The Pharmaceutics and Compounding Laboratory
Colloidal Dispersions

Laboratory Procedure

In the present experiment you will study some of the properties of colloids by examining a colloidal product which resembles Technetium-99m Sulfur Colloid Injection. This product is used daily in the practice of Nuclear Medicine. When used clinically the product is radioactive, but it may be prepared and studied without the use of radioactivity. The commercial product is sold as a radio-pharmaceutical kit that contains the necessary vials and syringes of pre-sterilized, non-radioactive reagents. The radioactive Tc-99m, as sodium pertechnetate, is added at the time of preparation.

In today's experiment, you will prepare Sulfur Colloid several different ways in test tubes to demonstrate the effect that various components have on the final product. Experiment 1 (Tubes A-C) will examine the protective colloid effect of gelatin, experiment 2 (Tubes D-L) examines a buffer incompatibility, and experiment 3 (Tubes M-R) demonstrates a way to resolve the buffer incompatibility.

Experiment 1 studies the effect of gelatin on the stability of hydrophobic colloids. Gelatin is available in two types depending on the method of preparation. Type A gelatin, derived from an acid-treated precursor, has an isoelectric point between pH 7 and 9; type B gelatin, derived from alkali-treated precursor, has an isoelectric point between pH 4.7 and 5.0. A protein at its isoelectric point has a net charge of zero and is least stable in solution; that is, it may precipitate if present in sufficient concentration. At pH values above or below the isoelectric point, gelatin will carry a net negative or positive charge, respectively.

Colloid Preparation

  • Prepare a rolling boiling water bath of approximately 500 ml of water in a 1000 ml beaker.

    Video
    View a video demonstration on a rolling, boiling water bath

  • In a smaller beaker, prepare 25 ml of an aqueous solution containing 9 mg/ml of gelatin. Submerge the beaker of gelatin and water in the water bath to dissolve the gelatin.
  • Label test tubes (16 x 150 mm) alphabetically from A to R.
  • Add the designated volume of reagents into each tube as indicated in Table 1. Add reagents from left to right; sodium thiosulfate first and 0.5M HCl last. Remember to rinse pipets thoroughly between reagents.
  • Following the addition of HCl, mix each tube well using a Vortex mixer.
  • Place the tubes into a rolling boiling water bath for 12 minutes.
  • Remove the tubes and allow to cool for 5 minutes.
  • Add buffer to the appropriate tubes as indicated in Table 1.
  • Mix each tube with the vortex mixer.

Table 1:Colloidal Dispersions
colloidal dispersions table

I. Experimental Protocols, Data, and Results

Experiment 1: Protective Colloid Effect, and Influence of pH on Protective Colloid Effect

  1. In a well lighted area, visually inspect Tubes A and B and C 30 minutes after preparation. Describe any difference in their appearance. (A true colloidal dispersion will appear uniformly hazy or cloudy, and may appear slightly blue when observed by fluorescent light).

    Visual Inspection: Record the appearance of each tube after 30 minutes.
  2. Do not shake the tubes. Using a glass stirring rod transfer a small drop of each solution to a glass microscope slide; Product A next to Product B next to Product C. Cover each drop with a glass cover slip. Observe each product using a light microscope under 100 and 400 - 430 power. Compare the products observing particle size, shape, number and movement. Describe the results.

    microscope

    Microscopic Inspection:

    Particle Description Product A Product B Product C
    Size (comparative)      
    Shape      
    Number/Field (comparative)      
    Movement (yes or no)      

  3. Measure the pH of Tube B________
       Sign of Charge on Gelatin _________
       Measure the pH of Tube C________
       Sign of Charge on Gelatin _________
  4. Allow Tubes A and B and C to stand for 2 hours and visually inspect them. Record the appearance of each tube. Comment on any differences observed.

    Allow Tubes A and B and C to remain standing for 18 - 24 hours and visually inspect them again. Record the appearance of each tube. Comment on any differences observed.

    Visual Inspection: Record the appearance of each tube after 2 hours and after 18-24 hours.

    Tube 2 hours 18-24 hours
    A    
    B    
    C    

  5. Attempt to resuspend the tubes' contents after 18 - 24 hours. What are your findings?

Experiment 2: Effect of Aluminum Ion on Colloid Stability

  1. Observe Tubes D through L after 2 hours. Record the appearance of each tube in #6 below.
  2. Select any tube with a precipitate and shake vigorously. Using a glass stirring rod transfer a small drop to a glass slide, cover with a cover slip and observe under 100 and 400 - 430 power using the light microscope. Compare the observation with the results found in Tubes A and B previously. What effect does aluminum ion have on the colloid dispersion?
  3. Measure the pH of the products in Tubes G and I. Record the result.
      pH of Test Tubes G _____ and I _____
      Sign of Charge on Gelatin __________
  4. Allow tubes to stand for 18 - 24 hours, and visually inspect them again. Record the appearance of each tube in #6 below.
  5. Attempt the resuspend the tubes' contents after 18 - 24 hours. What are your findings?
  6. Is there a concentration of Al3+ where the colloid dispersion is maintained?

    Tube µg Al3+(in tube) Buffer 2 hours 18-24 hours
    D        
    E        
    F        
    G        
    H        
    I        
    J        
    K        
    L        

  7. Compare Tubes G and H, I and J, and K and L, respectively. Explain the reason for the observed difference.
  8. Compare Tubes G, I, and K, with H, J, and L. Explain the reason for the observed difference.

Experiment 3: Stability of a Colloid Against Aluminum and Phosphate Ion Interaction

  1. Allow Tubes M through R to stand 18 - 24 hours and visually inspect them. Record the appearance of each tube. Comment on any difference in their appearance, and record the results.
  2. Calculate the molar ratios of EDTA:A13+ and express as whole number ratios (i.e., 1:x or x:1).

    Tube Mol A13+ Mol EDTA Molar Ratio EDTA:Al3+ Description at 18-24 hours
    M        
    N        
    O        
    P        
    Q        
    R        

  3. Measure the pH in Tube Q. Record the result.
      pH of Test Tube Q ______
      Sign of charge on gelatin ______
  4. Attempt to resuspend the tubes' contents after 18 - 24 hours. What are your findings?

II. SUMMARY

Complete the table. In the left hand column, indicate if the colloidal dispersion is clear or cloudy. In the right hand column, indicate the number of inversions needed to redisperse the colloid.

Tube 30 min 2 hours 18-24 hours # of Inversions Needed to Redisperse Additional Comments
A          
B          
C          
D XXX        
E XXX        
F XXX        
G XXX        
H XXX        
I XXX        
J XXX        
K XXX        
L XXX        
M XXX XXX      
N XXX XXX      
O XXX XXX      
P XXX XXX      
Q XXX XXX      
R XXX XXX      

III. DISCUSSION

  1. Explain any observed differences in tubes A, B, and C.
  2. Explain any observed differences in tubes D-H.
  3. Explain any observed differences in test tubes M through R.
  4. Based on your observations, what is the stoichiometric molar ratio of EDTA:Al3+ required for colloid stability?