1. INTRODUCTION 1.1 Introduction to Nestle
Nestlé was founded in 1867 on the shores of Lake Geneva in Vevey, Switzerland and its first product was “Farine Lactée Nestlé”, an infant cereal specially formulated by Henri Nestlé to provide and improve infant nutrition. From its first historic merger with the Anglo-Swiss Condensed Milk Company in 1905, Nestlé has grown to become the world’s largest and most diversified food Company, and is about twice the size of its nearest competitor in the food and beverages sector.
Nestlé’s activities contribute to and nurture the sustainable economic development of people, communities and nations. Above all, Nestlé is dedicated to bringing the joy of ‘Good Food, Good Life’ to people throughout their lives, throughout the world.
1.2 History Of Nestle

* The Nestlé Company was Henri Nestlé's search for a healthy, economical alternative to breastfeeding for mothers who could not feed their infants at the breast. * The Company formed by the 1905 merger was called the Nestlé and Anglo- Swiss Milk Company. The close of World War II marked the beginning of the most dynamic phase of Nestlé's history. Throughout this period, Nestlé's growth was based on its policy of diversifying within the food sector to meet the needs of consumers. * In 1947, Nestlé merged with Alimentana S.A., the manufacturer of Maggi seasonings and soups, becoming Nestlé Alimentana Company. After the agreement with L’Oreal in 1974, Nestlé's overall position changed rapidly. * Between 1975 and 1977, the price of coffee beans quadrupled, and the price of cocoa tripled. Taking such a step in a time of increased competition and shrinking profit margins required boldness and vision. * Nestlé approached the 1980s with a renewed flexibility and determination to evolve. Thus, between 1980 and 1984, the Company divested a number of non-strategic or unprofitable businesses. Nestlé managed to put an end to a serious controversy over its marketing of infant formula. * Nestlé opened the 20th century by merging with the Anglo-Swiss Condensed Milk Company to broaden its product range and widen its geographical scope. In the new millennium, Nestlé is the undisputed leader in the food industry, with more than 470 factories around the world

1.3 Nestle India

Nestlé set up its operations in India, as a trading company in 1912 and began manufacturing at the Moga factory in 1962. The production started with the manufacture of Milkmaid and other product was gradually brought into the fold. Nestlé India Limited was formally incorporated in 1978 prior to which the manufacturing license was issued in the name of the Food Specialties Limited. The corporate office is located at Gurgaon and the registered office at M-5A, Connaught Circus, and New Delhi.

Figure 1: Presence across India

Beginning with its first investment in Moga in 1961, Nestlé’s regular and substantial investments established that it was here to stay. In 1967, Nestlé set up its next factory at Choladi (Tamil Nadu) as a pilot plant to process the tea grown in the area into soluble tea. The Nanjangud factory (Karnataka), became operational in 1989, the Samalkha factory (Haryana), in 1993 and in 1995 and 1997, Nestlé commissioned two factories in Goa at Ponda and Bicholim respectively. Nestlé India has commissioned in 2006 its 7th factory at Pant Nagar in Uttarakhand.

At present Nestlé have 8 manufacturing units countrywide which are successfully engaged in meeting the domestic as well as the exports demand. In addition there are several co packing units. Presently Nestlé India employs over 3000 employees. In addition, thousands of people are associated with Nestlé, having indirect employment as Milk suppliers, Vendors, Contractors and Distributions etc.

The production group first incepted under the name Food Specialties Ltd. started production at the Nestlé Moga Factory in early 1962.At the time, with the help of Nestle International; the company shot into prominence as the country’s foremost producer of milk products, particularly baby foods.

Nestlé India manufactures products of truly international quality under internationally famous brand names such as NESCAFÉ, MAGGI, MILKYBAR, MILO, KIT KAT, BAR-ONE, MILKMAID and NESTEA and in recent years the Company has also introduced products of daily consumption and use such as NESTLÉ Milk, NESTLÉ SKIM Milk, NESTLÉ Fresh 'n' Natural Dahi and NESTLÉ Jeera Raita.

Nestlé India is a responsible organization and facilitates initiatives that help to improve the quality of life in the communities where it operates.

