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Description  |
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This invention concerns improvements in liquid chromatography, more
especially so-called high performance liquid chromatography.
The process of high performance liquid chromatography is well-known to
those skilled in the art, and will therefore not be described in detail.
One particularly effective technique used in this process is that of
gradient elution wherein the strength of the mobile phase is increased
during a chromatographic analysis. The formation of an appropriate
gradient is achieved in practice by varying the proportions of a mixture
of solvents supplied to the chromatographic column, so that the relative
proportions of the solvents in the mixture reaching the column varies with
respect to time, either stepwise or in a continuous manner.
The known processes for mixing solvents to achieve the required gradient
have a number of disadvantages.
In one arrangement, solvents are supplied to a mixing chamber on the
upstream side of a pump serving the chromatographic column by way of a
proportioning valve which periodically opens and closes conduits from each
solvent reservoir to the mixing chamber. The period of time for which each
responsive conduit is open is controlled electronically, thus controlling
the average composition of the eluent reaching the mixing chamber, and
thus the composition of the eluent reaching the pumping system. Such an
arrangement has the disadvantage that a relatively large mixing chamber is
necessary in order to average the mixture of eluent from the discrete
doses of solvent supplied via the mixing valve, which results in the
wastage of relatively large amounts of solvent, and the corresponding
increase in analysis time. Moreover, the arrangement is complex to operate
when more than two solvents are required and mixing problems can occur if
the operation of the system is not synchronised to match the
characteristics of the solvent pump.
In another system, the respective solvents are each supplied from a
reservoir via a separate pump which is controllable to vary the volume of
liquid pumped with respect to time. The outlets of the pumps are connected
to a common conduit via a T junction, so that mixing of the solvents
occurs on the downstream side of the pump, the proportions of the mixture
being determined by the relative flow rates of the two pumps. The flow
rates of the pumps are controlled electronically in such a manner that the
total flow of liquid is always constant, although the proportion of the
respective solvents can be varied. Such an arrangement has the
disadvantage that the pumps are required to be of very high accuracy in
order to provide the require accuracy of mixing, and are correspondingly
relatively expensive. The use of such a system is thus in practice limited
to a two solvent mixture, since the cost of multiple pumps for a greater
number of solvents would be prohibitive.
It is an object of the present invention to provide a novel system for the
accurate mixing of solvents in the process of gradient elution, which
avoids, or at least reduces, at least some of the disadvantages outlined
above.
In accordance with the present invention there is provided a method of
liquid chromatography utilising a mixture of liquid solvents of which the
mixed proportions are to be varied during the elution process, wherein the
respective solvents are supplied to the upstream side of a single pump
serving the chromatographic column, in each case from a separate liquid
reservoir, there is provided between each reservoir and a corresponding
supply conduit communicating with the suction side of the pump a control
valve providing a variable resistance to liquid flow, a predetermined
liquid pressure drop is maintained across each control valve for the
corresponding solvent, and the flow resistances presented by the
respective control valves are charged with respect to time, by
corresponding adjustment of said valves, in order to produce a desired
variation in the rate of flow of each solvent.
The process of the present invention is based upon the appreciation that,
for liquid with viscosities which are less than or close to that of water,
which is the case for all eluent liquids which are in practice used in
high performance liquid chromatography, the rate of flow of the liquid
through an orifice having a given resistance is substantially only
dependent upon the pressure difference across the restriction and the
density of the solvent. This relationship is set out in the following
formula
##EQU2##
where I=Flow Rate
.DELTA.H=Differential Pressure
S=Specific Gravity
L=Resistance to Fluid Flow
Thus, with appropriate control of the relationships between the fluid
pressures under which the respective solvents are supplied to the
corresponding control valves, and/or the relationship between the
corresponding adjustments of the respective control valves, any desired
elution gradient involving any desired number of eluents can be achieved
simply by corresponding adjustment of the respective control valves.
In practice the control valves may be simple needle valves coupled by an
appropriate mechanical transmission to electronically controlled stepping
motors, the stepping motors being controlled from an electronic
programming system in such a manner as to achieve the desired variation in
the solvent mixture.
The invention is illustrated by way of example in the accompanying
drawings, in which:
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a diagrammatic view of one liquid mixing arrangement in
accordance with the invention,
FIG. 2 is a similar view of another arrangement in accordance with the
invention, and
FIG. 3 is an illustration of a chromatograph produced in one example of a
process according to the invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Referring to FIG. 1, a plurality, for example, four, solvents to form an
eluent mixture are provided in corresponding liquid reservoirs 1 to 4,
within which the prevailing fluid pressure is maintained at some pressure
above atmospheric. Each liquid reservoir is arranged to supply a
corresponding control valve, for example, a needle valve, indicated
diagrammatically at 5 to 8, via a corresponding conduit 9 to 12. The
downstream side of the control valves 5 to 8 are coupled via a 5-way T
junction to a conduit 13 coupled to the inlet of a liquid pump, not shown,
which is arranged to serve a chromatographic column in a manner well-known
in the art. Each of the control valves 5 to 8 is arranged to be adjusted
in response to signals from an electronic control system 14. The broken
lines 15 to 18 indicate diagrammatically a servo control connection
between the control system 14 and each of the valves 5 to 8.
