By:

Bahram Hemmateenejad

Complexity in Chemical Systems

Unknown Components
Unknown Numbers
Unknown Amounts

Modeling Methods
Hard modeling
A predefined mathematical model is existed for the
studied chemical system (i.e. the mechanism of
the reaction is known)

Soft modeling
The mechanism of the reaction is not known

Basic Goals of MCR

1. Determining the number of components
coexisted in the chemical system
2. Extracting the pure spectra of the
components (qualitative analysis)
3. Extracting the concentration profiles of
the components (quantitative analysis)

Evolutionary processes

pH metric titration of acids or bases

Complexometric titration
Kinetic analysis
HPLC-DAD experiments
GC-MS experiments

The spectrum of the reaction mixture is

recorded at each stage of the process

 Data matrix (D)

Nsln

Nwav

Bilinear Decomposition
If there are existed k chemical components
in the system
Nwav

Nwav

k
S

Nsln

Nsln

D=

+ . + E

Mathematical bases of MCR

D= CS
D=UV

Real Decomposition
PCA Decomposition
Target factor analysis
D = U (T T-1) V
= (U T) (T-1 V)
C = U T, S = T-1 V
T is a square matrix called transformation
matrix
How to calculate Transformation matrix T?

Ambiguities existed in the

resolved C and S
Rotational ambiguity
There is a differene between the calculated T
and real T

Intensity ambiguity
D = C S = (k C) (1/k S)

How to break the ambiguities

(at least partially)
1. Combination of Hard models with Soft
models
2. Using of local rank informations
3. Implementation of some constraints

Non-negativity
Unimodality
Closure
Selectivity
Peak Shape

MCR methods
Non iterative methods (using local rank
information)
Evolving factor analysis (EFA)
Windows factor analysis (WFA)
Subwindows factor analysis (SWFA)

Iterative methods (using natural constrains)

Iterative target transformation factor analysis (ITTFA)
Multivariate curve resolution-alternative least squares
(MCR-ALS)

Mathematical Bases of MCR-ALS

The ALS methods uses an initial estimates
of concentration profiles (C) or pure spectra
(S)
The more convenient method is to use
concentration profiles as initial estimate (C)
D = CS
Scal = C+ D,
C+ is the pseudo inverse of C
Ccal = D S+
Dcal = Ccal Scal
Dcal
D

 Lack of fit error (LOF)

(LOF) =100 ((dij-dcalij)2/dij2)1/2
LOF in PCA (dcalij is calculated from U*V)
LOF in ALS (dcalij is calculated from C*S)

Kinds of matrices that can by

analyzed by MCR-ALS
1. Single matrix (obtained trough a single
run)
2. Augmented data matrix
Row-wise augmented data matrix: A single
evolutionary run is monitored by different
instrumental methods. D = [D1 D2 D3]
Column-wise augmented data matrix: Different
chemical systems containing common components
are monitored by an instrumental method
D = [D1;D2;D3]

 Row-and column-wise augmented data matrix:

chemical systems containing common components are
monitored by different instrumental method
D = [D1 D2 D3;D4 D5 D6]

Running the MCR-ALS

Program

1. Building up the experimental data matrix

D (Nsoln, Nwave)
2. Estimation of the number of components
in the data matrix D
PCA, FA, EFA
3. Local rank Analysis and initial estimates
EFA
4. Alterative least squares optimization

Evolving Factor Analysis

(EFA)
Forward Analysis

FA

1f, 2f

FA

1f, 2f, 3f

Backward Analysis

FA

1b, 2b

FA

1b, 2b, 3b

4.00

Log eigenvalues

2.00

0.00

-2.00

-4.00
1

11

Row Number

13

15

17

19

MCR-ALS program written by Tauler

[copt,sopt,sdopt,ropt,areaopt,rtopt]=als(d,x0,nex
p,nit,tolsigma,isp,csel,ssel,vclos1,vclos2);

