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User meeting 8 or 9 September 2015 | Sysmex
User Meeting 8 or 9 September 2015:
Laser diffractionData Quality: Do’s and Don’ts
1
User meeting 8 or 9 September 2015 | Sysmex
Things to consider during and after the measurement:
● Result – particle size distribution
● Background
● Raw data
● Calculation *) :
● Model● Fit
● Obscuration *)
● Repeatability *)
● Measurement settings *)
*) Optimized during method development but always good to check duringand after the measurement
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User meeting 8 or 9 September 2015 | Sysmex
Check the result
3
● Particle size and Particle size distribution:
● Logical, expected distribution?
User meeting 8 or 9 September 2015 | Sysmex
Check the result
4
● Mastersizer 2000: Result Analysis (M)
● Mastersizer 3000: Analysis
User meeting 8 or 9 September 2015 | Sysmex
Check the data
5
● Background and data
Sample dataBackground substracted
Background
User meeting 8 or 9 September 2015 | Sysmex
Check the data
6
● Background and data
Background
Sample data
User meeting 8 or 9 September 2015 | Sysmex
Check the data
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● Mastersizer 2000: Data (M)
● Mastersizer 3000: Data Background data
Sample data
User meeting 8 or 9 September 2015 | Sysmex
Check de Data and Background
Sample data
Background data
User meeting 8 or 9 September 2015 | Sysmex
Check the Background data and system cleanliness
● A good measurement requires a clean, stable background
● This should show progressive decrease across the detector range
Less than 20 scatteringunits by detector 20
Less than 100 units on detector 1
User meeting 8 or 9 September 2015 | Sysmex
Background example 1
● How does the background look like?
● What could be the reason?
● What could be done?
User meeting 8 or 9 September 2015 | Sysmex
Background example 1 - solution
● How does the background look like? Hump around detector 20
● What could be the reason? Material stuck to the windows
● What could be done? Extra Flush or clean the windows
User meeting 8 or 9 September 2015 | Sysmex
Background example 2
● How does the background look like?
● What could be the reason?
● What could be done?
User meeting 8 or 9 September 2015 | Sysmex
Background example 2 - solution
● Poor background – contaminated dispersant
● Particulate contamination causes fluctuations in the background over time
● Bubbles in the dispersant can also result in a similar background
User meeting 8 or 9 September 2015 | Sysmex
Background example 2 - solution
● View after background measurement, before sample addition
User meeting 8 or 9 September 2015 | Sysmex
Background example 3
● How does the background look like?
● What could be the reason?
● What could be done?
User meeting 8 or 9 September 2015 | Sysmex
Poor background – misaligned system.
● Castellation indicates a mis-aligned system.
● This may be caused by:
● Dirt on the cell windows. Address these and then re-align.● Changes in the temperature of the dispersant
Castellation
User meeting 8 or 9 September 2015 | Sysmex
Poor backgrounds: thermal gradients in organic dispersants
● The sample dispersion unit may be at 30°C+, while the solvent is generally much cooler
● This gives rise to thermal gradients resulting in fluctuations in the laser beam, alignment problems and poor backgrounds
Thermal gradient in dispersant
User meeting 8 or 9 September 2015 | Sysmex
Background example 3
● How does the background look like?
● What could be the reason?
● What could be done?
User meeting 8 or 9 September 2015 | Sysmex
Background example 3
In direct view: wild instability of the scattering on the first few detectors….
● After a few minutes the background should stabilise as the temperature differences equilibrate.
● If it does not stabilise, fit the cover of the dispersion unit to reduce surface evaporation.
User meeting 8 or 9 September 2015 | Sysmex
Check the data
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Data Graph - Light Scattering
1 3 5 7 9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 Detector Number
0
10
20
30
40
50
60
70
Ligh
t Ene
rgy
Background dataPharmaceutical in propanol, 22 September 2004 12:17:21
No Background Signal Data0 53545.938 46460.320 13.2331 67.011 53.034 -5.1102 56.543 40.361 -8.7003 34.427 22.086 -7.7854 31.487 16.457 -10.8635 36.039 19.474 -11.7976 30.898 13.803 -13.0067 31.508 13.554 -13.7858 28.469 10.780 -13.9219 25.682 9.428 -12.856
10 23.907 9.202 -11.54111 21.491 9.316 -9.33112 18.177 8.685 -7.08713 15.684 8.661 -4.948
● The data is already corrected for the background
● Negative data (avoid this!) unstable background
● Results are affected!
