**18. Foam fractionation (batch process)**

Initially, feed at a certain concentration was made by diluting stock whey with water to the desired concentration. To achieve the correct Protein Surfactant Ratio (PSR), the necessary amount of Sodium Lauryl Sulphate (SLS) was applied to the feed, which was then allowed to mix evenly with the help of an ultrasonic cleaner. The pH of the feed was then calculated and modified as required by adding

concentrated HCl or concentrated NaOH solution. After that, one litter of feed solution was added to the foam fractionation column, and nitrogen gas was moved through the feed at the required gas flow rate (GFR). An analysis of the percentage of gas hold up' The GFR was held between (100 to 200) cc/min. The frit first creates bubbles, which then rise through the liquid column. When bubbles leave a material, they form foam. Foam was absorbed constantly through the top outlet into a receiver as it moved up the column due to gas velocity. A foam breaker was added to the receiver. For the necessary amount of time, the foam was continuously extracted. The foam was then able to collapse using a stirrer until it broke down into foamate (collapsed foam). The total amount of foamate was weighed, diluted appropriately, and absorbance was recorded. The gas was turned off after the procedure was completed, and the residual liquid in the column was extracted. The volume of the residual liquid was calculated, as well as its concentration. The mass flow rate (MFR) was then calculated using a regression equation derived from a plot of protein volume versus time. Enrichment ratio (ER), separation ratio (SR), and recovery percent (percent RP) were all measured as performance parameters for foam fractionation.

## **19. Foam fractionation (continuous process)**

Feed was prepared by suitable dilution of Bovine Serum Albumin (BSA) to get the desired feed concentration. Required quantity of Sodium Lauryl Sulphate (SLS) was added to the feed to get the desired PSR, it was then allowed to mix uniformly with the help of an ultrasonic cleaner. Then the pH of the feed was measured and adjusted as per requirement. The foam fractionation column was then filled with 1 lit. of feed solution and Nitrogen gas was passed through the feed at desired gas flow rate (GFR). Feed was introduced from outside through an inlet into the column with the help of a peristaltic pump to maintain a constant volumetric flow rate (VFR), and the flowing effluent is constantly collected at intervals through a outlet from other side, the flow rate of the outgoing effluent is same as the incoming feed. Bubbles are formed initially which then rises to the top of the column leading to formation of foam. The foam is continuously collected for required period of time. Foam was then allowed to stir using a stirrer until the foam breaks down to form foam. The effluent was collected in a reservoir, the residual was also collected, then the collected material (effluent) was pumped into the second column, where it acts as feed for the second column. When the work with the first column is finished the gas flow into the first column was stopped and the valve is opened so that the gas now flows into the second column and samples were withdrawn at regular intervals assessed. After steady state was achieved, the effluent showed constant concentration. Whole procedure is repeated again as mentioned above. The volume of foam is measured, sample was suitably diluted and absorbance is noted. The total effluent and residual were collected and absorbance was noted, the total input amount, output amount, loss amount, recovery %, enrichment ratio was also calculated.

### **20. Results and discussion**

Binary protein mixtures were used in batch foam fractionation experiments to assess the enrichment ratio and percentage recovery for various feed concentrations at varying pH of the solutions, liquid pool heights, and air circulation rates. The obtained findings are discussed elaborately. The ratio of the concentration of the

*Separation of Bovine Serum Albumin (BSA) Protein by Foam Fractionation Technique DOI: http://dx.doi.org/10.5772/intechopen.99943*

foam (CP) to the concentration of the feed liquid (Cf) from which the foam was produced is known as the enrichment ratio or separation factor (E).

$$\begin{aligned} \text{Enriclment ratio (E)} &= \text{Concentration of Foam (Cp)} / \\ \text{Concentration of the feed solution (Cf)} \\ \text{Percentage removal (P.R96)} &= \text{Amount of recovered (Cf-Cb)} \ge 100 / \end{aligned} \tag{2}$$

Amount of metal ions in feed solution Cf ð Þ, (2)

where Cb is the concentration of metal ion in residual solution.

