Assist Data and Results Interpretation with an Expert Advice System

This article discusses the Expert Advice System available in 5.00 and subsequent versions of the Zetasizer Nano software. This offers information on the quality of the data and results obtained from both recorded and live measurements.

Earlier versions of the Zetasizer® Nano software contain several parameters and reports to help users with the interpretation of the data and results obtained from measurements. These include:

  • Size Quality Report which includes13 tests executed on a chosen record. If any test falls outside specific limits, a warning message is displayed along with advice on probable reasons for the warning.
  • Zeta Potential Quality Report which includes six tests executed on a preferred record.
  • Several Record View parameters (for instance, intercept, fit errors) which can be employed to analyze the quality of the data obtained.

These parameters and reports are available for post measurement analysis, that is, a measurement has to be completed before the data quality can be analyzed. The Expert Advice system is available for post and live measurement analysis.

Location of the Expert Advice System in the Software

In the Zetasizer Nano software, the Expert Advice system is available at several locations.

The Expert Advice Tab

During a live measurement, the Expert Advice Tab is available in the Measurement Display Window (Figure 1). The Expert Tab is a live report of the quality of the data being created for both a single measurement and a series of repeat measurements.

The Expert Advice tab is available during a live measurement in the Measurement Display Window

Figure 1. The Expert Advice tab is available during a live measurement in the Measurement Display Window

The single measurements employ the same standards used in the quality reports, with the difference being that the comments can be observed during the measurement. During a series of repeat measurements, the system searches for trends in the results.

Comments are provided in relation to variability in the results from variations in the sample due to, for instance, dissolution or aggregation, or where the first measurement is different because the sample has not had time to thermally equilibrate.

The Expert Advice System

This is accessed post measurement by clicking the right mouse on preferred records, or by choosing View, Expert Advice (Figure 2). This will show the tips dialogue of expert advice system, signifying the quality of the measurements preferred. An example dialogue which could be obtained for size measurements is shown in Figure 3.

The Expert Advice system can be accessed post measurement by right mouse clicking on selected records, or by selecting View, Expert Advice

Figure 2. The Expert Advice system can be accessed post measurement by right mouse clicking on selected records, or by selecting View, Expert Advice

An example of the Expert Advice system’s tips dialogue indicating the quality of size measurements

Figure 3. An example of the Expert Advice system’s tips dialogue indicating the quality of size measurements

The Expert Advice Report

The Expert Advice report is available in the suitable workspace for post measurement analysis of the quality of the data (Figure 4). It includes the same information displayed in the Expert Advice system studied in the earlier section.

The Expert Advice Report is available in the appropriate workspace for post measurement analysis of the data quality

Figure 4. The Expert Advice Report is available in the appropriate workspace for post measurement analysis of the data quality

The Expert Advice System in Dynamic Light Scattering Measurements

Single Measurements

In the case of sizing measurements, the Expert Advice system employs the several tests found in the Size Quality Report for testing data quality, and these are outlined in Table 1.

Table 1. Summary of the tests incorporated into the size quality report which are used in the Expert Advice system to look at the data quality during single sizing measurements.

Test Number Test Description Test Details Warning Message
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
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
3 Check appropriate lower size display limit used Is the z-average diameter < lower display limit? z-average is smaller than lower display limit
4 Check appropriate upper size display limit used Is the z-average diameter > upper display limit? z-average is larger than upper display limit
5 Check polydispersity index value Is the polydispersity index value > 1? Polydispersity index is very high
6 Check correlation function intercept value Is the correlation function intercept <0.1 or >1.0? Correlation function intercept out of range
7 Check first point selection for multimodal analysis Is the first correlation point >3 and the z-average diameter <5 nm? Check first correlation point selection for multimodal analysis
8 Check the in range value Is the in range value less than 95% The in range figure is low
9a Check for low mean count rate Is the mean count rate <20 kcps? Count rate is out of range
9b Check for high mean count rate Is the mean count rate >1000 kcps? Count rate is out of range
10 Check sufficient data collected Is the total number of photons collected <10000 K? Insufficient signal collected
11 Check the cumulants fit error Is the cumulants fit error >0.005? Cumulant fit error high
12 Check the distribution fit error Is the distribution fit error >0.005? Multimodal fit error high
13 Check if the cumulants and distribution fit errors Is the cumulants fit error >0.005 and the distribution fit error <0.0051? Sample too polydisperse for cumulant analysis - suggest rely on distribution analysis

When a measurement is performed, warning messages are displayed in the Expert Advice Tab in the Live Measurement Window (Figure 1) if any of the tests fall outside specific limits.

