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Laser Induced Breakdown Spectroscopy (LIBS) is an analytical chemistry technique that has the potential
to identify and measure the elements in a substance of interest. LIBS is applicable for any phase (solid,
liquid or gas). LIBS has gained importance in the fields of material identification, biomedical science,
forensics, military, art and archaeology in the recent years.

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Forensic Chemistry

journal homepage: www.elsevier.com/locate/forc

Anjali Gupta a,, James M. Curran a, Sally Coulson b, Christopher M. Triggs a

a

The University of Auckland, Auckland 1010, New Zealand

b

Institute of Environmental Science and Research, Auckland 1025, New Zealand

a r t i c l e i n f o a b s t r a c t

Article history: Laser Induced Breakdown Spectroscopy (LIBS) is an analytical chemistry technique that has the potential

Available online 25 January 2017 to identify and measure the elements in a substance of interest. LIBS is applicable for any phase (solid,

liquid or gas). LIBS has gained importance in the fields of material identification, biomedical science,

Keywords: forensics, military, art and archaeology in the recent years. In spite of its many advantages over other

LIBS instruments, it has a few drawbacks such as poor precision and repeatability. That is, different spectra

Variation may be observed for the same sample over successive runs. This indicates poor precision of the instru-

Experimental design

ment.

Normalization

Statistics

This paper presents an experiment designed to investigate the claim that there is higher variation in

the experiments conducted on different days as compared to the experiments conducted on the same

day. This study examines the variability in the spectra between the runs on the same day, and the vari-

ability between runs on different days using samples from a standard reference glass, and discusses the

conclusions that can be drawn from the results.

2017 Elsevier B.V. All rights reserved.

made on the same day and between runs made on different days.

Laser Induced Breakdown Spectroscopy (LIBS) is an atomic This variation has important consequences for forensic practition-

emission technique that has gained importance in the fields of ers and hence, understanding the nature of the variation is vital.

material identification, biomedicals, forensic science, military, art The following study has been conducted in order to investigate

and archeology in the past thirty years. the claim that inter-day variation in the spectra is higher than

LIBS possesses many advantages over other instruments such as the intra-day variation. Standard reference glass material (NIST

portability, relatively low cost and simpler sample preparation. It is 1831) has been used to perform the experiment.

a relatively non-destructive method and rarely consumes all of the

sample [16,5]. LIBS is important for trace evidence evaluation as it

is one of the elemental analysis methodology that strengthens the Background

value of association between the associated samples, reduces false

association between samples and references that come from differ- It is well-known that glass evidence may be used to associate a

ent sources. Elemental analysis also provides more discrimination suspect with a crime scene [7]. Glass is one of the most common

as compared to other methods like Refractive Index [3]. However, types of trace evidence found in cases of hit-and-run, burglaries,

it has a few drawbacks. These are - a high background continuum and can be easily recovered from a crime scene [3]. It is important,

[10], line-broadening, self-absorption [6], poor precision, and therefore, for forensic practitioners to be able to precisely, accu-

repeatability [11]. LIBS is known to generate different spectra for rately and repeatedly measure the physical characteristics of glass

the same sample when the instrument is run several times during (Refractive Index and elemental characteristics) and to understand

the day, and on various days. the limitations of any measurement technique. Thus, this study has

In this paper we have designed an experiment using the formal been conducted to explore the comparison of intra day variation in

principles of statistical experimental design [8] to explore intra- the intensities of elements in glass samples and the variation in

day and inter-day variation in LIBS measurements. That is, we intensities observed in runs on different days obtained using LIBS.

Winefordner et al. [16] stated that LIBS needs improvement as

the percent relative standard deviation (%RSD) in measurements

Corresponding author. from LIBS is higher than conventional techniques. Lee et al. [11]

E-mail address: anjaliscorpio@gmail.com (A. Gupta). also showed that the precision of LIBS (%RSD: 510%) is not

http://dx.doi.org/10.1016/j.forc.2016.12.002

2468-1709/ 2017 Elsevier B.V. All rights reserved.

A. Gupta et al. / Forensic Chemistry 3 (2017) 3640 37

comparable with the precision of other analytical techniques. shots was discarded. 100 shots were fired at each position on the

Almirall et al. [4] carried out a study using NIST SRM (Standard Ref- sample and the data for all the 100 shots was summed up to get

erence Material) 1831 and 610 which showed that reproducibility one spectra per position per sample. The spectra from each of the

of analysis conducted using LIBS on the same day was reasonably four positions on the sample was averaged to get one spectra per

good. %RSD for each emission line ratio for the experiments con- sample. This experiment provided with 12 spectra each day. It

ducted hourly on a single day for a single float glass sample was has been suggested previously that a few peaks be selected for fur-

recorded to vary from 0.8% to 15.0% [5]. Another study conducted ther statistical analysis instead of using the whole spectrum, thus,

experiments to measure %RSD for a set of 11 emission peak ratios the system software was used to acquire the intensities of a set of

on a single day and %RSD for the same peak ratios over a three days twelve peaks for each spectrum. Calcium, silicon and sodium are

period for the standard NIST SRM 610 glass sample. The results the main constituents of glass and the intensities for these ele-

(same day %RSD: 6:5 1:4%, 3 days %RSD: 24:5 29:2%) empha- ments have been included in the selected peaks for this study.

sized on the advantage of conducting the experiments of glass on The selected peaks were:

the same day [6].

