examples of sedimentation velocity experiments on ... scanning Schlieren optics for the use of six place rotors) or the MSE ... XL-A (the only Analytical Ultracentrifuge available after .... Schlieren optical system (see e.g. [11]) for the Beckman ... mode with a step size of 0.005 cm and only one average to ...... the axis of rotation.
Progr Colloid Polym Sci (1995) 99:167-186 © SteinkopffVerlag 1995
H. C61fen S.E. Harding
Received: 18 April 1995 Accepted: 5 June 1995
Dr. H. C61fen(1~) Max-Planck-Institute for Colloid & Interface Research Colloid ChemistryDepartment KantstraBe 55 14513 Teltow, Germany S.E. Harding National Centre for Macromolecular Hydrodynamics University of Nottingham Sutton BoningtonLE12 5RD United Kingdom
A study on Schlieren patterns derived with the Beckman Optima XL-A UV-absorption optics
Abstract This study describes how typical "Toepler" Schlieren patterns can be derived with the UVabsorption optics of the Beckman Optima XL-A ultracentrifuge without any modification of the optical system. Such a Schlieren effect can greatly enhance the application range of the XL-A ultracentrifuge which cannot yet be used to its possible full potential due to the present lack of a refractometric optical system and - not only for sedimentation velocity experiments - particularly the Schlieren optical system. It is shown that the Schlieren patterns detected with the UV-absorption optical system are caused by light refraction and not by light scattering or experimental artefacts. Two possible explanations for the generation of the Schlieren effect are given. Both explanations have in common that the refracted light cannot be detected by the photomultiplier system and is hence lost for detection. The intensity of the refracted light which can be registered with the photodetector of the optical system is dependent on the angle of incidence of the light, e.g., the local slope of the sedimenting boundary, and hence leads to a detection of the refractive index change with radial position: i.e.,
a Schlieren pattern. The advantages, disadvantages and differences of the XL-A Schlieren effect compared to conventional "Philpot-Svensson" Schlieren optics are discussed demonstrating that both optical systems deliver the same results for examples of sedimentation velocity experiments on several polysaccharides and bovine serum albumin. An estimation of the sensitivity of the Schlieren effect as a function of the wavelength selected is given as well as suggestions of how the Schlieren effect can be increased or suppressed. Considerations about the quantitative evaluation of XL-A Schlieren patterns show that the Schlieren effect is dynamic with respect to the concentration dependence. This creates some potential for new applications but is also a serious warning to those people who want to derive quantitative information from XL-A absorption traces as these can be superimposed by a Schlieren effect at certain wavelengths. Key words Schlieren optics absorption optics - analytical ultracentrifugation - sedimentation velocity - light refraction - optima XL-A ultracentrifuge
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Introduction
H. C61fenand S.E. Harding Schlieren patterns with the Optima XL-A
ing the sample in the cell. For a good description of the function of the optical elements of the Schlieren optical Throughout the several decades of Analytical Ultracen- system see [3]. This lack of provision of a reliable trifugation, the typical Schlieren peak has not only been Schlieren optical system for the Optima XL-A might be an the pattern most people associate Analytical Ultracen- effect of the considerably decreased optical path-length trifugation with, but Schlieren optics has furthermore due to the now significantly decreased instrument size as proved to be the most suitable optical system for sedi- Schlieren optics has up to now proved to be a long-range mentation velocity experiments and some other applica- optical system with large focal length lenses requiring long tions (e.g., density gradient experiments, experiments with optical paths in the instruments still in use (Model E, gels, etc.). This is especially valid for polysaccharides and MOM, etc.). Further improvements of the Schlieren opsynthetic polymers in UV-absorbing samples which have tical system with respect to its sensitivity and accuracy [4] not been detectable with a normal UV-absorption optical - which can be considerably enhanced - indicated that system up to now. All classical types of Analytical Ultra- Schlieren optics does not lend itself easily to miniaturicentrifuges, e.g., the Beckman Model E, the MOM ultra- zation because of decreased sensitivity. In contrast to the present commercial desire for apparatus miniaturization, centrifuge, the Heraeus AZ°9100 and the MSE Centriscan (the latter already equipped with a multiplexer controlled a study on an ultrasensitive Schlieren optical system [4] scanning Schlieren optics for the use of six place rotors) or clearly pointed out that a more sensitive Schlieren optical the MSE MK II are equipped with at least this type of system not only requires a larger phaseplate or knife edge, optical system as a standard. But the Beckman Optima but furthermore requires longer optical paths. This means XL-A (the only Analytical Ultracentrifuge available after that the application of lenses with