A “Data Meets Quality Criteria” message is displayed in the Expert Advice Tab if none of the tests fail.

Repeat Measurements

The Expert Advice system also searches for trends in the results if repeat sizing measurements are executed (three or more). Table 2 lists the tests executed for looking at these trends.

Table 2. Summary of the tests used in the Expert Advice system to look at trends in the size results obtained from repeat measurements (3 or more measurements).

Test Number Test Description Test Details Warning Message
1 For dual angle measurements, calculate an aggregation index value and check whether there is multiple scattering present (1) An aggregation index is calculated from ((ZD (13°)/ZD(173°) – 1) where ZD (13°) and ZD (173°) are the z-verage diameters determined at forward and back scatter angles.
(2) A calculated aggregation index of < - 0.05 is indicative of the presence of multiple scattering
(1) Aggregation index = “X” (see (1) below more detail)
(2) Indication of multiple scattering
2 Check if the first measurement deviates from the rest of the set Statistic tests are performed on the mean obtained from (1) all of the records and (2) with the first record omitted to determine whether the first record deviates significantly from the rest First measurement deviates from rest of set – exclude it or repeat measurement
3 Check if the mean size is trending from repeat measurements A model is fitted to the z-average diameters of the records to determine whether there is a statistically significant trend present Mean size is trending by (+/-) “Y”% (see (2) below more detail)
4 Check if the count rate and size are both decreasing from repeat measurements A model is fitted to the mean count rates and z-average diameters of the records to determine whether there is a statistically significant decreasing trend present Count rate and size are both decreasing – sample may be sedimenting or dissolving
5 Check if the count rate and size are both increasing from repeat measurements A model is fitted to the mean count rates and z-average diameters of the records to determine whether there is a statistically significant increasing trend present Count rate and size are both increasing – sample may be aggregating
6 Check if the count rate varies significantly from repeat measurements A model is fitted to the mean count rates of the records to determine whether there is a statistically significant variation present Count rate varies significantly – sample not stable or not clean

(1) where “X” = the aggregation index value determined from dual angle measurements
(2) where the direction (+/-) and “Y” percentage of any trend in the mean size is calculated and displayed in the warning message.

The Expert Advice System in Zeta Potential Measurements

Single Measurements

For zeta potential measurements, the Expert Advice system uses the several tests included in the Zeta Potential Quality Report for testing data quality, and these are outlined in Table 3.

Table 3. Summary of the tests incorporated into the zeta potential quality report which are used in the Expert Advice system to look at the data quality during single sizing measurements.

Test Number Test Description Test Details Warning Message
1 Check the intensity of the reference beam Is the reference beam intensity < 500 kcps Reference beam count rate too low. If you see this warning for a number of zeta potential results then please contact your nearest Malvern representative
2 Check the quality of the phase plot data Is zeta quality factor < 1.0? Phase data poor – signal to noise ratio low – increase number of sub runs per measurements
3 Check the quality of the distribution plot data Is the quality factor obtained from the Slow Field Reversal measurement < 1.0? Distribution data poor – increase number of sub runs per measurements
4 Check the limits of the zeta potential distribution are within the analyzed range Does the zeta potential spectrum fit in to the selected zeta range? Increase zeta potential analysis range
5 Check the conductivity and appropriate measurement protocol has been used Is the conductivity >10 mS/cm and has General Purpose been used? Suggest use of Monomodal analysis as conductivity is >10 mS/cm
6 Check for flare originating from the cell wall Is spectral quality factor >0.0 and <0.9? Flare from cell wall – check for bubbles, increase sample concentration
7 Check attenuator position used Is attenuator position selected 11? Sample concentration may be inappropriate (too high or low)

Repeat Measurements

The Expert Advice system also searches for trends in the results if repeat zeta potential measurements are executed (three or more). The tests which are executed for looking at these trends are outlined in Table 4. These tests are similar to those used for sizing measurements.

