plasma optical emission spectrometry by charles …

FUNDAMENTAL STUDIES AND APPLICATIONS IN MICROWAVE-INDUCED

PLASMA OPTICAL EMISSION SPECTROMETRY

BY

CHARLES BRYSON WILLIAMS, III

A Dissertation Submitted to the Graduate Faculty of

WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES

in Partial Fulfillment of the Requirements

for the Degree of

DOCTOR OF PHILOSOPHY

Chemistry

May 2019

Winston-Salem, North Carolina

Approved By:

Bradley T. Jones, PhD, Advisor

Michael D. Gross, PhD, Chair

Christa L. Colyer, PhD

George L. Donati, PhD

Scott M. Geyer, PhD

ii

This work is dedicated to my grandfathers:

LEWIS FRANKLYN SUTTON, PhD,

who taught me the value of knowledge;

CHARLES BRYSON WILLIAMS, Sr,

who taught me the value of hard work.

iii

ACKNOWLEDGMENTS

I wish to thank and acknowledge all those who have made this possible. First, my parents,

Laura and Bryson Williams, who raised me and supported me, my sister Meg, who put up

with me, and my aunt Nancy Sutton who has taken care of me in Winston-Salem. I also

could never have done this without my late grandfather, Lewis Sutton, who made much of

my education possible and who inspired my love of learning, as well as my living

grandfather Charles Williams, Sr, who inspired me by working his way out of poverty into

the American Dream.

I also wish to thank my advisors for all their support: Brad Jones for being willing to take

a student after a hiatus, and for securing a research assistantship for two years, and George

Donati for working tirelessly and putting up with me through melted torches, spilled

solutions, and sloppy calibrations, as I found my footing in the lab. I also greatly appreciate

my lab mates, Jake Carter and John Sloop, for their support and advice on projects and on

keeping the lab running smoothly, as well as for their friendship. And I thank Cliff

Calloway for his advice and perspective during the summer months. I also thank Tom

Whitmann for his hard work in our lab, as well as all the other undergraduate members.

I also thank those collaborators who have supported me through my years here, especially

Tina and Larry McSweeney, and everyone at Agilent Technologies, Paul Elliott and

everyone at CEM, Holly Peterson at Guilford College, and Daniel Goncalves and Renata

Amais from Brazil. I also thank Shiba Adhikari, Hui Li, Chang Lu, Jennifer Buchanan, and

iv

all of our collaborators within the Chemistry Department at Wake, as well as our

collaborators in the Physics Department.

I wish to thank my committee members, Christa Colyer, Michael Gross, and Scott Geyer

for their advice and feedback along the way. I also thank Al Rives and Jon Booze, the

members of the Teaching and Learning Collaborative, as well as my students, for their

support and guidance as I have developed my teaching skills. I also wish to acknowledge

Steve Creager and the Chemistry Department at Clemson University for setting me up with

a solid background in chemistry and teaching me that graduate school was possible.

I wish to thank David Pegg, the Rev. Lawrence Womack, the Rev. Ginny Wilder, and

everyone at St. Anne’s Episcopal Church for providing me a creative and spiritual outlet

in serving as their organist during my time in Winston-Salem.

Finally, and most importantly, I thank God, the Universal Mind and Source of all

knowledge and wisdom, without whose grace I would have been utterly unable to complete

this work.

v

TABLE OF CONTENTS

PAGE

LIST OF TABLES AND FIGURES vi

LIST OF ABBREVIATIONS xi

ABSTRACT xiii

CHAPTER I INTRODUCTION 1

CHAPTER II DETERMINATION OF CALCIUM, POTASSIUM AND SODIUM IN

SOFT DRINKS USING THE 4200 MP-AES 31

CHAPTER III DRY ASHING AND MICROWAVE-INDUCED PLASMA OPTICAL

EMISSION SPECTROMETRY AS A FAST AND COST-EFFECTIVE STRATEGY

FOR TRACE ELEMENT ANALYSIS 41

CHAPTER IV NATURALLY OCCURRING MOLECULAR SPECIES USED FOR

PLASMA DIAGNOSTICS AND SIGNAL CORRECTION IN MICROWAVE-

INDUCED PLASMA OPTICAL EMISSION SPECTROMETRY 66

CHAPTER VI CONCLUSIONS 124

APPENDIX A SUPPLEMENTARY INFORMATION FOR CHAPTER III 126

SCHOLASTICA VITA 136

vi

LIST OF TABLES AND FIGURES

TABLES

Table I. Recently described non-commercial MIP OES systems. ................................... 12

Table II. Recent sample introduction strategies and sample preparation procedures used in

MIP OES. .......................................................................................................................... 17

Table III. Element-Specific Operating Parameters.......................................................... 34

Table IV. Instrument Parameters ..................................................................................... 35

Table V. Soft drink analysis with the 4200 MP-AES. ..................................................... 38

Table VI. Ca, K and Na concentrations in soft drinks determined by the 4200 MP-AES.

........................................................................................................................................... 39

Table VII: Operating conditions used in MIP OES determinations. ............................... 49

Table VIII. Ashing parameters providing the best recoveries for determinations using the

HRA prototype and MIP OES. ......................................................................................... 53

Table IX. Evaluating the accuracy of the HRA-MIP OES procedure by analyzing a

standard reference material of Tomato Leaves (NIST SRM 1573a). ............................... 56

Table X. Comparison between ashing and acid extraction (HRA) with the traditional

microwave-assisted digestion (MAD). ............................................................................. 58

Table XI. HRA results presented as their percent portion of concentrations determined

using the MAD procedure. ................................................................................................ 60

Table XII. Instrumental operating conditions used in MIP OES. .................................... 74

Table XIII. Analyte percent recoveries for 2.0 mg L-1 solutions of Al, Ba, Mn, Sr and Zn

prepared in different matrices. .......................................................................................... 89

Table XIV. The two best signal correction strategies for each sample matrix. ............... 92

vii

Table XV. Comparison of MFC with EC using the same individual Q values while

determining Cu in River Sediment A.............................................................................. 107

Table XVI. Accuracy comparison between MFC and EC. ............................................ 111

Table XVII. Analyte percent recoveries (%) from spiked concentrations in water and food

samples analyzed by MIP OES using MFC or EC. ........................................................ 114

viii

FIGURES

Figure 1. Microwave cavity structure of the Hammer arrangement. ................................. 6

Figure 2. Representation of the Hammer cavity and resonant iris used in the commercial

MIP OES instrument by Agilent Technologies (used in models 4100, 4200, and 4210 MP-

AES). ................................................................................................................................... 7

Figure 3. Schematic representation of the detection system in a monochromator-based MIP

OES instrument (Agilent 4200 MP-AES)........................................................................... 9

Figure 4. Schematic representation of the pre-optics in an Agilent 4200 MP-AES

instrument. .......................................................................................................................... 9

Figure 5. Helios Rapid Ashing unit, courtesy of CEM Corporation................................ 47

Figure 6. Sample basket used with the HRA prototype. .................................................. 47

Figure 7. Sample “sandwich” setup using an aluminum foil at the bottom and a quartz fiber

pad on top loaded in the sample basket. ........................................................................... 52

Figure 8. Effects of nebulization gas flow rate on Mg II / Mg I (a), and N2+ / OH (b) in

MIP OES. .......................................................................................................................... 79

Figure 9. Effects of sodium concentration on the Mg II / Mg I and N2+ / OH signal ratios

at nebulization gas flow rates of 0.6 L min-1 (a) and (b), and 1.0 L min-1 (c) and (d). ..... 80

Figure 10. Effects of nebulization gas flow rate on (a) Mg II (280.271 nm) and Mg I

(285.213 nm), and (b) N2+ (391.439 nm) and OH (308.970 nm). .................................... 80

Figure 11. MIP OES spectra for the OH molecular species (band peak at 308.970 nm).

Each spectrum corresponds to a different plasma / sample introduction condition ......... 82

Figure 12. Correlation between the Mg II / Mg I and N2+ / OH signal ratios in the presence

of sodium at different nebulization gas flow rates (0.6 - 1.2 L min-1) .............................. 84

ix

Figure 13. Relationship between average analyte percent recovery and N2+ / OH signal

ratio in different matrices .................................................................................................. 87

Figure 14. Multi-flow calibration plots for determining (A) Cu in River Sediment A, and

(B) Mn in Tomato Leaves. .............................................................................................. 109

Figure 15. Long-term stability of MFC ......................................................................... 118

Figure S 1. Molecular emission spectra for CN recorded with the plasma off, or as a 1 %

v/v HNO3 solution was introduced into the MIP at a nebulization gas flow rate of 0.7 L

min-1. ............................................................................................................................... 128

Figure S 2. Molecular emission spectra for N2 recorded with the plasma off, or as a 1 %


min-1. ............................................................................................................................... 128

Figure S 3. Molecular emission spectra for N2+ recorded with the plasma off, or as a 1 %


min-1. .............................................................................................................................. 129

Figure S 4. Molecular emission spectra for OH recorded with the plasma off, or as a 1 %


min-1. ............................................................................................................................... 129

Figure S 5. Molecular emission spectra for CN recorded as 1 % v/v HNO3, distilled-

deionized water (NGFR = 0.7 L min-1), or no aqueous solution (no spray chamber) was

introduced into the MIP. ................................................................................................. 130

Figure S 6. Molecular emission spectra for N2 recorded as 1 % v/v HNO3, distilled-



x

Figure S 7. Molecular emission spectra for N2+ recorded as no aqueous solution (no spray

chamber), distilled-deionized water, or 1 % v/v HNO3 (NGFR = 0.7 L min-1) was


Figure S 8. Molecular emission spectra for OH recorded as 1 % v/v HNO3, distilled-



Figure S 9. Absolute emission signal percent change as Na concentrations in the 0 - 1000

mg L-1 range were introduced into the MIP at a NGFR of 0.6 L min-1. ......................... 133


mg L-1 range were introduced into the MIP at a NGFR of 1.0 L min-1. ......................... 133

Figure S 11. Absolute signal ratio percent change as Na concentrations in the 0 - 1000 mg

L-1 range were introduced into the MIP at a NGFR of 0.6 L min-1. ............................... 134


L-1 range were introduced into the MIP at a NGFR of 1.0 L min-1. ............................... 134

xi

LIST OF ABBREVIATIONS

Abbreviation Definition

ACS American Chemical Society

AES Atomic Emission spectrometry (synonymous with OES)

APER Average Percent of Recovery

CCD Charge-Coupled Device

CMP Capacitively-Coupled Microwave Plasma

CRM Certified Reference Material

EC External Standard Calibration

EDTA Ethylene Diamine Tetra-Acetate

EGCM External Gas Control Module

EIE Easily-Ionizable Element

FAAS Flame Atomic Absorption Spectrometry

FAES Flame Atomic Emission Spectrometry

FBN Flow-Blurring Nebulizer

FDA U.S. Food and Drug Administration

HPLC High-Pressure Liquid Chromatography

HRA Helios Rapid Ashing

HR-CS High-Resolution, Continuum Source

ICP Inductively-Coupled Plasma

IS Internal Standard

IUPAC International Union of Pure and Applied Chemistry

LED Light-Emitting Diode

LIBS Laser-Induced Breakdown Spectroscopy

LOD Limit of Detection

MAD Microwave-Assisted Digestion

MEC Multi-Energy Calibration

MFC Multi-Flow Calibration

MICal Multi-Isotope Calibration

xii

MICAP Microwave-Sustained, Inductively-Coupled, Atmospheric-Pressure

Plasma

MINDAP Microwave-Induced Nitrogen Discharge at Atmospheric Pressure

MIP Microwave-Induced Plasma

MP Microwave Plasma

MS Mass Spectrometry

MSC Multispecies Calibration

NGFR Nebulization Gas Flow Rate

NIST National Institute of Standards and Technology

OES Optical Emission Spectrometry

PID Proportional-Integral-Derivative

PN Pneumatic Nebulizer

Q Nebulization Gas Flow Rate

RF Radio Frequency

RSD Relative Standard Deviation

SA Standard Additions

SDA Standard Dilution Analyis

SRM Standard Reference Material

TIA Torche a Injection Axiale (Axial-Injection Torch)

TIAGO Torche a Injection Axiale sur Guide d’Ondes (Axial-Injection

Waveguide Torch)

USN Ultrasonic Nebulizer

VBPN V-Groove Babington-type Pneumatic Nebulizer

xiii

ABSTRACT

Microwave-induced plasma optical emission spectrometry (MIP OES) is a

technique within Atomic Spectrometry which is rapidly growing, following the relatively

recent introduction of a complete commercial instrument based on the Hammer-cavity

MIP. It offers advantages of low cost and simple operation, as well as the ability to run on

air, enabling it to be used in remote areas or places with underdeveloped infrastructure.

Due to the relative novelty of the technique, intensive method development for specific

sample types is necessary to expand the utility of the technique and compensate for some

of its limitations, such as its relatively low robustness and sequential detection. In addition,

developments in plasma diagnostics and novel calibration strategies are also important to

help increase the prominence and utility of the technique. The present research covers

several such applications and efforts to improve instrumentation performance, as well as

more fundamental studies of the properties of the plasma.

In the first project, a method was developed to determine concentrations of Ca, K,

and Na in various soft drinks. Addition and recovery experiments were used to evaluate

the accuracy of the method. This study shows the ability of the technique to withstand

relatively complex matrices, with no sample preparation other than simple dilution, and

produce accurate results with traditional calibration methods.

The second project involved development of a rapid dry-ashing technique to

prepare samples for analysis using MIP. Calcium, Fe, K, Mg, Na and Zn were determined

in complex-matrix samples such as tomato leaves, cheese, butter, peanut butter, infant

formula and biodiesel samples. General agreement was also found between MIP OES

xiv

results from samples decomposed either by the dry-ashing method or by a conventional

microwave-assisted digestion procedure, and accurate results were found when applying

the new, simpler method in certified reference material analyses.

In the third project, several molecular species, naturally occurring in the plasma,

were evaluated for their use as a plasma diagnostic tool. The N2+ / OH emission intensity

ratio was evaluated for identifying the best instrumental operating conditions in MIP OES.

Aluminum, Ba, Mn, Sr and Zn (analytes), and high concentrations of C, Na, Ca, HNO3 and

HCl (sample matrices) were used as models to investigate the effects of complex matrices

on analyte recoveries. The N2+ / OH signal ratio was more sensitive to changes in plasma

conditions than the traditionally-used Mg II / Mg I ratio. Some other advantages include

real-time monitoring capabilities, and the possibility of independently tracking variations

in both plasma and sample introduction. Significant improvements in accuracy were

achieved by employing the analyte-to-molecular species signal ratio, or their product, for

calibration.

In the final project, a novel calibration method, multi-flow calibration (MFC), was

proposed. This strategy involves the use of multiple nebulization gas flow rates in the

analysis to mitigate error caused by employing suboptimal sample introduction conditions,

and to eliminate the need for optimization of conditions. The new calibration method was

applied to the determination of Cr, Cu, Fe, and Mn in water and food samples. Addition-

recovery experiments and certified reference materials were used to validate the method.

Multi-flow calibration presents comparable or superior accuracy to the traditional external

standard calibration (EC), and offers simpler sample preparation than EC, requiring only a

single standard and no modification of the sample introduction equipment.

1

CHAPTER I

INTRODUCTION

REVIEW OF MICROWAVE-INDUCED PLASMA OPTICAL EMISSION

SPECTROMETRY

Charles B. Williams and George L. Donati

This chapter is based on unpublished material originally submitted as part of a

review article published in the Journal of Analytical Atomic Spectrometry, 2017, 32,

1283-1296. This material was excised because it was deemed off-topic from the main

subject of ICP OES. Stylistic variations are due to the requirements of the journal. First

authorship is shared between Charles B Williams and George Donati.

2

BACKGROUND: ATOMIC SPECTROMETRY

Atomic spectrometry involves the measurement of elemental concentrations in

various samples. Common methods used in this field include flame atomic absorption

spectrometry (commonly called AA), flame atomic emission spectrometry (FAES),

graphite furnace atomic absorption spectrometry (GFAAS), inductively-coupled plasma

optical emission spectrometry (ICP OES, also called ICP AES, for atomic emission

spectrometry), and inductively-coupled plasma mass spectrometry (ICP-MS).1 An

increasingly significant method in the field is microwave-induced plasma optical emission

spectrometry (MIP OES).2

The differences between these approaches generally break down to two factors: the

method of atomization/excitation, and the method of detection. Atomization/excitation

sources include flames, graphite furnaces, and plasmas, and detection can be accomplished

by either absorption, emission, or mass spectrometry. Generally, flame and graphite

furnace sources are associated with atomic absorption (using either a continuum-source or

line-source light), whereas plasma sources are associated with either atomic emission or

mass spectrometric detection. Flame and graphite furnace sources, as well as atomic

absorption and mass spectrometry will not be discussed in further detail in this text.

In a typical optical emission spectrometry (OES) system, atoms are excited in a

high-temperature environment such as a flame or plasma. As the electrons relax to a lower

energy state, a photon is emitted. Emitted photons from the analyte are measured using an

electronic detector and the intensity of the emission is directly proportional to the

concentration of the analyte in the sample.1 Each element has a characteristic set of

3

emission wavelengths, so with sufficient resolution, multielement samples can be analyzed

both qualitatively and quantitatively.

The most common atomization/excitation source in atomic spectrometry is the

inductively-coupled plasma (ICP). An ICP is generated from argon gas by a radio-

frequency electromagnetic field at either 27 or 40 MHz.3 This produces a highly stable

plasma with a high electron density and high temperature (7,500-10,000 K). This provides

a good source for atomization, ionization, and excitation of analytes. Because of the high

plasma temperature, most elements are ionized in an ICP.4 Some of its main limitations are

its relatively poor tolerance for organic solvents, and its expense, both in up-front cost and

running cost.

Microwave-induced plasma (MIP) can be generated from a variety of gases, but N2

is used most commonly. Microwave energy is generated from a magnetron, and then

transmitted and amplified by a waveguide cavity.2 Microwave-induced plasma is generally

lower in temperature than ICP, resulting in a predominance of atomic species, rather than

ionic ones. However, MIP is generally less expensive than ICP both in up-front and running

costs. In addition, it is more tolerant of organic solvents and other difficult matrices,

manifesting as matrix effects rather than extinguishing the plasma.

Matrix effects present one of the most significant challenges in atomic

spectrometry. They are caused by concomitant species within the sample which influence

the signal of the analyte, resulting in inaccurate analysis.5–7 Species which commonly cause

matrix effects include easily-ionizable elements (EIEs) such as Na and Ca, inorganic acids

at high concentrations, including HCl and HNO3, and high concentrations of carbon

species.

4

INSTRUMENTATION DEVELOPMENTS IN MIP OES

Microwave-induced plasmas have historically evolved as atomization and

excitation / ionization sources that could potentially be used as alternatives to traditional

ICPs in atomic spectrometry. There are two main approaches to generating microwave

plasmas. When microwave radiation is applied to an electrode, an electric field and a

perpendicular magnetic field are produced. In contact with a neutral gas and seeded

electrons, these fields will generate and sustain a so-called capacitively-coupled microwave

plasma (CMP) at the tip of the electrode. Alternatively, one can generate a microwave-

induced plasma by applying microwave radiation into a resonant structure filled with a

neutral gas. The energy associated with the standing wave inside the resonant cavity is

transferred to the gas, and the electric and magnetic fields generated produce and sustain

the plasma.8 The CMP was described in 1951 by Cobine and Wilbur,9 and the first use of

a 2.45 GHz microwave discharge as excitation source for emission spectrometry was

reported in 1965.10 The Beenakker cavity was introduced ten years later and used in MIP

OES for elemental analysis.11–13 For a historical perspective on the evolution of the

different instrumental arrangements used for microwave plasma generation and its

application in atomic spectrometry, the reader is referred to a thorough review recently

published by Jankowski and Reszke.8 Additional information can also be found in a book

by the same authors.2

The early instruments presented low sensitivity and had difficulty handling liquid

samples, which stemmed from the low microwave applied power (typically 200 - 300 W)

and the plasma non-toroidal shape. Later designs minimized some of these problems, but

low plasma temperatures and relatively poor interaction between plasma and liquid sample

5

often caused significant matrix effects.14–19 As discussed by Jankowski and Reszke,2 five

microwave plasma instruments have been commercially available in the past, but none of

them have generated enough interest to be widely adopted. A more successful system was

released in 2012 by Agilent Technologies (4100 MP-AES). The new MIP OES instrument

is based on a Hammer cavity with a resonant iris and runs on N2 gas.20,21 It has been

successfully used in several applications, and will be discussed in more detail in the next

paragraphs.

Microwave radiation is usually transmitted through a hollow rectangular structure

known as a waveguide. The electric field component of a typical microwave is aligned with

the short axis of the rectangular structure (i.e. height), while the magnetic field component

aligns with its longer axis (i.e. width). In both cases, the field is supported by induced

currents flowing on the walls of the metallic waveguide. The difficulties associated with

controlling the plasma central channel, as well as its non-toroidal shape, are directly related

to the fact that most MIPs are sustained by the microwave’s electric field component. In

the Hammer arrangement,22 this issue is resolved by positioning the magnetic field

component parallel to the torch. It then induces an electric field, which accelerates electrons

and ions causing collisions and generating and sustaining a doughnut-shaped MIP. To

improve sensitivity and minimize matrix effects, two additional modifications are adopted:

(i) N2 replaces monoatomic gases such as Ar and He as the plasma gas, and (ii) the torch

is placed in a resonant iris inside the waveguide. Plasmas composed of diatomic molecules

such as N2 have lower electron densities, which results in a larger skin depth (i.e., more

interaction between the plasma and the sample aerosol). The resonant iris further improves

coupling between the plasma outer layer and the cooler central channel by changing the

6

circular MIP into an elliptical shape. The iris structure also increases the intensity of the

magnetic field, which contributes to higher plasma temperatures. Figure 1 shows a picture

of the Hammer arrangement with the resonant iris, and Figure 2 shows a schematic

representation of the commercial MIP OES system based on this instrument design

(Agilent MP-AES). In the resonant iris, the axial magnetic field (small blue arrows in

Figure 2) and the transverse electric field (large red arrows in Figure 2) are combined to

render an elliptical form to the plasma.

