Note: Descriptions are shown in the official language in which they were submitted.
lOSOZ98
This invention relates to an improvement in photometric measuring
systems.
In analysis of very small quantities of liquids, it has been
recognized that the physical conditioning of the fluid must be done very
carefully. Thus, ~or example, in the field of liquid chromatography wherein
very small, continuously-flowing streams of liquid are measured, care is
taken to minimize mechanical and thermal disturbance of the liquid stream
between the chromatographic column and analytical apparatus in which the
liquid stream from the column is to be continuously analyzed. The primary
objective is to present, to a transparent sample cell, the precise sequence
of changing liquid composition that leaves the chromatography column.
The rationale and particulars of such apparatus are described in
the art. For example, see United States Patent 3,674,373 to Waters, Hutchins
and Abrahams which involves a refractometer particularly well adapted to
receive such a liquid stream. In general, the approach is to minimize the
conduit path through which the liquid to be analyzed must travel and to
provide a
L -1-
~'
.
42-041
1050Z98
maximum thermal-conditioning of the liquid within such a
minimized path. This generally illustrates the art-recognized
importance of careful handling of sample liquid between its
point of origin and the sample cell in which it is to be
5 ' subjected to analysis, usually analysis which measures an
effect of the sample liquid stream on some radiation directed
into a flow-cell through which the stream passes.
Investigators have also realized that some attention
must be given to the physical condition of the fluid even
after it enters the flow-cell. Consequently, flow-cells have
been made ever smaller to avoid mixing and peak-spreading
effects and, in some cases, a positive thermal equilibration
of the cell with the liquid has been sought in order to avoid
' light-shimmering effects along the cell walls. Moreover,
the cells are usually positioned with outlets so placed
that any entrained gas bubbles tend to be carried upwardly
out of the cell. It is noted that U.S. Patent 3,666,941
to Watson describes a conical bifurcated cell wherein the
, larger end of the cell faces the light source, thereby
forming means to gather a maximum amount of fluorescence-
exciting radiation. Applicant's discovery, to be detailed
below, is based upon a major improvement in flow-cell
construction which solves a problem quite different than that
described by Watson but which, like Watson's apparatus, is
25 I particularly useful in combination with liquid chromotograhy
applications.
'. I
.
- 42-041
., . _
!
1050~98
A recent patent, U.S. Patent 3,792,929, to Alpert, it
has been noted, seems to disclose a conical sample-holding cell. I
The patent related to static-sample devices and in no way involves
fluid lenses of any type; although the patent came to the atten- ¦
tion of the instant inventor after an error resulted in the word
"field" appearing as "fluid" in the title of the Alpert patent.
Moreover, the apparent and relative dimensions of the Alpert
cell would not allow its effective use in most continuous-flow
1 monitoring systems such as are encountered in liquid chromato-
; graphic work and the like.
! SummarY of the Invention
It is an object of present invention to provide an
improved liquid chromatographic system of the type utilizing ¦
a photometric analytical means.
It is a further object of the invention to provide a
means for operating a photometric process whereby it is possible
! to minimize the size of sample volume of a flow cell without
unduly affecting the performance of the photometer.
Another object of the invention is to provide a
novel process of analyzing liquid by photometric methods and
a novel photometer for carrying out such analysis. I
Another object of the invention is to provide a
novel and improved sample-receiving flow-cell.
Other objects of the invention will be obvious to
` those skilled in the art on reading the instant disclosure.
~ 42-041
~050298
The above invention is based on the discovery that
substantial spurious radiation signals are generated by a lens-
type effect caused by laminar-flow patterns at the interface
of compositions differing in refractive index; the effect is
particularly troublesome in small cylindrical photometer
sample-cells. These laminar flow patterns will sometimes be
called "dynamic liquid lenses" in this description. In general
the worst problems have been encountered in flow-cells in the
microliter range, say flow-cells having a diameter of less than
~ about 2 millimeters. In the usual situation the flow path of
an ultra violet absorptometer cell is selected to be one
centimeter in length, and a flow cell of 2 millimeters maximum
diameter will have a volume of less than about 32 microliters.
As the diameter increases the lens effect caused by a given
rate of laminar-flow tends to decrease; but a mere increase
in diameter of a cylindrical flow path to avoid the lens
effect is not practical because the increased diameter would
result in either (1) a large increase in the volume of the
tube or (2) a substantial decrease in length of the tube.
