Dec. 17; 1946. . 1.. A. CHAMBERS EI‘AL 2,412,602 ELECTROPHORES IS APPARATUS Filed Sept. 3, 1942 Qaafw guwiaow 2,412,602 Patented Dec. 17, 1946 UNITED STATES ‘PATENT oi-fncs) 2,412,602 ' ' ELECTROPHORESIS APPARATUS’ Leslie Addison Chambers, Drexel Hill, and Haldan Kelfer Hartline, Media, Pa., asslgnors to ‘The Trustees of the University of Pennsylvania, Philadelphia, Pa., a corporation of Pennsyl vania Application September 3, 1942, Serial No.v457,232 ' . 7 Claims. 1 > (Cl. 88-14) 2 - , , often found in the form wherein the concentra Our invention relates to optical interferom eters, and particularly to their adaptation to the analysis of the kinds and concentrations, relative tion gradients are produced by a ?xed voltage gradient (electrophoresis), it is not our intention to in any way limit our invention to this embodi ment, which we use below solely to illustrate our or absolute, of particles in suspension or solution in a liquid medium. Analysis is accomplished through interferometric measurement of local invention. - > Referring to the accompanying drawing, Fig changesvin the refractive index of the suspension or solution produced by the imposition of electri cal, centrifugal or gravitational ?elds, or by dif ure 1 is a side elevation of one possible arrange ment of the interferometer unit for the applica fusion. Any suspension or solution which can be 10 tion of a ?xed voltage gradient; Fig. 2 is a typical , schematic diagram of the essential components of caused by any of the above means to exhibit such the optical system; Fig. 3 illustrates the essential local changes in refractive index may be analyzed _ ‘light paths in the interferometer cell itself; Fig. according to the method of our invention. .These 4 shows‘ the appearance of a typical photograph local changes in refractive index are evidence ofv concentration gradients. . _ of the projected image of the interference fringes . In one of the methods of the practice of our showing a concentration gradient between por invention, concentration gradients are obtained by permitting the migration of charged particles of solute through a ?xed voltage gradient. The several kinds of particles move in direction and 20 with velocity determined by the nature of their tions of the solution being studied, the photograph charge. This usually results in a stratification of - the various kinds of solute particles. (If concen tration gradients are produced by centrifugal or gravitational ?elds, the rate and extent of strati ?cation are determined by the relative masses of the solute particles.) , The formation, rate ~01 being taken from the front of one of the cells; and Fig. 5 is a section taken on line 5—5 of the cell shownin Fig. 1. ' Measurement of the interference fringe spacing and displacement is correlated with other infor mation to disclose the mass, charge, size and con centrations of the solutes involved. 25 ' v ' The essential features of the illustrated em bodiment of_.our invention may best be ‘under stood by referring to Fig. 2, wherein I represents the interferometer-electrophoresis cell submerged movement. number and magnitude of the local in the temperature controlled bath 2. The inter refractive-index gradients which are caused by the concentration gradients are detected by the 30 ferometer cell is, illuminated and observed by the optical system shown. Light from the lamp 3 interferometric method (to be described). Infor (preferably a vapor lamp) passes through the mation may thereby be obtained concerning the ?lter l to render it monochromatic, thence mass, charge, size and/ or absolute or relative con through the condensing lens 5 whichibrings the centrations of the several solute constituents. The interferometric method of‘detection com 35 rays to convergence in a small‘ spot on the reflect ing surface-of'prism 8. The light is directed by bines sensitivity with compactness. the prim 6 to collimating' lens ‘I, which is located One of the objects of ‘our invention is to provide at-its focal distance from the point of ‘conver an apparatus which a?ords greatly increased gence. The collimated beam then falls‘ upon an sensitivity and a large reduction in the size of interferometer portion of cell i placed at approxi the necessary apparatus over other apparatus and mately twice the focal distance from lens 1. The‘ methods usually employed. re?ected beam-is directed through aperture‘! and Still another object of our invent-ion is to pro comes to focus on the photographic plat-e8, which vide an apparatus which will combine increased sensitivity and reduced size with means for actu is at approximately'twice the focal distance'from ally‘separati'ng and isolating the various ‘solutes 45 lens ‘I and is thus're'ceiving a real image of cell I. Alternatively, the cell I may be placed ‘closer to in complex mixtures, for further study outside the apparatus. I Another object of our invention is to provide an apparatus for the analysis of serum and of such complex body ?uids-as plasma, s‘ynovial ?uids, pleural exudates', urine, etc., as an adjunct ‘in the diagnosis of certain diseases, which combines sensitivity and‘ compactness through the use of the interferometric method of'measurement. _ lens "I and aphotographic-objective- lens placed‘ at 8 to'form- a r'ealimage of the=interferénce fringes on the plate 8.