Figure 1. Microwave cavity structure of the Hammer arrangement. Reprinted from Ref. 16. Copyright© 2017 Elsevier B.V. or its licensors or contributors. Reprinted with permission.

7

Figure 2. Representation of the Hammer cavity and resonant iris used in the commercial MIP OES instrument by Agilent Technologies (used in models 4100, 4200, and 4210 MP-AES). Small blue arrows and large red arrows represent the magnetic and electric fields, respectively. Copyright© 2017 Agilent Technologies, Inc. Courtesy of Agilent Technologies, Inc.. Reprinted with permission.

8

The commercial MIP OES instrument is based on a Czerny-Turner monochromator

(600 mm focal length), and a back thinned Peltier-cooled charge-coupled device (CCD)

detector. Figure 3 shows a schematic representation of the detection system. The

monochromator is composed of a fixed 2.5 mm high x 19 µm wide entrance slit, a spherical

parabolic collimating mirror (75 mm diameter), a 90 x 90 mm holographic diffraction

grating (2400 lines / mm and blazing angle optimized at 250 nm), and a spherical parabolic

focusing mirror (87 mm diameter). Considering, for example, an incident angle of 0o, this

system has a spectral bandpass of 0.011 nm and a resolving power of approximately 12,000

at 250 nm. The CCD detector is composed of a 532 wide x 128 high pixel array (each pixel

is 24 x 24 µm). It covers a spectral range between 176 and 1100 nm, and is Peltier-cooled

to 0 oC to minimize dark current noise. Figure 4 shows additional details of the pre-optics.

It is composed of a quartz entrance window (EW), two toric mirrors (M1 and M2), a

stepper-motor-positioned filter wheel assembly (FW), a stepper-motor-positioned flat

mirror (M3), and an exit slit (ES). The entrance window keeps the pre-optics separated

from the plasma and the exhaust system to minimize potential contamination. It also allows

for complete purging of the pre-optics system with N2 to prevent O2 absorption at the UV

region. Bandpass filters in the filter wheel assembly work in concert with the

monochromator to improve resolution. Wavelengths in the 160 - 320, 320 - 530, or 530 -

940 nm range are covered by moving the FW to positions zero (no filter), 1 (UV filter), or

2 (orange filter), respectively. A fourth position blocks the incoming light from the plasma,

and it is used in initialization routines to optimize the system’s dark current. The flat mirror

(M3) enables imaging of different regions of the plasma.

9

Figure 3. Schematic representation of the detection system in a monochromator-based MIP OES instrument (Agilent 4200 MP-AES). Copyright© 2017 Agilent Technologies, Inc. Courtesy of Agilent Technologies, Inc. Reprinted with permission.

Figure 4. Schematic representation of the pre-optics in an Agilent 4200 MP-AES instrument. Copyright© 2017 Agilent Technologies, Inc. Courtesy of Agilent Technologies, Inc. Reprinted with permission. M1, M2, M3 are mirrors, FW is the filter wheel, EW is the entrance window, and ES is the entrance slit to the optics.

10

Different from an echelle-CCD spectrograph, this monochromator-based system

detects analytical signals sequentially. As a consequence, the more analytes monitored, the

longer the analysis. On the other hand, while a compromise condition is used for all

elements in ICP OES, determinations using this sequential MIP OES instrument take place

at optimal nebulization gas flow rate and plasma viewing position for each individual

analyte. Plasma imaging is carried out axially, and is based on stepper motor positioning

of M3 (usually varying between -120 and 120 steps). Most determinations, however, are

carried out at position zero, which approximately corresponds to the center of the plasma.20

One of the main limitations of this MIP OES instrument is that the microwave applied

power is fixed at 1000 W and cannot be changed by the user. Depending on the position of

M3 and the nebulization gas flow rate, plasma temperature and electron number density

(ne) values vary between 4220 - 5360 K, and 0.47 - 3.72 x 1013 cm-3, respectively. Because

of the lower temperatures and ne values when compared with a typical Ar ICP, most

analytes are determined using atomic lines. As expected, the plasma is also less robust than

an ICP (Mg II / Mg I = 0.26 - 2.01), which requires matrix-matching calibration methods

(e.g. standard additions) to ensure adequate accuracy when analyzing some complex matrix

samples.20

Other non-commercial MIP instrumental arrangements have recently been

described in the literature.23–29 A list including a miniaturized, a low-pressure-operated,

and a portable system is presented in Table I. One of the most interesting new

arrangements is the microwave-sustained, inductively-coupled, atmospheric-pressure

plasma (MICAP).29 In this system, a typical ICP quartz torch is positioned concentrically

in a high-purity, high-density alumina (Al2O3) resonator ring. The microwave field

11

generated by a magnetron operating at 1000 W is directed toward the resonator by an

aluminum waveguide. It then reaches an inductive-coupling iris oriented perpendicularly

to the torch. Polarization currents are induced within the resonator as it couples with the

iris, and a corresponding oscillating magnetic field, oriented parallel to the torch, is

generated. The magnetic field induces an oscillating electric field that, similarly to a RF-

powered ICP, accelerates electrons and ions to generate and sustain a toroidal plasma. The

MICAP system runs on N2 or air and is tolerant to solvent loading. It can accept volatile

organic solvents and dissolved solids up to 3% w w-1. For a N2 plasma and determinations

in radial-view using an ultrasound nebulizer and a membrane desolvator, limits of detection

for Al, Ca, Cd, Co, Cr, Fe, Mg, Mn, and Pb were calculated in the 0.03 - 70 µg L-1 range,

with relative standard deviation (RSD) values ranging from 0.7 to 2.0 %. These analytical

characteristics are generally similar to a radial-viewing ICP OES. In fact, different from

the commercial MIP OES and closer to an ICP behavior, the most intense analytical signals

observed for the MICAP arrangement were recorded for ionic lines.

12

Tab

le I.

Rec

ently

des

crib

ed n

on-c

omm

erci

al M

IP O

ES sy

stem

s.

Mic

row

ave

syst

em

Plas

ma

gas

flow

ra

te

(L

min

-1)

Mic

row

ave

appl

ied

pow

er (W

)

Ana

lyte

s Sa

mpl

es

Com

men

ts

Ref

eren

ce

Mic

rost

rip M

IP

He,

0.2

5 40

Br

, C, C

l V

olat

ile h

alog

enat

ed

orga

nic

com

poun

ds

Min

iatu

re M

IP u

sed

as a

de

tect

or fo

r gas

ch

rom

atog

raph

y.

23

MIP

torc

h in

a T

E

rect

angu

lar c

avity

Ar,

0.2

- 1.0

10

0 - 1

80

Ba, C

a,

Cd,

Cu,

Fe, M

g,

Mn,

Ni,

Sr, Z

n

SRM

164

8 (U

rban

Pa

rticu

late

Mat

ter)

, IA

EA

336

(Lic

hen)

, SR

M 2

710

(Mon

tand

Soi

l), IN

CT-

SBF-

4 (S

oy B

ean

Flou

r)

Air-

cool

ed M

IP o

pera

ted

at

low

pre

ssur

e (0

.8 b

ar).

24

MIP

to

rch

in

a

cylin

dric

al c

avity

Ar,

0.4

- 0.5

15

0 A

s, C

d,

Hg,

Mn,

Ni,

P, P

b,

and

Sr

Aqu

eous

solu

tions

in 2

% v

v-1

HN

O3

OES

/ ca

vity

ring

dow

n sp

ectro

scop

y du

al m

ode.

25

MIP

tor

ch i

n an

Oka

mot

o ca

vity

N2,

14.0

; O2,

0 -

1.5

800

- 100

0 C

r A

queo

us so

lutio

ns

prep

ared

in d

ilute

d H

Cl

Ar a

t 0.5

L m

in-1

use

d as

ca

rrier

gas

. Stu

dy o

f pla

sma

char

acte

ristic

s and

com

paris

on

with

ICP.

27

13

MIP

tor

ch i

n an

Oka

mot

o ca

vity

N2,

14.0

; O2,

0 -

1.8

800

- 900

M

n A

queo

us so

lutio

ns

prep

ared

in d

ilute

d H

Cl

Ar a

t 0.5

L m

in-1

use

d as

ca

rrier

gas

. Stu

dy o

f pla

sma

char

acte

ristic

s and

com

paris

on

with

ICP.

26

MIP

tor

ch i

n an

Oka

mot

o ca

vity

N2,

14.0

80

0 - 1

500

Fe

Aqu

eous

solu

tions

pr

epar

ed in

dilu

ted

HC

l N

2 at 0

.5 L

min

-1 u

sed

as

carri

er g

as. I

nves

tigat

ion

of F

e ex

cita

tion

/ ion

izat

ion

mec

hani

sms i

n M

IP a

nd IC

P us

ing

the

Boltz

man

n pl

ot

met

hod.

28

Mic

row

ave-

sust

aine

d IC

P in

a

diel

ectri

c

reso

nato

r rin

g

N2 o

r air,

15.

0 -

18.0

1000

A

l, C

a,

Cd,

Co,

Cr,

Fe,

Mg,

Mn,

Pb, S

r

Aqu

eous

solu

tion

in 0

.1 M

H

NO

3; m

iner

al o

il,

kero

sene

, o-x

ylen

e,

tolu

ene,

ace

toni

trile

, he

xane

, met

hano

l

A ty

pica

l IC

P qu

artz

torc

h is

po

sitio

ned

conc

entri

cally

in a

hi

gh-p

urity

, hig

h-de

nsity

al

umin

a (A

l 2O3)

reso

nato

r rin

g.

The

mic

row

ave

field

indu

ces

pola

rizat

ion

curre

nts i

n th

e re

sona

tor a

nd g

ener

ates

a

mag

netic

fiel

d pa

ralle

l to

the

torc

h. T

he m

agne

tic fi

eld

indu

ces a

n el

ectri

c fie

ld th

at

sust

ains

the

plas

ma.

29

14

MIP OES APPLICATIONS FOCUSED ON SAMPLE INTRODUCTION AND

SAMPLE PREPARATION

Similar to ICP OES, many recent works on MIP OES describe new strategies for

sample introduction. Among these, most involve hydride generation and multi-channel

spray chambers, also known as multi-mode sample introduction systems.24,30–37 Typically,

in these systems, a multi-channel peristaltic pump introduces independent solutions into

the spray chamber, where efficient mixing and chemical reactions take place. Chemical

vapor generation is particularly useful in MIP OES because of the plasma’s lower

temperatures (ca. 5000 K)2,20 when compared with an ICP (6000 - 1000 K).3,4 Introducing

the analytes as gaseous species has three main beneficial effects on sensitivity and

accuracy: (i) sample introduction is significantly improved because it does not depend on

nebulization efficiency, (ii) with no solvent to vaporize, more plasma energy is available

for atomization and excitation, and (iii) negligible matrix effects.38,39 The simplest multi-

mode arrangements use one capillary channel to introduce the sample and a second one for

a solution containing the chemical-vapor-generating species.30–33 Due to its reducing

potential, NaBH4 (stabilized in solution with NaOH) is the most common reagent in HG

applications.38 LODs for this arrangement were calculated in the 1 - 10 µg L-1 range for

As, Ag, Bi, Cd, Cu, Ge, Hg, Mn, Ni, Pb, Rh, Se, Sn and Zn, with RSDs between 8 and 12

%. To improve sensitivity, triple-channel spray chambers typically introduce an acid

solution (e.g. HCl) in addition to the sample and the reducing agent (NaBH4 or

SnCl2).33–35 LODs and RSDs in this case were in the 0.3 - 9 µg L-1, and 5 - 9 % ranges,

respectively. In the quadruple-capillary sample introduction arrangement, acid, reducing

agent, sample, and internal standard solutions can be introduced separately into the spray

15

chamber to improve precision.36 By using internal standardization, RSDs were calculated

in the 2 - 3 % range, with LODs between 1 and 5 µg L-1 for As, Bi, Sb, Se and Sn. Most of

the recently described multi-mode systems use an USN, rather than the traditional

pneumatic nebulization (PN). Although more expensive and prone to memory effects, USN

produces smaller-diameter droplets and a more homogeneous tertiary aerosol, which

results in greater nebulization efficiency when compared with PN (ca. 20 % for USN cf.

ca. 5 % for PN).3 As observed in ICP OES applications, the increased nebulization,

combined with a finer aerosol, allows for more efficient contact and improved reaction

rates between analytes and reagents within the spray chamber. It also enables the analysis

of smaller sample volumes. Matusiewicz and Ślachciński, for example, used all four

channels of a quadruple-capillary system to introduce the sample solution. With more

sample effectively reaching the plasma, low-volume aliquots of liver, sediment, soil and

water samples were readily analyzed.37

To improve sensitivity and sample throughput, some procedures digest the sample

in-flow, or introduce it as a slurry or a powder.40–44 Jankowski et al., for example,

preconcentrated fluoride using a zirconia-based sorbent material, then dried the solid

mixture and introduce it into a lab-made continuous powder introduction chamber at the

base of a MIP torch.40 In a similar procedure, inorganic selenium was extracted from

aqueous samples and preconcentrated using bacteria immobilized on silica gel.41

Ślachciński determined several elements in liver, sediment, soil and coal ash by applying

ultrasound radiation to homogenize the sample slurry before introducing it into a lab-made

MIP OES using two different nebulizers: a V-groove Babington-type nebulizer (VBPN)

and a flow-focusing pneumatic nebulizer.42 To minimize matrix effects, Matusiewicz and

16

Ślachciński digested sample slurries of dogfish liver, milk powder, lichen, barley and

cinnamon immediately prior to analysis by using an in-line flow solubilization system

based on electromagnetic induction heating and a commercial ultrasonic nebulizer.43 In this

system, the sample slurry and nitric acid were pumped through Teflon tubing into a coil of

acid-resistant steel pipe, which was wrapped around a ferrite core. The mixture was then

heated and solubilized in-flow. A similar method is described by the same authors in

another paper, in which a UV micro-reactor is used to digest biological samples in-flow

prior to MIP OES analysis.44 As discussed by the authors, the main advantages of using

these strategies are the lower cost when compared with a conventional microwave-assisted

digestion, and potentially less sample contamination and analyte loss due to less sample

manipulation.

Even though MIP OES is not as sensitive as ICP-MS, it has also been recently used

in chemical speciation.45–47 Matusiewicz and Ślachciński used a microchip-based capillary

electrophoresis system to separate and determine As3+ and As5+, and Cu2+ and

Cu(EDTA)22- in water using a non-commercial MIP OES system.45,46 Barrientos et al.

determined Se and selenomethionine in yeast by coupling a commercial MIP OES system

with HPLC.47

Other sample preparation approaches such as dry-ashing, subcritical microwave-assisted

extraction with water, and ultrasound-assisted extraction were also used to improve

sensitivity and accuracy in MIP OES determinations.48–50 A summary of recent

developments in MIP OES involving sample introduction and sample preparation is

presented in Table II.

17

Tab

le II

. Rec

ent s

ampl

e in

trodu

ctio

n st

rate

gies

and

sam

ple

prep

arat

ion

proc

edur

es u

sed

in M

IP O

ES.

Sam

ple

intr

oduc

tion/

prep

arat

ion

Ana

lyte

s Sa

mpl

es

Com

men

ts

Ref

.

Dua

l cap

illar

y; u

ltras

onic

neb

uliz

er

/ vap

or g

ener

atio

n

Ag,

As,

Au,

Bi,

Cd,

Cu,

Ge,

Hg,

Mn,

Ni,

Pb, P

d,

Rh,

Sb,

Se,

Sn,

Zn

Biol

ogic

al ti

ssue

, hai

r,

soil,

food

, wat

er, w

ine

Seco

nd

capi

llary

in

trodu

ces

redu

cing

ag

ent

(NaB

H4

or

SnC

l 2) fo

r vap

or g

ener

atio

n.

30–3

3

Trip

le

capi

llary

; ul

traso

nic

nebu

lizer

/ va

por g

ener

atio

n

Ag,

As,

Au,

Bi,

Ge,

Hg,

Pd, P

t, R

h, S

b, S

e, S

n

Biol

ogic

al ti

ssue

, soi

l,

wat

er, w

ine,

ferti

lizer

Third

cha

nnel

int

rodu

ces

HC

l

to im

prov

e se

nsiti

vity

.

34,3

5,51

Qua

drup

le

capi

llary

; ul

traso

nic

nebu

lizer

/ va

por g

ener

atio

n

As,

Ba, B

i, C

a,

Cd,

Cu,

Fe,

Ge,

M

g, M

n, P

b, P

b,

Sb, S

e, S

n, S

r, Te

, Tl

, Zn

Wat

er,

Biol

ogic

al

tissu

e,

soil,

or

gani

c

solv

ents

Add

ition

al c

hann

els

used

for

vapo

r ge

nera

tion

and

inte

rnal

stan

dard

izat

ion.

36,3

7

Con

tinuo

us p

owde

r int

rodu

ctio

n F

Wat

er

Ana

lyte

pr

econ

cent

ratio

n on

zirc

onyl

nitr

ate.

40

Bact

eria

pr

econ

cent

ratio

n,

then

cont

inuo

us p

owde

r int

rodu

ctio

n

Se

Wat

er, b

eer

Bact

eria

im

mob

ilize

d on

sili

ca

gel.

41

18

Slur

ry m

icro

-sam

plin

g Ba

, Ca,

Cd,

Cu,

Fe,

Mg,

Mn,

Pb,

Sr,

Zn

Biol

ogic

al

tissu

e,

wat

er, s

oil

Cal

ibra

ted

by

stan

dard

addi

tions

, sl

urrie

s so

nica

ted

prio

r to

anal

ysis

.

42

Pres

suriz

ed

flow

so

lubi

lizat

ion

usin

g el

ectro

mag

netic

in

duct

ion

heat

ing

Ba, C

a, C

d, C

u, F

e, M

g,

Mn,

Na,

Pb,

Sr,

Zn

Biol

ogic

al ti

ssue

U

ses i

n-lin

e di

gest

ion.

43

Mic

roflu

idic

; m

icro

chip

-bas

ed

phot

o-m

icro

-reac

tor,

and

ultra

soni

c

nebu

lizat

ion

Ba, C

, Ca,

Cd,

Cu,

Fe,

Li, M

g, M

n, P

b, S

r, Zn

Urin

e,

wat

er,

Biol

ogic

al fl

uids

Sam

ple

flow

rate

of 9

µL

min

-1.

45

Mic

roch

ip c

apill

ary

elec

troph

ores

is

As3+

and

As5+

, and

Cu2+

and

Cu(

EDTA

) 22-

Wat

er

Buff

er:

Boric

aci

d +

CTA

B;

Sam

ple

Flow

Rat

e 0.

5 µL

min

-

1 .

45,4

6

Ion-

pair

reve

rsed

pha

se H

PLC

with

hydr

ide

gene

ratio

n

Se, S

eMet

Y

east

N

aBH

4 in

trodu

ced

in-li

ne.

Yea

st

sam

ples

re

cove

red

by

47

19

cent

rifug

atio

n, th

en r

eflu

xed

in

met

hane

sulfo

nic

acid

for 1

6 h.

Rap

id d

ry a

shin

g C

a, F

e, K

, Mg,

Na,

Zn

Food

, bio

dies

el

Sam

ples

ash

ed i

n A

l fo

il an

d

quar

tz fi

ber p

ads a

t 500

°C fo

r 3

min

, the

n ex

tract

ed in

10

% H

Cl

befo

re a

naly

sis.

48

Soni

catio

n /

Hea

ting

for

met

al

extra

ctio

n

Cd,

Cr,

Cu,

Mn,

Pb,

Zn

Inor

gani

c fe

rtiliz

er

Soni

cate

d in

50

% v

v-1

HC

l for

10 m

in a

nd h

eate

d at

85

°C

befo

re a

naly

sis.

49

Mic

row

ave-

assi

sted

su

bcrit

ical

wat

er e

xtra

ctio

n

Ba, C

a, C

u, F

e, M

g, M

n,

Na,

Pb,

Sr,

Zn

Plan

t tis

sue

Wat

er a

cidi

fied

with

100

µL

of

HN

O3.

Ana

lyte

ex

tract

ion

at

280

o C

and

90

bar.

Hig

h

pres

sure

ke

eps

wat

er

in

the

liqui

d ph

ase

(sub

criti

cal).