Inputs:
d: data matrix (r c)
Single matrix d=D
Row-wise augmented matrix d=[D1 D2 D3]
Column-wise augmented matrix d=[D1;D2;D3]
Row-and column-wise augmented matrix
d=[D1 D2 D3;D4;D5;D6]

 x0: Initial estimates of C or S matrices

C (r k), S (k c)
nexp: Number of matrices forming the data
set
nit: Maximum number of iterations in the
optimization step (default 50)
tolsigma: Convergence criterion based on
relative change of lack of fit error (default
0.1)

 isp: small binary matrix containing the

information related to the correspondence
of the components among the matrices
present in data set. isp (nexp k)
isp=[1 0;0 1;1 1]
csel: a matrix with the same dimension as C
indicating the selective regions in the
concentration profiles
ssel: a matrix with the same dimension as S
indicating the selective regions in the
spectral profiles

Nan Nan

Nan Nan

Nan

Nan Nan

Nan

Nan

Nan

Nan

 vclos1 and vclos2: These input parameters

are only used when we deal with certain
cases of closed system (i.e. when mass
balance equation can be hold for a reaction)
vclos1 is a vector whose elements indicate
the value of the total concentration at each
stage of the process (for each row of C
matrix)
vclos2 is used when we have two
independent mass balance equations

Outputs
copt: matrix of resolved pure concentration
profiles
sopt: matrix of resolved pure spectra.
sdopt: optimal percent lack of fit
ropt: matrix of residuals obtained from the
comparison of PCA reproduced data set
(dpca) using the pure resolved concentration
and spectra profiles.
ropt = T P CS

 areaopt: This matrix is sized as isp matrix

and contains the area under the
concentration profile of each component in
each Di matrix. This is useful for
augmented data matrices.
rtopt: matrix providing relative quantitative
information. rtopt is a matrix of area ratios
between components in different matrices.
The first data matrix is always taken as a
reference.

An example
Protein denaturation
Protein
(unfold)

denaturant

(intermediate)

denaturant

Protein
(denatured)

Metal Complexation
Complexation of Al3+ with Methyl thymol
Blue (MTB)

Applications
Qualitative MCR-ALS
Quantitative MCR-ALS

Nifedipine
1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5pyridine dicarboxilic acid dimethyl ester

selective arterial dilator

hypertension
angina pectoris
other cardiovascular disorders

NO2
COOMe

MeOOC
Me

N
H

Me

Nifedipine is a sensitive substance

UV light

daylight

4-(2-nitrophenyl)
pyridine

4-(2-nitrosophenyl)pyridine

NO2
COOMe

MeOOC
Me

Me

NO
COOMe

MeOOC
Me

Me

absorbance

2.5

1.5

0.5

0
225

275

325

wavelength (nm)

375

425

Data Analysis
Definition of the data matrix, D (nm)
n: No. of wavelengths
M: No. of samples

PCA of the data

D=RC

R is related to spectra of the components

C is related to the concentration of the components

Number of chemical components

Log (EV)

8
4
0
-4
-8

11

No. of factors

13

15

Score Plot
4

Score 1
Score2
Score 3

Score

-1

-2

-3
225

255

285

315

345

Wavelength

375

405

435

2.5

Nifedipin (resolved)
nitroso pyridine homologue
(resolved)
nifedipin (experimental)

Absorbance

mixture
1.5

0.5

0
225

275

325

Wavelength (nm)

375

425

1.2
Nifedipin

Fraction of components

Nitroso pyridine
homologue
0.8

0.6

0.4

0.2

0
0

50

100

150

Time (minute)

200

250

300

 Linear segment
CNIF = 1.181 ( 0.001) 10-4 4.96 (0.13) 10-9 t
r2 = 0.995
Exponential segment
CNIF = 1.197 ( 0.003) 10-4 Exp (-6.22 ( 0.10) 10-5 t)
r2 = 0.998
Zero order

4.96 (0.13) 10-9 (mole l-1 s-1)

First-order

6.22 ( 0.10) 10-5 (s-1)

