to improved separation.
It is simpler, however, to achieve defined
selectivities by skillful selection of the correct
stationary phase. A situation that often arises,
however, is that as soon as separation is achieved
between a particular critical pair of peaks, another
pair of peaks merge into one peak elsewhere in the
chromatogram.
To prevent this, it was proposed to use, as stationary
phase, a mixture of suitable stationary phases (mixed
bed) , with which, for a specific separation problem in
isocratic analysis, a selective separation can often be
achieved.

G.J. Eppert and P. Heitmann proposed, in LC.GC Europe,
p. 2, October 2003, optimization of mixed-bed columns
based on first determining, using three or four test
columns of different selectivity, the retention times
of a test substance mixture, and then calculating a
suitable mixed-bed column for the specific separation
problem.
However, these stationary phases as mixed bed have the
disadvantage that the column manufacturer must be
commissioned to pack a corresponding mixed-bed column
for each new separation problem. Thus, in the short
term, the user is quite unable to carry out an
isocratic analysis with the desired selectivity. The
column cannot be packed by the user, who does not have
the necessary special equipment, and lacks the relevant
know-how. Moreover, it is also relatively difficult to
pack a mixed bed in a separating column with good
reproducibility.
It is known, from multidimensional chromatography, to
provide columns branching from a main column, to be
connected at different times, so that individual
fractions are then "branched off" from the main column
at a defined point of time via the temporarily
connected column. Ultimately, separation of the mixture
in multidimensional chromatography only takes place to
a certain extent on the stationary phase of the
separating device, and the main separation is achieved
by switching to an additional separating device with an
additional supply of eluent.
Even if good separations can be achieved with
multidimensional chromatography, it is very expensive,
fault-prone and is only suitable for routine
measurements at considerable cost.

To solve special separation problems such as the
separation of a 20-component amino acid mixture, J.L.
Glajch et al. proposed, in "Journal of Chromatography",
318 (1985), page 23, improving the selectivity by
combining stationary phases along with four mobile
phases. However, this arrangement is based on purely
empirical tests and has therefore not been calculated
specifically.
For optimization of the mobile phase in liquid
chromatography, Sz. Nyiredy, B. Meier, C.A.J.
Erdelmeier, 0. Sticher proposed, in "High Resolution
Chromatogr. & Chromatogr. Communications" 8, (1985),
the so-called "prism" model. In this, the solvent
strength is plotted vertically, and the two-dimensional
representation of the proportions of the components,
which mainly influence the selectivity, is plotted in a
horizontal plane.
Later, in column liquid chromatography, the overall
optimum of the mobile phase was predicted from fifteen
individual measurements and the local optimum of the
mobile phase from twelve individual measurements (Sz.
Nyiredy, W. Wosniok, H. Thiele, 0. Sticher "Fluid
Chromatogr." 14, 3077 (1991)).
This model was also employed for optimization of the
extractants in liquid-liquid extraction, and in this
case optimized extractants could be determined from
twelve measured values (Sz. Nyiredy "Chromatographia"
51, 288 (2000)).
The present invention is based on the problem of
providing a device and a method with which a
chromatographic separation problem can be solved


quickly, efficiently and in an environment-friendly
manner with optimum selectivity and detectability.
This problem is solved by the features of claims 1 and
15.
According to the invention, a set of different
separating elements is provided, from which a
separating device with different stationary phases can
be constructed, construction being carried out by
coupling or connecting different separating elements
together.
Said set according to the invention comprises
separating elements of different stationary phases S1
(i ≥ 2) and at least three of these separating elements
containing different stationary phases Si, which can be
coupled or connected together.
Coupling or connection is carried out by coupling or
connecting the separating elements as such, in
particular coupling the carrier elements, which form
the separating element together with the respective
stationary phase. For example, column segments can be
coupled by coupling the column segments together on
their respective tube ends.
Coupling of the separating elements via additional
couplers or connectors is also possible.
Within the scope of the present invention, two
stationary phases SA and SB are different if they differ
from one another in their selectivity.
The minimal set thus comprises in each case three
separating elements or column segments SA and in each


case three separating elements or column segments SB,
where A and B are stationary phases of different
selectivity.
Preferably, a set comprises at least three separating
elements or column segments comprising different
stationary phases SA, SB and Sc, with the set containing
3 of each of these kinds of separating elements,
preferably at least 5, preferably at least 7 and
especially preferably in each case 10 or more.
The respective stationary phases should be selected so
that they possess different physiochemical properties.
Such a set can contain for example separating elements
with stationary phases, comprising saturated or
unsaturated alkyl groups (C1, C2, C3, C4, C6, C8, C12,
C14, C16, C18, C22, C27, C30), which can be mono-,
poly- or acyclic, ali- or heterocyclic, nitro, cyano,
carbonyl, carboxyl, hydroxyl, diol, or thiol groups,
glycidoxy groups, optionally etherified, amino or
chiral groups, amide groups, carbamates, urea,
perfluoralkyl groups or perfluoraryl groups and other
haloalkyl and haloaryl groups, polybutadiene groups or
other organic polymers, affinity-chromatographic
modifications or ion-exchangeable groups (cation
exchangers, anion exchangers, zwitterions), unmodified
support materials (e.g. silica gel), molecularly
imprinted polymers (MIPs) or combinations thereof, e.g.
multifunctional stationary phases, optionally
additionally to or instead of the aforementioned three
different types of separating elements.
In contrast to multidimensional chromatography, the
substances are separated on the separating device or
column coupled or connected on the basis of

