Centrifuging process for sample characterization

A process for characterizing at least one sample comprising at least one substance (S.sub.i), in which at least one substance (S.sub.i) in the sample is identified and in which at least one quantity (G.sub.ji) which characterizes a substance (S.sub.i) in the sample is determined, at least the following process steps being carried out successively or simultaneously in an analytical centrifuging device: PA0 a) centrifuging the sample in an analytical centrifuge, PA0 b) exposing the sample to monochromatic light, preferably laser light, PA0 c) detecting light scattered by the sample, PA0 d) identifying at least one substance (S.sub.i) by a spectral evaluation of the inelastically scattered fraction of the scattered light which was detected in step c), e.g. by means of Raman scattering, and PA0 e) determining at least one quantity (G.sub.ji) for a substance (S.sub.i) by evaluating the state of the sample which it has entered as a consequence of the centrifuging in step a).

BACKGROUND OF THE INVENTION
 (a) Field of the Invention
 The present invention relates to a process and a device for characterizing
 a sample which has at least one substance dissolved or dispersed in it, by
 a combination of analytical centrifuging and spectral evaluation of the
 inelastically scattered fraction of the scattered light emitted by the at
 least one substance. In particular, the invention relates to a process and
 a device for determining quantities characteristic of dissolved or
 dispersed substances in the form of particles, for example the density,
 molecular weight, molecular weight distribution and particle size
 distribution of the substances in the form of particles, providing extra
 information regarding the structure of the particles. In the context of
 the invention, "particle" is used to denote the investigated
 substances/materials in the form of dissolved and/or dispersed particles,
 this including substances both with low molecular weight and with high
 molecular weight.
 (b) Description of Related Art
 One known process for determining the characteristic quantities mentioned
 above is analytical centrifugation, although owing to the size of the
 substances which are generally to be investigated, in the form of
 particles, use is predominantly made of analytical ultracentrifugation.
 Moreover, the present application is to be understood such that other
 analytical centrifuges, for example a disc centrifuge, that is to say
 centrifuges in which only a comparatively low speed of rotation can be
 obtained, may also be used in the context of the process according to the
 invention. Nevertheless, the present invention will be explained below
 with reference to analytical centrifuges, and this being the case it
 should be noted that the term "ultracentrifuge" used in the context of the
 present invention always means an analytical ultracentrifuge (AUC).
 In an ultracentrifuge, that is to say a very fast centrifuge with which
 speeds of 60,000 rpm or more can be obtained, it is possible to separate
 (fractionate) mixtures of substances which have different density and/or
 size. This being the case, ultracentrifuges can be used in a variety of
 ways, an overview being found, for example, in an article by W. Machtle
 "Analysis of Polymer Dispersions with an Eight-Cell-AUC-Multiplexer: High
 Resolution Particle Size Distribution and Density Gradient Techniques" in
 S. E. Harding et al. (Ed.) "Analytical Ultracentrifugation in Biochemistry
 and Polymer Science", Royal Society of Chemistry, Cambridge England 1992,
 Ch. 10.
 The most important AUC measurement techniques are the sedimentation (S) run
 which fractionates the particles according to size, that is to say
 according to their molecular weight distribution or particle size
 distribution, the equilibrium run with which it is possible to determine
 the weight average of the molecular weight of the particles, and the
 density gradient (DG) run which fractionates according to density, that is
 to say according to chemical nonuniformity, number of components, degree
 of grafting, etc.
 As indicated above, centrifuging can take place in a variety of ways:
 1. Sedimentation run (S run)
 During the S run, the sample(s) to be analyzed is or are subjected to a
 constant or increasing speed of rotation, starting from about 600 rpm and
 increasing to about 60,000 rpm. Since the sedimentation rate of the
 particles inside the sample is proportional to the square of the
 rotational speed, particles of a given size sediment 10,000 times faster
 when the rotational speed is increased from, for example, 600 to 60,000
 rpm. Through measurement of a quantity, for example absorption,
 characteristic of the particles contained in the sample, it is possible to
 observe the kinetics of the particle sedimentation during an S run of this
 type. The speed at which the particles move to the bottom of the sample
 holder depends on their diameter. An S run therefore makes it possible to
 draw direct conclusions regarding the particle size and its distribution
 within a sample, since determination of a quantity characteristic of the
 sample at time T over the entire sample gives access to the "true" state
 of the sample in terms of the distribution of the particles within it. To
 this end, it is necessary to know particular auxiliary quantities, for
 example the particle density, the absorption coefficient or the specific
 refractive index increment.