### **20.1 Statistical analysis**

Each experiment was at least three times repeated. Graph Pad prism 5 was used to do an analysis of variance on the data. The t-test with (P < 0.05) was used to determine the difference between mean values.

### **20.2 The impact of airflow rate**

Experiments were carried out with different air flow concentrations at fixed other conditions such as a 30 cm liquid pool height, a feed concentration of 3 mg/ mL of Bovine Serum Albumin (BSA) and 2 mg/mL of Haemoglobin, a feed pH of 5.0, a drainage time of 4 minutes, and a foam height of 35 cm. The findings for the impact of air flow rate on enrichment ratio are as follows: The separation factor or enrichment ratio decreased from 2 to 0.568 as the air flow rate rose from 0.3 to 0.8 lpm. Brown et al. verified these findings. The enrichment ratio and percentage elimination increase as the air flow rate is increased from 0.4 to 0.6 lpm at first. However, as the air flow rate increased, the enrichment ratio and percentage elimination decreased. This is because, at low flow speeds, the bubble sizes are greater at first, resulting in more coalescence and drainage. As a result, the enrichment ratio and percentage elimination initially improved. As the air flow rate was increased higher, the foam bubble size declined, and coalescence and drainage reduced. As a result, both the enrichment level and the amount removed decrease.

### **20.3 Height of the liquid pool impact**

**Figure 4** shows the effect of liquid pool height on foam concentration and protein enrichment ratio at set other conditions of 0.2 Lpm air flow volume, 3 mg/ mL Bovine Serum Albumin (BSA) and 2.0 mg/mL haemoglobin, 5.0 pH of feed, 4 min drainage time, and 35 cm foam height. The enrichment ratio of metal ions improved as the height of the liquid pool increased from 5 to 25 cm, as seen in the **Figure 4**. When the liquid pool's height is greater, the residence time of bubbles in the liquid pool is longer. This results in a higher enrichment of metal ions on the bubble surface, and it may reach a point where enrichment cannot increase much more. This equilibrium is achieved in the current analysis at a pool height of 25 cm.

### **20.4 Effect of foam height**

**Figure 5** shows the experimental findings for the effect of foam height on foamate concentration and enrichment ratio. When the foam height is raised from 35 cm, the foam residence time increases, allowing for more liquid draining in the films. As a result, the foam is drier and the enrichment level is higher. The bubble

**Figure 4.** *Schematic diagram of foam fractionation apparatus operating (batch process).*

scale was found to be larger at the liquid-foam interface, indicating that the drainage is greater. Dry foams appear at the tip of the foam as the height is raised, indicating that optimum draining has already occurred. As a response, no substantial difference in enrichment ratio was observed above a certain foam height (**Figures 6**–**8**).

*Separation of Bovine Serum Albumin (BSA) Protein by Foam Fractionation Technique DOI: http://dx.doi.org/10.5772/intechopen.99943*

### **Figure 6.**

*Effect of air flow rate on foam concentration and enrichment ratio [liquid pool height = 30 cm, feed concentration = 3 mg/mL of BSA and 2 mg/mL of haemoglobin, pH of the feed 5, drainage time = 4 min, foam height = 35 cm]. Compared to the enrichment ratio of total protein with concentration of BSA in foam concentration, the p-value significantly changed (\*\*p < 0.05). As we air flow rate increases so the foam bubble size reaches big resulting in the waste product produce so significantly change observe 0.1 lpm to 0.8 lpm (litre per minute) height increase.*

### **Figure 7.**

*Effect of liquid pool height on foam concentration and enrichment ratio [air flow rate = 0.2 lpm, feed concentration = 3 mg/mL of BSA and 2 mg/mL of haemoglobin, pH of the feed 5.0, drainage time = 4 min, foam height = 35 cm]. Compared to the enrichment ratio of total protein with concentration of BSA in foam concentration, the p-value significantly changed (p < 0.05). As we foam height increase so the foam reaches dry so significantly change observe a 20 cm to 35 cm height increase.*