User meeting 8 or 9 September 2015 | Sysmex
Check the raw data, mastersizer 3000
21
● The data is already corrected for the background
● Negative data (avoid this!) unstable background
● Results are affected!
User meeting 8 or 9 September 2015 | Sysmex
Check the data● Idea about the particle size?
● First detectors numbers● Small angle Scatter● Higher light energy● Big particles
● Higher detector numbers● Big angle Scatter● Lower light energy● Small particles
User meeting 8 or 9 September 2015 | Sysmex 23
Check the fit : what is the fit?● The fit page shows the measured scattering data and the data predicted
by the scattering model● The precision of the overlay of these curves is known as the ‘data fit’● The goodness of the ‘data fit’ is quantified by the Residual
Data Graph - Light Scattering
1 3 5 7 9 11 14 17 20 23 26 29 32 35 38 41 44 47 50 Detector Number
0
50
100
150
200
Ligh
t Ene
rgy
Fit data(weighted) 10 August 2001 16:52:43
User meeting 8 or 9 September 2015 | Sysmex 24
How do we get the size distribution?
User meeting 8 or 9 September 2015 | Sysmex
Check the fit
● Weighted residual ≈ residual
● Weighted residual and residual < 2% (if possible)
● Bad fit could be caused by:
● Poor selection of Optical properties● Negative data● Too high obscuration (multiple scatter)● Wrong selection of the calculation model● The sample itself (mixture of components etc.)● Etc.
● Optimize the fit, but still look if you use logical optical properties and get a logical particle size distribution
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User meeting 8 or 9 September 2015 | Sysmex
Example of Unweighted & Weighted fit: Good
●
26
Example - Unweighted
Example - Weighted
User meeting 8 or 9 September 2015 | Sysmex 27
Assessing the Data Fit: Absorption (MS2000)
Data Graph - Light Scattering
7 20 23 26 29 32 35 38 41 44 47 50 Detector Number
TC 1 during 100% u/s, 11 January 2005 13:36:27
Detector 51 and 52: Absorption
● Misfits to the extinction detectors indicate an incorrect absorption value
● 51 in the red light● 52 in the blue light
User meeting 8 or 9 September 2015 | Sysmex 28
Assessing the Data Fit: RI (MS2000)
.D a ta G r a p h - L i g h t S c a tte r i n g
14 17 20 22 25 28 31 33 36 39 41 44 47 D e tec to r N um ber
C om pound A , 29 M arc h 2005 15 :16 :52
Other detectors: RI
● A poor fit to the focal plane detectors (<40) suggests an incorrect choice of refractive index
User meeting 8 or 9 September 2015 | Sysmex
Assessing the Data Fit: Absorption (MS3000)
● Misfits to the extinction detectors indicate an incorrect absorption value● 51 in the red light● 63 in the blue light
Poor data fit here indicates poor choice of
absorption
Poor data fit here indicates poor choice
of absorption
User meeting 8 or 9 September 2015 | Sysmex
Assessing the Data Fit: RI (MS3000)
● A poor fit to the focal plane detectors (<40) suggests an incorrect choice of refractive index
Poor fit indicates incorrect choice
of refractive index
User meeting 8 or 9 September 2015 | Sysmex
Check the fit : not good MS3000
● Residuals > then 1-2%; residual and weighted residual not same order.
● No good fit for detector 51 and 63 (extinction red and blue)
● optimize optical properties absorbance
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User meeting 8 or 9 September 2015 | Sysmex
Check the fit : not good MS3000
● Residuals > then 1-2%; residual and weighted residual not same order.
● No good fit for other detectors
● optimize optical properties refractive index
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User meeting 8 or 9 September 2015 | Sysmex
MS3000 development: The optical property optimiser (OPO)
● Offers a quick way to adjust optical properties and assess the fit and result
Optical Property Selection Data & Result views
User meeting 8 or 9 September 2015 | Sysmex
Check the obscuration
● Stable between measurements?