 A particularly useful way of separating a sample which contains particles
 with low and high molecular weights is to carry out a layering run using
 layering cells, as can be obtained from Messrs. Beckman for example. In
 addition to at least one analysis chamber, which contains the sample, a
 layering cell of this type contains at least one store chamber which
 contains solvent. During the ultracentrifuging run, this solvent is then
 released as a result of the fact that, above a certain ultracentrifuge
 speed, the partition between the chamber containing the sample and the
 store chamber for the solvent is removed, and the solvent can thus enter
 the chamber containing the sample.
 During the layering run, the ultracentrifuge is thus firstly started up,
 then above a certain speed the solvent in the store chamber enters the
 sample chamber and forms a layer on the solution which it contains. During
 this run, the large particles sediment in the direction of gravity, while
 the small particles, which are incapable of sedimenting on account of
 their small size, remain at the interface between the sample solution and
 the solvent forming a layer. The effect of the presence of the extra
 solvent is that the band attributable to these small particles broadens
 since the particles partly diffuse into the extra solvent.
 This method is extremely useful for separating samples which contain both
 small and large particles together.
 2. Equilibrium run
 In an equilibrium run, the weight average of the molecular weight Mw of
 particles can be determined. To do this, samples with different particle
 concentration c are centrifuged at the same time at one rotor speed until
 steady state conditions are reached. The relevant concentration
 distribution is determined from the radial concentration profile of the
 particles under steady state conditions within the measuring cell, and
 using the abovementioned auxiliary quantities which need to be determined
 separately, an apparent molecular weight M.sub.c is calculated. Plotting
 1/M.sub.c against c and extrapolating to c=0 gives M.sub.W. Using this
 method, it is possible to register particles with a weight average of the
 molecular weight of about 300 to 1.times.10.sup.8 g/mol. Optical
 techniques involving an interference or schlieren method are generally
 used for determining the radial concentration profile in the individual
 samples. When schlieren methods are used, however, all other things being
 equal, the Z average of the molecular weight Mz is determined instead of
 M.sub.W. By evaluating M.sub.Z and M.sub.W it is possible to draw
 conclusions regarding the molecular weight distribution and therefore the
 nonuniformity of a sample, for example a polymer.
 3. Density gradient run (DG run)
 A further option for ultracentrifuging involves mixing two solvents having
 different densities with the particles to be examined, and then
 centrifuging this mixture. The two differently dense solvents then form a
 density gradient in the sample, in which the particles are ordered
 according to their own density, which is to say they will stay at a fixed
 radial position where the density of the solvent mixture surrounding them
 corresponds to their own density. This method thus makes it possible to
 separate particles from one another according to their density, and thus
 according to their chemical nature. For example, polymer mixtures can in
 this way be broken down into their individual components. It is in this
 way possible, for example, to check that a block copolymerization has been
 brought successfully to its conclusion, or to determine the average and
 distribution of the degree of grafting in a graft dispersion.
 However, although ultracentrifuging is an elegant way of fractionating
 samples according to molecular weight, particle size and density of the
 particles which they contain, this method does not in principle allow
 chemical identification of the fractions.
 For this reason, ultracentrifuging has in the past been coupled with
 determination of absorption and fluorescence spectra during the
 ultracentrifuging run, in order to make it possible to draw conclusions
 regarding the "chemistry" of the particles contained in the sample.
 However, in particular if the particles contained in the sample are
 organic in nature, these methods provide only minor advantages, since the
 (usually) organic materials exhibit no, or at best very similar, UV/VIS
 absorption and/or fluorescence. Further, it is often necessary when
 recording fluorescence spectra to correspondingly label the particles to
 be examined, and this generally entails an additional working step.
 SUMMARY OF THE INVENTION
 The object of the present invention is accordingly to provide an
 ultracentrifuging process with which it is possible, in a simple and
 accurate fashion, to identify various particles in a sample during or
 after their separation in a centrifuge.