optimization, i.e. the components of the mixture pass
through the whole separating device. The separating
elements that are coupled together remain connected to
one another at least for a complete separation - a
coupling or decoupling during the separation process,
as is known in multidimensional chromatography, does
not take place.
Another important point is that the ratio of the
stationary phases of the coupled separating device to
one another is variable, since the separating device
can be dismantled again into the individual separating
elements and a separating device with a different ratio
of the stationary phases to one another and with a
different selectivity can be constructed from them.
In contrast to the known mixed-bed columns, the desired
column according to the invention is constructed by
mechanical coupling of individual ready-to-use
separating elements, i.e. generally by connecting or
coupling together the carrier elements (e.g. pieces of
tube as column segments). This does not alter the
stationary phases that remain unchanged in the
separating elements, in particular they are not mixed
together.
In contrast to the production of pre-calculated mixed-
bed columns, for which ultimately the column
manufacturer packs a column according to specific
customer requirements, i.e. has to prepare and mix a
special stationary phase, according to the invention,
the user can make the desired separating device in situ
by simple mechanical coupling of the necessary column
segments.

The invention also relates to a method of production of
an optimized device for separating mixtures of
substances.
First, with each of the at least two types of
stationary phases Si (i ≥ 2), a basis measurement of
the mixture to be separated in a mobile phase that is
to be established is carried out and the relevant
retention factor k of each component is determined from

(tR: retention time, tm: flow time, tmEc: extracolumn-
volume-correction). The extracolumn-volume-correction
takes account of the time contribution by the volume of
the feed lines, of the injector and of the detector
cell. It is recommended to specify the mobile phase so
that the K values lie between the substances that are
to be eluted first and last in the range from 1 to 20.
The basis measurement should preferably be performed on
each kind of separating element (e.g. SA, SB, Sc) or on
several similar or different coupled elements (for
example 10 coupled elements SA or 5 SA, 3 SB, 2 SC; 3 SA,
5 SB, 2 Sc and 2 SA, 3 SB; 5 SB) . The basis measurement
can also be carried out on a separate basis measurement
column.
From the k values determined for each component to be
separated and for each type of separating elements with
the stationary phase Si, the optimized combination of
separating elements and their dimension is then
calculated for the specific separation problem and the
optimized separating device is then coupled or
otherwise connected in the previously calculated manner

from the separating elements of the predetermined
dimension.
The dimension of a separating element means the volume
of the respective stationary phase in the separating
element. In the case of separating elements with a
constant diameter the dimension is proportional to the
length. If all separating elements have the same
diameter and the same length, the dimension is
proportional to the number N of separating elements to
be coupled.
The most suitable combination of the separating
elements for the specific separation problem is
preferably calculated using the prism model. Preferably
stipulating a predetermined number N of separating
elements to be coupled, for any possible combination of
stationary phases and their dimension, in particular
the number of separating elements of a predetermined
total number N=X+Y+Zof separating elements,
based on the retention factors ki determined with the
basis measurement, the respective retention factor of
the component to be separated at selectivity point XYZ,
i.e. the retention factor kxyz of the respective
heterogeneous combination of the stationary phases Si
is calculated

where X denotes the number of separating elements SA, Y
the number of separating elements SB and Z the
respective number of separating elements Sc.
From the large number of calculated coupling
possibilities XYZ, the coupling of the separating

elements or segments with the best selectivity a is
determined from

where tR1 is the retention time of the respective
compound and W1/2 1 is the half-height peak width of the
respective compound, and the separating device, in
particular column, optimized for this separation
problem, and comprising N separating elements, is then
coupled together from the respective elements or column
segments.
With specially calculated coupling of the separating
elements, in particular column segments, the stationary
phase can be optimized with respect to the particular
separation problem, so that it is no longer necessary
to perform elution with a continuously or stepwise
varying mobile phase or get the manufacturer to pack
special mixed-bed columns.
If the basis measurement already shows that the desired
separation can be achieved with only one type of column
segments, the rest of the calculation can be used for
optimizing the length of the column ' and for
constructing the column only from the one type of
column segments.
Otherwise the various possible combinations of the
column segments for a given number of column segments N
are calculated, until the desired separation is
achieved. Optimization and calculation can be


terminated when the desired selectivity acrit or
resolution Rs,crit of the critical peak pair or pairs is
accomplished.
If, within the scope of calculation of the various
possible combinations with for example three types of
column segments and a total number of N column
segments, sufficiently good separation is not
calculated, i.e. no Rs,Crit is obtained below the value
defined for the particular separation problem, the
number N of column segments is altered, in particular
increased, and the calculation is carried out once
again with this altered number N'.
If, within the scope of this calculation with N column
segments, no satisfactory separation is predicted, a
new calculation becomes necessary, adding one or more
additional stationary phases.
For this, basis measurements are carried out
additionally with the additional column segments Sj and
then a new calculation is performed.
If, for a given combination, the separation capacity is
insufficient, the resolution can be improved by
increasing the total number of column segments N at the
existing ratio. In practice, if separation is too good,
the number of segments can be reduced in the same
ratio, in order to shorten the total cycle time.
The separating device optimized for the particular
separation problem is then coupled or connected from
the separating elements in the calculated number and/or
dimension and the corresponding separation is carried
out.