50

20

RECENT APPLICATIONS USING THE HAMMER-CAVITY MIP OES

Although more prone to matrix effects, the N2 MIP is also more tolerant to organic

solvent load than an Ar ICP. Amais et al. have used the commercial Hammer-cavity MIP

OES instrument to determine Cr, Ni, Pb and V in gasoline and ethanol fuel, and Si in diesel

and biodiesel employing external standard calibration.52,53 No sample preparation other

than simple dilution in 1 % v v-1 HNO3 (ethanol samples) or ethanol (diesel and biodiesel

samples) was required for a sample introduction system composed of an inert FBN and a

cyclonic spray chamber. For gasoline, sample and reference standard micro-emulsions

were prepared in 1-propanol to minimize matrix effects. Air was introduced into the plasma

to prevent carbon deposition on the torch and the optics. Accuracy was checked by addition

and recovery experiments, with values in the 91 - 108 % range in most cases. Some

recoveries were outside the 10 % error range for Cr (86 %), Ni (123 %), and Pb (86 %) in

gasoline, and Ni (84 %) in ethanol fuel. The LODs were calculated as 20 µg L-1 for Si, and

between 0.3 and 60 µg L-1 for Cr, Ni, Pb and V. Nelson et al. used the same MIP OES

instrument to analyze several samples of crude oil, and compared the results with values

obtained with ICP OES and ICP-MS.54,55 Sample solutions were prepared by dilution with

o-xylene and homogenization in a mechanic shaker for 30 min. Matrix-matching and

internal standardization were adopted to minimize matrix effects. The standard reference

solutions were prepared with organosoluble standards diluted in o-xylene, mineral oil to

match the sample matrix, a dispersant (oronite) to ensure homogeneous and stable

solutions, and Sc as IS. A low flow of air was added to the N2 MIP to prevent carbon build-

up on the torch and ensure plasma stability. The LODs for Ca, Fe, K, Na, Ni and V were

all < 0.07 mg kg-1, which are comparable with values calculated for ICP OES (i.e. < 0.02

21

mg kg-1). Accuracies were also comparable with ICP OES and ICP-MS, with spiked sample

recoveries between 93 and 107 %, and between 102 and 110 % for a certified reference

material of fuel oil.

MIP OES has also been successfully used to analyze soil, geological samples,

vinegar, animal feed and fertilizers.56–59 Niedzielski et al. described a high throughput

procedure for assessing soil fertility based on Mehlich-3 extraction and Ca, K, Mg and P

determination by MIP OES.56 Up to 100 samples h-1 were analyzed, with LODs between

0.06 and 0.9 mg L-1, RSDs in the 1.0 - 4.6 % range, and accuracies between 92 and 107 %.

In another work, 23 elements in geological samples were determined by MIP OES after

extraction with HF (to assess total concentration), aqua regia (quasi-total concentrations),

or HCl (acid leachable fraction). The LODs were calculated between 0.001 and

0.1 mg L-1, with RSDs and accuracies in the 0.20 - 1.37 %, and 85 - 115 %, respectively.57

Ozbek et al. determined 10 elements in 35 vinegar samples, with LODs between 0.4 and

30 µg L-1, and accuracies in the 93 - 104 % range.58 Similar to behavior observed for other

complex samples, the MIP was stable and well tolerant to the direct introduction of diluted

vinegar samples. The commercial MIP OES system was also used to determine Cu, Fe, Mn

and Zn in animal feed and fertilizers.59 Samples were submitted to either microwave-

assisted digestion with HNO3 (animal feed), or HCl extraction on a hot plate (fertilizers).

A 40-fold dilution was required to minimize matrix effects and provide results comparable

with values obtained with ICP OES and FAAS. The LODs were comparable with ICP OES,

but significantly superior to FAAS: 2 - 4, 2 - 5, and 10 - 40 µg L-1 for MIP OES, ICP OES

and FAAS, respectively.

22

As discussed earlier, MIPs operate at relatively lower temperatures compared with

a conventional Ar ICP,3,20 and often require matrix-matching calibration to ensure accurate

results when analyzing some complex-matrix samples. A simple strategy to minimize

matrix effects in determinations using external standard calibration with aqueous solutions

was recently proposed by Lowery et al.60 In this work, emission signals from naturally-

occurring molecular species in the N2 MIP were used as internal standards to correct for

analytical signal variations due to the sample matrix. Emission signals from N2+ (0–0, B

2Σ+u → X 2Σ+g) and OH (0–0, A 2Σ+ → X 2πi), with emission band heads at 391.4 and 309

nm, were used as molecular internal standards to minimize matrix effects and determine

Ca, K, Mg and Na in biodiesel. Significant improvements in accuracy were observed by

using the Mg / OH and Na / OH signal ratios, and the Ca x N2+, and K x OH signal

relationships as the dependent variable (y-axis) in the calibration curve plot. Biodiesel

samples were simply diluted in 1-propanol, and recoveries for K and Mg employing

aqueous standards and external standard calibration went from 130 and 60 % without signal

correction to 82 and 92 %, respectively, using the molecular probe strategy. Solvent-

matching (i.e., standard reference solutions prepared in 1-propanol) was required for

determining Ca and Na, but recoveries were also improved from 74 and 84 % to 104 and

92 %, respectively, when molecular standardization was employed.

Another approach to minimizing matrix effects in MIP OES determinations is the

previously described SDA calibration method. Goncalves et al. determined 7 elements in

several complex-matrix beverage samples using a procedure based on simple sample

dilution in 1 % v v-1 HNO3 and determination by SDA and MIP OES.61 LODs for Al, Co,

Cr, Cu, Mn, Ni and Zn were in the 10 (Cu) - 500 (Zn) µg L-1 range, and average recoveries

23

for all analytes and samples evaluated were between 90 and 99 %. As a comparison,

average recoveries for the same samples and analytes using the standard additions method

provided values in the 101 - 122 % range.

CONCLUSIONS AND PERSPECTIVES

While MIP OES offers advantages of low cost and simple operation, at present it

cannot match the sensitivity or robustness of ICP OES, preventing MIP from expanding its

foothold in the field of atomic spectrometry. Further development of the technique is

required to improve accuracy and expand the types of samples that can be analyzed.

MIP OES has recently become a cost-effective option for trace multi-element

analysis. Its ability to run on air, combined with its relatively low cost of purchase and

operation show great potential for increasing access to atomic spectrometry to scientists

with limited budgets and limited infrastructure. Therefore, studies developing low-cost,

simple sample preparation strategies are critical to the development of MIP OES and

furthering its adoption. Two such studies are presented here, one focused on a simple,

dilute-and-shoot method for analysis of soft drink samples (Chapter II) and another focused

on the use of a rapid dry-ashing procedure to decompose samples prior to analysis by MIP

OES (Chapter III).

Studies associated with plasma diagnostics and analytical signal variation due to

sample matrix or type of solvent would significantly contribute to expanding the

capabilities of MIP OES. These studies would increase the understanding of the

characteristics of the MIP and would enhance the ability to use these plasma properties for

24

signal correction and for method development purposes, thereby increasing accuracy. A

study in plasma robustness based on the ratio of molecular lines N2+/OH is presented in

Chapter IV as a novel diagnostic tool developed specifically for MIP OES, as opposed to

being transferred from ICP. It contributes to increasing the understanding of the plasma

properties, as well as a method for optimization of operating conditions, and a preliminary

study of the use of molecular species for signal correction.

The development of novel calibration techniques is another strategy to improve the

ability of MIP OES to accurately analyze a broader variety of sample types. Calibration

methods tailored to the specific properties of MIP OES can address its shortcomings better

than more general techniques transferred from other instruments and techniques. In

Chapter V, multi-flow calibration (MFC) is presented as a novel strategy in MIP OES

which builds on the study of robustness presented in Chapter IV. By utilizing multiple

nebulization gas flow rates to perform analysis, the sample is exposed to a variety of

conditions, obviating the need to select a single set of conditions and simplifying method

development.

These studies together present a general advancement of microwave induced

plasma optical emission spectrometry as a technique. It can be coupled with simple, low-

cost sample preparation strategies to expand its analytical utility. In addition, molecular

species can be used to study and diagnose the plasma as well as for signal correction, to

improve accuracy. Finally, new calibration approaches such as multi-flow calibration can

be used to compensate for issues with robustness by exposing samples to a variety of

conditions, improving the quality of the analysis. Thus, the combination of sample

preparation, instrument optimization and calibration methods contributes to a more

25

efficient application of MIP OES and may bring it closer to the performance of traditional

methods such as ICP OES.

26

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31

CHAPTER II

DETERMINATION OF CALCIUM, POTASSIUM AND SODIUM IN SOFT

DRINKS USING THE 4200 MP-AES

Charles B. Williams, Tina McSweeney, Bradley T. Jones, and George L. Donati

This work was published as an Application Note by Agilent Technologies on

www.agilent.com on September 23, 2018 and is reprinted with permission (Appendix A).

Stylistic variations are due to the requirements of the publisher. In particular, the term

MP-AES is used instead of MIP OES to be consistent with the trade name of the

instrument. All of the work was performed by Charles B. Williams. The manuscript was

prepared by Charles B. Williams and edited by George L. Donati, Tina McSweeney, and

Neli Drvodelic.

32

INTRODUCTION

Soft drinks are among the most common beverages consumed in the United States.

Fifty percent of young adults report drinking one or more soft drinks per day [1].

Regulations by the United States Food and Drug Administration (FDA) require reporting

of Ca and Na content of packaged foods and beverages [2], while K content reporting is

optional. FDA regulations for package labeling dictate that K and Na concentrations in the

5-140 mg/serving range should be rounded to the nearest 5 mg. For values over 140

mg/serving, the label can be rounded to the nearest 10 mg. Calcium is reported to the

nearest 10 % of the “recommended daily value” of 1000 mg [2]. These labelling

requirements, as well as the typical quality control analyses carried out by manufacturers,

demonstrate the need for simple, cost-effective, sensitive, accurate, precise and high

sample throughput methods for soft drink analyses.

Current FDA guidelines suggest using microwave-assisted digestion (MAD) and

inductively coupled plasma optical emission spectrometry (ICP OES) for soft drink

elemental analysis [3]. MAD is recommended due to the complexity of this type of sample.

The combination of carbonation, dye additives, artificial flavoring, and high sugar content

contribute to significant matrix effects, which are difficult to overcome for most analytical

techniques [4]. Although effective, MAD is a labor-intensive, expensive, and time-

consuming process, especially due to sample handling and the time required for sample

cool down after digestion.

This application note describes a “dilute and shoot” method for soft drink elemental

analysis based on microwave-induced plasma optical emission spectrometry using

Agilent’s 4200 MP-AES. Sample preparation is not required, eliminating the need for an

33

expensive digestion apparatus as well as some time-consuming procedures associated with

them. The 4200 MP-AES is less expensive to acquire and operate than an ICP OES since

it runs on N2 rather than Ar. It can even run on air if compressed air is supplied to the

optional nitrogen generator. Because the N2 microwave-induced plasma is cooler than an

Ar ICP [5,6], background signals in the visible region of the spectrum are lower, which

allows for limits of detection (LODs) that are comparable to the ones obtained with ICP

OES. The 4200 MP-AES records emission signals sequentially. Therefore analytical

conditions can be optimized for each specific element within the same experiment, which

maximizes efficiency and may minimize potential interferences.

34

EXPERIMENTAL

Instrumentation

All determinations were carried out using the Agilent 4200 MP-AES. A liquid N2

Dewar was used to provide N2 gas to run the microwave-induced plasma. No sample

preparation other than simple dilution with 1 % v/v HNO3 was required. The sample

introduction system is composed of an SPS 4 automatic sampler, solvent-resistant tubing,

a double-pass cyclonic spray chamber, and an inert Flow Blurring nebulizer (OneNeb).

Nebulizer flow rate and plasma viewing position were adjusted for each individual analyte

to optimize recovery. Operating conditions were optimized by selecting one of the spiked

samples, then running the “optimize nebulizer flow” followed by “optimize viewing

position” features in MP Expert. One of the samples was chosen for optimizing the

instrumental operating conditions in order to match matrix conditions and improve

accuracy. The viewing position used in this case was 0 for all elements. The nebulizer flow

rates were 0.90, 1.00 and 1.00 L/min for Ca , K and Na, respectively. Table III and Table

IV list the instrument operating parameters.

Table III. Element-Specific Operating Parameters

Element Wavelength (nm) Nebulizer Flow Rate (L/min)

Ca 393.366 0.9

K 766.491 1

Na 588.995 1

35

Table IV. Instrument Parameters

Number of Replicates 3

Peristaltic Pump Speed 15 rpm

Uptake Time 45 s

Rinse Time 30 s

Stabilization Time 15 s

Background Correction None

No. of Pixels 3

Read Time (all elements) 3 s

Spray Chamber Double pass glass cyclonic

Nebulizer OneNeb Flow-Blurring

Sample Pump tubing Orange/Green

Waste Pump Tubing Blue/Blue

Autosampler SPS-4

Samples and sample preparation

All solutions were prepared using distilled-deionized water (18 M Ω cm, Milli-Q®,

Millipore, Bedford, MA, USA) and trace metal grade nitric acid (Fisher, Pittsburgh, PA,

USA). Single-element stock solutions containing 1000 mg/L of Ca, K or Na (SPEX

CertPrep, Metuchen, NJ, USA) were used to prepare standard reference solutions for

calibration and to carry out spike experiments.

36

Seven different soft drink samples were analyzed. Various popular beverages were

chosen to get a sample of different flavors and types (e.g. diet vs. regular, cola, orange,

ginger ale, etc.). Approximately 2.0-mL aliquots of each of these samples were weighed in

15-mL graduated polypropylene centrifuge tubes using an analytical balance (Mettler AE

100, Hightstown, NJ, USA). For K determination in Diet Dr. Pepper, and Na determination

in Diet Dr. Pepper, Mountain Dew and Schweppes, the same procedure was employed

using 0.2 mL sample aliquots. Sample mass was adopted rather than volume to minimize

any potential bias introduced by residual gas bubbles in the soft drink or inaccuracies due

to viscosity caused by high sugar content. The samples were then diluted to 10.0 mL with

1 % v/v HNO3. Five standard reference solutions (0.2-10 mg/L) and a blank, all prepared

in 1 % v/v HNO3, were used to build the calibration curves. The traditional external

standard calibration method was used in all determinations in order to simplify analysis.

Spike experiments were carried out to evaluate the procedure’s accuracy. Samples

used in this study were prepared in the same way as the unspiked samples. Adequate

volumes of stock solution were added to the samples such that the final concentrations were

1.00 mg/L for Ca, and 2.00 mg/L for K and Na. An intermediate stock solution of 10 mg/L

Ca and 20 mg/L K and Na was prepared from the same standards used to create the

calibration curve. 1 mL was added to the weighed 2.0 mL soft drink aliquots, which was

then completed to 10.0 mL using 1% HNO3.

37

RESULTS AND DISCUSSION

Limits of detection and accuracy

The limits of detection for determinations using Agilent’s 4200 MP-AES were

calculated according to IUPAC’s recommendations as 3 times the standard deviation of the

blank signal (SB) divided by the calibration curve slope (m): LOD = 3SB / m. Twelve

consecutive blank solution (1 % v/v HNO3) measurements were used to calculate SB for

each instrumental condition. The detection limits for Ca, K and Na were 30, 3 and 20 µg/L,

respectively.

The procedure’s accuracy was evaluated by spike experiments. Recoveries were

calculated by comparing expected (concentrations added) and measured (spiked -

unspiked) values. The results for each analyte and sample are presented in Table V.

Recoveries were within 91-110 % using the optimized conditions. The traditional external

standard calibration was used in all cases, which is much simpler and less labor-intensive

than standard additions or internal standardization. The spike recoveries were excellent

with simple external calibration, which are good indicators of the method accuracy. The

method also takes advantage of the MP’s tolerance to high carbon-content matrices, as the

regular (non-diet) drinks included a substantial concentration of sugar. The external gas

control module (EGCM) air injection was not used in this study, but significant carbon

buildup was not found to be a problem on the torch.

38

Table V. Soft drink analysis with the 4200 MP-AES. Values are the recoveries (%) for

spike experiments with 1.00 mg/L Ca, and 2.00 mg/L K and Na.

Sample Ca K Na

Cheerwine 95 91 105

Diet Dr. Pepper 100 106 102

Fanta 110 107 106

Mountain Dew 99 96 100

Pepsi 96 95 100

Schweppes 91 104 103

Sprite 91 106 108

Soft drink concentrations

The same procedure used in the addition and recovery experiment was used to

determine the concentrations of Ca, K and Na in the original (non-spiked) samples. The

results are shown in Table VI. Calcium concentrations were more varied across the

samples. Schweppes ginger ale presented a significantly higher Ca value, which may be

related to the high concentrations of Ca found in the Ginger root (Zingiber officinale) [6].

Sodium and potassium also had wider variations, which may be explained by some

products using a Na or K salt as a preservative, as disclosed on the individual labels.

39

Table VI. Ca, K and Na concentrations in soft drinks determined by the 4200 MP-AES.

Results are the mean ± 1 standard deviation (mg/L, n = 3).

Sample Ca K Na

Cheerwine 6.09 ± 0.10 6.07 ± 0.04 18.15 ± 0.14

Diet Dr. Pepper 3.06 ± 0.02 31.27 ± 0.41 58.58 ± 0.31

Fanta 6.17 ± 0.02 54.63 ± 0.46 3.28 ± 0.18

Mountain Dew 6.28 ± 0.06 21.20 ± 0.23 84.72 ± 0.52

Pepsi 3.30 ± 0.04 14.69 ± 0.20 20.08 ± 0.11

Schweppes

Ginger Ale

23.49 ± 0.07 3.16 ± 0.02 62.47 ± 0.58

Sprite 7.22 ± 0.12 1.03 ± 0.01 79.73 ± 0.51

CONCLUSIONS

The procedure described in this application note is an interesting alternative to the

MAD and ICP OES method recommended by the FDA. It is a fast, cost-effective and

efficient strategy that can be applied by manufacturing laboratories in routine quality

control analyses and determinations associated with package-labeling regulations It also

demonstrates the ability of MP AES to handle complex sample matrices without time-

consuming, expensive sample preparation steps.

40

ACKNOWLEDGMENTS

The authors would like to thank Agilent Technologies and the Department of

Chemistry at Wake Forest University for their support to this work.

REFERENCES

[1] E. Han and L. M. Powell, Consumption patterns of sugar-sweetened beverages in the

United States, J. Acad. Nutr. Diet 113(1) (2013) 43-53.

[2] United States Food and Drug Administration, Code of Federal Regulations: Title 21,

Chapter I, Subchapter B, Part 101: Food Labeling; Vol. Title 21: Food and Drugs § 101.9:

Nutrition Labeling of Food

[3] W. R. Mindak and S. P. Dolan, Inductively Coupled Plasma-Atomic Emission

Spectrometric Determination of Elements in Food Using Microwave Assisted Digestion,

United States Food and Drug Administration Elemental Analysis Manual for Food and

Related Products, 2010.

[4] R. E. S. Froes, W. B. Neto, R. L. P. Naveira, N. C. Silva, C. C. Nascentes and J. B. B.

Silva, Exploratory analysis and inductively coupled plasma optical emission spectrometry

(ICP OES) applied in the determination of metals in soft drinks, Microchem. J. 92(1)

(2009) 68-72.

[5] A. Montaser and D. W. Golightly (Eds.), Inductively Coupled Plasmas in Analytical

Atomic Spectrometry, 2nd ed., Wiley-VCH, New York, 1992.

[6] S. Adel P. R. and J. Prakash, Chemical composition and antioxidant properties of ginger

root (Zingiber officinale), J. Med. Plants Res. 4(24) (2010) 2674-2675

41

CHAPTER III

DRY ASHING AND MICROWAVE-INDUCED PLASMA OPTICAL EMISSION

SPECTROMETRY AS A FAST AND COST-EFFECTIVE STRATEGY FOR

TRACE ELEMENT ANALYSIS

Charles B. Williams , Thomas G. Wittmann, Tina McSweeney, Paul Elliott,

Bradley T. Jones and George L. Donati

The following manuscript was published in Microchemical Journal, 2017, 132, 15-

19, and is reprinted with permission. Stylistic variations are due to the requirements of the

journal. The presented research was conducted by Charles B Williams with assistance from

Thomas G. Wittmann. The manuscript was prepared by Charles B. Williams and edited by

George L. Donati.

42

ABSTRACT

Microwave-induced plasma optical emission spectrometry (MIP OES) is combined with a

simple dry ashing apparatus as a cost-effective alternative for trace element analysis. The

Helios Rapid Ashing (HRA) prototype can reach up to 750 °C using a ceramic radiative

heating element. It allows for sample decomposition in less than 5 min, and requires

inexpensive sample holder materials such as aluminum foil and quartz fiber pads. Samples

were decomposed at 500 oC and the analytes were extracted into a 10 % v/v HCl solution

before analysis by MIP OES. Limits of detection for Ca, Fe, K, Mg, Na and Zn were

calculated as 2, 20, 30, 0.6, 2 and 5 µg/L, respectively. These analytes were determined in

a certified reference material of Tomato Leaves (NIST SRM 1573a) and in challenging

samples such as cheese, butter, peanut butter, infant formula and biodiesel. No statistically

significant differences were observed between certified values and concentrations

determined by the HRA-MIP OES procedure (t-test at a 95 % confidence level). General

agreement was also found between MIP OES results from samples decomposed either by

the HRA or by a conventional microwave-assisted digestion procedure. MIP OES is an

efficient alternative to FAAS, with comparable linear dynamic ranges and significantly

improved sensitivity. It has short start/warmup times (ca. 20 min), runs on inexpensive N2,

and may be a perfect match to the HRA system. The HRA-MIP OES procedure can be a

simple, fast and accurate strategy for inexpensive and effective sample decomposition and

trace element analysis.

43

1. INTRODUCTION

Current United States Food and Drug Administration (US FDA) guidelines require

reporting of Ca and Na content of packaged foods [1]. Reporting of other elements such as

Fe, K, Mg and Zn is optional, but these are important nutrients in human diet, and such

information has become increasingly relevant for health-conscious consumers, particularly

parents selecting food products for their infants and children. Therefore, factors such as

marketing, consumer awareness, and commercial competitiveness has led food

manufacturers to seek reliable and cost-effective methods of determining trace elemental

nutrients in their products.

