Light Scattering - NBI

1 Light Scatteringa brief introductionLars gendalUniversity of Copenhagen 3rd May 2019iiContents1 is Light Scattering ? ..52 Light Scattering Light Scattering , SLS .. Light Scattering , DLS .. and comparisons ..403 Complementary , static Light Scattering .. , small angle X-ray Scattering .. , small angle neutron Scattering .. , mass spectrometry .. ultracentrifugation ..4812 ContentsPrefaceThe reader I had in mind when I wrote this small set of lecture notes is the absolutenovice. Light Scattering techniques are becoming increasingly popular but appar-ently no simple introduction to the field exists. I have tried to explain what thephenomenon of Light Scattering is and how the phenomenon evolved into measure-ment techniques. The field is full of pitfalls, and the manufacturers of Light scatter-ing equipment dont t emphasize this. For obvious reasons. Light Scattering equip-ment is often sold as simple-to-use devices, like a spectrophotometer.

2 Andthe apparatus software generally produces beautiful graphs of molecular weightor size distributions. Deceptively informative. But be warned! The informationshould often come with severalcave at sand the actual information content maybe smaller and more doubtful than it is pleasant to realize. Before you begin touse Light Scattering in your projects make sure to team up with someone who hasactually worked with it for some years. Dynamic Light Scattering , DLS, in particu-lar can be frustrating because it is a low resolution technique, a fact that is usuallyrecognized only after one or more minor more comprehensive set of lecture notes ( Light Scattering Demystified) explain-ing in more detail about the physical background for the Light Scattering methodsis also gendalCopenhagenMay What is Light Scattering ?When Light hits a small object (a particle or a molecule), and thereby changes itsdirection, the thing that happens is calledlight Scattering .

3 If, on the other hand,the Light disappears by the encounter with the particle we call the , in essence, you can say that Light like all other kinds of electromagneticradiation (radio waves, micro waves, heat radiation, ultraviolet radiation, X-rays,gamma radiation) em interacts with matter in two ways :1. Absorption: the photons (the Light ) duction2. Scattering : the photons change their directionHere we shall deal only with Scattering . And only Scattering from particles placed(and moving) randomly in relation to each other dissolved in a solvent. Iflight is being scattered fromorderedparticles this can cause such phenomena asreflection, refraction or of the above mentioned interactions will cause a Light beam to be attenuatedwhen passing through a solution1of particles (see figure ). It doesn t matterwhether Light is being attenuated by Scattering or absorption: In both cases thetransmitted intensity will decrease exponentially with the thicknessxof the thematerial the Light is passing through.

4 If the attenuation is due to absorption thetransmitted intensityIis usually writtenI=I0 10 xwhereas if the attenuation is due to Scattering the intensity is writtenI=I0 e xwhereI0is the incident intensity ( before attenuation). The quantities and are called the absorption coefficient and the turbidity, respectively. The two differ-ent bases (eand 10) for the exponential decays are merely a matter of , within the fields of physics or chemistry, one talks about doing Light scatter-ing measurements it is nearly always the case that thesystemunder investigationis asolutionof the in real life, what does the phenomenon, Light Scattering , actually look like?Let s look at some examples:Figure shows a turned off laser in a dark room and figure shows a laserthat is turned laser beam is vaguely visible in the dark room. But why? .. In order for usto see an object it needs to be hit by Light which then bounces off in different dir-ections, into our eyes.

5 The lens in the eye then forms an image of the objecton the retina by collecting the Light that was reflected from the object. But a xII-= 0 xIIe-= :Thetransmittedlightisweakenedbyeitherab sorption(top)orbyscattering(b ottom). :Laserswitchedo inadarkro :Laserswitchedoninadarkro om(asweenvisageitwilllo ok).Thelaserb ductionbeam is not an object or a thing . I is merely the name for a train of photonswithin a narrow portion of space, all moving in the same direction. Why then,can we see the laser beam? The answer is: We can t. At least not if the laseremits its beam in a rom where the air is totally clean, dust free. When we seea laser beam we actually see photons beingscatteredon dust particles along thepath of the beam (see figure ). Scattering (in all directions) by the solid :Alightb eamisnotaphysicalob jectscanb eseenwhentheyemitlightintooureyes,either b ecausetheob jectsemitlightbythemselvesorb ecausetheyre ectorscatterlightthatfallsup laserb eam isacollectionofphotonsthatmoveinthesamed irectioninanorderlyfashionwithinanarrowp eamisvisiblefromthesideisthatsomeoftheph otonshitdustparticlesintheairandarethere byde also the obvious explanation why we see an illuminated spot where the laserbeam hits a screen or the wall.