1.4 DIFFERENT FACTORIES OF NESTLE INDIA

MOGA Factory
Moga factory started production in 1962. Today, Moga is contributing almost 75% of Nestle India’s total production volume and manufacturing 109671 tons of food products. It employs around 1000 people. Dairy creamers, IMF, SCM, Cereals, Vending Mixes, Noodles, Ketchups, Bouillon are manufactured in Moga Factory. Moga is located in Punjab State about 400 kilometers North of Delhi.
MOGA Factory
Moga factory started production in 1962. Today, Moga is contributing almost 75% of Nestle India’s total production volume and manufacturing 109671 tons of food products. It employs around 1000 people. Dairy creamers, IMF, SCM, Cereals, Vending Mixes, Noodles, Ketchups, Bouillon are manufactured in Moga Factory. Moga is located in Punjab State about 400 kilometers North of Delhi.

Choladi Factory
The factory ion Choladi started production in 1967, Situated in South Asia, about 275 kilometers from Bangalore. The factory today has around 50 employees. It processes about 725 tons of soluble tea, which is all exported.
Choladi Factory
The factory ion Choladi started production in 1967, Situated in South Asia, about 275 kilometers from Bangalore. The factory today has around 50 employees. It processes about 725 tons of soluble tea, which is all exported.

Nanjangud Factory
Production in Nanjangud Factory started in 1989 with the manufacturing of Nescafe and Sunrise. Milo manufacture at Nanjangud began in 1996. It situated 160 kilometers south of Bangalore; the factory has around 200 employees. It manufactures 15500 tons of Nescafe mixes, Milo.

Nanjangud Factory
Production in Nanjangud Factory started in 1989 with the manufacturing of Nescafe and Sunrise. Milo manufacture at Nanjangud began in 1996. It situated 160 kilometers south of Bangalore; the factory has around 200 employees. It manufactures 15500 tons of Nescafe mixes, Milo.

Bicholim Factory
A satellite factory of Ponda at Bicholim for manufacturing of Noodles and Cold Sauces, It started their operational activity in 1997.
Bicholim Factory
A satellite factory of Ponda at Bicholim for manufacturing of Noodles and Cold Sauces, It started their operational activity in 1997.

Ponda Factory
Ponds Factory started production of Kit Kat in 1995. It is located 40 kilometers from Panji ‘the capital of Goa’. It manufactures Chocolates. Ponda currently employed around 250 people.
Ponda Factory
Ponds Factory started production of Kit Kat in 1995. It is located 40 kilometers from Panji ‘the capital of Goa’. It manufactures Chocolates. Ponda currently employed around 250 people.

Samalkha Factory
Samalkha Factory started production in 1993 situated 70 kilometers from Delhi. It has 260 employees and manufactures about 35000 tons of food products comprising IMFs, Infant Cereals, Noodles, Chilled dairy.
Samalkha Factory
Samalkha Factory started production in 1993 situated 70 kilometers from Delhi. It has 260 employees and manufactures about 35000 tons of food products comprising IMFs, Infant Cereals, Noodles, Chilled dairy.

Pant Nagar Factory: This is the one of the newly situated and the 7th factory of the Nestle in the India. Pantnagar Factory began production of Noodles in 2006. Pant Nagar Factory: This is the one of the newly situated and the 7th factory of the Nestle in the India. Pantnagar Factory began production of Noodles in 2006.

Tahliwal Factory: This factory is still in its nascent stage and is used for production of newly launched Alladin Chocolates. Factory began production of chocolates in 2012. Beside this Nestle, India has the co-packing arrangements also: * PAAM- Noida (Noodles) * Nijjer- Amritsar (Sauces) * SAJ- Kolkata (Noodles) * Brar- Moga (Small pouches coffee, Everyday etc.) 1.5 THE MOGA FACTORY Nestlé India Ltd. MOGA factory is their oldest factory in India. With a layout spread over nearly 57 acres & having plants within the factory, and it is also the largest factory among 510 Nestlé Factories worldwide. The company started milk collection in Moga area in 15 Nov 1961 and on the first day 510Kg of milk was collected as from four villages and 180 farmers. From that day onwards company is collecting milk in the morning and evening. The capacity rose from 40,000 liters. Of milk per day in 1962 to over 9, 00,000 liters of milk till date.
The factory consists of production plants as under: * Milk Operations * Cereals * Culinary
These plants are briefly described below.
1.5.1. Milk Operations:-