Most modern atomic spectrometry methods such as flame atomic absorption or

emission spectrometry (FAAS and FAES), inductively coupled plasma optical emission

spectrometry (ICP OES), microwave-induced plasma optical emission spectroscopy (MIP

OES), and inductively coupled plasma mass spectrometry (ICP-MS) require samples to be

introduced as liquid solutions, primarily aqueous solutions. For samples which already are

aqueous, this is trivial and usually involves simple dilution before analysis. On the other

hand, for the majority of samples, an additional time-consuming digestion step is

necessary. One of the most effective approaches to solubilizing complex samples is based

on microwave-assisted digestion (MAD) using acids [2]. Among the many advantages of

MAD are the typically low blanks, efficient digestion of a variety of sample matrices, and

relatively low consumption of reagents [3]. It involves using microwave radiation to

accelerate the decomposition of sample matrices by a strong acid at high pressures and

temperatures. In most MAD procedures, a mineral acid (usually HNO3) is added to a small

mass of sample (i.e. 0.1 - 0.5 g), and the mixture is heated up in a closed Teflon® vessel.

44

Diluted acid solutions may replace the concentrated reagent, and H2O2 may be added to the

digestion mixture to enhance the oxidative power of the acid [4, 5]. For trace element

analysis, MAD is used in association with ICP OES or ICP-MS, which results in highly

accurate and precise determinations.

Despite their many advantages, MAD and ICP-based systems are relatively

expensive to acquire and run. In addition, for a few high-temperature digestions, sample

throughput becomes an issue. Sample cool down after digestion and vessel

decontamination may represent one of the main time-limiting steps in a typical trace

element procedure. A less expensive alternative to MAD is based on dry and/or wet ashing

of samples [6]. With these methods, samples are heated in a crucible in a muffle furnace or

other conventional heating apparatus and reduced to a fine powder containing the residual

minerals from the original matrix. The main limitation of this approach is related to poor

sample throughput. Ashing may take up to several hours, with additional time for sample

cooling, and final solubilization of the residual powder in an aqueous medium before

analysis [7]. For certain applications, additional sample pretreatment is required, which can

lengthen an already long analysis by 12 hours or more [8]. Complete matrix decomposition

is easily achieved with sample ashing, however potential analyte contamination and low

sample throughput have prevented its use in routine trace element analysis.

Recently, a simple and efficient MIP OES system has become commercially

available [9]. This N2-plasma instrument is less expensive to acquire and maintain than

ICP systems. If a nitrogen generator and a conventional air compressor are used, it can

even run on air [10], which significantly reduces running costs and allows operation in

remote locations. For most elements, MIP OES has sensitivities which range between those

45

of FAAS and ICP OES, and it has been successfully used in many applications [10-15].

MIP OES is also advantageous when compared with low-cost flame-based methods (i.e.

FAAS and FAES) because of its improved safety (no flammable gases are required as with

FAAS) and multi-element capabilities.

In the present work, we describe the combination of MIP OES with a simple dry

ashing apparatus (Helios Rapid Ashing prototype, CEM Corporation) as a cost-effective

alternative for trace element analysis. The ashing prototype unit can heat samples up to 750

°C using a ceramic radiative heating element. It allows for sample decomposition in less

than 5 min, and utilizes very inexpensive materials such as aluminum foil and quartz fiber

pads. The efficiency of the dry ashing/MIP OES procedure is evaluated by analyzing

complex sample matrices of cheese, butter, peanut butter, infant formula and biodiesel.

These were chosen as test samples due to their high fat and high sodium contents, which

makes matrix decomposition challenging, and often results in severe matrix effects that can

compromise precision and accuracy [16, 17]. The procedure’s accuracy was evaluated by

determining Ca, Fe, K, Mg, Na and Zn in a certified reference material of tomato leaves

(NIST SRM 1573a). In addition, MIP OES results from samples decomposed either by the

ashing procedure or by a conventional microwave-assisted acid digestion were also

compared.

46

2. EXPERIMENTAL

2.1. Instrumentation

A prototype dry ashing apparatus (Helios Rapid Ashing, CEM Corporation,

Mathews, NC, USA) was used for sample preparation. A picture of the instrument showing

its touchscreen interface and user-programmable heating cycle is depicted in Figure 5.

This unit consists of a tubular furnace section oriented vertically into which a metallic

basket is introduced for sample ashing. The prototype is 25.4 cm high and 21.6 cm in

diameter, and its furnace is capable of sustaining temperatures up to 750 °C. The sample

basket consists of a steel grid at the bottom, onto which the sample holder (e.g. aluminum

foil) is placed, and an aperture-adjustable lid at the top, which controls air flow into the

furnace. A rubber-grip handle completes the sample basket setup (Figure 6). The Helios

Rapid Ashing (HRA) prototype is designed for ease of use as a manual operation. An LED

touchscreen panel on the unit has a timer and allows for temperature setting and control.

The interface relies on the PID control algorithm to quickly reach and hold the set

temperature. The timer is independent and is not synchronized nor does it control the

furnace temperature.

47

Figure 5. Helios Rapid Ashing unit, courtesy of CEM Corporation.

Figure 6. Sample basket used with the HRA prototype.

48

Approximately 7 cm2 sections of either aluminum foil (Reynolds Wrap Heavy

Duty, Reynolds Consumer Products, Lake Forest, IL, USA), or quartz fiber ashing pads

(CEM) were used as sample holders.

For comparison and accuracy evaluation, all samples were also submitted to

microwave-assisted acid digestion using an instrument with quartz digestion vessels

(Discover, CEM).

A MIP OES system (4200 MP-AES, Agilent Technologies, Santa Clara, CA, USA)

equipped with an SPS-4 automatic sampler, an inert Flow Blurring nebulizer (OneNeb),

and a double-pass cyclonic spray chamber was used in all determinations. Nitrogen gas

used by the instrument was provided by a liquid N2 Dewar. Default operating conditions

(Table VII) were used to analyze samples submitted to both dry ashing and MAD.

49

Table VII: Operating conditions used in MIP OES determinations.

Instrumental parameter Operating condition

Microwave frequency

(MHz)

2450

Applied power (kW) 1.0

Peristaltic pump speed (rpm) 15

Integration time (s) 3

Number of replicates 3

Plasma viewing positiona 0

Analyte (wavelength, nm) Nebulization gas flow rate

(L/min)

Ca (393.366) 0.60

Fe (371.993) 0.65

K (766.491) 0.75

Mg (285.213) 0.90

Na (588.995) 0.95

Zn (213.857) 0.45

a The plasma viewing position has no specific unit. It is based on stepper motor

positioning of a mirror. Position 0 approximately corresponds to the center of the plasma

[9].

50

2.2. Reagents, standard reference solutions and samples

Distilled-deionized water (18 MΩ cm, Milli-Q ®, Millipore, Bedford, MA, USA)

was used to prepare all solutions. Trace metal grade nitric acid (Fischer, Pittsburgh, PA,

USA) was used to prepare calibration standards, and to digest samples by MAD. ACS+

grade hydrochloric acid (Fischer) was used to extract the analytes from ashing residues

following sample decomposition by the HRA prototype. The standard reference solutions

used for calibration were prepared in either 1% v/v HNO3 (commercial samples) or 10 %

v/v HCl (certified reference material) by adequate dilution of single-element stock

solutions of Ca, Fe, K, Mg, Na and Zn (1000 mg/L, SPEX CertPrep, Metuchen, NJ, USA).

Various commercial samples were used to evaluate the efficiency of the dry

ashing/MIP OES procedure: cheese (Great Value low moisture part-skim mozzarella, Wal-

Mart Corp., Little Rock, AR, USA), butter (Land O Lakes, St. Paul, MN, USA), peanut

butter (Jif reduced fat creamy, J. M. Smucker, Orrville, OH, USA), infant formula (Parent’s

Choice, gentle, Perrigo Nutritionals, Charlottesville, VA, USA), and biodiesel (prepared

in-lab using soybean oil, methanol, and KOH) [18]. A Standard Reference Material from

the National Institutes of Standards and Technology (Tomato Leaves, NIST SRM 1573a)

was used to evaluate the procedure’s accuracy.

2.3. Sample preparation

Microwave-assisted digestion. Sample aliquots of 0.2 - 0.3 g were accurately weighed

using an analytical balance (Mettler, Toledo, OH, USA), and digested in quartz vessels

with 5 mL of HNO3 50% v/v. The MAD heating cycle was composed of a 4-min

51

temperature ramp to 200 °C, and a 3-min hold at 200 °C using 300 W of power. Samples

were allowed to cool, then quantitatively transferred into 50 mL polypropylene graduated

centrifuge tubes. The digested mixture was then diluted to 25 mL with distilled-deionized

water. Analytical blanks were prepared using the same procedure, and all samples were

digested in triplicate.

Dry ashing and analyte extraction

The HRA prototype was pre-heated to 500 °C to improve repeatability. Sample aliquots of

0.2 - 0.3 g were accurately weighed directly on square sections of either aluminum foil or

quartz fiber pads (sample holders) using an analytical balance (Mettler). Another ca. 7 cm2

section of aluminum foil or quartz fiber pad was placed on top of the sample, and the entire

“sandwich” set was transferred to the steel grid in the sample basket (Figure 7). With the

adjustable lid kept closed at all times, the basket was then lowered into the furnace, and the

timer was set for either 2 or 3 minutes depending on the sample. After the ashing was

complete, a set of forceps was used to remove the sample holder and place it into a 250 mL

snap-fit polypropylene flask (Corning, Corning, NY, USA). The analytes were extracted

using 25 or 30 mL aliquots of a 10 % v/v HCl solution. For quartz fiber pad sample holders,

the HCl solution was simply poured on top of the pad and the extraction vial was shaken

for a few seconds. Aluminum foil sample holders were held above the extraction vial with

forceps and rinsed with the HCl solution using a polypropylene syringe (Becton-Dickinson

& Co, Fraklin Lakes, NJ, USA). After the vial was shaken, the extraction mixture (i.e.

ashing residue, quartz fiber pads and HCl solution) was allowed to sit for approximately 1

hour. Finally, the mixture was gravity-filtered using a small disposable funnel and a 0.45-

µm-pore filter paper (CEM, Matthews, NC, USA). Samples were initially analyzed

52

undiluted. For elements with concentrations higher than 5 mg/L, a 10-fold dilution with

distilled-deionized water was carried out before MIP OES determination. Analytical blanks

were prepared using the same procedure and all samples were processed in triplicate. Table

VIII presents the specific ashing parameters which provided the best recoveries for each

sample (optimization not shown).

Figure 7. Sample “sandwich” setup using an aluminum foil at the bottom and a quartz fiber pad on top loaded in the sample basket.

53

Table VIII. Ashing parameters providing the best recoveries for determinations using the

HRA prototype and MIP OES.

Sample Ashing time (min) Sample holder Extracting

solution

volume (mL)a

Cheese 2 Two quartz fiber pads 30.0

Biodiesel 3 Two sections of aluminum foil 25.0

Butter 3 Two sections of aluminum foil 25.0

Peanut butter 3 Aluminum foil at bottom,

quartz fiber pad on top

25.0

Infant formula 3 Aluminum foil at bottom,


25.0

Tomato leaves

(NIST SRM 1573a)

3 Aluminum foil at bottom,


25.0

a Extracting solution: HCl 10 % v/v.

54

3. Results and discussion

3.1. Limits of detection and accuracies

Limits of detection (LOD) were calculated according to IUPAC’s recommendation

as three times the standard deviation of the blank solution (SB, n = 15) divided by the

calibration curve slope (m), i.e., LOD = 3SB / m. Similarly, the limits of quantification

(LOQ) were calculated as LOQ = 10SB / m. Considering the HRA extracting agent, the

blank used in these calculations was a 10 % v/v HCl solution. Using the operating

conditions listed in Table VII, LODs for Ca, Fe, K, Mg, Na and Zn were calculated as 2,

20, 30, 0.6, 2 and 5 µg/L, respectively. These concentrations, as well as the LOQs for the

same elements (i.e. 6, 60, 100, 2, 6 and 20 µg/L, respectively), are well below the reporting

ranges required by the US FDA [1]. In addition to the adequate sensitivities of MIP OES,

relatively low LODs are achievable due to the minimal sample dilution associated with

HRA. These values are comparable to a traditional MAD-ICP OES procedure for these

types of sample matrices [19], and significantly superior to FAAS [20].

The accuracy of the HRA-MIP OES procedure was checked by analyzing a

certified reference material of Tomato Leaves (NIST SRM 1573a). Nitric acid and HCl

solutions at 1, 10 and 20 % v/v were evaluated as extracting agent. In this study, HCl

provided better recoveries than HNO3. For the majority of analytes, a 1 % v/v acid

concentration resulted in low recoveries, while no significant differences were observed

between samples extracted with 10 or 20 % v/v HCl (results not shown). To minimize

potential matrix effects, and considering the best recoveries for all analytes evaluated, a 10

% v/v HCl solution was chosen as extracting agent in all subsequent HRA extractions. All

determinations were carried out using the external standard calibration method and

55

standard solutions prepared in 10 % v/v HCl. The results are shown in Table IX. As it can

be seen, no statistically significant difference was found between the reference values and

concentrations determined by HRA-MIP OES (t-test at a 95 % confidence level). Although

efficient and accurate, the procedure’s precision is relatively low. High relative standard

deviations (RSDs) are common in ashing procedures due to more sample manipulation and

less control of the digestion conditions [2]. In the specific case of this study, RSDs were

higher for the reference material than the commercial samples (see Table IX and Table X).

This fact may be related to the different consistencies of each sample and their behavior

during digestion. It is possible that the dry and powdery reference material was more

susceptible to spilling and analyte loss during the HRA heating cycle, which resulted in

higher RSDs and relatively lower concentrations for most analytes.

56

Table IX. Evaluating the accuracy of the HRA-MIP OES procedure by analyzing a

standard reference material of Tomato Leaves (NIST SRM 1573a). Reported values are the

mean ± 1 standard deviation concentrations in the original solid sample (mg/g, n = 3).

Analyte Certified Found

Ca 50.5 44.6 ± 8.1

Fe 0.368 0.337 ± 0.143

K 27.0 26.6 ± 3.5

Mg 12.0 9.7 ± 1.9

Na 0.136 0.122 ± 0.020

3.2. Application to commercial samples and comparison with MAD

Commercial samples of cheese, butter, peanut butter, infant formula, and a lab-

made biodiesel sample were digested by HRA or MAD and the concentrations of Ca, K,

Mg and Na were determined by MIP OES. The results were used to further assess the

efficiency and accuracy of the HRA-MIP OES procedure using challenging sample

matrices. Analyte concentrations determined with both digestion procedures were also

compared to product label information. In this study, calibration curve solutions were

prepared in HNO3 1 % v/v to evaluate the performance of the HRA-MIP OES procedure

in non-matrix-matching conditions. As can be seen in Table X, there is a general agreement

57

between concentrations determined in samples processed by the ashing and acid extraction

(HRA) procedure, and by the traditional microwave-assisted acid digestion (MAD). The

results also broadly agree with label values. The few discrepancies may probably be

explained either by uncertainty due to US FDA rounding requirements [21], or because

label values are based on a different sample lot.

As discussed previously, precision is generally similar for HRA and MAD. Based

on the results presented in Table X, HRA can be considered as efficient as MAD for

applications involving the matrices and analytes evaluated in this work. Table XI shows

the relationship between HRA and MAD results. Values in this table were calculated as

(HRA result / MAD result) x 100. For the majority of samples and analytes, HRA values

were within 90 - 110 % of the MAD results, and all values were in the 84 - 122 % range.

Considering the importance of micronutrients in infant formula, Fe and Zn were

also determined in this sample. For HRA, 0.104 ± 0.009 and 0.063 ± 0.003 mg/g were

found for Fe and Zn, respectively. These results are comparable to values obtained from

samples submitted to MAD, i.e. 0.102 ± 0.002 and 0.067 ± 0.001 mg/g, and slightly higher

than concentrations reported on the product’s label (0.08 and 0.045 mg/g for Fe and Zn,

respectively).

58

Tab

le X

. Com

paris

on b

etw

een

ashi

ng an

d ac

id ex

tract

ion

(HR

A) w

ith th

e tra

ditio

nal m

icro

wav

e-as

sist

ed d

iges

tion

(MA

D).

The a

naly

te

conc

entra

tions

wer

e det

erm

ined

by

MIP

OES

usi

ng th

e ext

erna

l sta

ndar

d ca

libra

tion

met

hod.

Val

ues a

re th

e mea

n ±

1 st

anda

rd d

evia

tion

(mg/

g, n

= 3

).

Sam

ple

Sam

ple

prep

arat

ion

met

hoda

Ca

K

Mg

Na

Che

ese

HR

A

4.70

± 0

.32

0.49

± 0

.03

0.21

4 ±

0.01

0 6.

57 ±

0.1

6

M

AD

4.

84 ±

0.1

3 0.

58 ±

0.0

1 0.

225

± 0.

010

7.09

± 0

.26

La

bel

7 ±

3 0.

89 ±

0.4

5 N

Ab

7.10

± 0

.40

Butte

r H

RA

0.

29 ±

0.0

2 0.

21 ±

0.0

1 0.

019

± 0.

003

6.32

± 0

.09

M

AD

0.

27 ±

0.0

2 0.

21 ±

0.0

2 0.

021

± 0.

001

5.24

± 0

.61

La

bel

NA

b N

Ab

NA

b 6.

40 ±

0.7

0

Pean

ut B

utte

r H

RA

0.

35 ±

0.0

3 3.

47 ±

0.1

9 1.

749

± 0.

232

4.12

± 0

.26

M

AD

0.

36 ±

0.0

2 3.

38 ±

0.0

8 1.

818

± 0.

104

4.00

± 0

.12

La

bel

0.56

± 0

.28

NA

b 1.

60 ±

0.6

0 5.

60 ±

0.3

0

59

Infa

nt F

orm

ula

HR

A

3.81

± 0

.17

5.32

± 0

.17

0.69

1 ±

0.00

8 1.

92 ±

0.0

6

M

AD

3.

82 ±

0.0

6 5.

69 ±

0.0

6 0.

683

± 0.

017

2.02

± 0

.01

La

bel

3.5

4.9

0.36

1.

2

Biod

iese

l H

RA

<

LOD

<

LOD

<

LOD

71

.72

± 3.

46

M

AD

<

LOD

<

LOD

<

LOD

84

.17

± 1.

52

a Lab

el v

alue

s ar

e ba

sed

on th

e pr

oduc

t pac

kagi

ng in

form

atio

n an

d th

e FD

A p

erce

nt re

com

men

ded

daily

val

ues

[22]

. Sta

ndar

d

devi

atio

ns fo

r ‘La

bel’

are

base

d on

the

FDA

roun

ding

rule

s for

pac

kagi

ng, r

athe

r tha

n st

atis

tical

ana

lysi

s [21

].

b Not

ava

ilabl

e.

60

Table XI. HRA results presented as their percent portion of concentrations determined

using the MAD procedure.

Sample Ca K Mg Na

Cheese 97 84 91 93

Biodiesel < LOD < LOD < LOD 85

Butter 107 100 90 122

Peanut Butter 94 103 94 102

Infant Formula 97 93 100 95

61

4. Conclusions

The HRA system is a simple and cost-effective alternative to MAD. It requires no

high-pressure-withstanding flasks, nor any other relatively expensive apparatus and

consumables. It also is a green approach to sample preparation since it uses few reagents,

generates no acid vapors, and produces fewer residues. When compared to traditional

methods of dry- and wet-ashing, the HRA procedure is faster, greener and more efficient.

Ten samples can be digested in less than two hours with no cooling step required. Dry-

ashing in a muffle furnace, for example, requires heating and subsequent cooling overnight

[8].

Similar to other open-system ashing procedures, HRA is more prone to

contamination and analyte loss than MAD. Although no severe contamination was

observed for the analytes evaluated in this study, memory effects may be particularly

critical, as the sample holder grid is not decontaminated between samples. In its current

state, the HRA prototype can only accommodate one sample at a time, and has no

automation capability, which may be expected of a prototype.

MIP OES is an efficient alternative to FAAS. It has short start/warmup times (ca.

20 min), and runs on inexpensive N2. MIP OES has also adequate sensitivities for

applications associated with US FDA requirements for food packaging, and may be a

perfect match to the HRA system. The HRA-MIP OES procedure can be a simple, fast and

accurate strategy for inexpensive and effective sample decomposition and trace element

analysis.

62

Acknowledgements

The authors would like to thank the Department of Chemistry at Wake Forest

University, as well as CEM Corporation and Agilent Technologies for their support to this

work.

References

[1] Code of Federal Regulations: Title 21, Chapter I, Subchapter B, Part 101: Food

Labeling. http://www.ecfr.gov/cgi-

bin/retrieveECFR?gp=1&SID=4bf49f997b04dcacdfbd637db9aa5839&ty=HTML&h=L&

mc=true&n=pt21.2.101&r=PART#se21.2.101_19 (accessed May 25, 2016).

[2] H. M. (Skip) Kingston and S. J. Haswell (Eds.), Microwave-Enhanced Chemistry:

Fundamentals, Sample Preparation, and Applications, 1st ed., American Chemical Society,

Washington, D.C., 1997, 800p.

[3] J. A. Nobrega and G. L. Donati, Microwave-Assisted Sample Preparation for

Spectrochemistry, In R. A. Meyers (ed.) and N. H. Bings (Assoc. ed.), Encyclopedia of

Analytical Chemistry, Wiley, Chichester, 2011, 23p.