The elution sequence and the resolution of the peaks
can be predicted on the basis of the calculation.
Thus, the separating device, in particular column,
optimized for the particular separation problem, can be
assembled quickly and efficiently from the set of
separating elements, namely generally on the basis of
only three (!) basis measurements.
Although often three different stationary phases are
sufficient to achieve a sufficiently effective, in
particular isocratic separation, the number of
stationary phases can of course be further increased to
four, five or more, and the formula for calculating the
heterogeneous retention factor K in the numerator and
denominator is extended correspondingly to n stationary
phases.
If the stationary phases or the separating elements
comprising the stationary phases do not have the same
dimensions (e.g. diameter and length of the packed
column segments), the retention factor for each
component can be calculated from the following equation

where a, |3, y correspond to the total internal volumes
of the separating elements packed with the stationary
phases A, B and C in the heterogeneous combination.
kfl;B,c are - as already described - the retention factors
determined by the respective basis measurements.
If, in the subsequent measurement, a protective column
must be provided before the separating device, this has
to be taken into account in the basis measurement and
in the calculation.


Another advantage is . that the first column segment can
also be regarded and used as a protective column and
can if necessary be replaced very easily. Accordingly,
use of an additional protective column is unnecessary.
The method according to the invention can moreover also
be applied in a variant, so that - without stipulating
particular dimensions of the separating elements - the
optimized separating device is calculated from the
basis measurements and the optimized column can then be
packed correspondingly by the manufacturer according to
the previously calculated proportions of the individual
stationary phases. In this case the calculation
provides the selectivity-optimized volumes of each
stationary phase, and the individual volumes determined
are then packed in a suitable carrier element, e.g. a
suitable column.
Generally, with the device according to the invention
and the method according to the invention, it is not
the mobile phase that is subject to a continuously
varying composition, but rather the separating device,
in particular column, assembled separation-specifically
by coupling the respective separating elements, has the
varying composition that is necessary for a selective
separation, based on separating elements or segments
with different selectivities, i.e. the separating
device has a gradient of the stationary phase.
Accordingly, separation with the device according to
the invention can also take place isocratically with
the desired selectivity, so that the disadvantages
described for gradient elution are avoided. Thus,
backwashing is not required, i.e. there is a saving of
solvent and the total cycle time is shortened. In
addition, the mobile phase or at any rate a large part


of the fractions that are not contaminated with
substances of different polarity in an isocratic
elution, can be recycled, giving an additional saving
on the costs for disposal and for the supply of fresh
solvents, and it is also beneficial for the
environment.
For this, preferably, individual regions of the mobile
phase, which are laden with the respective separated
substances, are removed and the rest of the solvent is
captured, recycled and reused. Especially in the area
of preparative chromatography, which uses considerable
amounts of solvents, reusability of the solvents
achieved with isocratic chromatography is not only of
great advantage on environmental grounds, but also
represents a considerable cost saving, as the costs of
disposal and supply of fresh solvents are greatly
reduced.
Another important advantage of the invention is that,
with isocratic elution, many more different detection
principles and therefore detectors can be used for
determining the respective substances.
Since the composition of the mobile phase remains
constant in isocratic elution, the differential
refractometer can again be used for universal detection
of the substances.
Often, the isocratic elution of the substances and
therefore the separation with the set according to the
invention can take place in an acetonitrile/water
mixture, for which the rise in absorption in the UV
does not begin until a wavelength of 190 nm (50%
transmission). Accordingly, substances with


chromophores can, as a result of isocratic elution, be
detected even in the short-wave UV region.
The separation that can be carried out isocratically
according to the invention thus makes it possible to
use other, often more suitable, more sensitive and/or
less expensive methods of detection.
As the composition of the mobile phase is constant over
time, in addition of course, in each method of
detection the base line is more stable and effects due
to mixing are avoided.
Isocratic separation also has a beneficial effect on
the quality of the mass spectra, as the ionization
remains constant and in addition no gradient-induced
secondary effects occur (stable, constant background).
Moreover, for detection in isocratic elution it is also
possible to use electrochemical detectors or
conductivity detectors.
Since, as a result of isocratic analysis, nearly all
detection principles are now available, there is the
possibility of coupling the various detectors in
series, for improved characterization of the
substances.
Furthermore, with the device according to the
invention, the fault-prone equipment required for
providing the gradient, such as additional pumps and
gradient mixers, is no longer required, leading not
only to lower acquisition costs for the chromatograph,
but also to reduced operating costs.