- " ~ - I I - _In Fig. 1 is shown one possible combination of interferometer and electrophoresis ‘cell: The interferometer portions [0, shown in ‘section as 1 Fig; 5, constitute the center sectionsof 'a‘closed U tube of rectangular cross-section designated ‘ 9,419,009 . the interferometer portions I 0. The cells com prise transparent plates "-23 separated by spacer members 21-28. Part II, made up of glass plates, forms the bottom of the U and the assembly l2 makes up the top of the ‘U. This section has two outlet tubes l2. and I4, which are used to communicate liquid in the U tube-under, examination with two appropriate glass vessels containing the electrodes for introduction of the ‘electric current. The joints between tubes l3 and I4 and the tubes on the electrode vessels are usually sealed by means of pieces of rubber tub ing. It will be noted that the entire assembly is 4 7 other about a vertical axis, thus enclosing a col umn of liquid having a wedge shaped cross-sec tion. The actual dimensional difference in thick ness from side 21 to side 28 is such as to give from '10 to 20 fringes (5 to 10 wavelengths) across the width of the cell with the solvent in the cell. In order to assure fringes which are straight and ‘ parallel, the inner faces of plates [8 and 22 are made ?at within a very small fraction of a light wave (e. g., 1/20). The fringes are held parallel to the sides of the cell by accurate control of the . dimensions of the spacer members 21 and 28 be tween the plates. The amount of the inclination of surfaces 20-22 is so slight that this does not in the familiar‘ form, in general, whereby the center portions It may be displaced laterally 15 show in the drawing. However, if lines were drawn through the planes of the surfaces 20-22 with respect to either or both of the-bottom and these lines would intersect in a point substantially top sections. In this way it is possible to isolate remote from the cell. Fig. 4 is an illustration of portions of the solution in the U tube whenever the appearance of the interference fringes at the ’ The construction of the interferometer por 20 boundary of two strata wherein exists a con tions of the cellis best considered by reference - centration gradient as is impressed on the photo graphic plate 9. to Fig. 3. This ?gure shows the construction of We have found the method of our invention what is known as the-“re?ecting"'or “autocolli highly practical and efficient in. operation. We mating" type of interferometer, and is used mere ly to illustrate the type of interferometer, and 25 realize that modifications may be made in carry ing out the method of our invention and we in not to limit (the interferometer to this particular tend no limitations upon our invention other design. The apparatus can incorporate the than may be imposed by the scope of the ap "transmission" interferometer as well. The lat pended claims. . ter type is so constructed that the light source and photographic apparatus are on opposite sides 30 What we claim as new and desire to secure by of the interferometer. It is not intended to limit - Letters Patent of the United States is as follows: 1. The method of analyzing solutions which detection of the interference fringes to a photo comprises con?ning the solution under observa graphic surface. Any suitable means may be tion between a pair of interferometer plates, sub employed, such as a telescope, scanning photo jecting the solution therebetween to electrophore - electric cell, etc. Returning to Fig. 3, number sis, passing rays of monochromatic light through 17 indicates a ray of light entering the interfer ~the solution, and recording the interference pat ometer from lens ‘I. This ray passes through the tern produced by said interferometer plates for transparent plate I! and strikes a semi-re?ecting determining the characteristics of the solution. coating I 9 on inner surface 20 of Plate l8. This coating I9 is of such thickness that it transmits 40 2. Apparatus for analyzing solutions compris ing a source of monochromatic light, a cell for part and re?ects part of the incident ray I ‘I in the solution having plane wall portions, one of a de?nite ratio determined by the theoretical which extends in a plane that is angularly dis analysis of the system. The transmitted portion posed with respect to the plane of the adjacent 2| of ray l'l passes through the solution being examined and strikes the totally re?ecting coat 45 wall portion to provide a con?ning cell for the so lution having slightly diverging walls, means for ing 