[4] G. C. L. Araujo, M. H. Gonzales, A. G. Ferreira, A. R. A. Nogueira and J. A. Nobrega,

Effect of acid concentration on closed-vessel microwave-assisted digestion of plant

materials, Spectrochim. Acta Part B 57 (2002) 2121-2132.

[5] E.I. Muller, J.P. Souza, C.C. Muller, A.L.H. Muller, P.A. Mello, C.A. Bizzi,

Microwave-assisted wet digestion with H2O2 at high temperature and pressure using single

63

reaction chamber for elemental determination in milk powder by ICP-OES and ICP-MS,

Talanta 156-157 (2016) 232-238.

[6] J. Tang, Y. Ying, X.-D. Pan, W. Jiang, P.-G. Wu, Elements analysis of infant milk

formula by ICP-OES: a comparison of pretreatment methods, Accredit. Qual. Assur. 19

(2014) 99-103.

[7] C.J. Amarasiriwardena, I. Jayawardene, N. Lupoli, R.M. Barnes, M. Hernandez-Avila,

H. Hu, A.S. Ettinger, Comparison of digestion procedures and methods for quantification

of trace lead in breast milk by isotope dilution inductively coupled plasma mass

spectrometry, Anal. Methods 5 (2013) 1676-1681.

[8] E. Poitevin, Official methods for the determination of minerals and trace elements in

infant formula and milk products: a review, J. AOAC Int. 99 (2016) 42-52.

[9] D. A. Goncalves, T. McSweeney and G L. Donati, Characteristics of a resonant iris

microwave-induced nitrogen plasma, J. Anal. At. Spectrom. 31 (2016) 1097-1104.

[10] G. L. Donati, R. S. Amais, D. Schiavo and J. A. Nobrega, Determination of Cr, Ni, Pb

and V in gasoline and ethanol fuel by microwave plasma optical emission spectrometry, J.

Anal. At. Spectrom. 28 (2013) 755-759.

[11] R. S. Amais, G. L. Donati, D. Schiavo and J. A. Nobrega, A simple dilute-and-shoot

procedure for Si determination in diesel and biodiesel by microwave-induced plasma

optical emission spectrometry, Microchem. J. 106 (2013) 318-322.

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[12] A. F. Lima, F. F. Lima, E. M. Richter and R. A. A. Munoz, Combination of sonication

and heating for metal extraction from inorganic fertilizers prior to microwave-induced

plasma spectrometry determinations, Appl. Acoust. 103 (2016) 124-128.

[13] N. Ozbek and S. Akman, Microwave plasma atomic emission spectrometric

determination of Ca, K and Mg in various cheese varieties, Food Chem. 192 (2016) 295-

298.

[14] P. Niedzielski, L. Kozak, M. Wachelka, K. Jakubowski and J. Wybieralska, The

microwave induced plasma with optical emission spectrometry (MIP-OES) in 23 elements

determination in geological samples, Talanta 132 (2015) 591-599.

[15] D. A. Goncalves, T. McSweeney, M. C. Santos, B. T. Jones and G. L. Donati, Standard

dilution analysis of beverages by microwave-induced plasma optical emission

spectrometry, Anal. Chim. Acta 909 (2016) 24-29.

[16] Z. Mester and R. Sturgeon, Sample Preparation for Trace Element Analysis, In D.

Barcelo (Ed.), Comprehensive Analytical Chemistry, Vol. XLI, Amsterdam, 2003, 1338p.

[17] K. O’Hanlon, L. Ebdon and M. Foulkes, Effect of easily ionizable elements on

solutions and slurries in an axially viewed inductively coupled plasma, J. Anal. At.

Spectrom. 11 (1996) 427-436.

[18] F. Motasemi, F. N. Ani, A review on microwave-assisted production of biodiesel,

Renew. Sust. Energ. Rev. 16 (2012) 4719-4733.

65

[19] T. Narukawa, E. Matsumoto, T. Nishimura, A. Hioki, Determination of sixteen

elements and arsenic species in brown, polished and milled rice, Anal. Sci. 30 (2014) 245-

250.

[20] C. Sola-Larrañaga, I. Navarro-Blasco, Optimization of a slurry dispersion method for

minerals and trace elements analysis in infant formulae by ICP OES and FAAS, Food

Chem. 115 (2009) 1048-1055.

[21] C. for F. S. and A. Nutrition, Labeling & Nutrition - Guidance for Industry: A Food

Labeling Guide (16. Appendix H: Rounding the Values According to FDA Rounding

Rules).

http://www.fda.gov/Food/GuidanceRegulation/GuidanceDocumentsRegulatoryInformati

on/LabelingNutrition/ucm064932.htm (accessed September 29, 2016).

[22] C. for F.S. and A. Nutrition, Labeling & Nutrition - Guidance for Industry: A Food

Labeling Guide (14. Appendix F: Calculate the Percent Daily Value for the Appropriate

Nutrients).

http://www.fda.gov/Food/GuidanceRegulation/GuidanceDocumentsRegulatoryInformati

on/LabelingNutrition/ucm064928.htm (accessed September 29, 2016).

66

CHAPTER IV

NATURALLY OCCURRING MOLECULAR SPECIES USED FOR PLASMA

DIAGNOSTICS AND SIGNAL CORRECTION IN MICROWAVE-INDUCED


Charles B. Williams, Bradley T. Jones and George L. Donati

The following manuscript was published in the Journal of Analytical Atomic

Spectrometry, 2018, 33, 1224-1232, and is reprinted by permission of the Royal Society of

Chemistry. Stylistic variations are due to the requirements of the journal. All of the

presented research was conducted by Charles B. Williams. The manuscript was prepared

by Charles B. Williams and George L. Donati. Supplementary material published online

for this chapter is presented in Appendix A.

67

ABSTRACT

In the present study, we evaluate the N2+ / OH emission intensity ratio as a

diagnostic tool for identifying the best instrumental operating conditions in microwave-

induced plasma optical emission spectrometry (MIP OES). This molecular species signal

ratio is compared with the traditional Mg II / Mg I ratio. Aluminum, Ba, Mn, Sr and Zn

(analytes), and high concentrations of C, Na, Ca, HNO3 and HCl (sample matrices) are

used as models to investigate the effects of complex matrices on analyte recoveries. The

N2+ / OH signal ratio is more sensitive to changes in plasma conditions than the Mg II / Mg

I ratio. Some other advantages include real-time monitoring capabilities, and the possibility

of independently tracking variations in both plasma and sample introduction. For less

complex matrices, the N2+ / OH signal ratio may be used for instrument optimization, which

ensures plasma conditions are as similar as possible when analyzing standard solutions and

samples. For analyses involving severe matrix effects, molecular species such as CN, N2,

N2+ and OH are used for signal correction. Significant improvements in accuracy are

achieved by employing the analyte-to-molecular species signal ratio, or their product, for

calibration. Both the use of the N2+ / OH signal ratio as a diagnostic tool, and of molecular

species for signal correction to minimize matrix effects are simple strategies that may

significantly contribute to expanding the analytical capabilities of MIP OES and facilitating

its application in routine analysis.

68

INTRODUCTION

Microwave-induced plasma optical emission spectrometry (MIP OES) is an

increasingly popular technique in atomic spectrometry, which has been thoroughly

reviewed in a monograph by Jankowski and Reszke.1 Such popularity is associated with

the relatively recent commercial introduction of a complete MIP OES instrument, which

provides adequate sensitivity for most elements as well as low costs of operation. The

commercial instrument features a resonant-iris, hammer-cavity MIP, which runs on

nitrogen, and a sequential monochromator and charge-coupled device (CCD) detector.2,3

Fundamental studies associated with plasma properties of this system are somewhat

limited. The literature has largely been application-based, with only two recent studies

focusing on topics associated with plasma fundamental properties. Goncalves et al., for

example, determined plasma robustness and temperature profiles for varying experimental

conditions.4 In another work, a comprehensive study by Chalyavi et al. characterized the

MIP using Thomson scattering and other techniques to determine electron number density

and temperature, among other properties.5

MIP OES has general features which are similar to the better-known inductively

coupled plasma (ICP) OES. However, the method is not as mature as ICP OES and it has

some marked differences. The primary distinction of relevance to matrix effects is that the

ICP is coupled to a radio-frequency-generated field, whereas the MIP is induced by a

standing wave generated by a magnetron. In an ICP, when the plasma impedance increases

during sample introduction, additional power is drawn to compensate such change. As a

consequence, the energy within the plasma remains relatively constant.6 In the

commercially available MIP OES, when the plasma impedance increases, the power put

69

out by the magnetron does not change, which results in a less energetic plasma.7 Because

of the fixed-power setup, MIP OES is significantly more prone to matrix effects than ICP

OES. Small variations in solution composition can lead to significant fluctuations in the

energy available for sample vaporization and analyte atomization and excitation, which, in

turn, will result in significant changes in emission intensity. Another major difference

between the two plasmas is that MIP runs on N2 rather than Ar. As noted by Chalyavi et

al., because N2 is a molecule, it consumes energy not only in translational degrees of

freedom, but also rotational and vibrational ones, while Ar, as an atom, has only

translational degrees of freedom.5 As a consequence, above ca. 5000 K, increased applied

power does not significantly result in higher temperatures for a N2 plasma, whereas

increased applied power continues to raise temperature in an ICP up to about 10,000 K.

Thus, plasma temperatures are necessarily lower in a N2 MIP than in an Ar ICP.

It is important to note that other MIP devices have been developed for OES

analyses, which can be based on a Beenakker cavity,8 the microwave-induced nitrogen

discharge at atmospheric pressure (MINDAP),9 an Okamoto cavity,10 a surfatron,11 the

torche a injection axiale (TIA) or torche a injection axiale sur guide d'ondes (TIAGO)

designs,12,13 and more recently, the microwave-sustained, inductively coupled,

atmospheric-pressure plasma (MICAP).14 However, given its increasing popularity and

cost-effective applications in different fields, we focus the present study on a commercially

available, Hammer-cavity-based MIP OES instrument.2 There have generally been no

plasma diagnostic tools developed specifically for commercially available MIP OES

systems, with most strategies transferred from ICP. For example, Goncalves et al. used the

concept of robustness as originally proposed by Mermet to describe the stability of the

70

plasma.4,15 According to Mermet’s definition, a robust plasma is one which is highly

tolerant to matrix effects, and which has a Mg ion-to-atom intensity ratio (Mg II / Mg I)

greater than 10. However, such Mg II / Mg I value is impossible to achieve with the

commercially available MIP, as the energy of the plasma is considerably lower. Goncalves

et al. found the Mg II / Mg I ratio to rarely exceed a value of 2, and to commonly be less

than 1 in MIP OES, pointing to a plasma in which most emitting species are neutral atoms

and molecules rather than ions.4 This notion may be confirmed by the analytical

wavelengths recommended by the instrument control software, which are mainly

composed of atomic lines rather than ionic ones. Considering the specific characteristics

of a N2 MIP, particularly the fact that most elements are determined as atoms rather than

ions, maximizing robustness as a strategy to optimize analytical accuracy may not be

effective. Therefore, a more nuanced approach than simply robustness may be appropriate

in MIP OES, possibly including monitoring variations in the plasma on a per-sample basis.

Another aspect associated with robustness is that it involves the addition of Mg as

a test element. It would be advantageous to be able to estimate robustness (or monitor

changes in the plasma) for any solution without having to add a test element. Thus,

naturally occurring molecular species, which are part of the MIP background, may

represent a promising source of information about plasma conditions. In the present study,

we evaluate some of these species as plasma diagnostic tools. In this context, a few studies

describing the use of molecular species (e.g. N2+ and OH) to estimate plasma temperature

can be found in the literature.16-18 However, they are rarely applied to MIP, nor are they

used in the context of atomic spectrometry. The molecular emission lines present in a MIP

were detailed by Jankowski and Reszke, and include peaks for N2, N2+, CH, CN, OH and

71

C2.1 Chalyavi et al. recently described the spectral background for the new commercial

MIP OES system, and note that it is dominated by several molecular species such as NO,

NH, OH, N2, and N2+.5 These species are usually treated as a nuisance, as they can interfere

with atomic emission measurements. Frentiu et al., for example, added methane gas to the

Ar supply of a capacitively-coupled plasma in order to quench interference from OH and

N2 emission bands.19 Chalyavi et al. note that a background modelling algorithm is

included in the instrumental control software of the commercial MIP OES in order to

remove spectral background due to molecular emission bands.5 On the other hand, Lowery

et al. have used molecular species to improve MIP OES’ performance in biodiesel

analysis.20 In this study, emission band peaks from OH and N2+, naturally occurring in the

MIP’s spectral background, were used as “molecular probes” to correct for changes in

emission intensity due to matrix effects. Significant improvements in accuracy were

observed when a ratio or multiplication between the emission signals from the analytes and

the molecular species were used for calibration rather than the analytical signals alone.

As discussed before, matrix effects can be severe in MIP OES determinations due

to typically lower plasma temperatures. Zhang and Wagatsuma investigated the effects of

Na, Ca and HNO3 on emission intensities in an Okamoto-cavity MIP OES.21 They found

that easily ionizable elements (EIEs) such as Na and Ca cause signal enhancement in most

atomic lines presenting lower excitation energies, and signal suppression in ionic lines and

atomic lines of higher excitation energies. The authors suggested that these effects were a

result of a shift in excitation equilibrium, which was caused by an increase in the plasma

electron number density (ne). They also found that HNO3 has a suppressive effect on most

analytical signals. In Zhang and Wagatsuma’s study, the sample was naturally aspirated

72

(i.e. no peristaltic pump used), which may explain part of the effects observed. It is also

important to note that signal intensity suppression due to HNO3 has been shown to be less

severe than the effects caused by the EIEs in that work.

In the present study, we investigate the use of the intensity ratio between the N2+

emission band peak at 391.439 nm and the OH band peak at 308.970 nm as a plasma

diagnostic tool for identifying the best plasma conditions to minimize matrix effects in MIP

OES. We evaluate a commercial MIP OES instrument (Agilent 4200 MP-AES), for which

results may be applicable to other MIP systems. The N2+ signal may be used as a proxy for

the energy available within the plasma, while the OH signal could be a proxy for the

efficiency of the sample introduction system. We examine the effects of high

concentrations of Na, Ca, HNO3, HCl and C on analyte recoveries for atoms and ions

presenting a wide range of excitation energies, i.e. Al, Ba, Mn, Sr and Zn. The N2+ / OH

signal ratio is compared to the traditional Mg II / Mg I ratio, and other plasma naturally

occurring molecular species (including CN, N2, N2+ and OH) are evaluated as signal

correction species to improve accuracy in complex matrix analysis by MIP OES.

73

MATERIALS AND METHODS

Instrumentation

All experiments were carried out on a commercial resonant-iris, Hammer-cavity

MIP OES instrument (4200 MP-AES, Agilent Technologies, Santa Clara, CA, USA).

Nitrogen gas was supplied from a liquid N2 Dewar (N2 99.998 % pure, Air Products and

Chemicals, Allentown, PA, USA). The sample introduction system comprised an SPS 4

autosampler, a double-pass cyclonic spray chamber and an inert OneNeb nebulizer. Each

solution was analyzed in three replicates of a 3-s integration time each. Emission band

peaks for CN (387.147 nm), N2 (337.097 nm), N2+ (391.439 nm) and OH (308.970 nm)

were monitored as potential candidates for plasma diagnostics and signal correction.

Emission spectra for these molecular species are presented in Appendix A (Figs. S1 - S8).

Because these specific molecules are not available as options in the instrument control

software, nearby lines for Fe I, Ti I, Nb I, and Tb II (where I and II represent atomic and

ionic species, respectively) were chosen, and the molecular emission peaks were adjusted

as described in a previously published work.20 Analytical emission lines at 396.152,

455.403, 403.076, 407.771 and 213.857 nm were used for Al I, Ba II, Mn I, Sr II and Zn I,

respectively.

Each solution was analyzed in a range of nebulization gas flow rates between 0.3

and 1.0 L min-1, at intervals of 0.1 L min-1. All emission signals were collected at the center

of the plasma (plasma viewing position zero, as identified by the instrument control

software).4 Atomic emission intensities at 280.271 nm (Mg II) and 285.213 nm (Mg I) were

used to calculate the Mg II / Mg I ratio. In this case, a detector correction factor of 1.1 was

employed according to the method outlined by Dennaud et al., and as previously

74

determined by Goncalves et al..4,22 The N2+ / OH and Mg II / Mg I signal ratios were

compared in various plasma conditions. General instrumental operating conditions are

listed in Table XII. Instrumental operating conditions used in MIP OES..

Table XII. Instrumental operating conditions used in MIP OES.

Instrumental parameter Operating

condition

Microwave frequency (MHz) 2450

Applied power (kW) 1.0

Peristaltic pump speed (rpm) 15

Integration time (s) 3

Number of replicates 3

Nebulization gas flow rate (L min-1) 0.3 - 1.0

Reagents and standard reference solutions

Concentrated acids were obtained from Fischer Scientific: HNO3 (Trace Metals Grade, ca.

16 M), and HCl (ACS+ Grade, ca. 12 M). All solutions were prepared in 18.2 MΩ cm-1

distilled-deionized H2O (Milli-Q, Millipore, Bedford, MA, USA). Single-element stock

solutions of Al, Ba, Fe, Mg, Mn, Sr and Zn (SPEX CertPrep, Metuchen, NJ, USA) were

used to prepare the working solutions. Solid urea (Fisher) was used to investigate the

75

effects of high carbon content in the plasma. In addition, 10,000 mg L-1 stock solutions of

Na and Ca (SPEX) were used to investigate the effects of EIEs on all analytes evaluated.

A series of solutions containing 2 mg L-1 of Al, Ba, Mn, Sr and Zn, and 1 % v v-1

HNO3 was prepared in various sample matrices: 1000 mg L-1 Na, 1000 mg L-1 Ca or 1000

mg L-1 C. Solutions containing 2 mg L-1 of these same analytes were also prepared in 20

% v v-1 HCl or 20 % v v-1 HNO3. Blank and calibration curve standard solutions containing

these analytes were prepared in 1 % v v-1 HNO3.


Determination of the detector response correction factor for calculating the N2+ /

OH signal ratio

We have evaluated four of MIP’s most intense background signal sources as part

of a strategy to monitor plasma conditions and improve accuracy in MIP OES analysis.

Individual signal intensities and different combinations of CN, N2, N2+ and OH signals

were examined, and as discussed in later sections, the best results were achieved when

employing the N2+ / OH signal ratio. To prevent any signal bias due to differences in

detector response at each wavelength, an experiment was carried out to determine the

extent of such a bias and correct it while calculating the N2+ / OH signal ratio. Goncalves

et al. used low-intensity Tb lines near 285.213 and 280.271 nm to minimize any

interference from residual Mg in solution when determining the detector response

correction factor used in Mg II / Mg I experiments.4 Because the molecular species used in

the current study are present in high concentrations as part of the plasma or the sample

76

solvent, no similar method could be used. Alternatively, a 150 W, 15 V light bulb (Osram

HLX 64633) was employed as a continuum source to determine detector sensitivity at

different wavelengths. With the plasma off and the torch removed, the radiation intensity

produced by a light bulb operated at 10.0 A was recorded at 308.958 ± 0.500 nm and

391.470 ± 0.500 nm (n = 3) using the “quick read” function of the instrument. Differences

in radiation intensity due to the blackbody emission from the tungsten filament were taken

into account using Plank’s law (Eq. 3), where 𝐵𝐵𝜆𝜆𝑏𝑏, λ and T represent spectral radiance in

W/sr·cm2·nm, wavelength in nm, and temperature in K, respectively.23 The tungsten

filament temperature used in blackbody radiation calculations was estimated using Eq. 4,

where T, ddp, L/A and i represent temperature (K), potential across the filament (V),

filament’s length-to-area ratio (m-1), and applied current (A).24 In this case, 12.81 V, 1.13

x 106 m-1 and 10.0 A were used to estimate a temperature of 3460 K, which is in agreement

with the bulb’s reported color temperature of 3450 K.

𝐵𝐵𝜆𝜆𝑏𝑏 = 1.190𝑥𝑥1016∙ 𝜆𝜆−5

𝑒𝑒1.438𝑥𝑥107/𝑇𝑇𝑇𝑇 −1 (3)

𝑇𝑇 = 𝑑𝑑𝑑𝑑𝑑𝑑𝐿𝐿𝐴𝐴 ∙ 𝑖𝑖

0.80548

∙ 2.1287 ∙ 108 (4)

The radiation source emits more intensely at 391 than at 309 nm (Eq. 3), so a blackbody

correction factor of 5.21 was used to correct radiation intensities recorded at 391 nm (i.e.

the signal intensity at 391 nm was divided by 5.21 to prevent bias). It was determined that

77

the detector is more sensitive at 391.439 nm than at 308.970 nm. Therefore, a correction

factor of 0.758 must be used when measuring the N2+ / OH signal ratio in our instrument.

This experimentally determined correction factor is in agreement with quantum

efficiencies reported at the respective wavelengths for the back-thinned CCD detector used

in the MIP OES instrument evaluated in this study. The detector response correction factor

calculated using values estimated from the detector’s quantum efficiency graph (available

for the commercial MIP OES) was estimated as approximately 0.8.

78

Effects of a changing plasma on the N2+ / OH and Mg II / Mg I ratios

As discussed earlier, we have evaluated individual signals and different

combinations of the emission intensities for CN, N2, N2+ and OH as plasma diagnostic

tools. Figs. S9 - S12 (Appendix A) show that the best results are achieved with the N2+ /

OH ratio. In addition to providing a consistently greater sensitivity to plasma changes

across all conditions evaluated (see Figs. S11 and S12), using a signal ratio (N2+ / OH in

this case) rather than individual signals allows for a broader application of the method.

