A Guide to Simplifying the Interpretation of Dynamic Light Scattering Data

The quality of data acquired from a dynamic light scattering (DLS) measurement has a vital role in the reliability of the obtained result. A size quality report has been created for the Zetasizer Nano range of instruments to simplify the interpretation of the data obtained from a dynamic light scattering measurement.

By default, this size quality report is displayed as one of the tabs in the size workspace. Various tests are carried out on a selected record, and in case any of the tests do not fall into the specified limits, a warning message is displayed along with the advice of probable reasons for the warning. In case there is no failure of any of the tests, a “Result Meets Quality Criteria” message is displayed in the Size Quality Report.

The size quality report includes 12 tests on any selected record. Table 1 summarizes the tests included and lists details and descriptions of the tests, the warning message displayed, probable reasons for the warning message and remedial actions.

Table 1. Summary of the tests incorporated into the size quality report listing descriptions and details of the tests, the warning message that is displayed, possible reasons for the warning message and possible actions that might remedy it.

Test Number Test Description Test Details Warning Message Possible Reasons for Warning Message Possible Actions
1 Check appropriate lower size analysis limit used in the distribution analysis Is the z-average diameter < lower analysis limit? z-average is smaller than lower size analysis limit The wrong lower size analysis limit used in the distribution analysis settings. Only applicable to research software Edit the lower size analysis settings in the research software
2 Check appropriate upper size analysis limit used in the distribution analysis Is the z-average diameter > upper analysis limit? z-average is larger than upper size analysis limit The wrong upper size analysis limit used in the distribution analysis settings. Only applicable to research software Edit the upper size analysis settings in the research software
3 Check appropriate lower size display limit used Is the z-average diameter < lower display limit? z-average is smaller than lower display limit The wrong lower display limit used. Edit the lower display settings
4 Check appropriate upper size display limit used Is the z-average diameter > upper display limit? z-average is larger than upper display limit The wrong upper display limit used. Edit the upper display settings
5 Check polydispersity index value Is the polydispersity index value > 1? Polydispersity index is very high Sample is very polydisperse and may not be suitable for DLS measurements Sample may not be suitable for DLS technique
Sample contains large particles/ aggregates/ dust Remove large particles/ aggregates/ dust by filtration or centrifugation
Wrong measurement position used (applicable only to Nano S/ZS) Seek for optimum measurement position should be selected
6 Check correlation function intercept value Is the correlation function intercept <0.1 or >1.0? Correlation function intercept out of range Sample concentration too high (multiple scattering) Dilute sample and re-measure
Sample concentration too low Increase sample concentration and re-measure
Sample fluorescence Use narrow band filter option
User different laser wavelength
Sample absorbance (colored sample) User different laser wavelength
Wrong measurement position selected (Nano S/ZS only) Allow instrument to seek for optimum measurement position
Sample contains very large particles (baseline definition problems) and may not be suitable for DLS measurements Remove large particles/ aggregates/ dust by filtration or centrifugation
7 Check first point selection for multimodal analysis Is the first correlation point >3 and the zaverage diameter <5 nm? Check first correlation point selection for multimodal analysis Only applicable to research software and is a check that the first correlation point selection for the distribution analysis is suitable for the particle size obtained Edit the first point selection for the distribution analysis and reanalyze the record
8 Check the in range value Is the in range value less than 90% The in range figure is low Presence of large or sedimenting particles Remove large particles/ aggregates/ dust by filtration or centrifugation
Sample fluorescence Use narrow band filter option
User different laser wavelength
Sample absorbance (colored sample) User different laser wavelength
9a Check for low mean count rate Is the mean count rate <20 kcps? Count rate is out of range Attenuator not set to automatic Allow instrument to seek for optimum attenuator position
Sample concentration too low Increase sample concentration and re-measure
Sample absorbance (colored samples) User different laser wavelength
Sample is not stable during measurement Sample may not be suitable for DLS technique
9b Check for high mean count rate Is the mean count rate >1000 kcps? Count rate is out of range Attenuator not set to automatic Allow instrument to seek for optimum attenuator position
Sample contains large particles/aggregates/dust Remove large particles/ aggregates/ dust by filtration or centrifugation
Sample is not stable during measurement Sample may not be suitable for DLS technique
10 Check sufficient data collected Is the total number of photons collected <10000 K? Insufficient signal collected Measurement duration not set to automatic Set instrument to automatic duration
Attenuator not set to automatic Allow instrument to seek for optimum attenuator position
Filter factor not set to default (50%). Only applicable to research software Edit filter factor setting in the research software
11 Check the cumulants fit error Is the cumulants fit error >0.005? Cumulant fit error high Data quality too poor for cumulant analysis Sample may not be suitable for DLS technique
Sample too polydisperse for cumulant analysis Sample more suitable for distribution analysis
Inappropriate cumulant analysis settings in research software Edit cumulant settings in research software
12 Check the distribution fit error Is the distribution fit error >0.005? Multimodal fit error high Data quality too poor for distribution analysis Sample may not be suitable for DLS technique
Sample too polydisperse for distribution analysis Sample may not be suitable for DLS technique
Inappropriate distribution analysis settings in research software Edit distribution settings in research software