 This object is achieved by the ultracentrifuging process according to the
 invention. This process represents a process for characterizing at least
 one sample comprising at least one substance (S.sub.i), in which at least
 one substance (S.sub.i) in the sample is identified and at least one
 quantity (G.sub.ji) which characterizes a substance (S.sub.i) in the
 sample is determined. This being the case, according to the invention, at
 least the following process steps are carried out successively or
 simultaneously in an analytical centrifuging device:
 a) centrifuging the sample in an analytical centrifuge, preferably an
 analytical centrifuge. In this context, an analytical ultracentrifuge is a
 centrifuge which rotates fast enough to separate even materials with
 relatively minor differences in density. In the scope of the invention,
 the duration and conditions of the centrifuging, for example the speed,
 the time profile of the speed, the duration of the run or the sample
 geometry may be tailored to the relevant working requirements. Likewise,
 the structure, for example the number of sample chambers and the way in
 which they are arranged, of the ultracentrifuge which is used may be
 tailored to the relevant requirements. Further, the centrifugation may,
 depending on the separation problem, be carried out as a sedimentation,
 density gradient or equilibrium run, as respectively defined above.
 b) Exposing the sample to monochromatic light, preferably laser light. This
 light is scattered by the sample.
 The frequency of the applied monochromatic light is chosen such that
 resonant amplification of the scattered light or fractions of the
 scattered light takes place. In this way, resonance can be created in the
 Raman effect discussed in the next step, and in many cases this is the
 only way in which it is possible to detect substances present only at
 trace levels.
 c) This light scattered by the sample is detected. To do this, it is
 possible to use any optical detection systems which permit detection
 according to spectroscopic bands, for example photomultipliers,
 photodiodes, two-dimensional detectors (CCD photodiodes, etc.) in
 conjunction with spectrographs, gratings, prisms and color or interference
 filters. In particular, all scattered light fractions should be picked up
 which have a wavelength shift relative to the applied light, and are thus
 due to inelastic light scattering from the sample, for example molecular
 or lattice modes. This wavelength shift is generally referred to as the
 Raman effect or Brillouin effect.
 Preferably, the light back-scattered from the sample is detected. This
 makes it possible to examine even samples which are opaque or weakly
 transparent, in particular optically thick polymer samples.
 d) Identifying at least one substance (S.sub.i) by a spectral evaluation of
 the inelastically scattered fraction of the scattered light which was
 detected in step c), i.e. the Raman scattered light. In this case, Raman
 spectroscopy is thus used to identify at least one substance (S.sub.i) in
 the sample, that is to determine its chemical composition. A number of
 Raman spectroscopy methods which are useful for the invention have been
 described in B. Schrader, Infrared and Raman Spectroscopy, Weinheim, 1995
 and L. Markwort, B. Kip, E. Da Silva, B. Roussel, Applied Spectroscopy, 49
 (1995) 1411. In this context, it is not absolutely necessary to determine
 the full chemical composition of the relevant substance. In many cases, it
 is sufficient for substances in a mixture to be discriminated
 qualitatively from one another in a sample. Nevertheless, it is in certain
 cases also necessary or desirable to determine not only the chemical
 composition of a substance in a sample, but also its bonding state on the
 basis of the measured molecular modes. A particular advantage with Raman
 spectroscopy is that it is not sensitive to polar compounds, and this
 permits measurements in an aqueous medium without the Raman spectra of
 other components of the samples having the water bands superposed on them.
 It is further possible, using new high-sensitivity spectrometers which
 work primarily with back-scattering, for example a combination of notch
 filters, simple spectrographs, CCD arrays and high-efficiency imaging
 optics, etc., for the measuring times to be shortened to seconds.
 Combining Raman spectroscopy with microscopes and fibre optics allows
 measurements with .mu.m spatial resolution, and also for the measuring
 head and spectrometer to be placed at different locations, a point which
 will be dealt with in further detail below.
 Further, the identification of a substance in the sample may also take
 place through evaluation of the intensity of the elastic scattered light
 fraction. The spectrum or intensity of fluorescent light emitted by the
 sample may also be used supplementarily, in order to determine the
 identity of substances in the sample. In this case, the determination of
 the fluorescence and light scattering may be determined in both the
 forward and back directions.
 For determining absorption, use is made of a dual beam spectrograph
 especially tailored to the equipment set-up in the ultracentrifuge, in
 which one beam of the applied monochromatic light is directed into the
 sample, and a parallel beam of the same light is directed into the
 corresponding pure solvent.
 The refractive index may be determined using an interference or schlieren
 optical method.
 Of course, the process discussed here, or the device used in this context,
 may be coupled with a corresponding database of, for example Raman
 spectra, and thus permit fast identification even of an unknown substance
 in the sample. In particular, this makes it possible for the process
 described here to be used for routine analysis of samples, in particular
 polymer dispersions. Examples which may be mentioned include, in
 particular: styrene, butadiene, acrylate, acrylonitrile, amide, etc.,
 homo- and copolymers, and inorganic or organic pigments.