Variations in the system will cause different effects in each individual signal. However,

such effects will be proportional in a signal ratio, which makes it more consistent over time

and across instruments, as well as more efficiently transferable between laboratories than

individual signals alone.

As noted in previously published works, the traditional definition of plasma

“robustness” may not fit well with a MIP.5,25,26 However, signal ratios such as the N2+ / OH

may still be used to monitor changes in the plasma due to instrumental fluctuations, or as

different matrices are introduced. Such strategy may then be employed to correct for signal

variation and improve accuracy when analyzing complex-matrix samples. In the present

study, we have compared the performances of the N2+ / OH signal intensity ratio and the

traditional Mg II / Mg I ratio to identify changes in the MIP. Figure 8 shows the effects of

nebulization gas flow rate (NGFR) on Mg II / Mg I and N2+ / OH. As it can be observed,

both signal ratios drop with an increase in NGFR, which is related to more solution

reaching the plasma, changes in electron number density (ne) and plasma cooling.4,5,21

However, the N2+ / OH ratio is more sensitive to these changes than the Mg II / Mg I ratio.

While the latter presents a quadratic relationship with NGFR (Figure 8a), the former has a

79

more intense power correlation with NGFR (Figure 8b). A similar trend is observed when

introducing increasing concentrations of sodium into the plasma at different NGFRs

(Figure 9). In this case, both signal ratios present a quadratic correlation with the

concentration of Na in solution. However, the N2+ / OH ratio is still more sensitive to

changes than Mg II / Mg I, as observed by comparing the curves’ gradients in Figure 9a

and b, and Figure 9c and d.

Figure 8. Effects of nebulization gas flow rate on Mg II / Mg I (a), and N2+ / OH (b) in

MIP OES.

80

Figure 9. Effects of sodium concentration on the Mg II / Mg I and N2+ / OH signal ratios

at nebulization gas flow rates of 0.6 L min-1 (a) and (b), and 1.0 L min-1 (c) and (d).

Figure 10. Effects of nebulization gas flow rate on (a) Mg II (280.271 nm) and Mg I

(285.213 nm), and (b) N2+ (391.439 nm) and OH (308.970 nm).

81

In addition to a potentially more sensitive diagnostic tool, the N2+ / OH ratio may

provide a more independent measure of change in both the N2 MIP conditions and the

sample introduction process than the traditional Mg II / Mg I ratio. The excitation

mechanism to produce N2+* involves species closely associated with the plasma, with the

promotion of N2+ from the ground state (X 2Σg, v) to the excited state (B 2Σu, v) by direct

electron impact and energy exchange reactions from collisions with vibrationally excited

N2 molecules.19,27 On the other hand, the OH radical is mainly produced from water, and

therefore, it is closely related to the nebulization process. In Figure 10b, for example, the

correlation coefficient between the NGFR and the OH signal intensity is R2 = 0.971.

Chalyavi et al. previously demonstrated that the OH emission bands are absent in a dry

MIP, but are easily detected upon introduction of 1% v v-1 HNO3.5 According to Frentiu et

al., OH radical species form following a reaction between dissociated O2 and water

molecules: O + H2O 2 OH.19 Additional evidence to this reaction is presented in Figure

11. When air is introduced into the plasma using the external gas control module (EGCM)

available in the commercial instrument, but water is not present (peristaltic pump speed at

0 rpm), the emission signal for OH at 308.970 nm is not distinguishable from the

background noise (Figure 11a). On the other hand, when no air is added, but distilled-

deionized water is introduced into the MIP at a peristaltic pump speed of 15 rpm and NGFR

of 0.7 L min-1, the emission band peak for OH can be easily identified (Figure 11b). In this

case, enough oxygen may be provided by the atmosphere, which is dragged into the plasma

by the N2 gas flow generating the MIP. As more oxygen gas is made available by

introducing air through the EGCM, with water uptake rate kept constant (peristaltic pump

speed of 15 rpm, and NGFR at 0.7 L min-1), the OH emission signal proportionally

82

increases (Figs. 4c-4e). These results are in agreement with the reaction mechanism

proposed by Frentiu et al..

Figure 11. MIP OES spectra for the OH molecular species (band peak at 308.970 nm).

Each spectrum corresponds to a different plasma / sample introduction condition: (a) no

water introduced (peristaltic pump speed at 0 rpm) and air added to the plasma at a medium

flow rate; (b) water introduced, with no air added to the plasma; (c) water introduced, with

a low air flow rate; (d) water introduced with a medium air flow rate; and (e) water

introduced with a high air flow rate. Conditions for water introduction: peristaltic pump

speed and NGFR of 15 rpm and 0.7 L min-1, respectively. No air flow rate specification

(other than low, medium and high) is available from the instrument’s EGCM.

83

Based on the mechanisms discussed for N2+ and OH formation, it is reasonable to assume

that combining the emission signals from these species into a ratio may be useful for

monitoring plasma and sample introduction variations in a more independent fashion than

with traditional plasma diagnostic tools such as the Mg II / Mg I ratio. As observed in

Figure 10a, especially at lower NGFRs, signals from both Mg lines generally increase with

the NGFR, as they are directly dependent on the amount of Mg going through the sample

introduction system. On the other hand, signals from N2+ and OH go in opposite directions

with increasing NGFR (Figure 10b), which may indicate more independence between their

original sources.

Using the N2+ / OH ratio to optimize instrumental operating conditions

The N2+ / OH and Mg II / Mg I signal ratios compare well, as observed in Figure

12. In this case, solutions containing 5 mg L-1 of Mg with increasing Na concentrations

(50, 100, 200, 500 and 1000 mg L-1) are introduced into the MIP under different

nebulization gas flow rates (0.6, 0.8, 1.0 and 1.2 L min-1). As discussed before, the N2+ /

OH ratio may present distinct advantages when compared with the traditional Mg II / Mg

I method. In addition to a potentially higher sensitivity to changes and the possibility of

more independently monitoring the sample introduction system and the plasma, it is also

convenient for routine applications. The N2+ / OH ratio method may be applied to any

sample, at any time, with no need for an additional sample preparation step (i.e. Mg does

not need to be added to the sample solutions). Thus, plasma conditions may be routinely

monitored (almost simultaneously since this is a fast sequential system) as different

samples are introduced into the MIP.

84

Figure 12. Correlation between the Mg II / Mg I and N2+ / OH signal ratios in the presence

of sodium at different nebulization gas flow rates (0.6 - 1.2 L min-1). Within each flow rate

group, Na concentrations in solution vary in the 50 - 1000 mg L-1 range (from right to left

on the graph).

85

The N2+ / OH signal ratio could also be used to determine the most favorable

conditions to minimize matrix effects. For example, one could monitor this signal ratio in

a range of NGFRs while running a standard solution, and then repeat the procedure for a

sample. The most favorable condition would be the one for which the N2+ / OH ratio

determined for the standard solution most closely matches the one from the sample. At that

specific NGFR, plasma conditions during sample introduction would be as close as

possible to those observed during the introduction of the calibration standard solutions,

which could improve accuracy in MIP OES measurements. Various sample matrices and

five elements with a wide range of excitation energies (i.e. Al, Ba, Mn, Sr and Zn) were

evaluated to investigate this hypothesis. To simulate a routine application, analyte

concentrations were determined using the external standard method, with no matrix-

matching, and calibration solutions prepared in 1 % v v-1 HNO3. Carbon (C as urea), Na

and Ca (all at 1000 mg L-1 each), and HNO3 and HCl (20 % v v-1 each) were separately

evaluated as matrices. Figure 13 shows the effects of each matrix, at various NGFRs, on

accuracy. For the analytes, the y-axis in Figure 13 represents the average percent error of

recovery, which is an average of absolute percent differences from 100 % recovery of all

analytes. For the N2+ / OH signal ratio, the y-axis represents the percent difference between

an average of N2+ / OH calculated for the standard solutions prepared in 1 % v v-1 HNO3

(calibration solutions), and values calculated for individual matrices. Note that, in general,

the lowest average percent errors of recovery coincide with the lowest percent difference

between N2+ / OH ratios calculated for standards and samples. Thus, without having

previous knowledge of the matrix, one could assume that the best overall accuracies may

be achieved at a NGFR of 0.9, 0.3 and 0.6 L min-1 for Al, Ba, Mn, Sr and Zn determination

86

in a matrix containing high concentrations of C, Na and HNO3, respectively (Figs. 6a, 6b

and 6d). On the other hand, the results show that this is not valid for a matrix with1000 mg

L-1 Ca or 20 % v v-1 HCl, in which case the best results would be found at the second lowest

N2+ / OH signal ratio difference (Figs. 6c and 6e). In Figure 13a, b, 6c, 6d and 6e, these

values are: 10.70 and 6.67 (1000 mg L-1 Na, NGFR = 0.3 L min-1); 0.664 and 0.446 (1000

mg L-1 Ca, NGFR = 1.0 L min-1); 1.25 and 1.20 (20 % v v-1 HNO3, NGFR = 0.6 L min-1);

and 0.664 and 0.655 (20 % v v-1 HCl, NGFR = 1.0 L min-1).

87

Figure 13. Relationship between average analyte percent recovery and N2+ / OH signal

ratio in different matrices: (a) 1000 mg L-1 C (as urea), (b) 1000 mg L-1 Na, (c) 1000 mg

L-1 Ca, (d) 20 % v v-1 HNO3, and (e) 20 % v v-1 HCl. Analytes evaluated: Al, Ba, Mn, Sr

and Zn at 2.0 mg L-1.

88

Signal correction using molecular species

Using the N2+ / OH signal ratio to identify the most favorable plasma conditions

and improve accuracy may be efficient for less severe matrix effects. For a matrix with

1000 mg L-1 C, for example, the average percent error of recovery went from 21.0 to 2.0

% by changing the NGFR from 0.4 to 0.9 L min-1 (Figure 13a). The recoveries for Al, Ba,

Mn, Sr and Zn went from 116, 112, 133, 107 and 126 % to 101, 98, 102, 97 and 102 %,

while the percent difference between standard solution N2+ / OH and sample N2

+ / OH went

from 18.0 to 0.4 %, respectively. On the other hand, some matrix effects may be too severe

to be corrected by just changing the NGFR. For Na at 1000 mg L-1, for example, the average

percent error of recovery at the most favorable plasma condition (i.e. NGFR at 0.3 L min-

1) was calculated as 77.0 % (Figure 13b), with Al, Ba, Mn, Sr and Zn recoveries of 112,

199, 130, 229 and 146 %, respectively. Table XIII. Analyte percent recoveries for 2.0 mg

L-1 solutions of Al, Ba, Mn, Sr and Zn prepared in different matrices. shows recoveries for

individual analytes at the best NGFRs, according to Figure 13. As previously observed by

Zhang and Wagatsuma, matrix effects due to EIEs are more pronounced than those due to

inorganic acids.21

89

Table XIII. Analyte percent recoveries for 2.0 mg L-1 solutions of Al, Ba, Mn, Sr and Zn

prepared in different matrices.

Sample matrix NGFR (L min-1)a Analyte Recovery (%)

1000 mg L-1 C 0.9 Al 101

Ba 98

Mn 102

Sr 97

Zn 102

APERb 2.0

1000 mg L-1 Na 0.3 Al 112

Ba 199

Mn 130

Sr 229

Zn 146

APERb 77.0

1000 mg L-1 Ca 1.0 Al 172

Ba 131

Mn 144

Sr 143

Zn 16

APERb 58.0

90

20 % v v-1 HNO3 0.6 Al 86

Ba 81

Mn 84

Sr 80

Zn 107

APERb 16.0

20 % v v-1 HCl 1.0 Al 93

Ba 90

Mn 94

Sr 87

Zn 96

APERb 9.0

a Nebulization gas flow rate (NGFR) providing the lowest average percent error of

recovery.

b Average percent error of recovery: absolute percent difference from 100 %

recovery of all analytes in a 2.0 mg L-1 solution.

91

A simple strategy to improve accuracy when severe matrix effects are present is the

use of MIP naturally occurring molecular species to correct for analytical signal bias.20 In

the present study, emission band peaks for CN (387.147 nm), N2 (337.097 nm), N2+

(391.439 nm) and OH (308.970 nm) were recorded in the same run as the analytical signals,

and then evaluated as signal correction species for improving accuracy while analyzing

different matrices. This strategy was investigated for NGFRs ranging between 0.3 and 1.0

L min-1. The analytical signals (A) were either divided or multiplied by the emission

intensity of the signal correction species (X), i.e. A / X or A · X. The calibration plot was

then built with A / X or A · X on the y-axis, and analyte concentration on the x-axis. The

results for the two best correction strategies for each sample matrix are presented in Table

XIV. The two best signal correction strategies for each sample matrix. The analyte percent

recoveries are associated with a 2.0 mg L-1 solution of each analyte..

For matrices causing less severe effects, a slight improvement in analyte recovery

was observed when using the molecular species correction strategy. The average percent

error of recovery (APER) for determinations in 1000 mg L-1 C and 20 % v v-1 HCl went

from 2.0 and 9.0 % (Table XIII) to 0.4 and 2.0 % (Table XIV), respectively. For the 1000

mg L-1 C matrix, recoveries for all analytes were in the 96 - 105 % when using A / X or

A · X, with X = N2, N2+ or OH. As expected, CN is not suitable for applications with high

C content matrices. Recoveries were in the 23 - 50 %, and 271 - 359 % for A / CN and A·

CN, respectively. For the 20 % v v-1 HCl matrix, analyte recovery was in the 92 - 105 %

range for all analytes by employing any combination of signal correction (i.e. A / X or A ·

X) and using either CN, N2, N2+ or OH.

92

Table XIV. The two best signal correction strategies for each sample matrix. The analyte

percent recoveries are associated with a 2.0 mg L-1 solution of each analyte.

Sample

matrix

Signal correction strategya Analyte recovery (%) APER

(%)b

Al Ba Mn Sr Zn

1000 mg

L-1 C

A / OH 99 100 100 100 100 0.4

NGFR (L min-1) 1.0 1.0 0.6 1.0 0.6

A · N2+ 99 99 100 99 100 1.0

NGFR (L min-1) 0.9 0.6 0.9 0.4 0.9

1000 mg

L-1 Na

A · CN 100 107 92 96 88 7.0

NGFR (L min-1) 0.4 0.4 0.5 0.5 0.3

A · N2 91 108 94 109 110 9.0

NGFR (L min-1) 0.5 0.6 0.6 0.6 0.3

1000 mg

L-1 Ca

A · N2+ 102 97 101 100 88 6.0

NGFR (L min-1) 0.6 1.0 0.9 0.8 0.3

A · CN 123 112 99 119 94 15.0

NGFR (L min-1) 0.6 0.9 0.7 0.8 0.3

20 % v v-1

HNO3

A / CN 101 99 102 98 106 3.0

NGFR (L min-1) 0.3 0.3 0.3 0.3 0.6

A / N2+ 100 100 100 99 109 4.0

NGFR (L min-1) 1.0 0.3 0.7 0.3 0.6

20 % v v-1

HCl

A / N2+ 100 98 98 98 99 2.0

NGFR (L min-1) 0.7 0.3 0.4 0.3 1.0

A · OH 105 99 103 99 99 3.0

NGFR (L min-1) 0.8 0.3 0.4 0.3 0.8 a A represents the analytical signal, and NGFR is the nebulization gas flow rate.

b Average percent error of recovery: absolute percent difference from 100 %

recovery of all analytes in a 2.0 mg L-1 solution.

93

For the other matrices, the molecular species signal correction approach allowed for significant

improvements in accuracy, as observed in Table XIII and Table XIV. In all cases, one of the two

best signal correction strategies, or a combination of them, can provide recoveries in the 90 - 110

% range for all the analytes and matrices evaluated in this study.

Conclusions

The use of the N2+ / OH signal ratio compares favorably with the traditional Mg II / Mg I

ratio as a plasma diagnostic tool. Among its advantages are the higher sensitivity to changes in

instrument conditions, and the quasi-simultaneous, sample-by-sample monitoring capabilities. No

additional solution preparation step is required since N2+ and OH are both plasma naturally-

occurring species. Considering the specific source of each of these molecular species, they may

also be used to optimize plasma / sample introduction conditions and improve accuracy when

analyzing less complex matrices. As the most useful analytical lines in MIP OES are primarily

atomic, maximizing robustness is not an effective method of optimizing accuracy. Instead, matrix

effects can be ameliorated by altering the instrumental operating conditions so that the plasma is

as similar as possible when introducing standard solutions and samples.

On the other hand, simply adjusting the instrumental operating conditions is not sufficient

to improve accuracy in analyses involving severe matrix effects. A simple and efficient strategy to

minimize matrix effects is the use of CN, N2, N2+ or OH for signal correction. The analyst may

just need to evaluate these molecular species in a range of NGFRs and identify the optimal

conditions and most effective signal correction operation as part of their method development.

94

Both the use of the N2+ / OH signal ratio as a diagnostic tool, and of molecular species for

signal correction to minimize matrix effects are simple strategies that may significantly contribute

to expanding the analytical capabilities of MIP OES and facilitating its application.

Conflicts of Interest

There are no conflicts of interest to declare.

Acknowledgements

The authors would like to thank the Department of Chemistry and the Graduate School of

Arts and Sciences at Wake Forest University for their support to this work.

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97

Multi-flow calibration applied to microwave-induced plasma optical

emission spectrometry


The following manuscript was accepted by the Journal of Analytical Atomic

Spectrometry on April 5, 2019, DOI: 10.1039/C9JA00091G. Stylistic variations are due to the

requirements of the journal. All of the presented research was conducted by Charles B Williams.

The manuscript was prepared by Charles B. Williams and edited by George Donati.

98

ABSTRACT

Multi-flow calibration (MFC) is based on a single calibration standard and multiple

nebulization gas flow rates (Q). Analytical signals are recorded at different Q conditions, and

intensities from calibration standard and sample are plot on the x and y axes, respectively. The

analyte concentration in the sample is calculated by multiplying the standard concentration by the

calibration plot slope. In the present work, MFC is used to determine Cr, Cu, Fe and Mn in water

and food samples by microwave-induced plasma optical emission spectrometry. Analyte percent

recoveries for certified reference materials and addition/recovery experiments were in the 91-

112% and 84-134% ranges for MFC and external standard calibration (EC), respectively. The

limits of detection (LODs) for Cr, Cu, Fe and Mn were 20, 5, 7 and 2 µg L-1 using MFC (0.6, 8,

20 and 1 µg L-1 for EC). Precisions were in the 0.9-12.2% and 1.9-23.9% ranges for MFC and EC,

respectively. MFC may minimize matrix effects as it exposes all solutions to a variety of plasma

conditions. This normalizing effect may be capable of improving accuracies compared with EC

for simple to moderately complex matrix samples. One of MFC’s main limitations is the potential

for systematic errors associated with solution preparation (a single calibration standard is used).

Variation in Q may also result in higher LODs and lower sample throughputs compared with EC.

On the other hand, neither negative effects due to a poor choice of Q nor additional experiments

to optimize it are required with MFC.

99

INTRODUCTION

Traditional calibration methods used in atomic spectrometry, such as the external standard

calibration (EC), typically involve preparing standard solutions at a range of known

concentrations, recording the respective analytical signal intensities for those solutions to

determine sensitivity, and then using the sensitivity value to calculate the analyte concentration in

a given sample.1-3 A series of standard solutions, rather than a single one, is generally employed

to minimize uncertainty in the mathematical model used for calibration.2,4 The method of standard

additions (SA) is based on a similar principle, with the difference of adding a series of standard

solutions directly to the sample to control for matrix effects.5,6

Recently, there has been an increased interest in alternative calibration methods to improve

accuracy and sample throughput in atomic spectrometry applications. Some of these methods have

been discussed in a recent review by Carter et al., which is primarily focused on inductively

coupled plasma mass spectrometry (ICP-MS), but also covers some applications involving ICP

and microwave-induced plasma (MIP) as atomization/excitation sources for optical emission

spectrometry (ICP OES and MIP OES, respectively).3 While traditional calibration methods use

analyte concentration as the independent variable and analytical signal intensity as the dependent

variable when building the calibration plot, some of the new strategies take advantage of multiple

variables such as wavelength (or transition energy),7-9 isotopes,10 or polyatomic species11 to

determine the analyte concentration in a sample. Multi-energy calibration (MEC), for example,

makes use of multiple transition energies (corresponding to different emission or absorption

wavelengths), combined with a matrix-matching approach, to improve accuracy in ICP OES, MIP

OES, high-resolution continuum source atomic absorption spectrometry (HR-CS AAS), and laser-

induced breakdown spectroscopy (LIBS).7-9 In ICP-MS, multi-isotope and multispecies calibration

100

(MICal and MSC) employ a similar strategy as MEC in which multiple analyte isotopes or multiple

polyatomic species containing the analyte ion are used to improve the accuracy of the calibration

procedure.10,11

MEC, MICal and MSC involve matrix-matching to compensate for matrix effects.

Therefore, although more efficient than the traditional SA method, the newly developed calibration

strategies still are more involved than EC. In each case, the sample is measured twice, i.e. once by

itself and once as a mixture containing added standard solution. Thus, similar to SA, these recently

described calibration strategies are more efficient at minimizing matrix effects, but present lower

sample throughputs when compared with EC. In the present work, we describe a method that

combines the simplicity of EC with the enhanced precision of employing multiple signal collection

conditions to carry out calibration. In multi-flow calibration (MFC), a single reference solution is

used, with no standard added to the samples. The analyte concentration is varied online by

modulating the amount of sample introduced into the plasma using the instrument’s nebulization

gas, i.e. the analyte concentration in the plasma varies as the nebulization flow rate (Q) changes.