 

In this article, example results are used to describe the warning messages and offer details of probable actions of ways to enhance the results.

Test 1

Is the z-Average Diameter < Lower Analysis Limit?

This test only applies to the research version of the Nano software where the limits of the distribution analysis have been manually selected incorrectly. The intensity size distribution acquired for a protein sample with a z-average diameter of 8.9 nm is illustrated in Figure 1. In the research software, the lower distribution analysis limit has been manually set to 10 nm. This gives the “z-average is smaller than lower size analysis limit” warning message in the size quality report. The problem can be rectified by selecting the record in the Record View and editing it (by clicking on Edit, Edit Result). This action leads to the Edit Result window.

An intensity size distribution of a protein sample with a z-average diameter of 8.9 nm where the lower distribution analysis limit has been manually set to 10 nm in the research software.

Figure 1. An intensity size distribution of a protein sample with a z-average diameter of 8.9 nm where the lower distribution analysis limit has been manually set to 10 nm in the research software.

On the Size Analysis tab, the Update Size Model option has to be selected. After selecting the suitable distribution analysis model (Multiple Narrow Modes, General Purpose, or CONTIN), it is necessary to access the Size Classes tab (see Figure 2). It is essential to either change the Lower Limit to an appropriate value or reset it to the Default value of 0.4 nm.

An appropriate value for the Lower Limit in the Size Classes tab needs to be selected.

Figure 2. An appropriate value for the Lower Limit in the Size Classes tab needs to be selected.

Calculation of a new record is performed by implementing these new settings and is displayed in the Record View. The intensity size distribution acquired for the edited record shown in Figure 2 is illustrated in Figure 3. Now, a “Result Meets Quality Criteria” message is displayed by the Size Quality Report.

The intensity size distribution obtained for the edited record displayed in Figure 2.

Figure 3. The intensity size distribution obtained for the edited record displayed in Figure 2.

Test 2

Is the z-Average Diameter > Upper Analysis Limit?

This test is only applicable for the research version of the Nano software in which the distribution analysis limits have been manually selected incorrectly. This test is similar to the one discussed in Test 1, and it is possible to rectify any warning message in the same manner as discussed above (see Figure 2). The only variation is the value used as the upper analysis limit.

Test 3

Is the z-Average Diameter < Lower Display Limit?

This test guarantees the selection of an appropriate lower display limit for the result. For this test, a warning message is obtained only if the user manually sets the lower display limit to an unsuitable value. The intensity size distribution of the protein sample used above as an example is illustrated in Figure 4; here, the lower display limit has been set as 15 nm. The left-hand side of the distribution has been cut off at the lower display limit that has been set. This results in the warning message “z-average diameter is smaller than lower size display limit” in the size quality report. It is necessary to edit the lower display limit in the record to a suitable value to rectify this problem.

The intensity size distribution obtained for a protein sample with a z- average diameter of 8.9 nm and a lower display limit set to 15 nm.

Figure 4. The intensity size distribution obtained for a protein sample with a z- average diameter of 8.9 nm and a lower display limit set to 15 nm.

