Hi,

   I  would first check the DLS quality report and the count rate, also the concentration of chitosan used for the preparation. At very low chitosan concentrations depending on the ratio we do observe this phenomenon. Which chitosan and concentration do you use?

Quality report was fine, there was no error or etc. for sample. Concentrations were relatively low about 2 mg/mL, i used variety of mass ratios said in literature, however, each sample is clear. 

If the system is small enough (

In case of CS-TPP nanoparticles, light opalescence must come..if it is not coming then check your pH of nanosuspension..CS-TPP nano are formed at specific range of pH between 6-6.5... 

Dear Himanshu Bhatt, 

But I see nanoparticles in DLS. PDI is about 0.2. Also, quality report was good. So, what do i see in DLS? 

If you check water sample in DLS, it will also give good size and PDI...As I observed in CS-TPP nanoparticles,  light opalescence must come and it indicates that nano has formed...

Actually, I double checked it for only water and only chitosan solution. PDI was 1.000 for each case. And quality report was awful. So, how would you interpret this?

Uğur-bey, do not listen to the advice provided by Himanshu.  A 40 wt % TiO2 of 30 nm particles in suspension will appear completely clear like water.  A 5 nm gold sol will be purple and transparent.  A 300 nm suspension of TiO2 will be like milk with a blue tinge.  Opalescence is a sign of material > 100 nm  (and this is, by definition, not nanoparticles).  Measurement of particle-free DI water by DLS will just generate noise-on-noise and you will not get a 'good size', PDI or quality report.  This is not a sensible thing to do.  The only circumstance where this could conceivably happen is if the DI water stream is highly contaminated with particles (or they come from other solutions and glassware) - maybe this is the cause of the opalescence in  Himanshu's systems?

A PDI of 1.0 would indicate that the system is too large and wide for DLS, that sedimentation would probably occur, and that standard laser diffraction is likely to be a better bet.

Dear Alan and Ugur

If you are talking about CS-TPP nanoparticles (whether you make it in triple distilled water or Millipore or MiliQ even), minute opalescence must come..CS-TPP system is formed at specific pH that indicates that specific moles of TPP polymer is completely interacted with CS ...Your CS M.W. is also crucial parameter...If that specific pH you are not getting, your nano will not be formed and your solution will remain clear and it will give good size and PDI as I practically observed...I will not bother in TiO2 case whether it is clear or not...but here your topic is CS-TPP...not TiO2 ...in CS-TPP nano I have vast practical experience....

So dear Ugur....just go through literature and try to apply it practically...you will get results in CS-TPP....I think Alan is only talking in general about nano...But it depends what polymeric systems you are using to form nano...for example, dendrimeric delivery, your dendrimer nano remains clear and it is nano in size....but CS-TPP it will never possible to remain clear... 

Dear Mr. Shajahan  and Mr. Rawle, thank you for your answers.

Dear Mr. Bhatt, thank you for your answers. 

I applied many formulations in literature, I saw blue opalescence in some of them. But samples that I saw opalescence had greater particles in size. On the other hand, samples that was clear solution had smaller in particle size, and quality report was fine. Thus, I am a bit confused, what do think I saw in DLS then for clear solutions? Because as I said, when I tried DLS for only water and chitosan solution, quality report was terrible and PDIs were 1. In addition, in some papers, it is not stated that blue opalescence is indication of nanoparticles, but many of them it is stated. 

Or  Would FT-IR be correct characterization technique to understand whether nanoparticles formed or not for clear solution?

Alan is saying that opalescence is a sign of material > 100 nm (and this is, by definition, not nanoparticles). If you prepare lipidic nanoparticles by high pressure homogenizer, you will get size in the range of 100 nm-150 nm (depends on homogenization cycles) WITH GOOD MILKY OPALESCENCE....so Alan wants to say that this size greater than 100nm is due to lipid impurity comes from glassware, other solutions and homogenizer machinery ...WHICH IS NOT POSSIBLE....even lipidic nanoparticles also give good milky appearance below 50 nm size..

My point is OPALESCENCE depends which carrier you are using to form nano...