smaller focal lengths a long time of stagnation in development) only at present and hence decreased optical paths as realized with the has UV-absorption optics although in the near future Rayleigh interference optics for the Optima XL-A simply there will be commercially available Rayleigh interference cannot be transferred to a Schlieren optical system without optics for it based on the prototype of Laue [-1]. Although significant loss of accuracy. This might be one of the this interference optical system - without doubt, being the reasons why a Schlieren optical system, although formally most accurate system for sedimentation equilibrium work identical to the already established Rayleigh interference is designed as an on-line system suitable for evaluation optical system, has not been launched for the Optima with computers [1] and hence suitable for differentiation XL-A up to now. Furthermore, problems in the picture leading to a Schlieren pattern or for the time derivative evaluation process of Schlieren patterns from a phase plate analysis of solutions with concentrations as low as (detection of the zero'th order of the Schlieren line in 0.02 mg/ml [2], many important applications - well estab- a non-uniformely illuminated greyscale picture) has relished in the past - still cannot be covered by this optical stricted their availibility for on-line picture evaluation, system due to its present physical limitations. One although some partly successful approaches have been example might be the detection in highly concentrated made [5, 6]. Nevertheless, none of these approaches leads solutions where no Rayleigh interference fringes are for- to universally applicable on-line picture evaluation bemed anymore, but Schlieren patterns are still detectable. cause the brightness values which must be used to define Another critical example might be steep concentration the relevant picture information (e.g., the Schlieren curve) gradients for which the detection accuracy of a CCD- are not that different in a Schlieren grey scale picture as camera based Rayleigh interference optics is considerably they are in a Rayleigh interference pattern. The high conlimited. Further, in systems where a z-average molecular trast of interference fringes has been exploited by Rowe et weight is desirable from sedimentation equilibrium, al. [7] using the Fresnel fringes in phase-plate Schlieren Schlieren optical records yield this directly. So there is still patterns to derive the zero'th order with increased accua demand for a Schlieren optical system for the Beckman racyA Such an evaluation technique is actually adapted to Optima XL-A ultracentrifuge. At present, this means that a knife edge based on-line Schlieren system for an MSEmany scientists need to keep their old "dinosaurs" with all MKII ultracentrifuge [8]. Another knife edge based their well known disadvantages, just because of the lack of Schlieren optical system principally suitable for on-line an alternative. To the authors' knowledge, no real com- evaluation is the scanning Schlieren system of the old MSE mercial progress in designing a Schlieren optical system Centriscan ultracentrifuge [9]. Furthermore, it was refor the Beckman Optima XL-A has been made up to now, ported that the UV-absorption optics with photoelectric although the Schlieren optical system is virtually identical scanner of the Model E can be modified by introducing to the Rayleigh interference optics with the addition of a knife edge as analyzer element so that it is possible to a knife edge or phase plate in the focal point of the scan up to five Schlieren patterns simultaneously when collimating lens which focuses the parallel light illuminat- using the multiplexer with the six place rotor [10]. This -
Progr Colloid Polym Sci (1995) 99:167 186 © SteinkopffVerlag 1995 system should as well be principally suitable for on-line evaluations. From the present point of view, it seems to be unlikely that first a classical phase plate or "Philpot-Svensson" Schlieren optical system (see e.g. [11]) for the Beckman Optima XL-A will be launched and second fully automatically and preferably on-line evaluation of Schtieren patterns can be maintained as is required by modern expectations. These considerations already show that new ways need to be explored if a Schlieren optical system can ever get a chance to be established as an on-line system available for computerized evaluation in the near future. Up to now, many of the benefits of Analytical Ultracentrifugation can still not be used by those scientists who definitely require refractometric optics [12], namely all scientists interested in the investigation of synthetic polymers in organic solvents, or polysaccharides or many colloidal systems with the Analytical Ultracentrifuge. But especially the latter field is steadily expanding. Hence the motivation for this study was to start some basic research on the generation of Schlieren patterns leading to a solution for all scientists working with the Beckman Optima XL-A with its present standard UV-absorption optical system. Hopefully, the results of this study will encourage others to further explore the Schlieren effect on the Beckman Optima XL-A and to carry out further studies. Perhaps the results given may even serve as basis to design a reliable Schtieren optical system for the XL-A ultracentrifuge.