This plant as the name suggests is engaged in the producing of milk and all the related activities that take place at MOGA Factory. This plant can be categorized into a number of sub-plants, which are discussed below in brief: * Fresh Milk Reception
The fresh milk, which is the fundamental constituent of various products, which are manufactured in the factor, is received in this area in tankers. There are 1100 agencies which supplies fresh milk. The fresh milk is supplied by various chilling centers including Nestlé’s four own chilling centers. Till now 500 FCT (farm cooling tanks) implemented for keep the quality of milk. Testing of fresh milk is done in fresh milk lab and is tested for FAT and SNF (Solid Non FATs) only. If any one of the tests is found to be positive then the tanker is rejected. * Ghee Plant:
The milk stored in soils D and E is used for manufacturing of Ghee, which is marked under the brand name EVERYDAY. In this plant milk is passed through two separations. A phase inversion from 40-45% cream in the 70-80% FAT in the second is obtained. The final concentration become 97% crude FAT. * Egrons:
Egron is a spray drier used to dry the milk, coffee liquid into power from by using hot air, in all there are four Egrons in MOGA Factory. Egron 2, 3, 4 are used for drying milk powders and No. 1 is used to drying coffee. The milk is then collected in tote bins and subsequent sent to power filling and packing.

* Powder Filling Plant:
The filling and the packing of milk like Everyday, Lactogens, Nestogen,Nestea, Badaam Milk and Cerelac Tin is done in this plant. There are five filling and packing lines for this purpose. Two of these are used in filling of Tins and other two are used to filling polypacks or bag-n-boxes required by production program and one line is for the bulk packing.

1.5.2. Cereals:-

The plant is engaged in the production of cereal-based baby food and infant formula. The production process consists of the addition of various Enzymes, Vitamins, Minerals and Fruit extracts to cereal base. There are three filling and packing line in the Cereal plant. On is for filling of 4000gm sachets and other for filling of 400gm tins. 5gm Everyday Creamer filling line machinery is also installed in the filling section. Though the manufacturing of products is a continuous three shift operation, only the Flexi runs for three shifts. It accounts for the filling of the 65% of the manufacturing products. The major products of cereal plant are Cereal Wheat, Apple, Orange and Vegetable.

1.5.3. Culinary:-

This plant is engaged in production of Noodles, Taste Makers, Soups, and Sauces etc. This plant is divided into two sections

* NOODLES The manufacturing of Noodles is a semi-automatic process. The Processes are:- i. Tipping of Wheat flour in the hoppers at start of line. ii. Mixing of dough releasing on the line. iii. Sheet formation with help of rollers. iv. Strand formation. v. Steaming Frying in oil. vi. Cooling vii. Wrapping cakes in sachets along with tastemaker. viii. Palestine of cases. * SEASONING The seasoning section is engaged it the manufacture of Taste marker soups cubs beside spice mixes for use in cold sauces. The major job in this area is the dry of various spices & powders and then packing them in the desired shape and from. Also the section is engaged in the manufacturing of hydrolyzed plant proteins a basic ingredient for most of seasoning products. The Products of this section are Maggi taste markers: Masala Chicken, Sweet & Sour, Maggi Soups (Chicken, Tomato, and Mushroom & Vegetable), Maggi Cubes, Maggi Super Seasoning, Mango Wonder-Mix, and Maggi Export Mixes. 1.6 Nestle Quality Assurance Centre (NQAC)

Nestle Quality Assurance Centre at Moga provides analytical service to various Nestlé factories & its Co-manufacturers/Co-packers in South Asia Region.
NQAC started in 1999. NQAC have 100% NABL (National Accreditation Board for Calibration & testing Lab) accreditation as per ISO-IEC17025.
IT Awarded Last 3 Years for Excellent in P- Test (proficiency Test) All Analysis done under Scope of ISO from April onwards it do 5 Day Tat ( Turn Around Time). It also provides support trials in any factory of nestle & also supporting to New Product Launching.
NQAC checks Approx. 50% release Criteria.
NQAC analyses the finished products manufactured by Nestlé factories & Co-manufacturers as well as raw materials.
In addition, microbiological checks are also performed on line & environmental samples taken from production area.
The products most commonly analyzed at NQAC are:
Finished products-
Dietetic and non-dietetic milk powder, Infant cereals
Beverages: instant coffee malted drinks, tea etc.
Liquid milks: sweetened condensed milk, sterilised and evaporated milk, UHT milk etc.
Culinary Product: Liquid Sauces, Noodles, Soups etc.
Confectionery Products
Drinking water etc.
Chilled dairy products: Yoghurt, butter, Cheese, Dahi etc.
Chocolates
Milkmaid-(pista kulfi, kesar kheer, badam kheer, eggless cake, instant besan ladoo)