The set of separating elements according to the
invention and the method according to the invention can
be used for all methods employing physicochemical
separation based on different interactions with at
least two phases, such as gas chromatography (GC) ,
liquid chromatography (LC) (column or planar),
chromatography with supercritical gases as the mobile
phase (supercritical fluid chromatography, SFC),
electrophoretic separation techniques, in each case in
the nano, micro, analytical and preparative areas of
application, and in the area of sample preparation
and/or purification by solid-phase extraction or
filtration.
In liquid chromatography, methods are also employed
that take place at reduced pressure (e.g. flash
chromatography) or elevated pressure (e.g. HPLC, MPLC)
or that utilize different mechanisms of interaction for
separation, e.g. reversed-phase liquid chromatography
(RP) , normal-phase chromatography (NP) , ion
chromatography (IC), size-exclusion chromatography
(SEC, GPC) or hydrophobic interaction chromatography
(HIC, HILIC).
The separating element of the stationary phase can be
for example a segment of a column, a segment of a TLC
plate or of an electrophoresis gel. Although the
present invention is described mainly with reference to
columns and column segments, what is said also applies
appropriately to other separating elements or segmented
or segmentable separating devices, for example TLC
plates, which can be adapted to any possible length of
the stationary phase in question.
The coupling of stationary phases is also independent
of the type of support material used (silica gels,

polymers, metal oxides, glasses, quartz glasses,
polysaccharides, agarose gels, carbon etc.) and also
applies to all orders of magnitude of the
chromatographic process, from miniaturized nano-methods
to the preparative range.
Furthermore, the stationary phase can be either
particulate, fibrous or fiberlike, or it can be
monolithic. An advantage in using monolithic stationary
phases is that they can be coupled very easily, as the
stationary phase does not have to be retained by frits
or sieves.
The present invention is also especially advantageous
when used in nanotechnology, because with such smaJl
amounts it is extremely difficult to provide gradients
in the mobile phase reproducibly.
Of course, the set according to the invention can also
be adapted to special separation techniques or can
contain special segments for special separation
problems, if necessary as additional segments.
Thus, in ion chromatography or ion-pair chromatography
it is also possible to construct the stationary phase
from column segments or for example also couple the
column segments for ion-pair chromatography with other
column segments, for example polar column segments.
A further advantage of using specially pre-calculated
and then coupled or assembled separating devices is
shortening of the total cycle time. The use of the
separating elements and calculation of the individual
separating elements that are to be coupled together as
well as their number ultimately also permits
optimization of the total cycle time, i.e. as short as


possible, but long enough to achieve the desired
separation.
A set can also comprise segments for targeted
adsorption of certain substances, as in solid-phase
extraction. From the mixture that is to be separated,
the substance in guestion is adsorbed almost
quantitatively in this specific adsorption segment and
is thus enriched in this segment, and is then desorbed
again. Instead of only in one segment, as customary
until now, a veritable cascade of segments could be
envisaged for targeted adsorption of different
substances in series.
The segments according to the invention can also be
used in other separation techniques, such as in
electrochromatography, in which mixtures of substances
are separated on the basis of electroosmotic flow, or
in electrophoresis.
The set according to the invention and the method
according to the invention are suitable for
continuously-operating online processes and/or methods
of separation with a mobile phase and/or constant rate,
for example HPLC, online-TLC and online-OPLC.
The set according to the invention can also comprise at
least two different types of TLC-plates, which should
then each be contained in the set in a larger number,
in particular at least three of each kind. The TLC-
plates can be adapted to the optimized length and the
various TLC-plate segments can then be coup]ed together
to a plate formed from the various segments and
therefore ultimately a plate with a gradient in the
stationary phase.


In particular, the set enables users to construct in
situ a separating device that is especially suitable
for their (routine) analysis, and then separation can
take place with the advantages of isocratic elution
already described.
The advantages of the optimized separating device are
achieved in particular when the separating device, once
optimized, is used in the continuous separation
process, namely isocratically and with a constant
stationary phase throughout the separation, i.e. even
without "switching" the columns.
In particular applications it is also possible of
course, in addition to optimization of the stationary
phase, to optimize the mobile phase as well for the
separation in question, for example also with a
gradient.
The specific design of the particular column segments
depends of course on the type of separating technique
and the amounts of substances to be separated using the
separating device prepared from the separating elements
that are to be coupled together.
The length of each individual separating element
depends on the subsequent total length of the device
comprising the stationary phase, in particular the
column, and the desired number of segments and the
specific separation problem. In the case of separating
elements that are segments of analytical LC columns,
the length of a segment is at least 0.5 cm, preferably
at least 1.0 cm, to provide simple mechanical coupling-
together of the segments. The length of a column
segment is preferably 2 cm, so that a column with a
length of 20 cm can be constructed from 10 coupled


segments. Especially preferably, the length of a column
segment is 1 cm or an integral multiple thereof and can
be up to 500 cm; preferably the length of a column
segment is not more than 12.5 cm. In capillary gas
chromatography (cGC) , column segment lengths up to
100 m can be envisaged, and the length is preferably
25 m.
The column segments can have either the shape of a
straight cylinder or of a spiral cylinder (especially
in the case of long column segments, for example for
GC) or can be of some other shape.
The inside diameter of the column varies considerably
depending on the amount of substance to be separated
and the particular chromatographic process and can be
between about 20 urn and 2 m, in the case of an
analytical column generally between 20 µm and 10 mm.
For isolation purposes a column with an inside diameter
between 2 mm and 1 m will be used.
It is in principle also possible for the set to
comprise additionally "multiple segments". These
"multiple segments" are column segments of a defined
stationary phase also present in the "single segment",
but the length of the "multiple segment" is an integral
multiple of the length of an individual segment. For
example, in addition to 10 individual segments with a
phase comprising phenyl groups of length 2 cm, the set
can also contain a "double segment" with a phase
comprising phenyl groups of length 4 cm and a triple
segment with the phase comprising phenyl groups of
length 6 cm, and with these multiple segments that are
additionally present, the time required for coupling
together and dismantling the segments can be reduced.