 When iodine dissolves in a binary mixture

of donating (D) and non-donating (ND)
solvents, preferential solvation indicates the
shape of iodine spectrum
Nakanishi et al. (1987) studied the spectra
of iodine in mixed binary solvents
Factor analysis was used to indicate the
number of component existed
No extra works were reported

Iodine spectra in dioxane-cyclohexane

1

Absorbance

0.8
0.6
0.4
0.2
0
400

450

500

550

Wavelength, nm

600

650

Iodine spectra in THF-cyclohexane

1.60

Absorbance

1.20

0.80

0.40

0.00
400

450

500

550

Wavelength, nm

600

650

Eigen-values Plot

Logarithm of eigen-value

8
5

THF
Dioxane

2
-1
-4
-7

-10
-13
1

5
Number of factors

Absorbance

1.00
0.80
0.60
0.40
0.20
0.00
400.00

450.00

500.00

550.00

600.00

650.00

Wavelength (nm)
Concentration x 10 3
M

1.00
0.80

0.60
0.40
0.20
0.00

0.00

0.20

0.40

XDioxan

0.60

0.80

1.00

1.20
0.80
0.40
0.00
400.00

450.00

500.00

550.00

600.00

650.00

Wavelength (nm)

1.00
3

Concentration x 10 M

Absorbance

1.60

0.80

3
1

0.60

0.40
0.20
0.00
0.00

0.20

0.40

XTHF

0.60

0.80

1.00

Dye aggregates

Dye monomer

Dye-Surfactant ion-pairing

Pre-micelle aggregate

Dye partitioned in the

micelle phase

Absorbance Spectra of MB
1.8
1.6

Absorbance

1.4
1.2
1
0.8
0.6
0.4
0.2
0
450

500

550

600

650

700

Wavelength (nm)

750

800

850

Absorbance

1.6

1.2

0.8

0.4

0
450

550

650

Wavelength (nm)

750

850

Resolved pure spectra of the

components

Absorbanve

2.00

D(m)

1.50

1.00

(S-D)n
0.50
0.00
500

S-D
550

600

650

700

Wavelength (nm)

750

800

Concentration Profiles

M ole fraction

1.00

S-D

0.80

D(m)

(S-D)n

0.60

0.40

0.20

0.00
0

0.002

0.004

0.006

[SDS]

0.008

0.01

 D+S

D-S

Ki = [D-S]/[D][S]

n D-S

(D-S)n

Kag = [(D-S)n]/[D-S]n

(D-S)n

n D(m)

Kd = [D(m)]n/[(D-S)n)

Log Kag = log [(D-S)n] n log [D-S]

log [(D-S)n] = Log Kag + n log [D-S]

-0.5

log[MS(n)]

-1

y = 4.0249x - 0.0576
2
R = 0.9844

-1.5

-2

-2.5
-0.7

-0.5

-0.3

log[MS]

n=4
log Kag = -0.058

-0.1

Interaction of MO with CTAB

1

Absorbance

0.8

0.6

0.4

0.2

0
330

380

430

480

Wavelength (nm)

530

580

Pure spectra of MO Components

Absorbance

0.75

D(m)

0.5

DS
0.25

0
330

(DS)n
380

430

480

Wavelength (nm)

530

580

Concentration Profiles
D(m)

Mole Fraction

1
0.8
0.6
0.4

(DS)n

0.2
0
0

0.001

0.002

0.003

[CTAB]

0.004

0.005

Mole Fraction

0.8

(DS)n

0.6
0.4

DS

0.2
0
0

[CTAB] / [MO]

 D+S
DS
Ki = [DS] / [D] [S]
CMO = 410-6 M
[D] = 0.49 CMO
[DS] = 0.51 CMO
CS = 2.5 10-5 M
[S] = CS [DS]

Ki = 4.92 104
4.64 104

Quinone reduction

In the presence of proton source

Q + e QQ- + HB QH + BQH + e QHQH- +HB QH2 + B-

(1)
(2)
(3)
(4)