Following the selection of the record and clicking on Edit, Edit Result, the Size Analysis Settings tab must be selected in the Edit Result window. The Advanced button can be accessed by selecting the Update Analysis Model (see Figure 5). It should be noted that the Size Analysis Settings are considerably simple in the normal software than in the Research software (refer to Figure 2). It is also worth noting that the Advanced button displays a red exclamation mark, suggesting that one of the settings is not at default value.

Selecting the Update Analysis Model on the Size Analysis Settings tab allows the Advanced button to be accessed Clicking on the Advanced button gives access to the Advanced Analysis Settings (Figure 6). This allows the Display Size Limits to be modified to appropriate values. Note that the default values for the lower and upper display limits are 0.6 and 6000 nm, respectively.

Figure 5. Selecting the Update Analysis Model on the Size Analysis Settings tab allows the Advanced button to be accessed Clicking on the Advanced button gives access to the Advanced Analysis Settings (Figure 6). This allows the Display Size Limits to be modified to appropriate values. Note that the default values for the lower and upper display limits are 0.6 and 6000 nm, respectively.

The Advanced Analysis Settings tab allows the Display Size Limits to be edited.

Figure 6. The Advanced Analysis Settings tab allows the Display Size Limits to be edited.

A new record is generated with the edited settings in the Record View. Figure 3 illustrates the intensity size distribution acquired for the protein sample with the help of default display limit settings. The Size Quality Report provides a “Result Meets Quality Criteria” message.

Test 4

Is the z-Average Diameter > Upper Display Limit?

This test is smiliar to the one discussed in Test 3, and it is possible to correct any warning message in the same manner as discussed earlier (see Figures 5 and 6).

Test 5

Is the Polydispersity Index Value >1?

The polydispersity index (PDI), along with the z-average diameter, is calculated using the cumulants analysis as described in ISO 13321. It is a dimensionless estimate of the distribution’s width, scaled from 0 to 1.

A PDI > 1 suggests that the size distribution of the sample is very broad and that the sample may consist of large particles or aggregates that could be slowly sedimenting. In such an instance, the sample may not be appropriate for a DLS measurement. A correlation function acquired using a sample with a broad size distribution and made of large, sedimenting particles is illustrated in Figure 7 (the noisy baseline must be noted here).

A correlation function from a sample consisting of a broad size distribution containing large, sedimenting particles.

Figure 7. A correlation function from a sample consisting of a broad size distribution containing large, sedimenting particles.

A PDI value of 1 was obtained from this sample, which is a good example of a sample not appropriate for the DLS method. It must be noted that the intercept value is greater than 1.0. This will be discussed further in Test 6 below. Usually, if a sample returns a PDI value of 1.0, cumulants and distribution fit errors also occur, which will be discussed further in Tests 11 and 12 below.

In Test 5, another probable reason for getting a warning message is the selection of an incorrect measurement position in a Zetasizer Nano S or ZS instrument. The default option is to enable the software to automatically request for optimum position. Yet, it is possible to manually select the measurement position. The Advanced Button on the Measurement Settings window can be clicked to access the default option of requesting for the optimum measurement position either while creating an SOP or setting up a Manual Measurement (see Figure 8).

Clicking the Advanced button on the Measurement Settings window allows access to the default option of seeking for the optimum measurement position.

Figure 8. Clicking the Advanced button on the Measurement Settings window allows access to the default option of seeking for the optimum measurement position.

Test 6

Is the Correlation Function Intercept <0.1 or >1.0?

The intercept value, which is the signal/background ratio, varies based on the sample and instrument optical configuration.

Suitable values are between 10% and 100% (i.e. 0.1 to 1.0). A value lower than the anticipated value indicates a very low or very high sample concentration, fluorescence, or absorption of the sample. A very high sample concentration could result in multiple scattering effects, which leads to a reduction in the intercept value. The NIBS configuration of the Zetasizer Nano S/ZS enables the laser beam’s path length to be reduced, which, consequently, will reduce any multiple scattering that exists. Possibilities are that the incorrect measurement position has been manually selected, leading to multiple scattering effects. The default setting of enabling the software to automatically request for the optimum measurement position can be accessed as described in Test 5 above (refer to Figure 8).