Theoretical considerations based on the prism model
show that, when in addition to column segments of
length 1 in each case a column segment of each type of
stationary phase with a smallest possible length (e.g.
1/2 or 1/4) is included in the calculation, a far wider
working range for selectivity optimization can be
covered with a reasonable number of calculations, than
if for example the total number N of column segments
were to be increased overall by one or two segments.
The separating elements should be coupled by means of
connections that are inert and free from dead space as
far as possible, for example by means of
• capillary joints
• directly with the aid of a coupler with small dead
space
• in the case of capillary columns, by joining the ends
of the capillaries using a coupler, with the ends of
the capillaries being butted together directly
• again in the case of capillary columns, by shrink-
fitting the ends of the capillaries via a shrinkable
sleeve
• by screwing the column ends together using a gasket
with a through-hole
• via a screw connection with a capillary hole
• directly by bringing together the packing end of one
separating element with the packing end of another
separating element


• by segmented packing of the stationary phase in a
column tube
Preferably, each separating element has the same
coupling means at each end, so that any end of one
column segment can be connected to any end of another
column segment.
Another preferred embodiment envisages providing
coupling means that are different from one another
(male, female) at either end of the column segment. In
this case the end of one column segment is inserted in
the start of the next one, maintaining the inside
diameter of the packing cross-section and therefore
keeping the linear flow rate constant. In this way, no
turbulence develops at the coupling points. Other
advantages of this system are the chambered seal at the
coupling points and the protected packing of the
individual separating elements. Moreover, the packing
is closed internally and protected. The simple
manipulation by inserting in one another is especially
advantageous. However, the most important point with
this coupling is that coupling is virtually free of
dead space, since the end of the separating segment is
pressed directly, with a thin frit, onto a sieve with
desired cross-filtration.
It is also possible to provide a monolithic seal, with
thickness preferably between 0.5 and 1 mm, at either
end of a column segment. These column segments are
coupled similarly to the manner described above (male,
female). In this case there is the advantage that it is
not necessary to use any metallic terminations such as
sieve and frit. Furthermore, the monolithic seal can be
modified chemically and can thus be adapted to the
particular stationary phase used. With this system it

is possible for two separating elements to be connected
together without a transition element (such as sieve
and frit).
If chromatographic plates shortened to the appropriate
length (dimension) are used as separating elements, for
example in TLC, OPLC or HPTLC, the separating elements
can be coupled by overlapping the stationary phases of
two plates at the plate ends, so that the stationary
phase of the first plate for example is directed upward
and the stationary phase of the next plate is directed
downward and so on, or the plates are placed next to
each other and then coupled via an inert bridge, which
then overlaps the stationary phases of both plates.
The method according to the invention will now be
explained in more detail with an example of
application.
I. Example of calculation of the optimized combination
of the separating elements (column segments) in
question on the basis of the prism model:
a) For a mixture of methylparaben, acetoph enone,
ethylparaben, dimethyl phthalate, 2,3-dimethylphenol,
methyl benzoate and anisole, determine the best
possible separation that is possible with a combination
of 10 column segments with cyano and/or phenyl and/or
C18 groups in acetonitrile/water 3C: 70 (v/v) as
solvent.
The set comprises in each case at least 10 column
segments with CN as stationary phase (SCN), 10 column

segments with phenyl groups as stationary phase SPH and
10 column segments with C18 as stationary phase (Sc18).
For each of these three different stationary phases,
the respective retention factor k, shown in the
following table, is determined according to (1) with.in
the scope of a basis measurement for each of the 7
different substances to be separated:

In the present example, the total number of column
segments N to be coupled together is set at 10. For
each heterogeneous coupling of the column segments,
i.e. X-times the stationary phase SCN, Y-times the
stationary phase SPH and Z-times the stationary phase
SC18 with N = 10 = X + Y + Z, the retention factor

of the heterogeneous coupling at the selectivity point
XYZ is calculated according to (2) . The selectivities
of all adjacent pairs of peaks are now calculated from
the calculated retention factors of the respective
couplings XYZ of separating elements. The optimum
selectivity point is the point XYZ at which all pairs
of peaks achieve a base line separation. If several
selectivity points achieve a base line separation, the
ideal point is the one with additionally the smallest
retention factor for the last eluting component.
The value for the base line separation, the point at

which a peak reaches the base line again, depends on
the particular chromatographic technique or on the
separation efficiency that the particular method of
separation can achieve. In the case of HPLC, to satisfy
the requirements for good resolution, this value should
be about 1.15, for GC it should be about 1.05 and for
TLC it should be greater than 1.3.
As shown by the following comparison of measured and
calculated retention factors for the example of the
selectivity point 442, i.e. 4 segments SCN, 4 segments
SPH and 2 segments SC18, the average deviation between
calculated and measured retention factors is on average
approx. 4%:

The selectivity of a separation depends on, among other
things, how many separating elements or column segments
with different stationary phases (or different polarity
of the latter) can be coupled together and on the total
number N of column segments to be coupled.
a2) For a mixture of 8 triazines, determine the best
possible separation that is possible with a combination
of in each case 10 column segments with cyan o and/or
phenyl and/or C18 groups and/or C18 with polar embedded
amide group in methanol /water 50:50 (v/v) as solvent._
The set comprises in each case at least 10 column
segments with CN as stationary phase (SCN), 10 column
segments with phenyl groups as stationary phase SPH, 10
column segments with C18 as stationary phase (Sc18) and

10 column segments with polar embedded C18 as
stationary phase (SpeC18).
For each of these four different stationary phases,
within the scope of a basis measurement, the respective
retention factor k is determined according to (1) for
each of the 8 different substances to be separated, and
» is shown in the following table:

The chromatograms for these values are shown in Fig. 1.
It can be seen that none of the individual separating
columns is able to solve the separation problem by
itself.


Fig. 1. Separation of triazines on the different
stationary phases
The optimum composition of the stationary phase can be
determined from the calculation of the k values and
selectivities of all possible combinations. In this
case it comprises 180 mm of the phenyl phase, 60 mm of
the C18 phase with polar embedded group and 60 mm of
the cyano phase.
The retention factors calculated for the optimum column
composition are compared against the measured values in
the following table. The deviations are on average
3.4%.

The calculated and measured chromatograms are compared
in Fig. 2. It can clearly be seen that they are in
excellent agreement.


prediction
b) Number_ of possible combinatjons
I) Starting from s = 3 different column segments (SA, SB
and Sc; here: SCN, SPH and SC18) of the same dimension and
a total number N = 10 of coupled column segments, we
get 27 possible combinations, if only two of the three
different column segments are coupled together, i.e.
only SA, SB or SA, Sc or SB, Sc. There are an additional
36 possible combinations if all three different column
segments SA, SB, Sc are coupled together.
Thus, in total, with three types of column segments of
different polarity and a total number of 10 segments to
be coupled, there are 63 possible combinations.
II) If the number of columns to be combined is not
given as exactly 10, but instead the total number N is
given as a range from 6 to 15, with n = 6 to 15, s = 3
the number of possible combinations increases to 625.
III) The use of an additional stationary phase SD, i.e.
s = 4, for the above calculation of the possible
combinations (I), i.e. N = 10, with three stationary
phases gives 960 possible combinations with 2, 3 or 4
stationary phases.
IV) If, on the basis of III), the total number is
extended to at least 6 and at most 15, then with s = 4
and the combinations of in each case 3 phases we get a
total of 2500 possibilities.
By extending the number of available stationary phases
for example to five or more, of combinations with one
another (for example combinations not more than three,
but of four different stationary phases in coupling),
there is a further increase in possible combinations.
Further "fine-tuning" can be achieved by taking into
account ½ or 1/4 segments in the calculation.



The invention is described below on the basis of a preferred example of application for the coupling
of the column segments.
The column comprises one or more column segments and a holder, which in its turn comprises
individual holding elements that can be screwed together, and can if required be screwed to the
connectors at the top and bottom of the respective column.
Independently of this especially preferred embodiment described in detail below, it is also possible
for the individual holding elements to be connected by means of bayonet joints or other types of
joints. Moreover, it is also possible to omit individual holding elements entirely and insert the
column segments into a guide sleeve of the appropriate length with threaded ends, and then press
together the individual column segments axially with the connectors that can be screwed on the
guide sleeve.
The column segments can also be pressed together axially by hydraulic means in an appropriate
device.
An important point in the following especially preferred embodiment is that the inside diameter of
the column remains constant even at the coupling points so that the flow rate is constant
throughout the column.
Furthermore, the column coupling described below can also be used in general for the coupling of
columns, for example of precolumns and main columns.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
The accompanying diagrams show:
Fig. 1: a) an exploded view of a column segment and
b) a schematic representation of the complete column segment,
Fig. 2: a schematic representation of the fitting

of a column segment from Fig. 1b) in a
holding element 30 (Step 1), the fitting of
a further holding element 30' (Step 2) and
the fitting of a further column segment 11'
(Step 3),
Fig. 3: a schematic representation of the coupling
of the top column segment 11 or of the top
holding element 30 and of the bottom column
segment 11" or of the bottom holding
element 30" to the respective adapters and
Fig. 4: a schematic representation of the column
segments and adapters coupled together to
form a column.
The column segment 11 (cf. Fig. 1) comprises a column
tube 13 with a stationary phase 12. The column tube 13,
which can be made for example of metal, plastic,
ceramic or combinations thereof, has a predefined
outside contour with steps 22, 21 and a constant inside
diameter di.
At the head end 14, the tube 13 is provided with a
defined recess 15, which must be larger in diameter d15
than the inside diameter di. The "recess 15 has a
shoulder, for the fitting seal 17. The seal 17 can be
made for example of plastic, metal or some other
material suitable for sealing. The recess 15 receives a
filter or a filter-sieve combination 16, which protects
the stationary phase, and has the ring seal 17 above
it. The wall 18 of the recess 15 serves simultaneously
as a guide during coupling with another column segment
11' or for fitting a male adapter 40, described later.
The tube 13 is shaped at its foot 19 according to a
male connector 21 and is closed by a porous frit 20,
which has the purpose of holding the stationary phase
12 in the tube 13. The porous frit 20 can be of metal,
plastic, ceramic, sintered silica gel, monolithic