Our data set

Vis. Spectra of 0.1 mM solution of 9,10anthraquinone at different applied potential
in DMF solution
Optically transparent thin layer electrode
(OTTLE)
The experiment was conducted in Arak
University

Absorbance

1.5

0.5

0
380

430

480

530

580

Wavelength (nm)

630

680

Table 1: Result of factor analysis of spectroelectrochemical data

No. of

Log (eigenvalues)

% Eigenvalue

Cumulative % of eigenvalue

factors
1

7.2847

85.9782

85.9782

5.0597

9.2918

95.2700

4.3647

4.6372

99.9072

-0.0273

0.0574

99.9645

-0.6388

0.0311

99.9957

-3.8141

0.0013

99.9970

-4.1098

0.0010

99.9980

-4.3311

0.0008

99.9987

-4.7288

0.0005

99.9992

10

-5.2691

0.0003

99.9996

11

-5.4931

0.0002

99.9998

EFA Plot
log (eigenvalue)

3.00
2.00
1.00
0.00
-1.00
-2.00
-3.00
1.00

3.00

5.00

7.00

Row Nnumber

9.00

11.00

Pure spectra
Absorbance

2.00

2
3

1.50

1.00
0.50
0.00
380

430

480

530

580

630

680

Wavelength (nm)

1) AQ-o

2) AQH-

3) AQ2-

730

Concentration Profiles
Fraction of components

1.00
2

0.80

0.60
0.40
0.20
0.00
-1.20

-1.40

-1.60

Potential (V)

-1.80

-2.00

 Conversion of AQ-o to AQH AQ-o + H+ AQH E = E - (0.0592/n) log ([AQH-]/[AQ-o][H+])

E = E - (0.0592/n) log(1/[H+])
- (0.0592/n) log ([AQH-]/[AQ-o])

Potential (V)

-1.30

-1.35

-1.40

-1.45
-0.80

-0.30

0.20

0.70

1.20

log ([AQH-]/[AQo- ])

R2 = 0.996

Slope = 0.0594

intercept = -1.37

MCR-ALS of polarographic data applied

to the study of the copper-binding ability
of tannic acid
R. Tauler et al Anal. Chim. Acta 424 (2000) 203209

Structures of tannic acid (TA) (a) and condensed tannin (b)

Cu+2 + TA
Cu+2

I = CV + E
DPP obtained for the system Cu(II) + TA during the titration of
a 1 10- 5 mol l-1 Cu(II) solution with TA in the presence of 0.01
mol l-1 KNO3 and 0.01 mol l-1 acetate buffer (pH = 5.0). The
thick line denotes the polarogram measured for the metal ions in
the absence of TA.

Singular value decomposition (SVD) for the data repre-sented

Concentration profiles (a, c, e) and normalised pure voltammograms (b, d, f), in

arbitrary units, obtained in the MCR-ALS decomposition of the data matrix of
Fig. 2 according to different assumptions: three components with selectivity,
non-negativity and unimodality constrains (a, b) (lof 8.1%); four components
with selectivity, non-negativity and unimodality (c, d) (lof 4.4%) or four
components with selectivity, non-negativity and signal shape (e, f) (lof 6.5%)

Study of the interaction equilibria between the ploynucleotide

poly (inosinic)-poly(cytidilic) acid and Ethidium bromide by
means of coupled spectrometric techniques
R. Tauler et al. Anal. Chim. Acta 424 (2000) 105-114

Activator of in vivo the interferon

biosynthesis

Fluorescent dye

Ethidium bromide (EtBr)

poly(I)-poly(C)

(3,8-diamino-5-ethyl-6-

phenylphenantridinium)

Techniques
Methods

Molecular absorption
Fluorscence
Circular dicroism (CD)

Continous variation
Mole-ratio

poly(I)-poly(C) concentration constant

EtBr concentration constant

Experimental conditions
37 oC, neutral pH, KH2PO4 0.021 M, Na2HPO4 0.029 M, and
NaCl 0.15 M, Itotal=0.26 M