Therefore, rather than preparing several calibration standards (as in EC), the same effect of

generating multiple signal levels to estimate analyte sensitivity and then determine the analyte

concentration in the sample is achieved with a single standard solution in MFC. The mathematical

basis of MFC is generally simple. Consider, for example, the parameters involved in signal

intensity (here represented as the measured output voltage, Eout) for an arbitrary emission line (eqn

(1)):12

𝐸𝐸𝑜𝑜𝑜𝑜𝑜𝑜 = 𝐶𝐶𝐶𝐶𝜖𝜖𝑎𝑎𝑄𝑄𝑒𝑒𝑓𝑓

𝑔𝑔𝑗𝑗𝑔𝑔0𝑒𝑒−𝐸𝐸𝑗𝑗0/𝑘𝑘𝐵𝐵𝑇𝑇 × 𝑉𝑉𝐸𝐸𝑗𝑗0𝐴𝐴𝑗𝑗0 × 𝑌𝑌𝑚𝑚 × 𝑇𝑇𝑜𝑜𝑑𝑑𝑅𝑅(𝜆𝜆)𝐺𝐺 (1)

101

where C, F, 𝜖𝜖𝑎𝑎, Q, ef, gj, g0, Ej0, kB, T, V, Aj0, Ym, Top, R(λ) and G represent analyte concentration,

solution flow rate, atomization efficiency, nebulization gas flow rate, gas expansion factor,

statistical weights of the excited state and the ground state, transition energy, Boltzmann constant,

plasma temperature, volume observed by the monochromator, rate of spontaneous emission,

monochromator collection efficiency, transmittance of the optics, detector responsivity, and gain

of the electronics. For a given instrument in a fixed condition and a given analyte, eqn (1) has

traditionally been simplified as:

𝐸𝐸𝑜𝑜𝑜𝑜𝑜𝑜 = 𝐾𝐾𝐾𝐾 (2)

with K representing a proportionality constant, which incorporates all parameters on the right hand

side of eqn (1) except for C. In the simplest calibration strategy, a single-point calibration,13 one

runs a standard solution for which Eout,std = KCstd (or Istd = KCstd to represent signal intensity rather

than voltage output). Then, the sample is run and Isam = KCsam. Because K is the same in both runs

(considering no matrix effects), the analyte concentration in the sample (Csam) may be determined

by:

𝐾𝐾𝑠𝑠𝑎𝑎𝑚𝑚 = 𝐶𝐶𝑠𝑠𝑠𝑠𝑠𝑠 𝐼𝐼𝑠𝑠𝑎𝑎𝑠𝑠𝐼𝐼𝑠𝑠𝑠𝑠𝑠𝑠

(3)

The primary issue with this simple strategy is the error associated with using a single

measurement for calibration. The analyte concentration in the sample is determined by

interpolation, and the greater the number of calibration points involved the lower the standard

deviation associated with the estimated concentration.4 Thus, working within the traditional

paradigm, several calibration standard solutions are prepared and run to minimize the error in

determining the unknown analyte concentration in the sample. This is done by re-arranging eqn

(3) into eqn (4) and plotting the analyte intensity collected from different standard solutions (which

102

obviously depend on the individual standard concentrations, Istd(Cstd)) versus the analyte

concentration in each standard (Cstd). The slope of that plot will equal Isam / Csam, and the unknown

analyte concentration in the sample can be found by recording Isam from the sample solution and

dividing that signal by the slope (eqn (5)).

𝐼𝐼𝑠𝑠𝑜𝑜𝑑𝑑 = 𝐾𝐾𝑠𝑠𝑜𝑜𝑑𝑑 𝐼𝐼𝑠𝑠𝑎𝑎𝑠𝑠𝐶𝐶𝑠𝑠𝑎𝑎𝑠𝑠

(4)

𝐾𝐾𝑠𝑠𝑎𝑎𝑚𝑚 = 𝐼𝐼𝑠𝑠𝑎𝑎𝑠𝑠𝑠𝑠𝑠𝑠𝑜𝑜𝑑𝑑𝑒𝑒

(5)

Alternatively, if one chooses to keep Cstd constant, re-arrange eqn (3) into eqn (6), and plot

Isam versus Istd as they change with Q, i.e. Isam(Q) vs. Istd(Q), the slope of that plot will be Csam / Cstd.

Therefore, Csam can be easily found by multiplying the concentration of analyte in the standard by

the slope, as represented in eqn (7).

𝐼𝐼𝑠𝑠𝑎𝑎𝑚𝑚 = 𝐼𝐼𝑠𝑠𝑜𝑜𝑑𝑑 𝐶𝐶𝑠𝑠𝑎𝑎𝑠𝑠𝐶𝐶𝑠𝑠𝑠𝑠𝑠𝑠

(6)

𝐾𝐾𝑠𝑠𝑎𝑎𝑚𝑚 = 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑒𝑒 ∙ 𝐾𝐾𝑠𝑠𝑜𝑜𝑑𝑑 (7)

In the present work, we evaluate the applicability of the MFC method in atomic

spectrometry by determining Cr, Cu, Fe and Mn in water and food samples by MIP OES. Certified

reference materials (CRMs) and addition and recovery experiments, as well as a comparison with

the traditional EC method, are used to validate the MFC strategy.

103

EXPERIMENTAL

Instrumentation

A MIP OES instrument (4200 MP-AES, Agilent Technologies, Santa Clara, CA, USA)

was used in all determinations. It was outfitted with a glass concentric pneumatic nebulizer

(Meinhard, Golden, CO, USA) and a glass double-pass cyclonic spray chamber (Agilent). A liquid

N2 Dewar (99.99% purity, Air Products, Allentown, PA, USA) was used to supply the MIP with

both plasma gas and nebulization gas.

The microwave applied power in this instrument is fixed at 1 kW. For all analytes, the

peristaltic pump speed was set to 15 rpm, the integration time was 3 s, and the plasma observation

position was set to 0 (which corresponds to the center of the plasma).14 Samples were analyzed to

determine Cr (at 425.433 nm), Cu (at 324.754 nm), Fe (at 371.993 nm) and Mn (at 403.076 nm).

In the present proof-of-concept study, the analytes were chosen based on their certified values in

the CRMs available and on the general MIP OES sensitivity. For MFC, each element was

measured at the following Q values: 0.4, 0.5, 0.6, 0.7, and 0.8 L min-1 N2. For EC comparisons,

default Q conditions recommended by the manufacturer were employed, i.e. 0.9 L min-1 for Cr and

Mn, and 0.65 L min-1 for Cu and Fe.

An Ethos UP microwave-assisted digestion system (Milestone, Sorisole, Italy) was used to

decompose the solid samples before analysis.

104

Reference materials, samples, and sample preparation

Distilled-deionized water (18 MΩ·cm, Milli-Q, Millipore, Bedford, MA, USA) and trace-

metal-grade nitric acid (Fisher, Pittsburgh, PA, USA) were used to prepare all working solutions.

All samples and standard solutions were prepared in 1% v/v HNO3. Trace-analysis-grade H2O2

(30% v/v, Sigma Aldrich, Atlanta, GA, USA) was used for sample digestion. Single-element stock

solutions containing 1000 mg L-1 Cr, Cu, Fe or Mn (High Purity Standards - HPS, Charleston, SC,

USA) were used to prepare the standard reference solutions and to carry out addition and recovery

experiments.

Three CRMs were used for method validation: Secondary Drinking Water Metals, River

Sediment A (HPS), and Tomato Leaves (National Institute of Standards and Technology - NIST,

Gaithersburg, MD, USA). Addition and recovery experiments were also carried out using Cheerios

(General Mills, Minneapolis, MN, USA), Oatmeal (The Quaker Oats Co., Chicago, IL, USA), and

two water samples from the Sea of Galilee and the Jordan River. The river water samples were

collected into new, clean 50-mL polypropylene centrifuge tubes and stored in 1% v/v HNO3 until

analysis. Secondary Drinking Water Metals was diluted 10-fold in1% v/v HNO3 before analysis,

and River Sediment A was diluted 5-fold or 100-fold, depending on the analyte, to adequate the

analyte concentrations to the calibration curve range. All the other samples were digested in

triplicate using 1 mL of concentrated HNO3 and 2 mL of H2O2. The heating program used for

microwave-assisted digestion included a 10-min step to reach 180 oC, a 15-min hold at 180 oC and

a 15-min cool down step.

105


MFC strategy

One of the key challenges in MIP OES and other spectrochemical analysis methods is

associated with the optimization of instrument operating conditions to improve accuracy. Because

applied power, plasma gas flow rate, and other conditions are fixed in the commercially available

MIP OES instrument, the chief operating condition which affects the state of the plasma and can

be changed on a per-wavelength basis is the nebulization gas flow rate.15-17 In this context, different

studies have evaluated the effects of operating conditions on accuracy, and sought to identify their

optimal setup to improve the performance of MIP OES.14,18-20 Plasma robustness, according to

several definitions, may be generally improved by reducing Q and adjusting the plasma viewing

position. However, such generally recommended conditions not always result in accurate results

for certain MIP OES applications.14,18 Extremely low Q values may lead to insufficient or

suboptimal amounts of analyte reaching the plasma and to a potential overpopulation of interfering

species. Thus, rather than seeking to select a single optimal condition for each sample matrix, the

MFC method simultaneously employs multiple Q values, which, on average, minimize the

negative effects of a poorly chosen condition on the overall accuracy of the analysis. In principle,

MFC can also be performed by modifying the rotation speed of the peristaltic pump (i.e. by varying

F rather than Q in eqn (1)). However, the commercially available MIP OES control software allows

for no modification of pump speed within a single run. Thus, a method based on such strategy

would require the use of multiple worksheets and large volumes of sample, resulting in

significantly longer analysis times and the generation of large volumes of waste.

Table XV demonstrates the multi-flow advantage when compared with EC. In this case, MFC and

EC carried out at several different Q conditions were used to determine Cu in a sample of River

106

Sediment A. As it can be seen, the MFC result is generally more accurate than any of the individual

values obtained by EC, and superior to the average 91.7% recovery calculated from all Q

conditions used with the traditional method. Because it is more accurate than EC at both 0.60 and

0.70 L min-1, it may be expected that MFC would also provide a more accurate concentration of

Cu than that obtained with EC at the default 0.65 L min-1 condition.

107

Table XV. Comparison of MFC with EC using the same individual Q values while determining Cu in River Sediment A. Analyte percent recoveries (%) refer to the 1 mg L-1 certified concentration in the CRM. Results are shown as mean ± 1 standard deviation (n = 3). MFC calibration standard: 5.00 mg L-1.

Calibration method, Q (L min-1) Recovery R2

MFC, 0.4 - 0.8 96 ± 2 0.99780

EC, 0.4 95 ± 4 0.99954

EC, 0.5 92 ± 1 0.99993

EC, 0.6 88 ± 1 0.99991

EC, 0.7 89 ± 1 0.99972

EC, 0.8 92 ± 1 0.99989

EC, 0.9 94 ± 2 0.99987

108

Typical MFC plots are shown in Figure 14. Copper in River Sediment A (Figure 14A)

and Mn in Tomato Leaves (Figure 14B) were determined using 5.00 mg L-1 standards. From eqn

(7), the concentration of Cu calculated for this particular sample replicate was 0.98 mg L-1, which

corresponds to a recovery of 98% from the certified value of 1 mg L-1. For Mn, considering an

initial sample mass of 0.2024 g and a final solution volume of 20.0 mL, the analyte concentration

in this sample replicate is calculated as 249 mg kg-1, which corresponds to a 101% recovery from

the certified value of 246 ± 8 mg kg-1.

When standard solution and sample present different matrices, the effects of each matrix

on the plasma mechanisms responsible for analytical signal generation may significantly

compromise accuracy in EC applications. The purpose of a matrix-matching calibration method,

such as SA and MEC, is to minimize such effects by preparing standard and sample solutions in

the same physical-chemical environment, i.e. the same matrix.5,7 An alternative approach to

minimizing matrix effects involves the normalization of the plasma rather than the matrix.

Although neither as specific nor as effective as matrix-matching, one may minimize the effects of

different matrices on the plasma by deliberately exposing standard and sample solutions to a

variety of plasma conditions, which is achieved in MFC by employing multiple nebulization gas

flow rates. Such strategy may be compared to the calibration method known as extrapolation to

infinite dilution, with the advantage of requiring no complicated sample preparation procedures

and no additional sample introduction apparatus.21 In addition to its plasma normalizing effects,

MFC may also contribute to mitigating matrix interferences on the nebulization process itself, as

it employs a range of conditions rather than restricting the analysis to a single Q value that may be

more prone to interference.22

109

Figure 14. Multi-flow calibration plots for determining (A) Cu in River Sediment A, and (B) Mn in Tomato Leaves.A 5.00 mg L-1 calibration standard was used in both determinations. The calibration plots refer to a single sample replicate.

110

It is important to emphasize that MFC can never be as efficient as matrix-matching

methods, and may be considered an intermediary strategy between EC and SA, for example. It is

even more straightforward than EC, as it only requires one calibration standard. In addition, it

should perform as well as, and mostly better than, EC for simple and moderately complex matrix

samples such as the ones presented in Table XVI. However, it probably needs to be replaced with

a matrix-matching alternative to ensure accuracy when analyzing complex-matrix samples capable

of producing severe matrix effects. Similar to MEC, MICal and MSC (which use a multivariate

calibration strategy not directly associated to multiple standard concentrations), MFC is more

prone to systematic errors related to solution preparation. Because a single standard calibration

solution is used, any inaccuracy with that solution produces biased results. On the other hand, an

advantage of MFC over MEC, MICal and MSC is that a certified stock solution can be used

directly (i.e. without dilution) if available, whereas the other methods require the addition of a

standard solution to an aliquot of each sample.7-11

T

able

XV

I. A

ccur

acy

com

paris

on b

etw

een

MFC

and

EC

. Ana

lyte

con

cent

ratio

ns a

re re

porte

d as

mea

n ±

1 st

anda

rd d

evia

tion

(n =

3).

Ana

lyte

per

cent

reco

verie

s (%

) fro

m th

e cer

tifie

d va

lues

are s

how

n in

par

enth

esis

. MFC

calib

ratio

n st

anda

rd: 5

.00

mg

L-1 fo

r all

anal

ytes

ex

cept

for C

u in

Tom

ato

Leav

es w

hich

was

0.0

500

mg

L-1.

a Sec

onda

ry d

rinki

ng w

ater

met

als

(HPS

). C

ertif

ied

valu

es:

50,

100

and

50 m

g L-1

for

Cu,

Fe

and

Mn,

res

pect

ivel

y.

b Ri

ver S

edim

ent A

(HPS

). C

ertif

ied

valu

es: 3

00, 1

, 120

0 an

d 8

mg

L-1 fo

r Cr,

Cu,

Fe

and

Mn,

resp

ectiv

ely.

The

sta

ndar

d de

viat

ions

calc

ulat

ed fo

r thi

s sam

ple

are

base

d on

inst

rum

enta

l rep

licat

es.

c Tom

ato

Leav

es (

NIS

T). C

ertif

ied

valu

es (m

g kg

-1):

1.99

± 0

.06,

4.7

0 ±

0.14

, 368

± 7

and

246

± 8

for

Cr,

Cu,

Fe

and

Mn,

resp

ectiv

ely.

d NA

= N

ot a

vaila

ble.

Sam

ple

Cr

Cu

Fe

Mn

MFC

E

C

MFC

E

C

MFC

E

C

MFC

E

C

SDW

M a

NA

d N

A d

50 ±

1

(100

%)

52 ±

1

(104

%)

91 ±

2

(91%

) 92

± 5

(9

2%)

48 ±

1

(96%

) 49

± 1

(9

8%)

CR

M-R

S-A

b 29

1 ±

8 (9

7%)

309

± 11

(1

03%

) 1.

01 ±

0.0

5 (1

01%

) 1.

11 ±

0.1

0 (1

11%

) 12

12 ±

15

(101

%)

1368

± 1

31

(114

%)

8.4

± 0.

2 (1

05%

) 10

.7 ±

2.4

(1

34%

)

NIS

T 15

73a

c <L

OD

1.

67 ±

0.0

6 (8

4%)

5.02

± 0

.21

(107

%)

5.75

± 0

.31

(122

%)

400

± 9

(109

%)

339

± 7

(92%

) 26

2 ±

12

(106

%)

277

± 8

(113

%)

111

112

Limits of detection

The limits of detection (LODs) were based on eqn (7) and calculated as 3 times the standard

error of the MFC calibration curve slope (Sslope), times the concentration of the standard, i.e. LOD

= 3·Sslope·Cstd. A solution of 1% v/v HNO3 was treated as sample in LOD calculations, with 60

data points used for determining Sslope (12 samples measured at 5 different Q conditions). A 5 mg

L-1 standard solution was used in this experiment. The LODs calculated for Cr, Cu, Fe and Mn

were 20, 5, 7 and 2 µg L-1, respectively. Except for Cr, these values are comparable with those

obtained with EC (calculated as three times the standard deviation of a blank solution, n = 12,

divided by the calibration curve slope): 0.6, 8, 20 and 1 µg L-1, respectively.

To investigate the discrepancy in Cr LODs, a 1% v/v HNO3 solution was analyzed at Q

values of 0.4, 0.5, 0.6, 0.7 or 0.8 L min-1, and the respective BG spectrum at the 425.433, 324.754,

371.993 and 403.076 nm regions (i.e. Cr, Cu, Fe and Mn) were evaluated. Percent relative standard

deviations (RSDs) associated with BG signals collected at the different Q conditions were then

calculated for each wavelength. In this case, BG signals recorded at each Q condition were used

to calculate mean, standard deviation and RSD for each of the analytes’ wavelengths. The results

indicate that higher MFC LODs (when compared with EC) are related to higher BG signal RSDs.

The highest values were found for Cr and Mn, with 52 and 25%, while Cu and Fe presented 15

and 18%, respectively. For Cr, for example, there was a 68% decrease in BG signal intensity when

Q was changed from 0.4 to 0.8 L min-1. For Cu, the signal reduction was 30% for the same change

in Q. These results suggest that the BG signal at 425.433 nm is more sensitive to changes in Q

when compared to the other wavelength regions evaluated, which may have resulted in a relatively

high LOD for Cr when applying MFC.

113

The upper limit of the calibration curve, for which analyte concentrations can be accurately

determined using a single standard of 5 mg L-1, was not experimentally determined. However,

preliminary results suggest MFC may be successfully applied within an order of magnitude from

the standard concentration used. In addition, the introduction of samples with concentrations

higher than 100 mg L-1 resulted in the saturation of the instrument’s detector.

Accuracy

The method’s accuracy was evaluated by analyzing CRMs and by addition and recovery

experiments. The results, along with a comparison with values obtained with EC, are presented in

Table XVI and Table XVII. It is important to note that the different recoveries for Cu in River

Sediment A between Table 1 and Table XVI are due to different analysis days and the use of a

single-element (Table 1) or a multi-element (Table XVI) standard for calibration. In addition, the

results shown for River Sediment A (Table XVI), Sea of Galilee and Jordan River (Table XVII)

are based on instrumental replicates, rather than true replicates, due to the limited amount of these

samples available.

114

Tab

le X

VII

. Ana

lyte

per

cent

reco

verie

s (%

) fro

m sp

iked

con

cent

ratio

ns in

wat

er a

nd fo

od sa

mpl

es a

naly

zed

by M

IP O

ES u

sing

MFC

or

EC

. The

resu

lts ar

e pre

sent

ed as

mea

n ±

1 st

anda

rd d

evia

tion

(n =

3).

Ana

lyte

conc

entra

tion

adde

d to

the s

ampl

es =

5.0

0 m

g L-1

. MFC

ca

libra

tion

stan

dard

= 5

.00

mg

L-1.

a The

stan

dard

dev

iatio

ns c

alcu

late

d fo

r the

se sa

mpl

es a

re b

ased

on

inst

rum

enta

l rep

licat

es.

Sam

ple

Cr

Cu

Fe

Mn

MFC

E

C

MFC

E

C

MFC

E

C

MFC

E

C

Sea

of G

alile

e a

107

± 1

118

± 4

109

± 1

113

± 12

10

7 ±

2 10

9 ±

26

112

± 5

103

± 4

Jord

an R

iver

a 10

9 ±

2 12

0 ±

6 10

2 ±

2 11

4 ±

6 95

± 1

10

7 ±

12

103

± 2

106

± 8

Che

erio

s 99

± 3

11

0 ±

8 92

± 7

92

± 5

98

± 1

2 94

± 1

2 97

± 4

10

5 ±

6

Oat

mea

l 97

± 7

10

9 ±

6 93

± 8

91

± 6

10

1 ±

9 10

5 ±

6 94

± 9

10

0 ±

7

115

As expected considering the early discussion on plasma normalization, MFC results are

similar, and often more accurate than those from EC. MFC’s superior performance becomes

evident as a more complex-matrix sample such as Tomato Leaves is analyzed. From Table XVI,

no statistically significant difference between certified and determined values were found for most

analytes (Student’s t-test at the 95% confidence level), except for Fe in both Secondary Drinking

Water Metals and Tomato Leaves for MFC, and all analytes in Tomato Leaves for EC. Analyte

percent recoveries were in the 91-109% and 84-134% ranges for MFC and EC, respectively. In

addition and recovery experiments (Table XVII), analyte percent recoveries were in the 92-112%

and 91-120% ranges for MFC and EC, respectively. No statistically significant difference was

found between MFC and EC results (two-mean Student’s t-test at the 95% confidence level),

except for Cu in Tomato Leaves and Jordan River, and Cr in Sea of Galilee and Jordan River. It is

interesting to note that in all these four cases, EC’s analyte recoveries were higher than 110%,

which may be additional evidence of MFC’s relatively higher accuracy.