silica gel or monolithic polymer, optionally surface-
modified .
The holding element 30 (Fig. 2) receives the column
segment 11 and its coupling. The holding element 30 can
be of metal, plastic, ceramic or a combination thereof.
It has an internal thread 32 at the head end 31 and an
external thread 34 at the foot end 33. Inside the
holding element 30 there is a through-hole 35 with a
step 36, into which the column segment 11 fits. The
step 22 of the column segment 11 bears on the step 3 6
of the holding element 30.
The outside surface 37 of the holding element 30 can be
knurled diagonally or axially for easier manipulation
or can have surfaces for using a wrench.
Assembly of the column segments is illustrated in Figs.
2 and 3:
1. Put the column segment 11 in the holding element
30 in the manner described previously (arrow 1 in
Fig. 2).
2. Screw another holding element 30' fully into the
first holding element 30 at the head end 31. This
locks the column element 11 in the holding
element 30 (arrow 2 in Fig. 2) .
3. Put another column segment 11' into the threaded
holding element 30' (arrow 3 in Fig. 2).
4 . Repeat steps 2 and 3 until the desired number of
column segments 30 is reached.
5. Insert a male adapter 40 into the recess 15 in
the top column segment 11 in Fig. 3 and screw the
adapter screw 41 into the holding element 30.
6. At the bottom end 19 of the bottom column segment

11", fit the female adapter 42 on the projection
21" and screw on the holding element 30 with the
adapter nut 43.
7. The resultant segmented column can now be
connected by means of a capillary connection
(stainless steel, plastic or fused silica) to the
chromatographic system.
If, in the above preferred embodiment, for example a
double-length column segment is to be connected to
other column segments, the double-length column segment
can be held in two holding elements joined together and
can then be coupled.

WE CLAIM
1. Set of elements for the production of a device for separating substances by distributing between
a stationary and a mobile phase, wherein the set includes at least six individual separating
elements coupleable together to form a separating device, wherein the separating elements are
columns segments and each comprise any stationary phase, a carrier element and coupling
means and wherein at least two different stationary phases Si (i ≥ 2) and of each type of
separating element Ti with a defined stationary phases Si, at least three are in each case present
in the set.
2. The set as claimed in claim 1, wherein the set comprises separating elements with at least three
stationary phases of different selectivities SA, SB and Sc and of each of these three types of
separating elements, at least 3, preferably at least 5, preferably at least 7, and especially
preferably 10 or more are contained in the set.



3. The set as claimed in one of claims 1 to 2, wherein the stationary phases of the set
are selected from the group comprising the saturated or unsaturated alkyl groups (C1,
C2, C3, C4, C6, C8, C12, C14, C16, C18, C22, C27, C30), which can be mono-, poly- or
acyclic, ali- or heterocyclic, nitro, cyano, carbonyl, carboxyl, hydroxyl, diol, or thiol
groups, glycidoxy groups, optionally etherified, amino or chiral groups, amide groups,
carbamates, urea, perfluoralkyl groups or perfluoraryl groups and other haloalkyl and haloaryl
groups, polybutadiene groups or other organic polymers, affinity-chromatographic modifications
or ion-exchangeable groups (cation exchangers, anion exchangers, zwitterions), unmodified
support materials (e.g. silica gel) and molecularly imprinted polymers (MIPs).
4. The set as claimed in one of the preceding claims, wherein the length of a separating element, in
particular a column segment, is between 0.5 cm and 50 m, preferably between 1.0 and 12.5 cm
and especially preferably between 1.5 and 2.5 cm.
5. The set as claimed in one of the preceding claims, wherein the set additionally comprises
separating elements or separating devices for carrying out the basis measurement, with the
number of basis measurement elements preferably corresponding to at least the number of
separating elements Ti of different stationary phases Si contained in the set.



6. The set as claimed in one of the preceding claims, wherein the set also comprises multiple
segments or multiple elements, the length or dimension of which corresponds to a multiple of
the length or dimension of a single separating element.
7. The set as claimed in one of the preceding claims, wherein the column segment is essentially
without any dead space.
8. The set as claimed in one of the preceding claims wherein coupling means (15, 21) that are
different from one another, in particular a male and a corresponding female coupling means,
are provided at the top and bottom of the column segment, so that the bottom end of one
column segment can be inserted into a top end of another column segment.

9. The set as claimed in one of the preceding claims, wherein the coupling of the column segments
(11) takes place via holding elements (20), which the column segments receive.
10. A method of production of an optimized device for separating mixtures of substances, wherein
separating elements are coupled or connected, said separating elements containing different
stationary phases Si (i ≥ 2) and being of identical or different dimension, with the optimized
combination of the selectivities and of the respective dimension of the separating elements
being determined from the determined retention factors ki of each of the stationary phases Si (i
≥ 2) that are present, and the separating elements are then coupled or connected to form the
optimized separating device.