Raw Material-
Apple crunches
Corn crunches
Spinaches
Carrot flasks
Corn flakes
Spice mix
Barley crunchy
Noodle cake
Lactose
Skimmed milk powder
Dry tapioca starch
Cinnamon powder
Vitamin premix
Garlic flakes
DW flour

LINE SAMPLES-
Cerelac line products
Brar line samples
Startup samples of different products
Line samples of all nestle factories of SARC region

ENVIORNMENT SAMPLES-
Swabs from different areas of the factory and lab
The basic functions of Nestle Quality Assurance Centre: * To provide analytical services to Nestlé factories & Co-manufacturers / Co-packers in South Asia Region * To co-ordinate & to provide assistance to Nestlé factories & Co-manufacturers / Co-packers in South Asia Region regarding proficiency tests. * To provide assistance to Nestlé factories & Co-manufacturers/ Co-packers in South Asia Region regarding Good Laboratory Practices (GLP) and to perform regular audits. * To co-ordinate Nestlé proficiency tests for external labs also takes audit of Factory. * To undertake any technical projects as required by the organisation without any potential conflict of interest. * It also checks minor parameter which not analysed in factory QA lab. * It take part P test , to make sure its Quality System more Reliable
NQAC includes 3 Main Sections: * Contaminants ; * Nutrition ; * Microbiology.
It is the Nutrition section, where the Multi-elemental Mineral analysis is carried out.

MINERALS | SERIAL No. | PARAMETER | UNIT | INSTRUMENT | 1 | Sodium | mg/100g | ICP-OES | 2 | Potassium | mg/100g | ICP-OES | 3 | Calcium | mg/100g | ICP-OES | 4 | Magnesium | mg/100g | ICP-OES | 5 | Iron | mg/100g | ICP-OES | 6 | Zinc | mg/100g | ICP-OES | 7 | Copper | mcg/100g | ICP-OES | 8 | Manganese | mcg/100g | ICP-OES | 9 | Phosphorus | mg/100g | ICP-OES |

2. REVIEW OF LITERATURE

2.1 Minerals and their Requirements

Dietary minerals (also known as mineral nutrients) are the chemical elements required by living organisms, other than the four elements carbon, hydrogen, nitrogen, and oxygen present in common organic molecules.
Examples of mineral elements include calcium, magnesium, potassium, sodium, zinc, and iodine. Most minerals that enter into the dietary physiology of organisms consist of simple chemical elements. Larger aggregates of minerals need to be broken down for absorption.
The following play important roles in biological processes: Dietary element | RDA/AI | Description | Category | Insufficiency | Excess | Potassium | 4700 mg | Quantity | A systemic electrolyte and is essential in coregulating ATP with sodium. Dietary sources include legumes, potato skin, tomatoes, and bananas | Hypokalemia | Hyperkalemia | Chlorine | 2300 mg | Quantity | Needed for production of hydrochloric acid in the stomach and in cellular pump functions. Table salt is the main dietary source. | Hypochloremia | Hyperchloremia | Sodium | 1500 mg | Quantity | A systemic electrolyte and is essential in coregulating ATP with potassium. Dietary sources include spinach, vegetables. | Hyponatremia | Hypernatremia | Calcium | 1300 mg | Quantity | Needed for muscle, heart and digestive system health, builds bone, supports synthesis and function of blood cells. | Hypocalcaemia | Hypercalcaemia | Phosphorus | 700 mg | Quantity | Component of bones, cells, in energy processing and many other functions. In biological contexts, usually seen as phosphate. | Hypophosphatemia | Hyperphosphtemia | Magnesium | 420 mg | Quantity | Required for processing ATP and for bones. Dietary sources include nuts, soy beans, and cocoa mass. | Hypomagnesemia | Hypermagnesemia | Zinc | 11 mg | Trace | Pervasive and required for several enzymes such as carboxypeptidase, liver alcohol dehydrogenase, and carbonic anhydrase. | Zinc deficiency | Zinc toxicity | Iron | 18 mg | Trace | Required for many proteins and enzymes, notably hemoglobin to prevent anemia. Dietary sources include red meat, leafy green vegetables, fish (salmon, Tuna), eggs, dried fruits and beans | Anaemia | Iron overload disorder | Manganese | 2.3 mg | Trace | A cofactor in enzymefunctions. | Manganese deficiency | Manganism | Copper | 900 µg | Trace | Required component of many redox enzymes, including cytochrome c oxidase. | Copper deficiency | Copper toxicity |
Table 1: Various nutritional elements, their daily requirements, functions and diseases related to them
Dietitians may recommend that dietary elements are best supplied by ingesting specific foods rich with the chemical elements of interest. The elements may be naturally present in the food or added to the food.
Dietary supplements & Infant Foods can be formulated to contain several different chemical elements, a combination of vitamins and other chemical compounds, or a single element, such as calcium or magnesium, chromium.
Appropriate intake levels of certain chemical elements have been demonstrated to be required to maintain optimal health. Diet can meet all the body's chemical element requirements, although supplements can be used when some requirements are not adequately met by the diet, or when chronic or acute deficiencies arise from pathology, injury, etc.
2.1.1 Need for Mineral Analysis

