*9.1.2 Surface charge*

The surface charge of liposomes in dispersion can be known by zeta potential [82]. The zeta potential is the total charge obtained by liposomes in liposomal dispersion [83]. The liposomes stability always depends on zeta potential [84]. The liposomal dispersion is always stable when vesicles remain separate without any aggregation. When vesicles have charge then repulsion is seen between vesicles in dispersion with repulsive forces and become stable. For stable liposomal dispersion there will be maximum vesicle charge. Liposomal dispersion with zeta potential of greater that 30 mV or lesser than 30 mV are considered to be more stable.

#### *9.1.3 Lamellarity*

The lipid bilayers present in liposomal vesicles represents the lamellarity. This lamellarity of liposomes is mostly applicable in encapsulation efficiency, drug release, fate of drug, and applications [85]. Lamellarity is identified using nuclear magnetic resonance (NMR) spectroscopy. In this 31P NMR method, paramagnetic ion Mn2+ or Co2+ or Pr3+ are added into liposomal dispersion [86, 87]. These ions quenches 31P signal from outer part of phospholipids on reaction with negative phosphate groups. This causes disturbance in spin relaxation and decreases 31P resonance signal. The lamellarity is calculated from comparing signal before and after addition of reagent.

## *9.1.4 Liposomal dispersion phase behavior*

Liposomal dispersion phase behavior can be identified by using differential scanning calorimetry (DSC) [88]. Differential scanning calorimetry method depends on temperature measurement at excess heat capacity of liposomes [89].

#### *9.1.5 Encapsulation efficiency*

Percentage encapsulation efficiency (% EE) can be determined by ultracentrifugation method [90, 91]. To find out the entrapment efficiency the liposomal dispersions are to be centrifuged at 5° C at 18,000 rpm for 1 h. The sediment portion of the mixture containing liposomes will be separated and lysised using methanol. Then the concentration of drug from lysised liposomes after suitable dilution was estimated by using UV Visible Spectrophotometer at respective wave length. The entrapment efficiency can be calculated by using following formula.

$$\text{Entropyed Efficiency} = \frac{\text{Entropyed Drug Content}}{\text{Total Drug Content}} \times 100\tag{1}$$

#### **9.2 Chemical characterization**

Chemical characterization studies gives results for identification of purity in liposomal constituents.

#### *9.2.1 Phospholipids concentration*

Phospholipids concentration can be known by using barrlet assay and its principal depends on colorimetric method by inorganic phosphate measurement. The concentration of phospholipid in liposomes is identified by addition of perchloric acid and that gives inorganic phosphate. On adding ammonium molybdate, inorganic phosphate will be converted into phospho-molybdic acid. On adding 4-amino-2-napthyl-4-sulfonic acid to phospho-molybdic acid under hot condition its gives blue color complex which can be determined calorimetrically at 830 nm.

#### *9.2.2 Cholesterol concentration*

The adequate separation of cholesterol and its oxidation products in liposomal dispersion can be analyzed by HPLC method. This is mainly studied in stability tests for liposomal formulations.

## *9.2.3 Fatty acid composition in phospholipids*

The fatty acid composition in phospholipid or liposomal dispersion is analyzed by gas chromatography. This method is suitable in estimation of fatty acids oxidation. Two types of column are used in gas chromatography. One is packed column in which liquid phase is coated on granular support and packed into a coiled tube of glass or stainless steel. Other is capillary column which is much narrower in bore, longer and made of glass or fused silica capillary and contains no packing but the liquid phase is coated directly on to inner capillary wall.