ABSTRACT

ELEMENTS FOR SEPARATING SUBSTANCES BY DISTRIBUTING BETWEEN A
STATIONARY AND A MOBILE PHASE. AND METHOD FOR THE PRODUCTION OF A
SEPARATING DEVICE
The invention relates to elements for separating substances by distributing between a stationary
and a mobile phase. Said separating elements comprise any stationary phase and a support
element. The separating elements are part of a set that in provided with at least two separating
elements Ti (i ≥ 2) encompassing different stationary phase Si (i ≥ 2). The set comprises at least
three pieces of each type of separating element Ti with a specific stationary phase Si. The
separating elements can be coupled to each other or interconnected in another manner so as to
form a separating device. The invention further relates to a method for producing an optimized
device for separating substance mixtures.

Documents:

04517-kolnp-2007-abstract.pdf

04517-kolnp-2007-claims.pdf

04517-kolnp-2007-correspondence others.pdf

04517-kolnp-2007-description complete.pdf

04517-kolnp-2007-drawings.pdf

04517-kolnp-2007-form 1.pdf

04517-kolnp-2007-form 2.pdf

04517-kolnp-2007-form 3.pdf

04517-kolnp-2007-form 5.pdf

04517-kolnp-2007-international publication.pdf

04517-kolnp-2007-pct request form.pdf

4517-KOLNP-2007-(09-05-2012)-ABSTRACT.pdf

4517-KOLNP-2007-(09-05-2012)-AMANDED CLAIMS.pdf

4517-KOLNP-2007-(09-05-2012)-DESCRIPTION (COMPLETE).pdf

4517-KOLNP-2007-(09-05-2012)-DRAWINGS.pdf

4517-KOLNP-2007-(09-05-2012)-EXAMINATION REPORT REPLY RECEIVED.pdf

4517-KOLNP-2007-(09-05-2012)-FORM-1.pdf

4517-KOLNP-2007-(09-05-2012)-FORM-2.pdf

4517-KOLNP-2007-(09-05-2012)-FORM-3.pdf

4517-KOLNP-2007-(09-05-2012)-OTHERS.pdf

4517-KOLNP-2007-(09-05-2012)-PA.pdf

4517-KOLNP-2007-(09-05-2012)-PETITION UNDER RULE 137.pdf

4517-KOLNP-2007-CORRESPONDENCE 1.2.pdf

4517-KOLNP-2007-CORRESPONDENCE 1.3.pdf

4517-KOLNP-2007-CORRESPONDENCE OTHERS 1.1.pdf

4517-KOLNP-2007-EXAMINATION REPORT.pdf

4517-KOLNP-2007-FORM 18 1.1.pdf

4517-kolnp-2007-form 18.pdf

4517-KOLNP-2007-FORM 26.pdf

4517-KOLNP-2007-FORM 3.pdf

4517-KOLNP-2007-FORM 5.pdf

4517-KOLNP-2007-GRANTED-ABSTRACT.pdf

4517-KOLNP-2007-GRANTED-CLAIMS.pdf

4517-KOLNP-2007-GRANTED-DESCRIPTION (COMPLETE).pdf

4517-KOLNP-2007-GRANTED-DRAWINGS.pdf

4517-KOLNP-2007-GRANTED-FORM 1.pdf

4517-KOLNP-2007-GRANTED-FORM 2.pdf

4517-KOLNP-2007-GRANTED-SPECIFICATION.pdf

4517-KOLNP-2007-INTERNATIONAL PRELIMINARY EXAMINATION REPORT.pdf

4517-KOLNP-2007-INTERNATIONAL SEARCH REPORT .pdf

4517-KOLNP-2007-OTHERS.pdf

4517-KOLNP-2007-PRIORITY DOCUMENT.pdf

4517-KOLNP-2007-REPLY TO EXAMINATION REPORT.pdf


Patent Number 254140
Indian Patent Application Number 4517/KOLNP/2007
PG Journal Number 39/2012
Publication Date 28-Sep-2012
Grant Date 24-Sep-2012
Date of Filing 23-Nov-2007
Name of Patentee BISCHOFF ANALYSENTECHNIK UND-GERATE GMBH
Applicant Address BOBLINGER STRASSE 23, D-71229 LEONBERG
Inventors:
# Inventor's Name Inventor's Address
1 NYIREDY, SZ. (DECEASED) C/O. RESEARCH INSTITUTE FOR MEDICAL PLANTS, LUPASZIGETI ST. 4, H-2011 BUDAKALASZ
2 BISCHOFF, KLAUS TUTTLINGER STRASSE 9, D-71229 LEONBERG
3 SZUCS, ZOLTAN C/O. RESEARCH INSTITUTE FOR MEDICAL PLANTS, LUPASZIGETI ST. 4, H-2011 BUDAKALASZ
PCT International Classification Number B01D 15/08
PCT International Application Number PCT/EP2006/004744
PCT International Filing date 2006-05-18
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 10 2005 024 154.9 2005-05-23 Germany