A correlation function acquired from a sample that exhibits fluorescence is illustrated in Figure 9, showing an intercept value of less than 0.1. Optimized data can be obtained by making use of the narrow band filter option available for the Zetasizer Nano series. In case the data quality is not adequately enhanced using this, then an instrument with a distinct wavelength of laser would have to be used.

A correlation function obtained from a sample exhibiting fluorescence. Note the very low intercept value of 0.011 obtained.

Figure 9. A correlation function obtained from a sample exhibiting fluorescence. Note the very low intercept value of 0.011 obtained.

The correlation function illustrated in Figure 7 was achieved by using a sample made of large, sedimenting particles which lead to interference in the measurement (number fluctuations). When such large particles are present, it leads to an ill-defined baseline that results in an intercept value of more than 1.0. Moreover, in range, possibilities are that cumulants and distribution fit error messages are obtained (for further information, refer to Tests 8, 11, and 12). This sample will be appropriate for measurement using DLS only if it will be possible to suitably remove the large particles present by, for instance, centrifugation or filtration.

Test 7

Is the First Correlation Point >3 and the z-Average Diameter <5 nm?

This test is only applicable for the research version of the Nano software and verifies whether the first correlation point used in the distribution analysis is appropriate for the obtained particle size. For this test, a warning message would be received only if the user manually sets the first correlation point to an incorrect value with the z-average diameter less than 5 nm. This problem can be overcome by selecting the record in the Record View and editing it (by clicking on Edit, Edit Result). This action leads to the Edit Result window.

It is necessary to select the Update Size Model option on the Size Analysis tab. Following the selection of the suitable distribution analysis model (Multiple Narrow Modes, General Purpose, or CONTIN), it would be possible to access the Points Selection section on the selected distribution analysis tab (see Figure 10). It is vital to either change the First Channel of the Point Selection to a suitable value or to reset it to the Default value of 3. A new record can be calculated by implementing the new settings and is displayed in the Record View.

The points selection section can be accessed on the chosen distribution analysis tab in the Edit Result window.

Figure 10. The points selection section can be accessed on the chosen distribution analysis tab in the Edit Result window.

Test 8

Is the in-Range Value Less than 90%?

The in-range value specifies the overall quality for good data acquired from a measurement. The maximum value that can be achieved is 100%, and a majority of the effects that contrive to spoil a measurement will minimize this value.

In this test, the principal reason for obtaining a warning message is that the sample includes large, sedimenting particles. Figure 7 illustrates a typical correlation function from such a sample, where the sample is highly polydisperse and the large particles that exist cause baseline definition problems. Without removing the large particles by centrifugation or filtration, this sample is not appropriate for DLS measurement.

Other probable reasons for low in-range values are sample absorbance or fluorescence due to which definition of the baseline could be difficult and/or the intercept value of the correlation function could be very low (see Test 8 and Figure 9 above).

Test 9a

Is the Mean Count Rate <20 kcps?

This test verifies whether adequate scattering is detected at the time of a measurement.

There are different reasons for this warning message, most are sample-related. These include a sample exhibiting strong absorbance of the laser beam, a very low sample concentration, or a sample not stable at the time of the measurement and not appropriate for the DLS method.

The Zetasizer Nano is equipped with an automatic attenuator that modifies the intensity of the laser that enters the sample cuvette. This then modifies the intensity of the detected scattered light. In the case of samples that do not scatter a considerable amount of light (e.g. samples of low concentration or very small particles), it is necessary to increase the amount of scattered light being detected. Hence, the attenuator will automatically enable more light to pass through the sample. In the case of samples that considerably scatter light (for example, samples at high concentration or large particles), the attenuator automatically minimizes the amount of light passing through to the sample.

The attenuator equipped in the Zetasizer Nano includes 11 positions covering an attenuator range of 99.997% (Position 1) to 0% (Position 11). Hence, if the automatically selected attenuator position is 11 (0% attenuation or 100% transmission) and the mean count rate is below 20 kilo counts per second (kcps), it indicates either a very low sample concentration or a strong sample absorbance. If low concentration is the issue, then it is necessary to increase the sample concentration if possible and perform re-measurement. If strong absorbance is the issue, the only successful method to optimize the results could be to use a distinct wavelength of laser.