6 If the screen is replaced by a mirror or a polishedsurface we will not se a bright spot where the laser beam hits: The photons are allscattered ( reflected, in this case) in the same direction so it is unlikely that anyof them will reach our eyes. (figur ). If one holds a test tube (or even better,a Light Scattering cuvette) with skim milk diluted 1000 times in the laser beam itwill look something like figure even though the liquid looks perfectly clearand transparent to the eye. The same phenomenon can be seen if the test tube con-tains a solution of molecules of sufficient high molecular weight (and :Laserturnedoninadarkro eamhitsthescreenwithinasmallareathatligh tsupasabrightsp , oth,p olishedsurface, ,nobrightsp othsurfacedo esnotscatterthephotonsintoal ldirectionsbutonlyintoonesingledirection whichisprobablynotdirectlyintooureye(s). :Alaserb eampassingasolutionof particles willpro (ormolecules) ductionhigh concentration).

7 Compounds of sufficiently high molecular weight are usuallypolymers that may be of biological origin, proteins or polysaccharides. Thesebiological polymers constitute vast classes of molecules with molecular weightsranging from approx. 1000 g/mol up to or beyond 1,000,000 g/mol. The higherthe molecular weight the less dissolved material is needed to produce Light scatter-ing of a given intensity. If one has a thin solution of regular milk (not skim milk) inthe test tube the Scattering will look as shown in figure The fact that the :Ascreenb ehindthesolutionofmoleculesthatscatterth elightdo esnotonlyshowthesmallintensesp otwherethelaserb the screen is not confined to a small, intense spot is exactly Light scattered Light has the highest intensity near the centre and gets progressivelyweaker away from the centre. Had the solution been diluted skim milk instead ofregular full fat milk the scattered Light on the screen would not have weakened asrapidly with increasing distance from the centre.

8 It turns out that the reason forthis is that the particles responsible for the Light Scattering in the two types of milkare of different size: In skim milk the scatterers arecasein micelles(spherical structures with a diameter of approximately 300 nm) whereas the scatterersin regular milk are fat globules (spheres) with a diameter of approximately3 m, ten times higher. This is an example of the principle that small particles tendto scatter Light more evenly in all directions whereas larger particles tend to favourscattering in the forward direction. This indicates that the precise way in whichthe intensity of scattered Light changes as a function of the angle through whichthe Scattering takes place contains information about the size of the particles thatscatter the Light . Moreover, if the concentration of the particles is known, also themolecular weight can be determined in this way. In order to make any practicaluse of Light Scattering a screen is of course not sufficient.

9 Instead one uses a lightdetector that can measure the intensity of the scattered Light at specified duction2 Light Scattering Static Light Scattering , SLSWhat can be measured by static Light Scattering ? Below is a list (precise numbersshould be taken with a grain of salt. Everything depends ..): Molecular weight. Range: 1000 g/mol 109g/mol Size. Range: 10 nm 1000 nm Interactions. The second virial are the limitations of static Light Scattering ? The volume of the sample has to be higher than with some other on the specific setup but is typically 1 ds The concentration of the sample has to be high enough. The lower themolecular weight the higher the necessary concentration. The sample solution must be completely transparent (non-turbid). The sample solution should not absorb Light of the wavelength used. (Maybe compensated for to some degree) The molecules in solution should have a refractive index which is differentfrom that of the that need to be known about the solution/system: The weight concentration ( g/L) of the solute molecules.

10 The refractive index incrementdndcof the solute/solvent shows a sketch of a setup for measuring Light Scattering , so-calledstaticlight scatteringor SLS. Figure shows how a typical SLS instrument looks inreal sample (the solution of molecules under investigation) is in acuvette which is normally of cylindrical shape. A monochromatic1light source -usually a laser - shines Light on the sample. Before 1960 the Light sources used inlight Scattering were incandescant lamps or mercury or sodium vapour lamps inconjunction with optical filters, collimator slits and lenses. Since the advent of thelaser it has been the nearly universal Light source in Light Scattering instrumentsbecause of its monochromatic, and inherently collimated beam. The intensity(or the power) of the Light scattered from the sample as well as the power of thelight source are recorded continuously. Dividing one by the other eliminates thepossible effect of variations in the laser power.