A large increase in volume is untenable because the ability
;j of the apparatus to detect very small samples would be
substantially limited by dilution factors. The length of the
cell cannot be mar}cedly reduced without proportionately decreas-
ing the magnitude of light absorbed by a given solution flowing
through a cell. Still other conceivable tube configurations
would give disadvantageous liquid flow patterns.
,
_ 42-041
lOSOZ98
Because the problem of these dynamic fluid lenses
is primarily encountered at the point of changing compositions,
its solution has been found io enhance both the quantitative and ¦
qualitative analytical capabilities of liquid chromatographic
systems and like analytical systems where constantly changing
compositions are inherent in the method. However, the apparatus
is useful in other lens-inducing situations encountered in the
process industry; e.g., where the dynamic fluid lens may be
induced by temperature change or other phenomena that result in
; formation of a refractive index gradient within the flow-cell.
On discovering the nature of the problem associated
with such small flow-cells, applicant has devised a simple con-
structional solution which substantially eliminates the problem:
I; he has provided a flow-cell whereby the lens effect is rapidly
dissipated by a progressive increase in the crossectional area
of the flow-cell along the flow path. Thus, the wall of the
flow-cell advantageously forms a diverging surface of rotation
whereby the walls form an angle of divergence of at least
about one angular degree with the axis of the cell. An angle
of about 1.5 or slightly greater provides sufficient widening
to substantially dissipate the undesirable effect of the
dynamic liquid lens formed at the interface of water and most
organic solvents. The improvement is laxgely achieved by
collecting refracted light, which would have otherwise been
_ 5_
4~-041
(~
lOSOZ98
absorbed on the wall of the cell, but it is also believed
the reduction in velocity of the stream during its transit
through the cell--usually a reduction of over 50%--causes a
l! dissipation of the lens effect itself which reduces the amount
1 of refracted light directed against the walls of the cell.
Angles of divergence between the axis of the flowpath and the
wall of the cell of 1 to 3 are most advantageous; larger
angles only become problems because they usually dictate a larger
Il cell size.
l, In liquid chromatographic applications, best results
will be achieved if the apparatus to be used with the flow-
cell is selected to achieve the most ideal flow pattern
possible, i.e., the flow pattern most nearly achieving plug
Il flow. This is true of all flow in a liquid chromatographic
system: flow from sample injection to the column and flow
between the column and the analytical component of the system.
Such apparatus is available: an injector advantageously used
is that available under the trade description Model U6K Injector
~ by Waters Associates, Inc. A pumping system, advantageously
1 used to feed liquid into a high pressure column, is that
available from the same source under the trade designation
Model 6000 Solvent Delivery System~ However, as will
be obvious to those skilled in the art, other such apparatus
will be generally useful in many applications in which the
-6-
~ 1~ ~, 42-041
1050~98
~ instant invention is advantageously used.
¦~ It will also be obvious to those skilled in the art
¦~ that a number of modifications can be made in the shape of the
wall structure of the flow-cell. For example, further enlarge-
ment of the cell conduit over that defined minimal conical
shape will yield an operable cell that will avoid the effect
of the dynamic liquid lens but will also be larger in size and
therefore less favorable for many applications. Such enlargement
is nonfunctional with respect to the present invention. However
other such shapes including such as catenoidal horns, hyperbolic
horns, parabolic and hyperbolic suraces as well as similar
surfaces of revolution are all intended to be covered by the term
"generally truncated cone" as used in this application. Such
shapes may on some occasions be favorable in view of effects
caused by special flow properties of the fluid components which
form the dynamic lens, temperature profiles across the cell,
friction effects along the surface of the wall or the like.
"Generally conical", therefore, is meant to include any flow-cell
wherein the inlet port is smaller than the outlet port and the
cross section of the cell is progressively larger as measured
closer to the outlet port.
¦ It is to be realized that the most important structura:
aspect of the invention relates to the relationship of the con-
¦ ical cell to the direction of the lightpath: the larger end of
the cone must be toward the detector. It is possible, however,
lOSOZ98
to reverse the direction of flow of the liquid to be analyzed through the
ce]l. Best practice is to avoid this situation or, if for some reason it is
desirable, to arrange the attitude of the cell so that any minute gas
bubbles can be displaced upwardly toward the outlet port of the cell.