 Further, the process according to the invention is particularly useful for
 examining samples of unknown composition and the distribution of known
 substances within a sample.
 e) Determining at least one quantity (G.sub.ji) for a substance (S.sub.i)
 by evaluating the state of the sample which it has entered as a
 consequence of the centrifuging in step a). In this process step, the
 characteristic quantities of a substance (S.sub.i) which are determined
 are those which can in any case be determined using ultracentrifuging
 processes, cf. W. Machtle, S. Harding, AUC in Biochemistry and Polymer
 Science, Cambridge, 1991; W. Machtle, Angewandte Makromolekulare Chemie,
 162 (1988) 35; W. Machtle, Colloid and Polymer Science, 262 (1984) 270.
 Preferably, the quantities (G.sub.ji) determined are the density or the
 sedimentation rate or sedimentation coefficient of a substance (S.sub.i)
 in the sample.
 From the characteristic quantities determined in this process step, it is
 then possible in an additional evaluation step to determine the desired
 values, for example molecular weight distribution or particle size
 distribution.
 Preferably, a difference in the refractive index at various points in the
 sample is used to determine the quantity (G.sub.ji) of a substance
 (S.sub.i). It is in this way, for example, possible for domains of
 substances in the sample to be determined after the ultracentrifuging.
 Under certain circumstances, the substance of interest must first of all
 be found in the sample, because it is not optically distinguishable from
 the rest of the sample, but is separated by the ultracentrifuging in terms
 of density, size, etc. and subsequently it is possible for the individual
 fractions to be examined spectroscopically. This is often the case with
 samples of a mixture of polymers having different structures but the same
 basic chemical composition.
 The measurement of the differences in refractive index along the sample
 may in this case be carried out using any known process for determining
 the refractive index of a sample. As a rule, a separate light source is
 provided for these measurements, in addition to the monochromatic light
 source mentioned in step b). It is, however, also conceivable to deviate a
 fraction of the monochromatic light for this case, so that only one light
 source is required. In the context of the invention, it is not absolutely
 necessary to determine absolute values of the refractive index. In many
 cases, it is sufficient to determine variations in refractive index merely
 qualitatively, in order for example to recognize a domain of a substance
 in the sample by means of a sudden jump in the refractive index. This
 substance can then be identified using Raman spectroscopy, so that the
 variations in the refractive index are used merely as a probe.
 A preferred process is one in which use is made of an optical system which
 shifts the focal point of the monochromatic light applied in step b)
 inside the sample, for example confocal optical imaging in which moving
 the source to and fro makes it possible to adjust the observed depth
 segment within a sample. In this case, it is possible to achieve an
 accuracy of about 1 to 3 .mu.m in terms of the focal point which is set,
 that is to say of the desired depth segment. The depth sharpness can be
 set by using a diaphragm or an optical fiber with appropriate internal
 diameter, which is to say this method makes it possible to scan a sample
 both vertically, i.e. in the direction of the light beam (depth profile)
 and radially, i.e. in the direction of gravity. If fairly large lateral
 regions are to be observed, then it is further possible in the context of
 the present invention to translate the entire optical system, for example
 using a traveling table or a piezo mechanical device. Using arrangements
 of this type is, for example, advantageous if sample chambers are used in
 the ultracentrifuge which are divided radially and/or vertically into a
 plurality of parallel subchambers. This makes it possible to scan a
 plurality of samples at the same time during a single ultracentrifuging
 operation.
 Shifting the focal point for the incidence cone of the monochromatic light,
 as well as a corresponding shift in the observation cone for detecting the
 scattered light, also allows the successive examination of a plurality of
 samples, which are contained in parallel chambers within a cell, without
 providing separate optical systems for each chamber. In general, the
 preferred process makes it possible to scan a sample locally.
 A further preferred process is one in which it is possible to shift the
 point of application of the monochromatic light in step b) and, in step
 c), shift the point of detection of the scattered light along the sample.
 This makes it possible to examine the sample during the centrifuging using
 a single Raman spectroscopy system, since this is simply moved to and fro
 along the sample. Nevertheless, depending on the intended purpose, it is
 also possible to measure the sample statically at a single point, and for
 example to observe the motion of the particles through this point.
 The present invention can be applied particularly advantageously using
 fiber optics. For example, the monochromatic light used to generate the
 scattered light can be applied to the sample using an optical fiber. The
 detection may then likewise take place using optical fibers. This makes it
 possible to arrange the elaborate Raman technology in a different place
 from the ultracentrifuge. Both parts of the measuring set-up may for
 example be in separate laboratories, and may be connected using fiber
 optics and, where appropriate, a control system for the abovementioned
 preferred movement of the light source and the Raman detector along the
 sample.
