Implementation of chemometric techniques in chromatographic data station software
description
Transcript of Implementation of chemometric techniques in chromatographic data station software
![Page 1: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/1.jpg)
Implementation of chemometric techniques in
chromatographic data station software
Yuri Kalambet, Ampersand Ltd., Moscow, Russiawww.ampersand.ru
![Page 2: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/2.jpg)
2
Data processing• Noise level estimation• Noise filtration and/or data bunching• Peak search• Peak quantification (area, height, asymmetry, etc.)• Table of components (qualitative analysis)• Calibration graph (quantitative analysis)• Peak shape analysis and deconvolution• Periodic noise filtration• Multichannel (spectral) data analysis
![Page 3: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/3.jpg)
3
Noise• High-frequency noise estimation using the whole
time series data set• Noise filtration could cause questions from GLP
rules• Bunching never causes questions from GLP, but
can significantly reduce observed noise• Noise is taken into account while:
– peak detection; – approximation quality evaluation– multichannel/spectral analysis
![Page 4: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/4.jpg)
4
Peak Quantification
• Baseline Y position using weighted Savitsky-Goley approximation with evaluation of approximation quality
• Primary peak parameters using adaptive polynomial approximation, where parameters of polynomial depend on target peak width and asymmetry
• Criterion for algorithm selection: Peak parameters should be resistant to bunching operation
![Page 5: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/5.jpg)
5
Noise definition
•1st derivative point DOES NOT belong to “noise pool of points” if both of the neighboring derivative points have the same sign
•1st derivative point DOES NOT belong to “noise pool of points” if its absolute value is outside 5 sigma of absolute values distribution (iterative procedure)
![Page 6: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/6.jpg)
6
Calibration
Predictive relationship between input and detector response
• Input: calibration samples – concentrations of components
• Output: peak area or height• Prediction: Predict unknown input looking
at response
![Page 7: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/7.jpg)
7
ESTD Calibration• Response (Area or Height) versus
Quantity• Quantity is provided without error - false• Response is measured with random
normally distributed error – sometimes true
![Page 8: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/8.jpg)
8
Calibration Curve
• Axes: Q – Quantity (NOT Concentration), R – Response (Area or Height)
• Independent variable: Typically Q, sometimes R
• Calibration curve: polynomial interpolation• Prediction: either solution of polynomial
equation (independent Q) or value of polynomial (independent R) – we denote either of them W(R)
![Page 9: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/9.jpg)
9
QuantificationAbsolute Concentration
• Quantity of injected substanceQx = W(Rx)
• Concentration of initial sampleCx = Qx/V = W(Rx)/(Vinj*A/D)
• Vinj – injection volume• A – amount• D – dilution
![Page 10: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/10.jpg)
10
ISTD Targets
Reason Axis• Sample-size variations Q• Effect of sample preparations Q• Instrument drift R
All reasons are acting always together
![Page 11: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/11.jpg)
11
ISTD tricks
• Add component with known concentration to sample
• Add component with known concentration to calibration samples
Usually it’s done together, but who told that you can’t do it separately?
![Page 12: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/12.jpg)
12
ISTD with ESTD calibration = Relative Concentration
• Accounts for systematic error due to sample-size error and sample losses while preparation.
• Assumption: volume is unknown and is calculated using known concentration of the internal standard
V = Qxistd/Cxistd = Wistd(Rxistd)/Cxistd, • Qxistd is calculated using calibration curve of
Internal standard from Rxistd, Cxistd – declared concentration of standard in sample
• Relative ConcentrationC = Qx/V = Cxistd×Wx(Rx)/ Wistd(Rxistd)
![Page 13: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/13.jpg)
13
“Universal” ISTD Calibration• If calibration is nonlinear, we must measure this
nonlinearity, i.e. we MUST know ESTD calibration curve of the Internal Standard component
• In the case we learned this curve somehow, we can use this curve to change positions of calibration points of other components using the same trick as was used while calculating Relative Concentration:
Qn = Cn×V = Cn×Wistd(Ristd)/Cistd
• So, for Internal Standard predefined curve is in use, all other components get curves constructed conditionally, condition being known calibration curve of the Internal Standard component
![Page 14: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/14.jpg)
14
Pro et Contra
Advantages: • Only one type of axes• Calibration curves are suitable for
calculation of both Absolute and Relative concentrations
Disadvantages:• ESTD Calibration curve of Internal
Standard is required
![Page 15: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/15.jpg)
15
Qn = Cn×V = Cn×Wistd(Ristd)/Cistd
• Multiplication of Q axis of Internal Standard to any number will multiply Q coordinates of all corrected points of all components to the same number, hence causing “affinity” change of all calibration curves. Calibration curve will change, absolute concentration also, but not Relative Concentration
C = Cxistd×Wx(Rx)/ Wistd(Rxistd) • If all calibration dependencies are linear through origin
Q = K×R, it is possible to select multiplication factor so, that Kistd = 1 and we will get relative response factors for all other components.