22 on the inner surface of plate 23. The ray directing monochromatic light through said last is re?ected by this surface through the solution mentioned wall for re?ection by each of said once more and impinges on the partial mirror walls, optical means for directing the re?ected ll, where part of it is re?ected and part trans mitted to form ray 2|. The re?ected portion of 60 light to a light sensitive photographic plate, and means for effecting a migration of the particles _ the ray suffers a number of multiple re?ections in said cell for recording interference fringes es between the two mirror surfaces. Each time it tablished in said cell according to the character strikes surface l9, however, it loses part of its istics of the solution therein. energy as a returning ray. These rays help to increase (or decrease) the fringe intensity and 55 3. The method of analyzing solutions which comprises con?ning the solution under observa thus contribute to the contrast ratio or resolving tion between a pair of interferometer plates, sub power of the interferometer. There is a theo .iecting the solution therebetween to electrophore retical optimum ratio of re?ectance to transmit sis, passing rays of monochromatic light through tance of ?lm is at which value the interference fringes are the brightest and sharpest. The por 60 the solution, and optically observing the inter ference pattern produced by said‘ interferometer tion of the original ray H which is re?ected plates for determining the characteristics of the from coating is is designated by 25. ‘With the solution. conditions of illmnination as outlined herein, rays it is so desired. 4. Apparatus for analyzing solutions compris 24 and 25 are in‘ a state and position to cause ing a source of monochromatic light, a cell for the interference with each other. Whether they ac solution having plane wall portions, one of which tually interfere or reinforce each other depends extends in a plane that is angularly disposed with entirely upon the path difference of rays 24 and respect to the plane of the adjacent wall portion 25, and in effect depends only upon the time re to provide a con?ning cell for the solution hav quired for ray 2| to traverse the solution in both directions. This time is controlled directly by the 70 ing slightly diverging walls, means for directing monochromatic light through said last mentioned index of refraction of the solution at that par ticular spot. In order to obtain interference fringes which wall for re?ection by each of said walls. optical means for directing the re?ected light to an op tical observing means, and means for effecting a will be useful for measurement and recording purposes, surfaces 2|! and 22 are inclined to each 1g migration of the particles in said cell for optically 2,412,802 5 observing interference fringes established in said 6 ' . a totally re?ecting surface on the interior face of the other 01' said walls, a source of monochromatic cell according to the characteristics of the solu tion therein. light directed through the semi-re?ecting surface 5. A system of analysis comprising a, cell for of said cell upon the re?ecting surface thereof, solution under examination having a horizontally 5 and means for observing the interference pattern tapered chamber for the formation of optical in resulting from the re?ection of the monochro terference fringes, a source of monochromatic matic light between the cell walls. light directed upon said chamber, the surface of 7. A system of analysis comprising a cell for the rear wall of said tapered chamber being pro solution under examination, said cell comprising a vided with a re?ector for said monochromatic 10 pair of plane walls disposed in diverging relation light after the passage thereof through said cham with respect to each other, a semi-re?ecting sur ber, and means optically aligned with said cham face on the interior face of one of said walls, a ber in substantially the same transverse plane totally re?ecting surface on the interior face of through said light source and said tapered cham the other wall, a source of monochromatic light di ber for observing the interference pattern result 15 rected upon the totally re?ecting surface of said ing from the re?ection of the monochromatic light last mentioned cell wall through the semi-re between the cell walls. ?ecting surface of the ?rst mentioned wall of said 6. A system of analysis comprising a cell for cell, and means optically aligned with said source solution under examination, said cell comprising a of monochromatic light for recording the inter pair of plane walls, one of said walls being dis 20 ference pattern resulting from the re?ection of posed in a plane at a slight angle with respect to the monochromatic light between the cell walls. the plane of the opposite wall, a semi-re?ecting LESLIE ADDISON CHAMBERS. surface on the interior face of one of said walls, HALDAN KEFFER. HARTLINE.