Materials and Methods A Beckman Optima XL-A (Beckman, Spinco Division, Palo Alto, California, USA) Analytical Ultracentrifuge equipped with standard UV-absorption optics as described elsewhere [-13] was used to investigate the Schlieren effect. The basic experiments for this study have been carried out on an XL-A from 1991 (one of the original four prototype or "beta site" units) in Nottingham which does not have a blocking filter in the monochromator. Control experiments were performed on an XL-A from 1992 in Dr. A.J. Rowes' laboratory in Leicester, UK, and on an XL-A from 1993 in Dr. A. Seiferts laboratory (Potsdam, FRG) already equipped with the blocking filter, and even a newer XL-A from 1994 in Dr. H.G. Mfillers' laboratory (Leverkusen, FRG) with blocking filter. For the controlexperiments on the newer XL-A's, the blocking filter was disabled to maintain comparable experimental conditions on all machines. All experiments were performed at 20 °C at various speeds of 12 600, 30 000, 50000 and 60 000 rpm depending on the sample and the type of experiment. All runs have been of the sedimentation velo-
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city type with the exception of one sedimentation equilibrium experiment on bovine serum albumin. The buffer was used as reference solvent in every case. Generally, a wavelength scan was first performed at 3000 rpm. Afterwards, three wavelengths have been selected (where the sample shows little or no absorption) to derive the Schlieren patterns. The only exception is the bovine serum albumin sample which has been used to demonstrate equivalence between the conventional absorption optical traces (scanned at an absorbance wavelength) and the Schlieren patterns. To maintain short scanning times at the cost of noise, scans were performed in continuous mode with a step size of 0.005 cm and only one average to get scanning times of about 2 min/scan. t h e
The samples and buffers used in this study
w e r e :
bovine serum albumin 96-99%, Sigma bovine serum albumin partially degraded - Chitosan, practical grade from crab shells, Sigma x-carrageenan, No. C-1013, Sigma Na-alginate, Fisons Dextran 20 (Mw ~ 20000 g/mol), Pharmacia Fine Chemicals, Uppsala, Sweden Dextran 150 (Mw ~ 150 000 g/mol), Pharmacia Fine Chemicals, Uppsala, Sweden Xanthan Keltrol RD, Kelco International -
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-
-
-
-
-
The buffers used were a phosphate/chloride buffer, pH 7, I = 0.3 for all samples except chitosan which was dissolved in an acetate/chloride buffer, pH 4.5, I = 0.1. The loading concentrations of the samples were 1 or 2 mg/ml with the exception of one run employing high bovine serum albumin (BSA) concentrations of 5, 10 and 20 mg/ml. To compare the results of the XL-A Schlieren effect, a Beckman Model E Analytical Ultracentrifuge equipped with conventional Schlieren optics and working with a phase plate as analyzer element has been used applying a phase plate angle of 70 ° for all velocity runs. The sensitivity of the XL-A Schlieren effect, was estimated by performing an equilibrium experiment at 12600rpm with a 1 mg/ml BSA solution (degraded BSA) at 19 °C simultaneously on the XL-A and a Model E with calibrated Schlieren optics 1-14] at the phase plate angles 40-85 ° in 5 ° steps. For all Model E experiments, the same samples and experimental conditions have been applied as for the XL-A experiments. For the Model E experiment, a 2 ° monosector 12 mm KEL-F centerpiece was used, whereas in the XL-A a 12 mm 2.5 ° KEL-F double-sector center-piece was employed. Both cells were filled with 120/al BSA solution. The slight difference in the filling heights resulting from different volumes due to the different sector angles of both centerpieces could be neglected because the filling height was small.
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H. C61fen and S.E. Harding Schlieren patterns with the Optima XL-A
To investigate if the Schlieren effect is a general effect occurring on UV-absorption optical systems or if it is related to the special optical arrangement in the Optima XL-A, a control experiment with a 2 mg/ml BSA solution in phosphate/chloride buffer was performed at 50 000 rpm using a MSE Centriscan ultracentrifuge equipped with scanning absorption and scanning Schlieren optics. The Schlieren photos of the Model E ultracentrifuge have been evaluated using a method similar to that reported by Gauglitz et al. [15]. The photos were scanned into a computer with a high resolution and then evaluated using image analysis software for electron micrographs (Zeiss AnalySIS 2.0, Soft Imaging Software GmbH, D48153 Miinster). All XL-A scans were evaluated with the Microcal Origin software (Beckman, Palo Alto, California, USA) using the data pointer to derive radial positions etc. The sedimentation coefficients have been evaluated using self-written software for all experiments (XL-A absorption, XL-A Schlieren and Model E Schlieren) [16].
1.5 --
Bovine serum albumin 2 mg/ml I
1.0-
cu o
o ~ .13