Different food products contain a range of components procured from various natural sources contributing to the heterogeneity in the Nutrient Composition of the final processed food merchandise.

The PFA & Nestlé has set specific norms regarding the concentration of individual nutrient in any food commodity. The processing & fortification must be hence, carried out in a way that the declared norms are strictly followed & there are no alterations in the specifications.
Hence, chemical analysis of random samples has to be carried out from different batches of manufactured food products to evaluate the nutritional specifications quantitatively.
In case, the processed food is known to deviate from the specified norms the batch is held from release into the market as the consumption of such products may lead to serious tribulations as a result of lack or excess of specific nutrients.

2.1.2 Various Methods used for Mineral Analysis

There are many methods through which analyze minerals quantitatively. Some of these methods are mentioned below * Classical Wet Chemical Methods * Atomic Emission Spectrometry * Atomic Absorption Spectrometry * X-Ray Fluorescence Analysis * Neutron Activation Analysis * Mass Spectrometry * Beam Methods * Secondary Ion Mass Spectrometry * Electron Microprobe Analysis * Proton Induced X-Ray Emission * Gamma Ray Emission
Inductively Coupled Plasma-Optical Emission Spectroscopy is based on the principle of Atomic emission spectroscopy, which is explained in detail in the next section.

2.2 Atomic Spectroscopy
By using atomic spectrometry techniques, meaningful quantitative and qualitative information about a sample can be obtained. In general, quantitative information (concentration) is related to the amount of electromagnetic radiation that is emitted or absorbed while qualitative information (what elements are present) is related to the wavelengths at which the radiation is absorbed or emitted.

An affiliated technique to atomic emission or absorption spectrometry is atomic mass spectrometry. In mass spectrometry, instead of obtaining analytical information from the radiation of atoms or ions, ions introduced into a mass spectrometer are separated according to their mass to charge ratio and are either qualitatively or quantitatively detected.

2.2.1 Nature of Atomic or Ionic Spectra

The measurement of absorption and emission of electromagnetic radiation can be more easily described once the nature of atomic and ionic spectra is understood. Consider the Bohr model of an atom shown in Figure 1. The atom is depicted as a nucleus surrounded by electrons which travel around the nucleus in discrete orbitals. Every atom has a number of orbitals in which it is possible for electrons to travel. Each of these electron orbitals has an energy level associated with it. In general, the farther the nucleus from an orbital, the higher its energy level.

When the electrons of an atom are in the orbitals closest to the nucleus and lowest in energy, the atom is in its most preferred and stable state, known as its ground state. When energy is added to the atom as the result of absorption of electromagnetic radiation or a collision with

Figure 2 Bohr model of an atom. As energy is absorbed by an atom, an electron jumps to an orbital with a higher energy level. The atom may decay to a lower energy state by emitting a photon, hv.

another particle (electron, atom, ion, or molecule), one or more of several possible phenomena take place. The two most probable events are for the energy to be used to increase the kinetic energy of the atom (i.e., increase the velocity of the atom) or for the atom to absorb the energy and become excited. This latter process is known as excitation.