You can use FTIR as well as DSC for characterization...It is little bit difficult to separate correctly formed CS-TPP nano....so just make sure that samples should be correct...otherwise you will get only chitosan or TPP peak in DSC and FTIR 

You can control your opalescence by choosing volume and concentration of TPP and CS..

When I was working on Chitosan TPP nanoparticle previously, I have optimized Chitosan concentration and volume as well as TPP concentration and volume. In my findings, I have optimized (TPP concentration 0.5 mg/mL and volume 1.5 mL) and Chitosan (M.W. 50 kD, 75% deacetylated) concentration at 1 mg/mL and 5 mL volume. The final pH of suspension was 6-6.5. I got particle size 100-120 nm after drug loading. If you increase chitosan concentration, volume of TPP will be increased with same concentration and your particle size will also be increased. Always add TPP solution into chitosan solution. Do not do vice versa.

How to make chitosan and TPP solution: First add chitosan polymer into water. Then add Concentrated HCl in that solution untill the pH of solution comes to 3. At pH 3, your polymer will be dissolved easily. Avoid acetic acid to dissolve.

TPP will be easily dissolved in water with pH around 8-8.5.

These nanoparticles are made by ionic gelation technique. TPP molecules will interact with surface terminal groups of chitosan molecules by crosslinking. So each particles are like nanogel like particles. If you centrifuge this nanoparticles, you will get gel like pellets which could be difficult to re-disperse but with vortexing those gelly pellets you can get again re-disperse particles with water. If you want to find encapsulation, it is better to check it by indirect method.

Just general talk about nano, I agree with Alan...but in case of CS-TPP, I disagree...

Dear Azeez

If you use acetic acid to make it soluble, at lower pH some visible particles of chitosan (even if you purchase CS from good source like sigma aldrich, Himedia) will be seen..just to make them soluble and to make chitosan in fully protonated state, I add few microliter of Conc HCl ...nothing else....

DLS data can be misleading in this case at times. check the pH of the chitosan solution before addition of TPP . A acidic chitosan solution requires more amount of TPP to fold. The optimum pH for getting nanoparticles of average 100nm would be around 6.4-6.8 for which you can visibly observe opalescence. please remember chitosan should be solubilized before making cs-tpp nanoparticles with acetic acid only not HCL 

Deepanjallee  You are correct with "The optimum pH for getting nanoparticles of average 100nm would be around 6.4-6.8 for which you can visibly observe opalescence"  (My italics).  At these sizes one would expect opalescence (except that great than 100 nm isn't nanomaterial by ISO and ASTM definitions - 'sub-micron' would be a better descriptor.

Dear Ms. Dutta,

You say optimum pH to get nanoparticles is between 6.4-6.8. However, I can provide you number of articles that obtained nanoparticles below 100 nm around pH 5. I attached one of them. Please take a look at it. 

Article Ultrafine chitosan nanoparticles as an efficient nucleic aci...

Dear Ugur

Definitely you can provide number of articles that obtained nanoparticles below 100 nm around pH 5. If you change the concentration of chitosan or TPP, the final point of pH at which nano are formed would be changed..that is obvious thing..the chitosan gets precipitated above final point pH (at which nano are formed) even if you add very few microlitre of TPP...

Dear Ugur, i never said at pH 5, u wont get it u will definite get nanoparticles at pH 5 too provided u have to use higher concentration of TPP

Agree with deepanjalee's previous discussion about pH at which nano are formed..pale opalescence must come

The appearance of pearlescence or opalescence occurs in particulate systems that are monodisperse, not polydisperse.  The particle size will determine the color. 

Usually the color will be observed at interfaces, including liquid-air.  The color observed is not usually a bulk phenomenon, because of the rapid Brownian motion of particles in suspension.  It is only when they adsorb at interfaces (thus becoming immobilized) that the color appears.  The most common observation is in a glass container, where the adsorption occurs on the glass walls of the container. 

I personally spent years dealing with monodisperse TiO2 and p-styrene latex.  Both show similar behavior, but only when monodisperse.  TiO2 is not usually perfectly monodisperse, but rather contains some higher order clusters of primary particles which interfere with the packing regularity of the resultant particles.  Latex, on the other hand, if properly stabilized readily form the color mentioned. 

Stability is important for the regular packing of particles.  Unstable particles will form extended clusters and chains that will not form the regular array and thus color.