300 400
400 500
500 600
600
300
Et

DUV-Vis

11

0.2
0.2
00

CD
CD(a.u.)
(a.u.)
700
700

800
800
Et

00

300 400
400 500
500 600
600
300
Et

DDC

00

-1
-1

600
600

700 800
800
700
poly

300 400
400 500
500 600
600
300
poly

Dfluor

DDC

0.2
0.2

0.1
0.1
00

-0.2
-0.2

11

0.2
0.2

300 400
400 500
500 600
600
300
Wavelength(nm)
(nm)
Wavelength

00

Dfluor

11

0.3
0.3

Fluor.
Fluor.int.
int.(a.u.)
(a.u.)

Absorbance
Absorbance(a.u.)
(a.u.)

300 400
400 500
500 600
600
300
poly

DUV-Vis

0.4
0.4

600
600

0.5
0.5

0.5
0.5
00

00

CD
CD(a.u.)
(a.u.)

Absorbance
Absorbance(a.u.)
(a.u.)

1.5
1.5

0.2
0.2

0.1
0.1

Fluor.
Fluor.int.
int.(a.u.)
(a.u.)

00

DDCvar

CD
CD(a.u.)
(a.u.)

0.5
0.5

Dfluorvar

0.2
0.2

Fluor.
Fluor.int.
int.(a.u.)
(a.u.)

Absorbance
Absorbance(a.u.)
(a.u.)

11

DUV-Visvar

00

-0.2
-0.2

600 700
700 800
800
600
Wavelength(nm)
(nm)
Wavelength

300 400
400 500
500 600
600
300
Wavelength(nm)
(nm)
Wavelength

Data matrices arrangement: (a) analysis of a single spectroscopic data matrix;

(b) simultaneous analysis of several spectroscopic
data matrices corresponding
to different
spectroscopic techniques and different experiments.

Cvar

SUV-Vis

-5

Sfluor

x 10

x 10

x 10

SCD

x 10

0
0

0.1

0.2

0.3

0.4

0.5
Xpoly

0.6

0.7

0.8

0.9

CEt

-5

-2

250

300

350

400
450
Wavelength (nm)

500

550

600

550

600

650

700
Wavelength (nm)

750

800

850

-4

220

240

260

280

300
320
Wavelength (nm)

340

360

380

poly(I)-poly(C)

x 10

EtBr

1.5

C oncentration (M )

CD (a.u.)

Absortivity

2.5

Fluorescence (a.u.)

Concentration (M )

poly(I)-poly(C)-Et

0.5

0
0

x 10

3
r poly:dye

Cpol

-5

Kapp = [EtBr poly complex]

/[Poly(I)-poly(C)
EtBr]
RESULTS

2.5

C oncentration (M )

1.5

0.5

0
0.75

0.8

0.85

Xpoly

Poly(I)-poly(C) + EtBr EtBr poly complex

0.9

0.95

The intercalation sites occur every 2-3 base pairs and

the value for the log Kapp was 4.6 0.1 M-1

400

Study of conformational equilibria

of polynucleotides
R. Tauler, R. Gargallo, M. Vives and
A. Izquierdo- Ridorsa
Chemometrics and Intelligent Lab
Systems, 1998

Poly(adenylic)-poly(uridylic) acid system

Absorbance

Melting data

Wavele
ngth

( n m)

m
Te

pe

re
u
t
ra

C
(

Melting data recorded at = 260 nm

Absorbance

(univariate data analysis)

Temperature (C)

Melting Curve

Absorbance

Melting recorded at = 280 nm

Temperature (C)

Melting Curve

Poly(A)-poly(U) system. Two different melting experiments

Relative concentration

ALS recovered concentration profiles

poly(A)-poly(U) ds

poly(U) rc

poly(A) rc
poly(A)-poly(U)-poly(U) ts

Temperature (C)

poly(A) cs

ALS recorded pure spectra