 For characterizing a plurality of samples at the same time, with a
 plurality of samples being centrifuged simultaneously using a multichamber
 rotor and simultaneously examined using Raman spectroscopy, the process
 according to the invention may be carried out as follows.
 Light pulses, for example from a pulsed laser or a modulated continuous
 wave laser are focused on the individual cells in a rotor, for example a 4
 or 8 hole rotor, using optical triggering. The Raman light detected under
 back-scattering conditions is analyzed in a spectrometer.
 Using a two-dimensional detector, for example a CCD array, makes it
 possible on the one hand to detect the back-scattering from the individual
 samples (for example in horizontal lines) and, on the other hand, to
 allocate to specific yet different chambers (for example in vertical
 lines) using, for example, a tilting mirror driven synchronously, for
 example by an electrically controlled suspension, or piezo-translators or
 acousto- and electro-optical beam guide systems. This permits simultaneous
 characterization of the samples within the chambers.
 The measurement may, of course, also be carried out sequentially, for
 example with the AUC running for a sufficiently long time, in which case
 it is not necessary to have the extra optical imaging system.
 With this measuring arrangement, the process according to the invention is
 very useful for routine analysis.
 The present invention also provides a device for characterizing at least
 one sample which contains at least one substance (S.sub.i). According to
 the invention, this device has at least the following interacting
 components:
 an analytical centrifuging device having at least one chamber for holding
 the sample,
 a device for exposing the sample to monochromatic light and for detecting
 light scattered by the sample, and
 a device for determining a quantity G.sub.ji which characterizes the
 substance S.sub.i.
 This device can be used to carry out the process according to the invention
 presented above. It may also be supplemented by further elements which are
 necessary or useful for carrying out the preferred or advantageous
 variants of the process according to the invention which have been
 presented above.

DETAILED DESCRIPTION
 FIG. 1 shows a schematic representation of a sample characterization device
 according to the invention. It contains a rotor 1 with an axis of rotation
 2 and a cell, having at least one chamber which is intended to hold a
 sample and is located in a bore in the rotor (3, 4), a laser light source
 5, which may or may not have an optical imaging system and can apply light
 to the cell 4, and a detection system 6 which can record back-scattered
 light from the sample chamber 4 with spectral discrimination. Further, a
 light source 9 may be projected onto the cell 3 using a notch filter 10.
 The back-scattered light passes through the notch filter 10 and is
 spectrally analyzed in the detection system 11. The light source 5, 9 and
 the detection system 6, 11, or its optical imaging system, may be fitted
 on separate supports or on a common support (not shown). In both
 arrangements, it is possible for them to be moved synchronously along the
 arrows 7a or 12a, radially, and along the arrows 7b or 12b, vertically. It
 is therefore possible to scan the cells 3, 4 in the radial direction 7a,
 12a and vertical direction 7b, 12b.
 While the rotor 1 is turning about the axis of rotation 2, the light source
 5 can apply monochromatic light to the cell 4 just when it is moving past
 it in the course of a revolution. The elastically and inelastically
 scattered light is registered under a back-scattering geometry by the
 detection system 6, 11 or its optical imaging system. The detection system
 forwards the recorded values to the evaluation unit 8 which undertakes the
 evaluation. The characteristic quantities of a substance which has been
 found and identified, for example density, sedimentation rate,
 sedimentation coefficient, molecular weight distribution or particle size
 distribution, can be determined with the usual processes (not represented
 or described here) for conventional ultracentrifuges.
 FIG. 2 shows a detail of a preferred embodiment of the sample
 characterization device according to the invention. A cell 3 inside the
 rotor 1 is here divided in the vertical direction, parallel to the axis of
 rotation 2, into four chambers 3a to 3d. It is therefore possible to
 measure a plurality of samples simultaneously and under identical
 conditions. According to the invention, a light source 14 is used in a
 focal optical configuration with a focal point 15, it being possible for
 the focal point 15 to be shifted inside the cell 3 between the subchambers
 3a to 3d, vertically along the arrow 16, and where appropriate radially
 with respect to it. It is therefore possible for all four subchambers to
 be examined successively without using separate optical systems. This is
 beneficial, above all, in the case of a light source 14 which applies
 monochromatic light to the sample for Raman scattering, and for a
 corresponding detection system.