Using the Nicomp for Automatic Dilution in Sample Preparation

Dynamic light scattering (DLS) can be a challenging or easy technique based on the sample. Occasionally, the challenge comes from data interpretation1 and sometimes from sample preparation2. The dilution step in sample preparation is where a number of users make mistakes that result in repeatability, poor accuracy or both. The Nicomp automatic dilution option eradicates the chance of operator error and enables the generation of optimum results.

Introduction

Classical DLS experiments are performed at low concentrations for several reasons including the ease of using the solvent viscosity and the avoidance of concentration effects. Concentration effects comprise of multiple scattering and particle-particle interactions. The algorithms and theory used in DLS systems believe that light interacts with a single particle and it gets scattered onto the detector.

As concentrations increase, the likelihood of the scattered light to interact with additional particles before reaching the detector increases. This effect decreases the reported particle size. Higher concentrations also impact particle-particle interactions and can lead to restricted diffusions — also introducing errors into the final result. Measuring at lower concentrations also refers to the fact that the viscosity of the diluent/solvent can be used instead of performing a viscosity measurement before the particle size analysis.

The lower concentration limit of the Nicomp DLS system (Figure 1) is influenced by the scattering properties of the sample; see TN 718 - 0.1 mg/mL Lysozyme3. Measurements at extremely low concentrations can be noisy, reducing repeatability and accuracy. The upper concentration limit is based on both the concentration and size of the sample, with improved high concentration measurements possible for extremely small than for bigger size particles.

Nicomp DLS system

Figure 1. Nicomp DLS system.

Ideally, the sample concentration is adjusted so that the count rate on the detector is approximately 300 kHz. This scattering intensity is considered to be “the sweet spot” for standard DLS size measurements. For a number of samples, the Nicomp can simply automatically adjust the neutral density (ND) optical filter in order to adjust incident laser intensity to decrease or increase the count rate to the optimum range. However, a pre-dilution step may be required for other samples in order to adjust to the ~300 kHz range. A word of caution to users; just because the ND filter reduced counts to the proper range, this does not promise that all concentration effects have been eliminated from the experiment.

Nicomp Autodilution Option

The PSS Nicomp is considered to be the only DLS system to offer an auto-dilution option. This technology was patented a number of years ago. The fluidics that allow the auto-dilution include a flow pump (Figure 2), mixing chamber, diluent source and flow through cell (Figure 3). Additionally, a multi-position valve and syringe is mounted in front of the unit in order to facilitate sample introduction (Figure 4).

Flow pump and inlet/outlet tubing.

Figure 2. Flow pump and inlet/outlet tubing.

Flow through cell.

Figure 3. Flow through cell.

Injection syringe and valve on front panel

Figure 4. Injection syringe and valve on front panel Note: the button on the left of the display is used to open and close the sample valve.

Experimental

A high concentration pharmaceutical emulsion was employed for testing the Nicomp autodilution feature. These emulsions are regularly tested in order to determine if they meet the size criteria provided in USP 729, Globule Size Distribution in Lipid Injectable Emulsions4,5. The samples are labeled “Sample 1” for the newer sample and “Sample 2” for the older sample. Both samples were over one year beyond the shelf life date on the container packaging.

The Nicomp settings used for the measurements are displayed in Figures 5, 6 and 7.

Nicomp Control Menu.

Figure 5. Nicomp Control Menu.

System Setup menu.

Figure 6. System Setup menu.

Nicomp Auto Print/Save Menu.

Figure 7. Nicomp Auto Print/Save Menu.

The flow pump was initialized and all tubing was checked for bubbles (none found) prior to making measurements. The syringe seen in Figure 4 was filled with 5 mL of filtered DI water. Drops of the emulsion samples were then added to the open end of the syringe. First one drop, followed by two drops, three drops, etc. The valve open/close control button was turned on until 2 mL of sample was dispensed into the system. The Start Autodilution icon was clicked and the system started to dilute the sample. The protocol must first exceed the preferred count rate by about 20% (Intensity Overshoot Factor in Figure 6) and then it must dilute down in order to attain the Intensity Setpoint of 300.

This procedure was followed for Sample 1 by adding one through nine drops of emulsion and Sample 2 by adding one through four drops of emulsion. A standard disposable pipette was used for transferring the drops. Two measurements were made for each sample and number of drops. For each sample, the intensity mean and Polydispersity Index6 (PI, or variance) was recorded.

Results

A plot of five overlays from Sample 1 is displayed in Figure 8 and a table of the recoded values is displayed in Table 1. Note: overall reproducibility/repeatability is calculated by showing the coefficient of variation (COV) defined as:

Overlay of five results from Sample 1.

Figure 8. Overlay of five results from Sample 1.

Table 1. Intensity mean and PI for Sample 1.

Sample 1 Mean PI
1 drop R1 282.3 0.032
1 drop R2 281.3 0.049
2 drops R1 277.3 0.036
2 drops R2 278 0.049
3 drops R1 280.2 0.047
3 drops R2 281.1 0.069
4 drops R1 280.7 0.042
4 drops R2 281.3 0.044
5 drops R1 279.3 0.06
5 drops R2 280.4 0.034
6 drops R1 280.2 0.037
6 drops R2 279.3 0.04
7 drops R1 280.2 0.064
7 drops R2 280 0.056
8 drops R1 278.2 0.05
8 drops R2 279.3 0.066
9 drops R1 280.6 0.083
9 drops R2 278.8 0.065
Mean 279.92
St dev 1.29
COV 0.46

 

A plot of five overlays from Sample 2 is revealed in Figure 9 and a table of the recoded values is shown in Table 2.

Overlay of five results from Sample 2.

Figure 9. Overlay of five results from Sample 2.

Table 2. Intensity mean and PI for Sample 2.

Sample 2 Mean PI
1 drop R1 289.8 0.042
1 drop R2 288.4 0.056
2 drops R1 289 0.048
2 drops R2 290.7 0.035
3 drops R1 287 0.043
3 drops R2 285.9 0.05
4 drops R1 286.8 0.049
4 drops R2 287 0.043
Mean 288.08
St Dev 1.67
COV 0.58

References

1. PSS Technical Note - 730 - DLS Data Interpretation

2. PSS Technical Note - 722 - DLS Sample Preparation

3. PSS Technical Note - 718 - 0.1 mg per mL Lysozyme

4. USP 729—Globule Size Distribution in Lipid Injectable Emulsions

5. PSS Application Note - 736 - USP 729

6. PSS Technical Note - 730 - DLS Data Interpretation

This information has been sourced, reviewed and adapted from materials provided by Particle Sizing Systems, An Entegris Company.

For more information on this source, please visit Particle Sizing Systems, An Entegris Company.

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