When an atom becomes excited, an electron from that atom is promoted from its ground state orbital into an orbital further from the nucleus and with a higher energy level. Such an atom is said to be in an excited state. An atom is less stable in its excited state and will thus decay back to a less excited state by losing energy through a collision with another particle or by emission of a "particle" of electromagnetic radiation, known as a photon. As a result of this energy loss, the electron returns to an orbital closer to the nucleus.

If the energy absorbed by an atom is high enough, an electron may be completely dissociated from the atom, leaving an ion with a net positive charge. The energy required for this process, known as ionization, is called the ionization potential and is different for each element. Ions also have ground and excited states through which they can absorb and emit energy by the same excitation and decay processes as an atom.

Figure 2, shows the excitation, ionization and emission processes schematically. The horizontal lines of this simplified diagram represent the energy levels of an atom. The vertical arrows represent energy transitions, or changes in the amount of energy of an electron. The energy transitions in an atom or ion can be either radiational or thermal.

The difference in energy between the upper and lower energy levels of a radiative transition defines the wavelength of the radiation that is involved in that transition.

The relationship between this energy difference and wavelength can be derived through Planck’s equation

E = hv

Figure 3 Energy level diagram depicting energy transitions where a and b rep- resent excitation, c is ionization, d is ionization/excitation, e is ion emission, and f, g and h are atom emission.

Where, E is the energy difference between two levels, h is Planck’s constant, and v is the frequency of the radiation. Substituting c/ λ for n, where c is the speed of light and is λ, the wavelength, we get, E = hc/ λ

This equation shows that energy and wavelength are inversely related, i.e., as the energy increases, the wavelength decreases, and vice versa. Using Figure 1-2 as an example, the wavelength for emission transition f is longer than the wavelength for emission transition g since the energy difference for f is less than for transition g.

Every element has its own characteristic set of energy levels and thus its own unique set of absorption and emission wavelengths. It is this property that makes atomic spectrometry useful for element-specific analytical techniques.

The ultraviolet (UV)/visible region (160 - 800 nm) of the electromagnetic spectrum is the region most commonly used for analytical atomic spectrometry. This is also the region of the electromagnetic spectrum that we generally refer to as "light", although technically, all electromagnetic radiation can be considered as light. For further discussions in this book, the term "light" will often be used in place of "UV/visible electromagnetic radiation".

The principal reasons for the popularity of analytical techniques that use the UV/visible region are that these techniques are accurate, precise, flexible, and relatively inexpensive compared to techniques which use other regions, such as gamma ray spectrometry and X-ray spectrometry. Many of the devices used in UV/visible atomic spectrometry, such as photomultipliers and gratings, are relatively inexpensive since they were developed for and are commonly used in high-volume applications. Also, unlike gamma rays and X-rays, UV/visible radiation is not ionizing radiation. This lessens the expenses associated with shielding and licensing of the laboratory and with disposal of analyzed samples.

2.2.2 Analytical Techniques Based on Atomic Spectrometry

In the atomic spectrometry techniques most commonly used for trace element analysis, the sample is decomposed by intense heat into a cloud of hot gases containing free atoms

Figure 3 Atomic spectrometry systems

and ions of the element of interest. Figure 3 shows the instrumental arrangements for four different techniques used to detect these atoms or ions.

In atomic absorption spectrometry (AAS), light of a wavelength characteristic of the element of interest is shone through this atomic vapor. Some of this light is then absorbed by the atoms of that element. The amount of light that is absorbed by these atoms is then measured and used to determine the concentration of that element in the sample.

In optical emission spectrometry (OES), the sample is subjected to temperatures high enough to cause not only dissociation into atoms but to cause significant amounts of collisional excitation (and ionization) of the sample atoms to take place. Once the atoms or ions are in their excited states, they can decay to lower states through thermal or radiative (emission) energy transitions. In OES, the intensity of the light emitted at specific wavelengths is measured and used to determine the concentrations of the elements of interest.