In chromatographic related analytical operations and other such
operations which monitor microliter quantities of a flowing sample, the
length-to-average diameter ratio of the flow cell is advantageously at
least 5 to 1. It is primarily the monitoring of such small samples, rather
than inherent optical considerations, which make angles of divergence
greater than 3 undesirable for many applications.
One additional advantage of the apparatus disclosed herein is
the fact that, for some applications, it allows the light source to be
brought (physically, or by optical means) closer to the sample cell without
undue losses of light by refraction and light scattering occuring primarily
at the interfaces of gas-lens and liquid-lens interfaces.
Thus, in accordance with one broad aspect of the invention there
is provided, in a photometer of the type utilizing a light source, a sample
cell adapted to transmit a continuously-flowing liquid to be analyzed from
an inlet port near one end thereof through a flowpath to an outlet port near
the other end thereof, and a means for measuring the absorption of light in
said sample cell, the improvement wherein said photometer comprises a light
detector forming means to receive substantially all non-absorbed light trans-
mitted from said sample cell, means for eliminating effects of liquid lenses
comprising a generally conical sample cell, a smaller end of said sample
; cell being nearer said light source, such that there is substantially reduced
loss of light refracted by said liquid lenses on walls of said sample cell.
`In accordance with another aspect of the invention there is provid-
` ed, in a process for measuring the radiation absorptivity of a flowing
liquid sample which comprises a plurality of sequential liquid compositions
-8-
1050Z98
in a laminar flow mode, the improvement comprising substantially eliminating
the interference of dynamic liquid lenses with said measuring by a) feeding
sai.d liquid into a generally conical sample cell proximate a smaller cross-
sectional end thereof, b) removing said liquid from said sample cell at a
larger cross-sectional end thereof, c) and measuring the radiation
absorptivity of said liquid through said cell, said measurement being
carried out by detection at said larger end of said cell, substantially all
of the non-absorbed radiation from a source proximate the smaller end of
said cell.
In accordance with a further aspect of the invention there is
provided a sample cell having an inlet port, an outlet port and a flowpath
therebetween, said flowpath formed of a truncated, generally conical chamber
forming means for overcoming refraction of radiation onto walls of said
chamber during analysis of liquid passing therethrough, which cell has
transparent end members adapting it to serve also as a path for transmitting
light, said flowpath having a length-to-average diameter ratio of at least
5: 1.
; According to still another aspect of the invention there is
provided, in a liquid chromatographic analytical apparatus of the type having
a liquid chromatographic column adapted to emit a liquid stream comprising a
series of sequentially-arranged liquid compositions, and means for conducting
said stream to a photometer of the type comprising a sample flow-cell form-
; ing a conduit for said liquid stream~ a means to provide a source of
radiation, and a radiation detector arranged with respect to said conduit
to form a radiation path therethrough~ the improvement including means for
eliminating distortion of said radiation by dynamic liquid lens effects
comprising a flow-cell which forms a truncated, generally conical, flowpath,
a smaller end of said cone being nearer said radiation means, such that there
is no substantial loss of refracted radiation on walls of said flow-cell.
-8a-
lOSOZ98
Illustrative Example of the Invention
In this application and accompanying drawings there is shown and
described a preferred embodiment of the invention and suggested various
alternatives and modifications thereof, but
''
;~
,.,
. .
~, :
:.~
:
~ 8b-
.
l~ 42-041
j., I
i 1050298
it is to be understood that these are not intended to be
exhaustive and that other changes and modifications can be
made within the scope of the invention. These suggestions
are selected and included for purposes of illustration in order
that others skilled in the art will more fully understand the
invention and the principles thereof and will be able to modify
it in a variety of forms, each as may be best suited in the
condition of a particular case.
,,''"
" In the drawinqs:
¦ Figure l is a schematic diagram of an analytical, lO apparatus constructed according to the invention.
, Figure 2 is a section of a flow-cell constructed
; according to the invention.
Figure 3 is a graph illustrating the output signal
"; of an ultra-violet absorption-measuring apparatus using a
~;' 15 ¦ conventional cylindrical flow-cell.
' Figure 4 is a graph illustrating a chart similar to
: .
that shown in Figure 3 but obtained utilizing a flow-cell
constructed according to the invention.