Rodriguez-Celis et al. [15], inspired by the aforementioned Al 394 Na 330

research, conducted a study which attempted to minimize inter- Sr 460 K 766

day variation. In one of the studies described in the paper, the Ca 534 Ca 643

atomic line at 288.16 nm (Si) was selected and tracked regularly Na 818 Fe 373

for a standard glass sample NIST 612. The analysis was not contin- Al 309 Ba 493

ued if the variation in the intensity of this line exceeded the Sr 407 Si 288

expected variation of 15%. McIntee et al. [12] conducted a study

in 2010 where the researchers aim was to compare the LIBS spec-

tra for float glass samples using non-parametric permutation test.

Based on the results of prior studies, the researchers collected the The actual elemental ratios for the selected peaks for NIST 1831

spectra for the samples on the same day to avoid the day-to-day are stated below (Table 1):

variation amongst the LIBS spectra.

It is clear from the potentially conflicting conclusions of previ- Software

ous research, as well as research into attempts to mitigate

observed variation, that understanding intra-day and inter-day Data acquisition was performed using Applied Spectras Axiom

variation is pivotal in any further effort to use LIBS measurements software. Statistical calculations performed on the collected spec-

for forensic purposes. tra were done in R [1].

Statistical analysis

Experimental

The amount of glass ablated when laser is fired on the sample

Instrumentation and data acquisition cannot be controlled which results in different levels of intensities

for the spectra. These different levels of recorded intensities for the

The LIBS instrument used in this research was an Applied Spec- selected peaks can be corrected by taking ratios of the recorded

tra (Fremont, CA), model J200 along with an Aurora LIBS spectrom- intensities with the intensity of an element that is present in abun-

eter. This instrument used 266 nm Nd:YAG laser for ablating dance in glass such as Silicon at 288 nm (Almirall, personal com-

samples. The laser pulse was focused on the sample with a munication, 2016). The ratios of the intensities range from

3.5 cm focal length lens. The lens could accommodate samples of 0; 1 and these ratios do not exhibit normal behavior. In order

distinct thickness as it was flexible along the vertical axis. Experi- to change the scale of the observations to 1; 1 as per the

ments have been conducted in the past to optimize the precision of range of normal distribution, logarithm of the ratios were taken.

LIBS that has led to setting the spectrometer delay as 1 ls and gate This transformation is known as additive log ratio transformation

width as 1.05 ms. The use of Argon gas in the sample chamber is and the transformed data is known to have similar properties as

known to enhance the spectral intensity [5], due to which Argon normal distribution [2,14].

gas was used to purge the sample chamber at a rate of approxi- It is known that variance components models are used to esti-

mately 1 L/min and the laser output level was set at 35%. The laser mate the variability defined by each of the levels in the model.

repetition rate was 10 Hz and the spot size was set to 100 lm. The These models may consist of fixed and random intercepts, com-

plasma-generated emission intensities (1901040 nm) were col- monly known as mixed intercept models. Fixed effects are the ones

lected by a unique light collection optics design connected to the where the sources of variation can be controlled, whereas random

system detector module (six linear CCD array spectrometer). effects are the sources of variation that cannot be controlled.

Randomized Complete Block Design (RCBD) is one of the tradi- Since, this study was conducted to compare the inter-day and

tional experimental designs where the experimental units are intra-day variation in the intensities recorded using LIBS, thus,

arranged in blocks or groups such that the units within blocks variance component models, as given in Eq. (1), were fit to the

are homogeneous and there is heterogeneity between the blocks. transformed intensities of each of the 11 peaks (except Si 288) to

The results of the previously conducted research were used while

designing this experiment. Since, the prior studies suggested that

the intra-day variation is less than the inter-day variation for LIBS Table 1

Certified mass fraction ratios for selected peaks.

spectra, each of the days formed blocks of the experiment. Four

pieces of glass were taken from a box of standard glass NIST Elemental ratios Ratio of certified mass fractions

1831 for data acquisition. The experiment was conducted for five Al/Si 0.017

days, where three spectra were saved for each sample each day. Ca/Si 0.112

The order in which the samples were run each day was decided Fe/Si 0.001 (Fe2 O3 ); 0.000 (FeO)