It is possible for particles in a sample to dissolve gradually, creaming or sedimenting at the time of the measurement. In each of these cases, the mean count rate reduces with time. An unstable sample such as this might not be appropriate for measurement by DLS. A more appropriate solvent might eliminate dissolution and therefore stabilize the sample.

Elimination of the sedimenting particles by centrifugation or filtration will enhance the appropriateness of the sample to DLS measurement. As described in Test 8 above, an in-range warning message is also given by particle sedimentation.

The final reason for this warning message could be that the attenuator setting is not set to automatic and an incorrect setting has been used where the attenuation of the laser beam is very high. It is recommended for the instrument to be enabled to select the optimum attenuator position. This default option of choosing the optimum attenuator position can be accessed by clicking the Advanced Button on the Measurement Settings tab either while creating an SOP or while setting up a Manual Measurement (see Figure 11).

Clicking the Advanced button on the Measurement Settings tab allows access to the default option of seeking for the optimum attenuator position.

Figure 11. Clicking the Advanced button on the Measurement Settings tab allows access to the default option of seeking for the optimum attenuator position.

A count rate as close as 300 kcps can be used (if this is achievable) by verifying the Seek For Optimum Attenuator option in the Advanced–Measurement Positioning and Attenuation window.

Test 9b

Is the Mean Count Rate >1000 kcps?

This test is similar to Test 9a with the exception that it verifies whether the scattering detected is very high.

One reason for a warning message is that an incorrect attenuator setting has been used (very low attenuation). This can be corrected in the same manner as described in Test 9a above and illustrated in Figure 11.

Other reasons for obtaining a warning message with this test are sample-related. The sample might include large particles that lead to high levels of scattering and these will have to be eliminated by enabling them to naturally sediment with time or by centrifuging or filtering the sample. On the other hand, the sample might slowly aggregate with time, resulting in an increase in the scattering intensity. In that case, the dispersion must be somehow stabilized (e.g. adjustment of pH, addition of surfactant, etc.).

Test 10

Is the Total Number of Photons Collected <10,000 K?

This test verifies whether adequate data has been collected. There are three probable reasons for receiving a warning with this test, which are all related to inappropriate settings in the software.

First, the duration of the measurement may have been manually set to an incorrect value. It is recommended to use the default option of automatically enabling the software to ascertain the optimum measurement duration. This is performed by choosing the Automatic Measurement Duration in the Measurement Settings window while setting up a Manual Measurement or creating an SOP (see Figure 12).

Automatic Measurement Duration can be selected in the Measurement Settings window when setting up a Manual Measurement or creating an SOP.

Figure 12. Automatic Measurement Duration can be selected in the Measurement Settings window when setting up a Manual Measurement or creating an SOP.

Second, the attenuator position might be set at an incorrect value. This can be modified as described in Test 9a above (see Figure 11).

Ultimately, the Filter Factor may not be set to the default value of 50%. This is only applicable to the research software. Each of the size measurements carried out by the Nano are divided into a sequence of sub-runs. The default Filter Factor value of 50% suggests that only half of the sub-runs taken are employed in the final data analysis. The criterion for determining the sub-runs that are selected is the mean count rate. In the analysis, the 50% of sub-runs with the lowest mean count rates are used. This approach is chosen to reduce the effects of any dust contamination that may be present. In case the Filter Factor is manually set to an incorrect value, inadequate data may have been used in the analysis.

It is possible to edit a record such that more of the sub-run data is used. This is performed by choosing the relevant record in the Record View and editing it (by clicking on Edit, Edit Result). This action leads to the Edit Result window. It is necessary to select the Update Size Model option on the Size Analysis tab. The Advanced Analysis Settings can be accessed by clicking on the Advanced button (see Figure 13). The Filter Factor can be modified to a suitable value by clicking on the Update Filtering option (see Figure 14). A new record is calculated by implementing these new settings and is displayed in the Record View.

