NMR spectroscopy should be the method of choice here. I would measure 1H NMR spectra of the isolated compounds and of equimolar mixtures after an appropriate time of mixing and waiting (equlibrium is reached). Then I would compare the spectra of pure compounds and mixtures to see if there are differences in chemical shifts. Maybe different solvents can be used (D2O, CD3OD, DMSO, ...). The pH can be also changed to study this influnece. I would look for isolated signals to see if changes in the chemical shifts occur. From the changed and unchanged signals you can determine directly the equilibrium constant.

Hello Tobias,

thanks for you helpful answer.

is there any other way?

You could try to crystalize a mixture with slow evaporation of an water/Acetonitril solvent. Then you would see how many molecules are bound in the cavity and how but you couldnt gain Information about the extend.

Dear Tobias:

if it is spray dried?

Most of the common spectroscopic techniques depend on solutions. Maybe you could measure solid state NMR?

I think that you can measure the peptide concentration in solution before and after of the encapsulation process. Before, there is no problem because you are going to have a solution of the peptide. But after encapsulation, you need to centrifuge or separate the capsules to measure the concentration of peptide in the supernatant solution.

After this, you can calculate the total mass in the used volume and generate the efficiency trough:

% efficiency = mass after encapsulation / mass before encapsulation X 100.

Ideally, to measure the concentration you can use a LC method with UV or MS detection using your purified peptide as standard. If you don't have this type of equipment, the other option is using Bradford method (classical or modified) with a common spectrophotometer with BSA (bovine serum albumin) as standard.

There are two aspects to check:

1. encapsulation efficiency that is the percent of the substance encapsulated versus the amount added to the process. (for me less relevant)

2. the percent of substance encapsulated referred to the dry microcapsules collected (more important).

You have to use a technique that identifies and quantifies the amount encapsulated. So you need to separate the capsules from the solution before to determine the substance content.

If you have a spray dried product, you must separate the two species,i.e., encapsulated and not encapsulated, in advance

I completely agree Paolo about the importance of the second point he talked about.

Regarding such point, microphotography is a great interest in order to observe the presence of cracks. This is really important because through cracks, many elements (water, compounds, microorganisms, etc.) can go in and reduce the encapsulation strength and its by-pass value. This is also important because is an indirect indicator to measure stability under heat stress (heat and freezing) during transport and storage.

I read one article on measuring EE using Bichinchoninic acid micro protein assay kit by Sigma Aldrich. Test the supernatant (drug/actives that is not encapsulated) of your formulations after the centrifugation. Use this equation for calculation :

EE(%) =

[(Total amount of drugs - Untrapped untrapped drugs)/ Total amount of drugs x 100]

But I prefer cheaper protein assay kit if possible.