One of the most important advantages of OES results from the excitation proper- ties of the high temperature sources used in OES. These thermal excitation sources can populate a large number of different energy levels for several different elements at the same time. All of the excited atoms and ions can then emit their characteristic radiation at nearly the same time. This results in the flexibility to choose from several different emission wavelengths for an element and in the ability to measure emission from several different elements concurrently. However, a disadvantage associated with this feature is that as the number of emission wavelengths increase, the probability also increases for interferences that may arise from emission lines that are too close in wavelength to be measured separately.

In atomic fluorescence spectrometry (AFS), a light source, such as that used for AAS, is used to excite atoms only of the element of interest through radiative absorption transitions. When these selectively excited atoms decay through radiative transitions to lower levels, their emission is measured to determine concentration, much the same as in OES. The selective excitation of the AFS technique can lead to lesser spectral interference than in OES. However, it is difficult to detect a large number of elements in a single run using AFS, as the number of spectral excitation sources and detectors that can be used at one time is limited by the instrument.

Another technique, called atomic mass spectrometry, is related to three atomic spectroscopy techniques described above. Instead of measuring the absorption, emission or fluorescence of radiation from a high temperature source, such as a flame or plasma, mass spectrometry measures the number of singly charged ions from the elemental species within a sample. Similar to the function of a monochromator in emission/absorption spectrometry that separates light according to wave length, a quadruple mass spectrometer separates the ions of various elements according to their mass-to-charge ratio in atomic mass spectrometry.

2.2.3 Atomization and Excitation Sources

In general, there are three types of thermal sources normally used in analytical atomic spectrometry to dissociate sample molecules into free atoms: flames, furnaces and electrical discharges. High-power lasers have also been used for this purpose but tend to be better suited for other uses such as solids sampling for other atomization sources.

The first two types of sources, flames and furnaces, are hot enough to dissociate most types of molecules into free atoms. The main exceptions are refractory carbides and oxides, which can exist as molecules at the upper flame and furnace temperatures of 3000 - 4000 °K. When configured properly, flames and furnaces can also be used to excite many elements for emission spectrometry. Because most of the free atoms in typical flames and furnaces are in their ground states, however, absorption spectrometry is the preferred method to detect the presence of elements of interest. The exceptions are those elements whose lowest excited state is low enough in energy that it can be easily populated by a flame or furnace. Examples of such elements are lithium, sodium and potassium. In fact, flame emission spectrometry is still widely regarded as the preferred method for detecting the alkali elements.

Electrical discharges are the third type of atomization sources used in analytical optical emission spectrometry. For many years, dc arcs and ac sparks were the mainstay of OES. These electrical discharges are created by applied currents or potentials across an electrode in an inert gas and typically produce higher temperatures than traditional flame systems.

More recently, other types of discharges, namely plasmas, have been used as atomization/excitation sources for OES. Strictly speaking, a plasma is any form of matter that contains an appreciable fraction (>1%) of electrons and positive ions in addition to neutral atoms, radicals and molecules. Two characteristics of plasmas are that they can conduct electricity and are affected by a magnetic field.
The electrical plasmas used for analytical OES are highly energetic, ionized gases. They are usually produced in inert gases, although some work has also been done using reactive gases such as oxygen. These plasma discharges are considerably hotter than flames and furnaces and, thus, are used not only to dissociate almost any type of sample but also to excite and/or ionize the atoms for atomic and ionic emission. The present state-of-the-art in plasma sources for analytical optical emission spectrometry is the argon-supported inductively coupled plasma (ICP). Other plasmas currently being used include the direct current plasma (DCP) and the microwave induced plasma (MIP).

Because the argon ICP can efficiently generate singly charged ions from the elemental species within a sample, it makes an ideal ion source to use synergistically with mass spectrometers. This combination of an ICP and mass spectrometer is called ICP-MS.

2.3 Inductively Coupled Plasma- Optical Emission Spectroscopy

This instrument was developed by Stanley Greenfield 1964 and is an analytical tool used for the determination of 75 elements and their states (oxidation, isotopic, etc.) in a wide range of sample matrices. Elements that are not determined are those already in the plasma from sources not in the sample, argon, carbon dioxide, hydrogen, oxygen, and nitrogen that are in the air surrounding conventional plasma, and elements that require very high energy, such as the halogens. The argon gas used to generate this high temperature has many advantages in that it forms a stable and chemically inert environment, which eliminates many of the interferences encountered with combustion flames. Plasmas are used for emission because the temperature is high enough to excite most of these elements. Detection of elements at its wavelengths is usually by photomultiplier tube(s) (PMT) or by charge coupled device (CCD). More expensive ICPs are designed to direct individual emission lines from different elements in the plasma to individual detectors. Such instruments allow simultaneous multi-elemental analyses that are rapid and carry out considerably more analysis in a shorter analysis time.