The Advanced button can be clicked on the Size Analysis tab in the Edit Result window to give access to the Advanced Analysis Settings window (Figure 14).

Figure 13. The Advanced button can be clicked on the Size Analysis tab in the Edit Result window to give access to the Advanced Analysis Settings window (Figure 14).

Clicking on the Update Filtering option allows the Filter Factor to be changed to an appropriate value.

Figure 14. Clicking on the Update Filtering option allows the Filter Factor to be changed to an appropriate value.

Test 11

Is the Cumulants Fit Error > 0.005?

In the cumulants analysis, the z-average diameter and polydispersity index are derived as described in the International Standard ISO 13321. The cumulants fit error is a measure of the quality of the cumulants adjusted to the measured data showing whether the polydispersity and z-average diameter obtained for a specific measurement are reliable values.

If the fit error is more than 0.005, a warning message is obtained with this test. This could be as a result of the very poor data quality or the sample being highly polydisperse for the cumulants analysis. A correlation function acquired from a sample with a broad size distribution containing large, sedimenting particles is illustrated in Figure 7. This is a good example of a sample that is inappropriate for the DLS method. With this sample, a cumulants fit error message is obtained.

The intensity size distribution and the correlation function obtained for a bi-modal sample are illustrated in Figure 15. The size quality report gives a cumulants fit error indicating that in this example, the z-average diameter, of 301 nm, is not reliable. Figure 16 shows the cumulants and distribution fits.

The correlation function and intensity size distribution (inset) obtained for a bi-modal sample.

Figure 15. The correlation function and intensity size distribution (inset) obtained for a bi-modal sample.

The cumulants fit and distribution fit (inset) obtained for the bi-modal sample shown in Figure 15.

Figure 16. The cumulants fit and distribution fit (inset) obtained for the bi-modal sample shown in Figure 15.

Although the bimodality of the sample results in a poor cumulants fit, the distribution fit is exceptional. Hence, despite the fact that the z-average diameter for this record is not reliable, the intensity size distribution acquired from the distribution analysis is accurate.

Another reason for the cumulants fit error is the use of incorrect settings in the cumulants analysis. This is only applicable for the research software. In such a case, it would be possible to edit the record by choosing the relevant record in the Record View and by clicking on Edit, Edit Result. It is necessary to select the Update Size Model option on the Size Analysis tab in the Edit Result window. The Advanced Settings of the Cumulants Analysis can thus be accessed (see Figure 17). Once the settings are edited to more suitable values, a new record is created with the edited settings in the Record View.

The Advanced Cumulants Settings can be edited by clicking on Update Size Model on the Size Analysis tab in the Edit Result window.

Figure 17. The Advanced Cumulants Settings can be edited by clicking on Update Size Model on the Size Analysis tab in the Edit Result window.

Test 12

Is the Distribution Fit Error > 0.005?

The distribution fit error is a measure of the quality of the selected distribution analysis fit to the measured data. Multiple Narrow Modes and General Purpose are the distribution analyses that can be used in the standard Nano software. CONTIN and Discrete Exponential Analysis are also available in the research version of the software.

If the fit error is greater than 0.005, a warning message is received with this test. This could be as a result of the very poor data quality or the sample being highly polydisperse indicating that the sample might not be appropriate for the DLS method. Figure 7 illustrates an example of such a sample. This sample provides warning messages for the in-range test, the cumulants fit, the correlation function intercept test, the distribution fit, and the polydispersity index test.

Incorrect settings in the selected distribution algorithm can also result in a distribution fit error message.

However, this is only applicable for the research software. It would be possible to select more suitable settings by editing the relevant record in the Record View (by clicking on Edit, Edit Result). It is necessary to select the Update Size Model option on the Size Analysis tab in the Edit Result window. After selecting the suitable distribution analysis model (Multiple Narrow Modes, General Purpose, or CONTIN), it would be possible to edit more suitable settings in the selected distribution analysis tab (see Figure 10) and a new record will be created in the Record View.

This information has been sourced, reviewed and adapted from materials provided by Micromeritics Instrument Corporation.

For more information on this source, please visit Micromeritics Instrument Corporation.

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