K/Si 0.005

randomly. Each sample was ablated at four positions. 20 warmup

Na/Si 0.182

shots were used to ablate sample surface and the data from these

38 A. Gupta et al. / Forensic Chemistry 3 (2017) 3640

explore the variation due to runs on the same day, and runs on Maximum Likelihood (ML) and Restricted/Residual Maximum

k th th Likelihood (REML)

different days. Let yij be the intensity on i day, j run and for

th Maximum Likelihood method is the most popular method for

k sample for each of the peaks.

point estimation of the population parameters. There is a similarity

lk di eij

k between ML method and method of moments in terms of the num-

yij 1

ber of parameters being estimated, but the estimation procedures

where i 1; . . . d; j 1; . . . r; k 1; . . . ; s, for both the methods are different.

As the name suggests, for ML method, a likelihood function is

lk : k fixed sample effect

th

determined, and the values of the estimators are computed for

th ~ r2

di : i random day effect N0; which the likelihood of the observed data is the highest. These

d

~ r2

eij : random runs within days effect N0; maximum likelihood estimators (MLE) may not necessarily by

e

unbiased estimates.

In the above mentioned model, the variation due to days, and REML, on the other hand, produces unbiased estimates or less

different runs within days cannot be controlled, hence, di and eij biased estimates than the ML estimates. REML takes into account

have been considered as random effects. the loss of degrees of freedom during estimation, and provides bet-

Here, r2d and r2e are defined as the experimental variation in ter estimates than ML approach. Thus, REML approach was applied

the intensities recorded for d days (inter-day variation) and the to the transformed intensities for each of the selected peaks.

residual variance respectively. Residual variance is defined as The above mentioned procedures, method of moments, maxi-

the variance in the experimental units that cannot be explained mum likelihood and REML fall under the category of Frequentist

by the factors under consideration. In this scenario, any unex- analysis. The estimated variances were computed using both,

plained variance in the observations that cannot be explained method of moments and REML methodology, and the estimated

by different days, is attributed to variance due to r runs each variances were used for testing the below hypothesis.

day (intra-day variation), r2r , and can also be termed as residual

variance. H0: The experimental variation in the intensities measured from

Several methods can be used to compute the estimates of vari- runs conducted over a few days is the same as the experimental

ance components r2d and r2e . The obtained estimates of the vari- variation in the intensities recorded from successive runs on the

ance components are then used for hypothesis testing for same day

Frequentist analysis, and in calculating posterior probability of Ha: The experimental variation in the intensities measured from

the desired statistic in case of Bayesian analysis. runs conducted over a few days is greater than the experimen-

tal variation in the intensities recorded from successive runs on

Estimation methods the same day

Method of moments The quantity of interest for testing the hypothesis is the theo-

r2

Method of moments is the oldest method for obtaining point retical parameter F rd2 . The variance estimates obtained using

r

estimates of the population parameters. As the name suggests, the above mentioned procedures were used to compute the esti-

the first k sample moments are equated to the first k population b

r2

moments in order to compute the desired population estimates. mator F 0 d2 . When the null hypothesis is true, this test statistic

b

rr

Variance estimator can be estimated by using the first and the sec- follows an F distribution with 4,52 degrees of freedom. If we adopt

ond sample moments, m1 and m2 respectively. a 5% level of significance, then the critical value for an F 4;52 distri-

r2 EX 2 EX2 2 bution is 2.55. The test statistic, F 0 , is then compared with the crit-

ical value. If F 0 > critical value, it may be assumed that there is

rb 2 m2 m1 2 3 significant evidence at 5% level against the null hypothesis, hence,

rejecting H0 . If F 0 critical value, it can be concluded that signifi-

Since, the method of moments estimator of variance is a differ- cant evidence is not present at 5% level to reject H0 .

ence of m2 and m1 2 , this method may sometimes yield a negative

estimate for variance.

From Eq. (1), the total sum of squares (TSS) in the intensities, Bayesian method

k

yij , for each of the selected peaks can be divided into days sum Bayesian methodology refers to a method where the unknown

parameters in the model are assumed to follow prior distributions

of squares (Days SS), and residuals sum of squares (Residuals SS)

and the observed data is then used to update the distributions of

as shown below:

the unknown parameters. The updated distributions for the

TSS Days SS Residuals SS 4 unknown parameters are known as posterior distributions. This

analysis was run using software JAGS, which was run in R using

This facilitates the computation of the variance estimates using

Rs package rjags.

the formulae given below:

For Eq. (1), appropriate priors were assumed for the unknown

rb 2e Mean Residuals SS 5 parameters in the model. Half-t prior distribution was used for

days variance component as recommended by Gelman [9]. As the

Mean Days SS Mean Residuals SS bayesian methodology is applied to the model Eq. (1), the posterior

rb 2d 6

s r distribution for the unknown parameters is obtained. This is fur-

ther used to calculate the same ratio as in the case of Frequentist

rb 2r rb 2e 7 r2

analysis, rd2 . The comparison of this ratio in Bayesian analysis is car-

r

where s is the number of glass samples, r is the number of runs con- ried out on similar lines as Frequentist analysis. The posterior

ducted each day and r b 2r is the estimate of the experimental varia- probability of this ratio being P 1 is monitored and relates to

tion in the intensities recorded from successive runs r on a single inter-day variation being greater than or equal to intra-day

day. variation. The higher the probability, higher the chances are for

A. Gupta et al. / Forensic Chemistry 3 (2017) 3640 39

Table 2

Estimated variance components using Method of Moments, Bayesian posterior probabilities of the ratio of the variance components being P1.

Al309 Al394 Ba493 Ca534 Ca643 Fe373 K766 Na330 Na818 Si288 Sr407 Sr460

r

b 2

d

0 3 3 28 26 12 63 8 47 2 57

r

b 2

r

23 25 29 74 55 122 123 62 89 41 119

F0 r

b 2d = r

b 2r 0.14 0.09 0.38 0.47 0.10 0.51 0.13 0.53 0.04 0.48

r r P 1

P b 2d = b 2r 0.02 0.09 0.05 0.17 0.26 0.02 0.24 0.07 0.27 0.02 0.23

the variation to be greater on different days as compared to varia- emission intensities data is not significantly more, at 5% level, than

tion on the same day. the intra-day variation. Method of moments approach, restricted

maximum likelihood and Bayesian methodology provides no sig-

nificant evidence in favour of high day-to-day variation in the mea-

Results and discussions

surements from LIBS machinery. It can be deduced from this study

that the evidences may be analysed on different days using LIBS

The estimated variance components computed using method of

and the spectra from different samples may be archived in a data-

moments (104 ) for the selected peaks are shown in Table 2. The

base for future use. This establishes the fact that LIBS may be a reli-

ratio of the variation in the intensities recorded for five days and

able instrument that can be used for comparison of recovered with

the intra-day variation is computed for each of the peaks and dis-

the control samples from the crime scenes.

b

r2

played in the table in third row d2 . The Bayesian posterior probabil-

b

rr

ities for the ratio P 1 are displayed in fourth row in the table Acknowledgements

br2

P d2 P 1.

brr The authors acknowledge the helpful suggestions provided by

Method of moments estimate for r2d for aluminium at 309 nm Dr. Jos R. Almirall. The experiments for this work were performed

was computed as negative. Due to this, the estimated inter-day at The Institute of Environmental Science and Research (ESR). This

variance component for Al309 was replaced with zero as it can work was supported under The University of Auckland Doctoral

be seen in Table 2 [13]. REML approach was also used to compute Scholarship.

the variance estimates and it was observed that This project was supported by Award No. 2015-DN-BX-K049,

r

b 2d 6:4 1018 0 for Al309. This resulted in the ratio of the awarded by the National Institute of Justice, Office of Justice Pro-

variance components for Al309 as very close to zero grams, U.S. Department of Justice. The opinions, findings, and con-

(2:8 1015 ). The variance estimates, r b 2d ; r

b 2r for all the other clusions or recommendations expressed in this publication/

selected peaks were exactly the same for method of moments program/exhibition are those of the author(s) and do not necessar-

approach and REML. ily reflect those of the Department of Justice.

b

r2

As illustrated in Table 2, third row, d2 < 2:55 critical value for

b

rr References

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[4] J.R. Almirall, S. Umpierrez, W. Castro, I. Gornushkin, J. Winefordner, Forensic

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[5] C.M. Bridge, J. Powell, K.L. Steele, M. Williams, J.M. MacInnis, M.E. Sigman,

the variance components being greater than or equal to one is close

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818 nm. This can be explained by the fact that calcium and sodium spectrometry, Appl. Spectrosc. 60 (10) (2006) 11811187.

[6] C.M. Bridge, J. Powell, K.L. Steele, M.E. Sigman, Forensic comparative glass

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[8] R. Fisher, The design of experiments, 1935.

that the inter-day variation is greater than the intra-day variation [9] A. Gelman, Prior distributions for variance parameters in hierarchical models

0, further implying that the variation in the experiments run on (comment on article by browne and draper), Bayesian Anal. 1 (3) (2006) 515

different days is either equal or less than the variation in the exper- 534.

[10] M. Kuzuya, M. Murakami, N. Maruyama, Quantitative analysis of ceramics by

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40 A. Gupta et al. / Forensic Chemistry 3 (2017) 3640

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