~;, Figure 1 illustrates an analytical system 10 com-
¦ promising a source 12 of a liquid to be analyzed, a liquid
j chromatography column 14, and an ultra-violet absorbtometer
~:~
, . I
',~
~ 9_
... '11 ,
42-041
!
lOSOZ98
16 comprising a light source 18, an interference filter 20,
a lens system 22, front windows 23, main housing wall of a
sample cell 24, a rear window 26 and photoelectric detector 28.
Signals from photo detector 28 and a reference detector 28a are
processed according to known techniques to provide a suitable
electronic signal which may be used as a control means or as is
more frequent, to provide a visible recording on a recorder
means 30.
The single novel feature in Figure 1 is the sample
cell 24 which incorporates the conical flowpath 32. However,
this innovation directly enhances the performance of the entire
system by providing means to take the liquid output from chroma-
tographic column 14 and process it in the ultra-violet absorption
apparatus so that the resulting light reaching detector 28 is
substantially free of detrimental loss of light due to the
influence of dynamic liquid lenses.
In the apparatus of Figure 1, the light source is rated
at 2.4 watts and has principal wave length of 253.7 nanometers.
The volume of the sample cell, best seen in Figure 2, is about
12.5 microliters: it is about 0.04 inches in diameter at the
inlet end, about 0.06 inches in diameter at the outlet end and
- ¦ about 0.394 inches in length. A reference flo~-cell 34 is posi-
tioned within cell assembly 36, as is common in the photometric
¦l analysis of liquids. This cell may be empty, full of a stagnant
- 25 ¦ liquid or have a flowing reference fluid therein.
., 11
¦1 -9a-
Il
il 42-041
Il
. 105(1 ;~98
¦ Figure 3 illustrates graphically the type of detection
problem which can be encountered in radiation-absorption
I'lanalysis because of interference in ultra-violet transmittance
¦Iby dynamic liquid lens as they move through a thin cylindrical
~sample cell.
I In each of Figures 3 and 4, there is an initial peak
¦60 caused by a calibration fluid - a standard dichromate solution
flowing through the cells at a rate of one milliliter per
l minute. The next rise 61 in each curve, is merely an adjustment
¦ of the zero level of the recorder. At this point, each curve
has a relatively flat reference level indicative of the low
ultra-violet absorption of water.
This reference level is flat for the continuous feed
¦ in Figure 3 but interrupted by abrupt drops in light transmission
1 when injections of aqueous methanol solution are introduced into
the column. These apparent increases absorptivity in absorption
are caused by the refraction from dynamic fluid lens formed by
the methanol-water interface and the interfaces of various
Imixtures thereof. Once refracted, a substantial portion of light
is absorbed on the parallel walls of the conventional flow-cell.
The valleys 64 of Figure 3 illustrate the effect caused
¦Iby a transition from water flow of .3 ml/minute to a flow of
1-3 ml per minute of a 10% aqueous solution of methanol. This
¦ solution is added through a sample loop over a period of about 3.3
minutes. Then, as water returns flushing the loop, there is an
¦lupward displacement 65 of the curve caused by the dynamic
~ 42-041
,. . I,
1050298
liquid lens ncw being formed of the water flush flowirg behind
¦ the methanol solution. After the flushing with water is com-
pleted, fluid-lens induced displacement subsides until another
I injection of water-methanol solution is started.
I Equivalent injections made in the same system, except
for the use of a flow-cell as shown in Figure 2 result in no
reduction in transmission, when methanol is added. Nor is
there any substantial increase in transmission when the water
~ flush occurs. Such points are identified as 64a and 65a in
Figure 4.
¦ It is stressed that it is intended to cover the appara-
¦ tus of the invention, whether or not it exists in non-assembled
parts, wherein some intrinsic or extrinsic system is so related
; ¦ to such parts that the system facilitates the collection of the¦ parts for assembly at a particular place or places. Such a
system could include co-ordinated shipping instructions, a co-
ordinated parts-packaging system, assemply instructions or any
other system which facilitates assembly of apparatus into a
¦ functioning system as defined in claims explicitly relating to
¦ assembled systems.
It is to be understood that the following claims are
intended to cover all of the generic and specific features of
; i the invention herein described and all statements of the scope
of the invention which might be said to fall therebetween.
.1 1
., ~
.,:, I ,