To further evaluate the effect of exposing sample and calibration standards to different

plasma conditions on accuracy, the MFC results presented in Table XVI may be compared with

values obtained from a single-point calibration13 using a 5.00 mg L-1 standard and determinations

at the default Q conditions. For Secondary Drinking Water Metals (HPS), recoveries for Cu, Fe

and Mn were 120, 121 and 104%, respectively (100, 91 and 96% for the same elements using

MFC, Table XVI). Similar results were found for River Sediment A (HPS) and Tomato Leaves

(NIST), with 100 and 101% recovery for Cr, 113 and 117% for Cu, 115 and 95% for Fe, and 128

and 108% for Mn, respectively. From Table XVI, the MFC results for these same CRMs were

97% and < LOD for Cr, 101 and 107% for Cu, 101 and 109% for Fe, and 105 and 106% for Mn,

respectively. In comparison with MFC, a single-point calibration using the same reference

116

standard solution generally produces overestimated values, which reinforces the hypothesis of the

plasma normalizing effect associated with the new calibration strategy.

As noted earlier, MFC performs better when analyte concentrations in the sample and the

calibration standard are within one order of magnitude of each other. Therefore, when applying

MFC in a routine analysis, if the analytical signals recorded for the sample are too low or too high

in comparison with those from the calibration standard (i.e. more than one order of magnitude

difference), sample dilution or a higher-concentration standard must be adopted. A similar

procedure is expected in EC applications, i.e. an estimate of analyte levels in the sample is required

before deciding on the range of concentrations covered by the calibration curve. Alternatively, if

the analytical signal is too high, sample dilution is required to fit the calibration curve’s

concentration range. In this context, a 0.0500 mg L-1 standard solution was used with MFC to

determine Cu in Tomato Leaves, given the relatively lower levels expected in this case (Table

XVI). To highlight the effects of standard concentration on MFC accuracy, the same Tomato

Leaves sample replicates used to produce the values shown in Table XVI were analyzed using a

1.00 mg L-1 Cu standard (ca. 20-fold higher than the expected Cu concentration in the sample).

The Cu value found in this case was 5.80 ± 0.30 mg kg-1, which corresponds to a 123% recovery

and is obviously less accurate than the 107% value obtained with a 0.0500 mg L-1 calibration

standard.

117

Precision

Precision for both MFC and EC may be assessed by calculating RSDs from the results in

Table XVI and Table XVII. RSD values are generally lower for MFC when compared with EC,

which may be associated to fewer sources of error when running a single calibration solution in

MFC. In Table XVI, RSDs are in the 1.2-5.0% and 1.9-9.6% ranges for MFC and EC, respectively.

In Table XVII, RSD values are calculated between 0.9 and 12.2% for MFC, and between 3.4 and

23.9% for EC.

Long-term stability

In EC applications, it is generally necessary to re-run the calibration curve after a certain

period of time to minimize the effects of signal drift on accuracy. Thus, a long-term stability

experiment was carried out to evaluate the applicability of the MFC method over time. A solution

containing 5 mg L-1 of each Cr, Cu, Fe and Mn was introduced into the plasma and measured every

10 min for a period of 90 min using MFC. The first measurement (minute 0) was used as the

calibration standard and the subsequent measurements were considered samples. As observed in

Figure 15, accurate results are expected for periods of up to 90 min without the need for

recalibration, except for Cr, which requires recalibration approximately every 30 min. These

results suggest MFC’s recommended procedure for minimizing signal drift is not significantly

different from that routinely adopted for EC. The signal behavior observed for Cr suggests higher

instability. As discussed earlier, Cr results may be related to changes in BG signal over time and a

relatively higher sensitivity of the BG signal at the 425.433 nm region to plasma changes.

118

Figure 15. Long-term stability of MFC. Analyte percent recoveries (%, n = 3) are based on a 5.00 mg L-1 solution measured every 10 min over a period of 90 min. Average values are the mean percent recoveries of all four analytes at each time point.

119

Sample throughput

Considering an analysis involving twenty samples, four analytes, five calibration points for

EC, and the analytical parameters used in the present work, the sample throughputs calculated for

EC and MFC are 27 and 13 samples h-1, respectively. Therefore, EC is significantly faster than

MFC, which is especially due to the different Q conditions required for the new method. However,

these estimates are based on time spent during instrumental measurements, with no consideration

of solution preparation. Thus, for an analysis involving a small number of samples, in which

preparing the calibration standard solutions has a more significant effect on the final sample

throughput, MFC’s speed may be comparable to EC. This is especially valid considering that the

detection step is fully automated and most of the analyst’s time is spent on solution preparation.

Analyte concentrations in the samples

For most samples evaluated, the original analyte concentrations (non-spiked) were lower

than the respective LODs. Detectable values were found only for Fe in Cheerios, with 255 ± 20

and 279 ± 24 mg kg-1 (n = 3) determined by MFC and EC, respectively. In this case, no statistically

significant difference was observed between the different calibration methods (two-mean

Student’s t-test at a 95% confidence level). Both values are also in agreement with the product

labelling, which indicates an approximate Fe mass equivalent to 45% of the recommended daily

value in a 28 g serving size. According to the product’s label and information from the U.S. Food

and Drug Administration,23 the approximate Fe concentration in this sample should be 280 ± 30

mg kg-1.23

120

CONCLUSIONS

Multi-flow calibration is a novel strategy for use in MIP OES and other spectrochemical

analysis methods that allow for variation of nebulization gas flow rates during the analysis. It is an

efficient method, as it employs a single calibration standard and provides accuracies and precisions

comparable to, and often better than, the traditional EC method. Although not as effective as SA

and other matrix-matching strategies, MFC may minimize matrix effects. It exposes samples and

standards to a variety of plasma conditions, which may have a normalizing effect capable of

improving accuracies, in comparison with EC, for analysis involving samples with simple to

moderately complex matrices.

As demonstrated in this proof-of-concept work, the MFC method is an effective alternative

to EC for applications involving Cr, Cu, Fe and Mn, but additional studies are required to evaluate

other analytes and sample matrices. One of its main limitations is the potential for systematic errors

associated with solution preparation, especially considering a single calibration standard is used to

determine analyte concentrations in the samples. Although less time is spent with solution

preparation, MFC presents a lower sample throughput than EC when analyzing a large number of

samples. In addition, signal fluctuations due to the required changes in Q may result in higher

LODs when compared with EC. On the other hand, no additional experiments are required to

optimize Q conditions, and no negative effects on accuracy due to a poor choice of Q are expected

with MFC.

121

CONFLICTS OF INTEREST

There are no conflicts of interest to declare.

ACKNOWLEDGEMENTS

The authors would like to thank High Purity Standards, and the Department of Chemistry

and the Graduate School of Arts and Sciences at Wake Forest University for their support to this

work.

122

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123

12. J. D. Ingle and S. R. Crouch, Spectrochemical Analysis, Prentice Hall, Englewood Cliffs, 1988.

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16. N. Ozbek, H. Tinas and A. E. Atespare, Microchem. J., 2019, 144, 474-478.

17. S. M. Azcarate, L. P. Langhoff, J. M. Camiña and M. Savio, Talanta, 2019, 195, 573-579.

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19. N. Chalyavi, P. S. Doidge, R. J. S. Morrison and G. B. Partridge, J. Anal. At. Spectrom., 2017,

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20. K. L. Lowery, T. McSweeney, S. P. Adhikari, A. Lachgar and G. L. Donati, Microchem. J.,

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23. Center for Food Safety and Applied Nutrition, A Food Labeling Guide: Guidance for Industry,

Food and Drug Administration, College Park, p. 127.

https://www.fda.gov/downloads/Food/GuidanceRegulation/GuidanceDocumentsRegulatoryInfor

mation/UCM265446.pdf, (accessed March 12, 2019).

124

CHAPTER VI

CONCLUSIONS

Microwave-induced plasma optical emission spectrometry (MIP OES) is a rapidly-

growing technique in the field of atomic spectrometry. Its chief advantages are its simple,

low-cost operation for routine trace element analysis, and its ability to be used in remote

locations by virtue of its ability to run on air. With intensive method development and

further understanding of the nature of the plasma, the method can be applied to increasingly

complex sample types and matrices, expand access to elemental analysis in remote areas,

and better compete with established techniques such as ICP OES.

When combined with other simple, low-cost sample preparation techniques, the

utility of MIP OES for routine analysis of complex samples can be greatly expanded. MIP

OES coupled with a simple, dilute-and-shoot procedure with soft drink samples shows the

ability of the technique to perform routine elemental analyses in aqueous samples with high

levels of organic concomitants, further enabling the field to expand and potentially

increasing access to safety and quality control testing of food and other consumer products.

Similarly, MIP OES was combined with a prototype rapid dry-ashing unit to facilitate

sample preparation. This expands the utility of the technique to cover even more complex

sample types and further improves access to low-cost elemental analyses.

The development of plasma diagnostic tools is an important factor in both

increasing the understanding of the fundamental properties of the MIP and in expansion of

the analytical utility of the plasma. The development of the N2+/OH ratio as a plasma

diagnostic tool is one of the first strategies developed specifically for MIP OES, as opposed

to being transferred from another technique. This development not only opens up a path

125

for better understanding the MIP, but also enables real-time analysis and signal correction.

It offers a short-cut to extensive trial-and-error method development by characterizing the

change in plasma conditions caused by a specific sample and allowing the analyst to

modulate operating conditions or sample preparation steps accordingly. While further

development of their use as signal-correction species is still required, these molecular

species present potential for use in a successful approach for further improving accuracy

in MIP OES determinations.

Novel calibration strategies are another potentially efficient means of improving

the accuracy of MIP OES. In this context, multi-flow calibration is well-adapted to the

limitations of MIP OES and may contribute to expanding its analytical utility. Although

not as effective as standard additions and other matrix-matching methods, MFC has the

ability to compensate for matrix effects, which improves on the shortcomings of combining

MIP OES with traditional external standard calibration. Because the properties of the

plasma change significantly with the flow rate of the solution entering the MIP,

nebulization gas flow rate can be used in the analyst’s favor. In particular, the performance

of the combined MFC-MIP OES strategy at low concentrations (i.e. ppb-level) is an

important avenue for further development.

The developments described in this dissertation represent a significant step forward

in the expansion of MIP OES as a mainstream technique in atomic spectrometry. The future

of MIP OES as an important tool for routine elemental analysis is promising. While it still

requires improvements relative to other, more established techniques, the gap is closing

and the niche for MIP OES in the atomic spectrometry market is becoming wider and more

secure.

126

APPENDIX A

SUPPLEMENTARY INFORMATION FOR CHAPTER III

NATURALLY OCCURRING MOLECULAR SPECIES USED FOR PLASMA

DIAGNOSTICS AND SIGNAL CORRECTION IN MICROWAVE-INDUCED



This appendix presents material published online as Electronic Supplementary

Information to accompany Chapter III, by the Journal of Analytical Atomic

Spectrometry, 2018, 33, 1224-1232. All of the presented research was conduced by

Charles B. Williams. The supplementary information section was prepared by Charles B.

Williams and George L. Donati.

127

EMISSION SPECTRA FOR CN, N2, N2+ AND OH

Figs. S1 - S4 show emission spectra recorded in wavelength regions

corresponding to CN, N2, N2+ and OH, respectively. The spectra were collected with the

microwave-induced plasma (MIP) either on or off to demonstrate the origin of these

molecular species in the N2 plasma. The CN emission band corresponds to the B(2∑+) -

X(2∑+) electronic transition, with a band peak at 387.147 nm. The N2, N2+, and OH

emission bands correspond to the C(3∏u) - B(3∏g), B(2∑u+) - X(2∑g

+), and A(2∑+) - X(2∏i

+) electronic transitions, with band peaks at 337.097, 391.439, and 308.970 nm,

respectively.1

Figs. S5 - S8 show the spectra collected for these same molecular species when the

instrument’s spray chamber was removed (i.e., no aqueous solution was being introduced

into the plasma), as well as when distilled-deionized water or 1 % v v-1 HNO3 were

introduced into the MIP. Note that N2+ is originally part of the MIP (Fig. S7), while OH is

mainly produced as an aqueous solution is introduced into the plasma (Fig. S8).

128

Figure S 1. Molecular emission spectra for CN recorded with the plasma off, or as a 1 % v

v-1 HNO3 solution was introduced into the MIP at a nebulization gas flow rate of

0.7 L min-1.

Figure S 2. Molecular emission spectra for N2 recorded with the plasma off, or as a 1 % v

v-1 HNO3 solution was introduced into the MIP at a nebulization gas flow rate of 0.7

L min-1.

129

Figure S 3. Molecular emission spectra for N2+ recorded with the plasma off, or as a 1 %

v v-1 HNO3 solution was introduced into the MIP at a nebulization gas flow rate of 0.7 L

min-1.

Figure S 4. Molecular emission spectra for OH recorded with the plasma off, or as a 1 % v

v-1 HNO3 solution was introduced into the MIP at a nebulization gas flow rate of 0.7

L min-1.

130

Figure S 5. Molecular emission spectra for CN recorded as 1 % v v-1 HNO3, distilled-


introduced into the MIP.

Figure S 6. Molecular emission spectra for N2 recorded as 1 % v v-1 HNO3, distilled-



131

Figure S 7. Molecular emission spectra for N2+ recorded as no aqueous solution (no spray

chamber), distilled-deionized water, or 1 % v v-1 HNO3 (NGFR = 0.7 L min-1) was


Figure S 8. Molecular emission spectra for OH recorded as 1 % v v-1 HNO3, distilled-



132

EVALUATION OF INDIVIDUAL EMISSION SIGNALS AND COMBINATIONS

OF CN, N2, N2+ AND OH INTENSITIES AS PLASMA DIAGNOSTIC TOOLS

To evaluate the efficiency of some of the MIP’s naturally occurring species as

diagnostic tools, individual emission signals for CN, N2, N2+ and OH, as well as their

combination in the form of signal ratios (i.e. N2+/OH, N2

+/N2, N2+/CN, N2/OH, N2/CN

and CN/OH) were studied. The performance criterion was the sensitivity to plasma

changes caused by the introduction of high concentrations of Na into the MIP at different

nebulization gas flow rates (NGFR). The molecular species were also compared with Mg

II and Mg I lines, and with the Mg II/Mg I signal ratio. As shown in Figs. S9 - S12, the

N2+/OH signal ratio was the most sensitive to plasma change across all conditions

evaluated. For example, when the Na concentration increased from 200 to 500 mg L-1, at

a NGFR of 0.6 L min-1, the N2+/OH signal ratio dropped more than 43 %, from 1.173 to

0.666 (absolute percent change of 43.2 %). For comparison at the same conditions, the

Mg II/Mg I, N2+/N2, N2

+/CN, N2/OH, N2/CN and CN/OH ratios changed 22.3, 34.1, 21.9,

13.9, 18.6 and 27.4 %, respectively. If a more drastic change takes place, as for example

with the Na concentration increasing from 0 to 500 mg L-1 (not shown in Figs. S9 - S12),

the N2+/OH signal ratio will change 65.2 %, as opposed to 33.2, 51.6, 32.1, 28.1, 40.2 and

48.7 % for Mg II/Mg I, N2+/N2, N2

+/CN, N2/OH, N2/CN and CN/OH, respectively.

Based on these results, and considering Na as a model for EIEs, which are

responsible for some of the most severe matrix effects observed in MIP OES,2 the

N2+/OH signal ratio was further studied as a diagnostic tool in the present work.

133


mg L-1 range were introduced into the MIP at a NGFR of 0.6 L min-1. Species 1 - 6

correspond to Mg II (280.271 nm), Mg I (285.213 nm), CN (387.147 nm), N2 (337.097

nm), N2+ (391.439 nm) and OH (308.970 nm), respectively.


mg L-1 range were introduced into the MIP at a NGFR of 1.0 L min-1. Species 1 - 6

correspond to Mg II (280.271 nm), Mg I (285.213 nm), CN (387.147 nm), N2 (337.097

nm), N2+ (391.439 nm) and OH (308.970 nm), respectively.

134


L-1 range were introduced into the MIP at a NGFR of 0.6 L min-1. Here, 1 - 7 correspond

to Mg II/Mg I, N2+/OH, N2

+/N2, N2+/CN, N2/OH, N2/CN, and CN/OH, respectively.


L-1 range were introduced into the MIP at a NGFR of 1.0 L min-1. Here, 1 - 7 correspond

to Mg II/Mg I, N2+/OH, N2

+/N2, N2+/CN, N2/OH, N2/CN, and CN/OH, respectively.

135

REFERENCES

1. Z. Zhang and K. Wagatsuma, Spectrochim. Acta Part B, 2002, 57, 1247-

1257.

2. K.P. Huber, G. Herzberg, Constants of Diatomic Molecules, in: P.J.

Linstrom, W.G. Mallard (Eds.), NIST Chemistry WebBook, NIST Standard Reference

Database, Num. 69, National Institute of Standards and Technology, Gaithersburg, MD,

USA, 2016 (Available at http://webbook.nist.gov (retrieved May 4, 2018)).

136

SCHOLASTICA VITA

CHARLES BRYSON WILLIAMS, III

BORN: December 17, 1992, Winston-Salem, NC

UNDERGRADUATE STUDY: Clemson University

Clemson, South Carolina

B.S. Chemistry, 2015

GRADUATE STUDY: Wake Forest University

Winston-Salem, North Carolina

PhD. 2019

SCHOLASTIC AND PROFESSIONAL EXPERIENCE:

Graduate Teaching Assistant

Wake Forest University, 2015-2017

Graduate Research Assistant

Wake Forest University, 2017-2019

Organist-Choir Director

Ascension Episcopal Church, 2013-2015

Organist

St. Anne’s Episcopal Church, 2016-2019

PROFESSIONAL SOCIETIES:

Society for Applied Spectroscopy, 2018-Present

137

HONORS AND AWARDS:

Merck Index Award, 2015

Glaxo-Smith-Kline Assistantship, 2015-2016

Alumni Student Travel Award, 2017, 2018

American Institute of Chemists Graduate Student Award, 2019

PUBLICATIONS:

G.L. Donati, R.S. Amais, C.B. Williams, Recent advances in inductively coupled plasma optical emission spectrometry, Journal of Analytical Atomic Spectrometry. 32 (2017) 1283–1296. doi:10.1039/C7JA00103G. C.B. Williams, T.G. Wittmann, T. McSweeney, P. Elliott, B.T. Jones, G.L. Donati, Dry ashing and microwave-induced plasma optical emission spectrometry as a fast and cost-effective strategy for trace element analysis, Microchemical Journal. 132 (2017) 15–19. doi:10.1016/j.microc.2016.12.017. A.G. Althoff, C.B. Williams, T. McSweeney, D.A. Gonçalves, G.L. Donati, Microwave-Induced Plasma Optical Emission Spectrometry (MIP OES) and Standard Dilution Analysis to Determine Trace Elements in Pharmaceutical Samples, Applied Spectroscopy. 71 (2017) 2692–2698. doi:10.1177/0003702817721750. C.B. Williams, G.L. Donati, Multispecies calibration: a novel application for inductively coupled plasma tandem mass spectrometry, J. Anal. At. Spectrom. 33 (2018) 762–767. doi:10.1039/C8JA00034D. C.B. Williams, B.T. Jones, G.L. Donati, Naturally occurring molecular species used for plasma diagnostics and signal correction in microwave-induced plasma optical emission spectrometry, Journal of Analytical Atomic Spectrometry. 33 (2018) 1224–1232. doi:10.1039/C8JA00086G. H. Li, Q. Li, P. Wen, T.B. Williams, S. Adhikari, C. Dun, C. Lu, D. Itanze, L. Jiang, D.L. Carroll, G.L. Donati, P.M. Lundin, Y. Qiu, S.M. Geyer, Colloidal Cobalt Phosphide Nanocrystals as Trifunctional Electrocatalysts for Overall Water Splitting Powered by a Zinc-Air Battery, Advanced Materials. 30 (2018) 1705796. doi:10.1002/adma.201705796. D.R. Onken, S. Gridin, R.T. Williams, C.B. Williams, G.L. Donati, V. Gayshan, S. Vasyukov, A. Gektin, Investigating the origins of double photopeaks in CsI:Tl samples through activator mapping, Nuclear Instruments and Methods in Physics

https://doi.org/10.1039/C7JA00103G

https://doi.org/10.1016/j.microc.2016.12.017

https://doi.org/10.1177/0003702817721750

https://doi.org/10.1039/C8JA00034D


https://doi.org/10.1002/adma.201705796

138

Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment. 893 (2018) 151–156. doi:10.1016/j.nima.2018.03.028. C.B. Williams, T. McSweeney, B.T. Jones, G.L. Donati, Determination of Ca, K and Na in Soft Drinks Using MP-AES, (2018). C.B. Williams, B.T. Jones, G.L. Donati, Multi-flow calibration applied to microwave-induced plasma optical emission spectrometry, Journal of Analytical Atomic Spectrometry. (2019). doi:10.1039/C9JA00091G.

CONFERENCE PRESENTATIONS

“Use of N2+ Emission Intensity To Estimate Plasma Robustness in

Microwave-Induced Plasma Optical Emission Spectrometry (MIP OES),” Oral

Presentation, Winter Conference on Plasma Spectrochemistry, Amelia Island,

Florida, January 13, 2018

“Multi-Flow Calibration as a Novel Strategy in MIP OES,” Poster

Presentation, FACSS SCIX, Atlanta, GA, October 24, 2018

https://doi.org/10.1016/j.nima.2018.03.028