![Page 16: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/16.jpg)
16
“Classic” ISTD
• Calibration dependence in coordinates Response Ratio vs. Concentration ratio
• Calibration curve: polynomial, typically straight line through origin
• Prediction: from Response Ratio predict Concentration Ratio
• Peculiarities: no calibration curve for Internal Standard component
![Page 17: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/17.jpg)
17
Example of “Classic” ISTD failure
Response AResponse B(Standard) Concentration Area Ratio
Concentration Ratio
0 0 0 0 0
18.1269247 9.516258 1 1.904837 1
32.9679954 18.12692 2 1.818731 1
55.0671036 32.968 4 1.67032 1
79.8103482 55.0671 8 1.449329 1
95.9237796 79.81035 16 1.201897 1
![Page 18: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/18.jpg)
18
ESTD/”Universal” ISTD Calibration
ESTD Calibration
y = 0.0089x3 - 0.45x2 + 9.9125x
y = 0.0406x3 - 1.4697x2 + 19.118x
0
20
40
60
80
100
120
0 2 4 6 8 10 12 14 16 18
Concentration
Res
pons
e Substance BSubstance APoly. (Substance B)Poly. (Substance A)
![Page 19: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/19.jpg)
19
“Classic” ISTD calibration curve
ISTD Classic
y = 1.609xR2 = 0.8686
0
0.5
1
1.5
2
2.5
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Concentration Ratio
Res
pons
e R
atio
ISTD Calibration PointsLinear (ISTD Calibration Points)
![Page 20: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/20.jpg)
20
Device Drift
• Drift model: R = K×F(Q)• Drift can be compensated completely, if
exists such k, thatK×F(Q)=F(k×Q)
• Particular case: linear through origin calibration; K = k
![Page 21: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/21.jpg)
21
Example
![Page 22: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/22.jpg)
22
![Page 23: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/23.jpg)
23
Conclusions• ISTD is split in two parts: ISTD quantification (Relative
Concentration) and ISTD Calibration. Parts can be applied separately
• Relative Concentration is applicable for both ESTD and “Universal” ISTD calibrations
• Full “Universal” ISTD calibration can be used for both ESTD and ISTD calculations
• Simplified “Universal” ISTD calibration can be used in the case of linear through origin calibrations instead of “Classic” ISTD calibration including imitation of user interface
• Full “Universal” ISTD solution still works where “Classic” already fails, i.e. it allows to get into account wide concentration range of Internal Standard in the case of nonlinear calibrations
![Page 24: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/24.jpg)
24
Difficulties
• User mind. The more users are used to “Classic” ISTD method, the more difficult it is to change their minds. Typical argument: -“Old approach works. We typically use linear through origin calibrations. Why should we change the way how we work?”.