### **Figure 8.**

*Effect of foam height on foam concentration and enrichment ratio of total proteins [air flow rate = 0.2 Lpm, liquid pool height = 30 cm, feed concentration = 3 mg/mL of BSA and 2 mg/mL of haemoglobin, pH of the feed = 5.0, drainage time = 4 min]. Compared to the enrichment ratio of total protein with concentration of BSA in foam concentration, the p-value significantly changed (\*\*p < 0.05). As we liquid pool height increase so the foam reaches dry so significantly change observe a 5 cm to 30 cm height increase.*

### **20.5 Effect of pH of feed**

In **Figure 5** the impact of feed pH on formats concentration and protein enrichment ratio is seen. It can be shown that the highest enrichment ratio is obtained at a pH of 5.0. These were induced by the protein's increased hydrophobicity at its isoelectric point. Between proteins adsorbed on the air-liquid interface, an electrostatic repulsive force and the Vander Waals attractive force act. The dissociation of amino acid residues causes the surface charge on the protein molecule. Because this electrostatic repulsion between protein molecules adsorbed on the bubble surface is thought to be lowest at the isoelectric point, proteins should be adsorbed more compactly on the bubble surface at that point (**Figure 9**).

### **20.6 Effect of feed concentration**

Impact of haemoglobin and BSA concentrations in feed on foamate concentration and enrichment ratio seen changes. The concentration of haemoglobin in the feed solution was increased from 0.6 to 1.6 mg/L by maintaining the BSA concentration at 3 mg/L, and it was discovered that as the concentration of haemoglobin in the feed solution was increased, its adsorption decreased, but BSA adsorption increased. That's because the inclusion of haemoglobin in the bulk solution facilitates the adsorption of BSA on the bubble surface. In the presence of haemoglobin, the BSA concentration in the foam is clearly increased. As the feed haemoglobin concentration exceeds 1.5 mg/mL, it ceases adsorbing on the bubble surface entirely. The enrichment ratio and the foam concentration of BSA decrease as the feed concentration of BSA is increased from 2 to 2.9 mg/mL at fixed other conditions of 2 mg/mL of haemoglobin in feed, 0.2 lpm of air flow volume, 30 cm of liquid pool height, 5.0 pH of feed, drainage time of 4 min., and 35 cm of foam height. This may be because the surface tension reduces as the feed concentration

*Separation of Bovine Serum Albumin (BSA) Protein by Foam Fractionation Technique DOI: http://dx.doi.org/10.5772/intechopen.99943*

### **Figure 9.**

*Effect of pH of feed on foam concentration and enrichment ratio of total protein [air flow rate = 0.2 Lpm, liquid pool height = 30 cm, feed concentration = 3 mg/mL of BSA and 2 mg/mL of haemoglobin, drainage time = 4 min, foam height = 35 cm]. Compared to the enrichment ratio of total protein with concentration of BSA in foam concentration, the p-value significantly changed (\*\*\*p < 0.05) from pH 4 to pH 8.*

increases. As a result, more stable bubbles form with less coalescence, resulting in decreased drainage. As a result, the wetness of the foam is greater, lowering the enrichment ratio and percentage reduction.

### **21. Conclusion**

The effects of parameters like air flow rate, liquid pool height, feed concentration, pH of the feed, and foam height on the foam concentration and enrichment ratio were studied in experimental studies on batch foam separation of binary proteins such as Bovine serum albumin (BSA) and haemoglobin. The perfect pH for maximal separation was discovered to be 5, perhaps owing to enhanced hydrophobicity of proteins. The amount of BSA adsorbed on the foam increases as the concentration of haemoglobin in the feed increases. This is because the inclusion of haemoglobin in the feed liquid enhances the adsorption of BSA on the bubble surface. At the optimal operating conditions of 0.2 Lpm air flow rate, 30 cm liquid pool height, feed concentration of 3 mg/ml BSA and 2 mg/mL haemoglobin, 5.0 pH of feed, and 35 cm foam height, an enrichment ratio was achieved. As a result, the foam separation technique of pure BSA focused on the foam will successfully separate binary proteins Bovine serum albumin (BSA) and haemoglobin.

*Bovine Science - Challenges and Advances*