As already explained above, the rate of flow of each of the solvents from
the reservoirs 1 to 4 through the corresponding valves 5 to 8 will depend
in each case upon the resistance provided by the needle valve, which is in
turn a function of the degree of adjustment thereof effected by the
control system 14, the specific gravity of the respective solvent, and the
pressure drop across the respective valve, that is to say the difference
in the fluid pressures prevailing in the respective one of the conduits 9
to 12, on the one hand, and the conduit 13 on the other hand. If this
pressure drop is arranged to be same in each case, for example by
arranging the liquid reservoirs 1 to 4, the valves 5 to 8 and the liquid
pump all on the same level so that there is effectively no head of liquid
on the upstream side of the pump, then the rate of flow of liquid in each
case will be a function solely of the specific gravity of the respective
liquid and the flow resistance presented by the respective control valve.
Therefore, the liquids can be mixed in the desired proportions simply by
correspondingly controlling the adjustment of the respective needle valve
5 to 8 after adjusting the degree of opening in accordance with a
correction factor determined by the density of the respective liquid.
Referring now to FIG. 2, there will be described one specific example of
the process in accordance with the invention. Two solvent reservoirs
indicated diagrammatically at 101 and 102 were arranged on the same level
as a liquid pump (not shown) feeding a chromatographic column, so that
there was no head of liquid on the upstream side of the pump. The pump
utilised in this example was a known chromatographic solvent pump supplied
by H. P. L. C. Technology Limited, Type No. R R 065. The solvent
reservoirs 101 and 102 comprise closed containers of which the free space
unfilled by liquid was coupled to a source of gas pressure at 10 p.s.i.
(0.67 bar) via a conduit 120. Liquid supply conduits 109 and 110 coupled
the solvent reservoirs to the inlet side of corresponding needle valves
105 and 106 of which the outlets were coupled via a tee piece to a conduit
113 coupled to the upstream side of the liquid pump. Check valves 121 and
122 were provided between the needle valve and the tee piece to avoid
syphoning of the solvent back into reservoirs 101 and 102 with the liquid
pump in the idle condition. The needle valves 105 and 106 were coupled by
a gearing indicated diagrammatically at 123 and 124 to stepping motors 125
and 126 of which the position could be controlled by way of a
microprocessor 127 via an interface 128. The microprocessor was associated
with a keyboard 129 and video display unit 130.
In the examples to be described, the solvent contained in the reservoirs
101 and 102 was methyl alcohol of which one sample was colourless, and the
other was provided with an ultraviolet light absorbing dye. The
microprocessor 127 was caused to adjust the stepper motors 125 and 126
with respect to time according to a software programme which caused the
settings of the needle valves 105 and 106 to be adjusted relatively to one
another in order to change the composition of the solvent reaching the
conduits 113 from one percent to 99% of the dye containing solvent in one
percent steps. The absorbancy of the resulting eluent at 300 nanometers
wavelength was monitored by an ultraviolet detector type LC3 supplied by
Pye Unicam Limited, the output signal from the detector being utilised to
control a chart recorder in known manner.
Since the solvent contained in both reservoirs 101 and 102 was the same, no
adjustment of the control programme to take account of relative specific
gravity of the solvent was necessary, and the needle valves 105 and 106
were simply adjusted so that the respective flow resistances varied
inversely with respect to one another to give a constant liquid flow rate
to the pump of 6 ml per minute. The resulting chromatograph is indicated
in FIG. 3. Thus it will be seen that simply by servo-controlled actuation
of the needle valves 105 and 106, an accurate adjustment of the relative
proportions of the solvent in the mixture was obtained, without the
requirement for the use of a large volume mixing chamber, and without the
need for expensive variable volume pumps to control the flow of each
liquid.
It will be appreciated that various modifications to the invention as
described may be made without departing from the scope thereof. For
example, although in the example illustrated the pressure drop across each
needle valve was the same for each liquid, it will be appreciated that
these pressures may differ provided that a corresponding allowance is made
for this factor when adjusting the control valve. In a particularly
advantageous arrangement, the need to correct the settings of the control
valves to compensate for the use of liquid of different density may be
avoided when the liquid supply reservoirs are so arranged that a positive
head of liquid is present on the upstream side of each control valve, the
head of liquid being maintained the same in each case, so that the
pressure drop across each control valve is directly proportional to the
specific gravity of the corresponding liquid. An appropriate mechanism is
thus arranged to compensate for the different volumes of solvent supplied
from the respective reservoirs. For example, a float valve may supply
solvent to each reservoir at the same rate as it is withdrawn therefrom.
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