![Page 25: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/25.jpg)
25
Peak deconvolution by shape
Peak shape1. Gaussian2. Exponentially modified Gaussian3. Corresponds to reference peakTuning, specific to chromatography• Peak width
– individual for every peak;– the same for all peaks– proportional to retention time– asymmetry of the same sign (2)
Optimization criterion• quadratic (sum of squares of residuals)• weighted quadratic (weight = response)
![Page 26: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/26.jpg)
26
Gaussian peak shape analysis
![Page 27: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/27.jpg)
27
Gaussian peak shape analysis
![Page 28: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/28.jpg)
28
Gaussian peak shape analysis
![Page 29: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/29.jpg)
29
EMG peak shape analysis
Problems: product of zero to infinity for some range of parameters
Solution:
or “standard” Gaussian function, depending on parameter values
22
22 erfc
2erf1
22
2
2
2
t
t
t
t
ehehtF
22 erfcx2
2
2
tt
ehtF
![Page 30: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/30.jpg)
30
Fourier-domain periodic noise filtration
• Median filter in Fourier space
![Page 31: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/31.jpg)
31
Periodic noise filtration – mains frequency
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185 190 195 200 205 210 215 220 225 230 сек
-5
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
mV
ch1
1
2
3
4
5
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185 190 195 200 205 210 215 220 225 230 сек-5
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
res1
1
2
3
4
5
![Page 32: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/32.jpg)
32
Periodic noise filtration – pump pulsations
1 2 3 4 5 6 7 8 9 10 11 12 13 14 мин
580 mV
ch3
1
2
3
45
6
7 мин
3.4 mV
res1
res2
![Page 33: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/33.jpg)
33
Multichannel chromatogram questions
• Which channel is better?• How to detect peaks?• How to identify peaks?• How to access substance purity?
![Page 34: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/34.jpg)
34
Multichannel chromatogram answers
• Calculated channels• Using weighted PCR: Noise-normalized detector
response space for spectra comparison• Measure of difference for spectra: angle• “Local” spectrum quality: angle with neighbors• Factor analysis with missing (overloaded) points
allowed• Qualitative and quantitative spectra comparison
![Page 35: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/35.jpg)
35
Weighted PCA
• Channel weighting• Point weighting
pEwpCSwpRwECSR
![Page 36: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/36.jpg)
36
Calculated channels Summary.
Types of Calculated channels:
• Total• Average• Difference• Integral• Derivative - up to 3-rd order• Angle• «Response/Time» channel• Spectral ratio channel
![Page 37: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/37.jpg)
37
Spectral chromatogram
with angle profile.
![Page 38: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/38.jpg)
38
Best purity spectrum advantage
0.01 0.1 1 10amount, mL
0
10
20
30
40
50
elg na,
s eerged0.01 0.1 1 10
DAD100 - "Etomidate"
Average Best Purity Peak Top
0.01 0.1 1 10amount, mL
0
10
20
30
40
50
elg na,
s eerged0.01 0.1 1 10
DAD100 - "Etomidate"
Average Best Purity Peak Top
![Page 39: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/39.jpg)
39
Spectrum identificationStep 1:Spectrum calculation.
![Page 40: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/40.jpg)
40
Step 2:Performing recognition.
![Page 41: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/41.jpg)
41
Step 3:Conflicts resolving.
![Page 42: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/42.jpg)
42
Step 4:View and print report.
![Page 43: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/43.jpg)
43
Calculated channels in “Chrom&Spec”. Summary.
Types of Calculated channels:
• Total• Average• Difference• Integral• Derivative - up to 3-rd order• Angle• «Response/Time» channel• Spectral ratio channel
![Page 44: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/44.jpg)
44
Main spectral window.
![Page 45: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/45.jpg)
45
3D multi-channel chromatogram view.
![Page 46: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/46.jpg)
46
2D multi-channel chromatogram view.
![Page 47: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/47.jpg)
47
Factor analysis example.
Picture shows the “sum of s/n” channel of multi-channel chromatogram.Usual peak-detection procedure
failed to resolve peaks. What about factor analysis?
![Page 48: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/48.jpg)
48
Factor analysis. Step 1. Software makes PCR
of the region selected and suggests number of components in the analyzed region (Rank).
Overlapping only 2 peaks means that software does not look for overlapping of 1st and 3rd components, only 1st and 2nd and then 2nd and 3rd.
![Page 49: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/49.jpg)
49
Factor analysis. Step 2
On the left panel we can choose any channel to view peak deconvolution results.
Rotation is selected using “local purity” attribute, resulting in “Pure component” approach
![Page 50: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/50.jpg)
50
Factor analysis. Step 3
The resolved peaks are displayed together with the original channel.
The chromatogram with additional newly generated channels can be saved into separate file. Each generated channel represents the detector response, corresponded to particular component.
When user finishes the wizard, the original peak is split in the proportions, corresponding to component’s fractions.
![Page 51: Implementation of chemometric techniques in chromatographic data station software](https://reader030.fdocuments.in/reader030/viewer/2022020716/56815e6e550346895dccef10/html5/thumbnails/51.jpg)
51