Table 4. Summary of the tests used in the Expert Advice system to look at trends in the zeta potential results obtained from repeat measurements (3 or more measurements)

Test Number Test Description Test Details Warning Message
1 Check if the first measurement deviates from the rest of the set Statistic tests are performed on the mean obtained from (1) all of the records and (2) with the first record omitted to determine whether the first record deviates significantly from the rest First measurement deviates from rest of set – exclude it or repeat measurement
2 Check if the mean zeta potential is trending from repeat measurements A model is fitted to the mean zeta potential values of the records to determine whether there is a statistically significant trend present Mean zeta potential is trending by (+/-) “Y”% (see (1) below for more details)
3 Check if the count rate and zeta potential are both decreasing from repeat measurements A model is fitted to the mean count rates and zeta potential values of the records to determine whether there is a statistically significant decreasing trend present Count rate and zeta potential are both decreasing – sample may be sedimenting or dissolving
4 Check if the count rate and zeta potential are both increasing from repeat measurements A model is fitted to the mean count rates and zeta potential values of the records to determine whether there is a statistically significant increasing trend present Count rate and zeta potential are both increasing – sample may be aggregating
5 Check if the count rate varies significantly from repeat measurements A model is fitted to the mean count rates of the records to determine whether there is a statistically significant variation present Count rate varies significantly – sample not stable or not clean

(1) where the direction (+/-) and “Y” percentage of any trend in the mean size is calculated and displayed in the warning message

The Expert Advice System in Molecular Weight Measurements

For molecular weight measurements, the Expert Advice system only analyzes the data from single measurements, that is, no information on trending from repeat measurements is offered. Table 5 lists the tests employed for molecular weight measurements.

Table 5. Summary of the tests used in the Expert Advice system to look at quality of data in a molecular weight measurement

Test Number Test Description Test Details Warning Message
1 Check that the scattering from the various sample concentrations are appropriate Is the count rate obtained from the various sample concentrations at least 10% more than the scattering obtained from the solvent? The “A” g/ml concentration is producing less excess scattering (“B” kcps) than is advised. Ideally the scattering should be more than “C” kcps. Use a higher concentration of sample (see (1) below for more details)
2 Check that the scattering from the various sample concentrations are not too high Is the count rate obtained from the various sample concentrations greater than 1000 kcps? The “D” g/ml concentration is producing more excess scattering (“E” kcps) than is advised. Ideally the scattering should be less than 1000 kcps. Use a lower concentration of sample (see (2) below for more details)
3 Check the value of the correlation coefficient (R2) Is the correlation coefficient (R2) < 0.9 Low correlation coefficient (“F” < 0.9). Either remove outlying points or remeasure the samples at those concentrations. Use the Debye plot to identify outlying points (see (3) below for more details)

(1) where “A” is the concentration of the sample and “B” is the mean count rate in kcps of that sample and “C” is the minimum ideal scattering in kpcs calculated from ((Scattering of solvent) + 10%)
(2) where “D” is the concentration of the sample and “E” is the mean count rate in kcps of that sample
(3) where “F” is the correlation coefficient (R2) calculated for the selected record

Below, each of these tests is discussed in more detail.

Test 1: Check that the Scattering from the Various Sample Concentrations are Appropriate

The preparation of samples with very low concentration is the common mistake that users make during molecular weight measurements. In such cases, the excess scattering produced by the analyte over that of the solvent will be very low to acquire good quality data. This test is intended to underline unsuitable scattering levels for the sample concentrations prepared.

The mean count rate from the solvent is measured during a molecular weight measurement on the Zetasizer Nano. The mean count rates obtained for the different sample concentrations (i.e. analyte dissolved in the solvent) should be higher than that for the solvent, that is, there is excess scattered light produced. A reasonable minimum excess level of scattering is regarded to be 10%. This test thus guarantees that the mean count rates for each sample concentration are at least 10% greater than the mean count rate of the solvent.

Test 2: Check that the Scattering from the Various Sample Concentrations Are Not Too High

The operating range of the avalanche photodiode detector (APD) used in the Zetasizer Nano series is linear up to 1000 kcps. Therefore, Test 2 underlines any sample concentrations which produce a mean count rate of higher than 1000 kcps and necessitates such samples to be diluted before being re-measured.

Test 3: Check the Value of the Correlation Coefficient (R2)

R squared (R2), also called the Pearson’s coefficient or correlation coefficient, is a calculated parameter that is used to explain how well graphical data fit an applied model. Values for R2 range from 0 to 1, with a value of 1 signifying that there is zero deviation of the data from the model or best fit line (or curve).

R2 is calculated for the Debye plot and a value below 0.9 is regarded to be a poor fit. It is recommended to either re-measure the samples at those concentrations or remove outlying points. Any evident outliers in the Debye plot can be identified in the Molecular Weight report and can be eliminated by editing the record.

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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