● Compare obscuration red/blue
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User meeting 8 or 9 September 2015 | Sysmex
Check the obscuration: red and blue obscuration
35
Particle Size Distribution
0.01 0.1 1 10 100 1000 3000 Particle Size (µm)
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6
6.5
7
7.5
Volu
me
(%)
testx, vrijdag 7 mei 2010 12:02:41
Particle Size Distribution
0.01 0.1 1 10 100 1000 3000 Particle Size (µm)
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
6
6.5
7
Volu
me
(%)
testy, vrijdag 7 mei 2010 13:09:24
Particle Size Distribution
0.01 0.1 1 10 100 1000 3000 Particle Size (µm)
0 0.5
1 1.5
2 2.5
3 3.5
4 4.5
5 5.5
6 6.5
7 7.5
8 8.5
9 9.5 10
10.5 11
Volu
me
(%)
testz, dinsdag 29 juni 2010 12:12:01
X Y
Z
User meeting 8 or 9 September 2015 | Sysmex
Check obscuration
● Stable between measurements?
● Compare obscuration red/blue (indicative information)
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User meeting 8 or 9 September 2015 | Sysmex
Check the obscuration
● Obscuration too high:
● Multiple scatter● Obscuration too low:
● Signal/noise ratio low● Bad reproducibility
● Blue>Red:
● Small particles, risk for multiple scatter.● Avoid blue > ca. 15%● Test right obscuration with an
obscuration titration
37
User meeting 8 or 9 September 2015 | Sysmex
Guideline Obscuration Ranges for Sizes
Dv(50) Value Obscuration Range
Submicron 2-5%
1-10um 5-10%
Over 10um 5-15%
User meeting 8 or 9 September 2015 | Sysmex
Obscuration guidelines
● Obscuration is tested during method development.
39
Method Distribution Particle size Obscuration
Dry Wide Coarse 0.5 - 6%
Dry Narrow Fine 0.5 - 3%
Wet Narrow Nano 5 - 6%
Wet Narrow <4 µm 10%
Wet Wide Coarse 15 - 25%
User meeting 8 or 9 September 2015 | Sysmex
MS3000 development: Data Quality
40
User meeting 8 or 9 September 2015 | Sysmex
Check the Repeatability
● A good measurement is repeatable
41
Particle Size Distribution
0.01 0.1 1 10 100 1000 3000 Particle Size (µm)
0 2 4 6 8
10 12 14
Volu
me
(%)
Solvent1, 11 May 2010 12:43:33 Solvent1, 11 May 2010 12:44:10 Solvent1, 11 May 2010 12:44:48Solvent1, 11 May 2010 12:45:25
User meeting 8 or 9 September 2015 | Sysmex
Check the Repeatability
● If not repeatable:
● Did the measurement settings change?● Did the calculation change?● Did the dispersion change?
42
Particle Size Distribution
0.01 0.1 1 10 100 1000 3000 Particle Size (µm)
0
2 4
6 8
Volu
me
(%)
product1, 13 January 2011 13:17:38 product1, 13 January 2011 13:18:24product1+, 13 January 2011 13:19:50 product1+, 13 January 2011 13:20:35product1+, 13 January 2011 13:21:21 product1++, 13 January 2011 13:22:40product1++, 13 January 2011 13:23:25 product1++, 13 January 2011 13:24:11
User meeting 8 or 9 September 2015 | Sysmex
Check the Repeatability – ISO guide line
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● Limits suggested in ISO13320:
● Dv(50): RSD < 3%● Dv(10) and Dv(90): RSD < 5%● “Below 10µm, these maximum values should be doubled.”
● In ideal conditions● 0.5% COV on parameters >1μm● 1% COV on parameters <1μm
● Limits suggested within USP<429> and EP 2.9.31:
● Dv(50): RSD < 10%● Dv(10) and Dv(90): RSD < 15.0%● “Below 10µm, these maximum values should be doubled.”
User meeting 8 or 9 September 2015 | Sysmex
Check the Repeatability
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User meeting 8 or 9 September 2015 | Sysmex
Check Repeatability
● A good measurement is repeatable
45
User meeting 8 or 9 September 2015 | Sysmex
Check Repeatability
● If not repeatable:● Did the measurement settings change? ● Did the calculation change?● Did the dispersion change?
46
User meeting 8 or 9 September 2015 | Sysmex
Summary - data quality● Background data
● Make sure that:● Material is not stuck to the cell windows● There is no dispersant contamination● There are no thermal gradients● That the system has been properly aligned ● The inner detector data is free from castellation
● Sample data● Check that
● There are reasonable signal to noise levels● There is no multiple scattering● There is no negative data● There is no noisy data ● There is no beam steering
User meeting 8 or 9 September 2015 | Sysmex
Questions?
Please contact:Sandra Remijn – Application [email protected]
Tineke Mink – Application Specialist [email protected]
www.sysmex.nl www.sysmex.be
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