2.3.1 The ICP Discharge

The inductively coupled plasma discharge used today for optical emission spectrometry is very much the same in appearance as the one described by Velmer Fassel in the early 1970’s. Argon gas is directed through a torch consisting of three concentric tubes made of quartz or some other suitable material, as shown in Figure 4. A copper coil, called the load coil, surrounds the top end of the torch and is connected to a radio frequency (RF) generator.

When RF power is applied to the load coil, an alternating current moves back and forth within the coil, or oscillates, at a rate corresponding to the frequency of the generator. In most ICP instruments this frequency is either 27 or 40 megahertz (MHz). This RF oscillation of the current in the coil causes RF electric and magnetic fields to be set up in the area at the top of the torch. With argon gas being swirled through the torch, a spark is applied to the gas causing some electrons to be stripped from their argon atoms. These electrons are then caught up in the magnetic field and accelerated by them. Adding energy to the electrons by the use of a coil in this manner is known as inductive coupling. These high-energy electrons in turn collide with other argon atoms, stripping off still more electrons. This collisional ionization of the argon gas continues in a chain reaction, breaking down the gas into a plasma consisting of argon atoms, electrons, and argon ions, forming what is known as an inductively coupled plasma (ICP) discharge. The ICP discharge is then sustained within the torch and load coil as RF energy is continually transferred to it through the inductive coupling process.

The ICP discharge appears as a very intense, brilliant white, teardrop-shaped discharge. Figure 5 shows a cross-sectional representation of the discharge along with the nomenclature for different regions of the plasma as suggested by Koirtyohann et al. At the base, the discharge is toroidal or "doughnut-shaped" because the sample-carrying nebulizer flow literally punches a hole through the center of the discharge. The body of the "doughnut" is called the induction region (IR) because this is the region in which the inductive energy transfer from the load coil to the plasma takes place. This is also the area from which most of the white light, called the argon continuum, is emitted. Allowing the sample to be introduced through the induction region and into the center of the plasma gives the ICP many of its unique analytical capabilities.

Figure 4 Cross section of an ICP torch and load coil depicting an ignition sequence. A - Argon gas is swirled through the torch. B - RF power is applied to the load coil. C - A spark produces some free electrons in the argon. D - The free electrons are accelerated by the RF fields causing further ionization and forming a plasma. E - The sample aerosol-carrying nebulizer flow punches a hole in the plasma.

Most samples begin as liquids that are nebulized into an aerosol, a very fine mist of sample droplets, in order to be introduced into the ICP. The sample aerosol is then carried into the center of the plasma by the inner (or nebulizer) argon flow. The functions of the ICP discharge at this point are several folds. Figure 6 depicts the processes that take place when a sample droplet is introduced into an ICP.

The first function of the high temperature plasma is to remove the solvent from, or desolvate, the aerosol, usually leaving the sample as microscopic salt particles. The next steps involve decomposing the salt particles into a gas of individual molecules (vaporization) that are then dissociated into atoms (atomization). These processes, which occur predominantly in the preheating zone (PHZ) shown in Figure 4, are the same processes that take place in flames and furnaces used for atomic absorption spectrometry.
Once the sample aerosol has been desolvated, vaporized and atomized, the plasma has one, or possibly two, functions remaining. These functions are excitation and ionization. As explained in earlier, in order for an atom or ion to emit its characteristic radiation, one of its electrons must be promoted to a higher energy level through an excitation process. Since many elements have their strongest emission lines emitted from the ICP by excited ions, the ionization process may also be necessary for some elements. The excitation and ionization processes occur predominantly in the initial radiation zone (IRZ) and the normal analytical zone (NAZ). The NAZ is the region of the plasma from which analyte emission is typically measured.

Figure 5 Zones of the ICP. IR - Induction Region, PHZ - Preheating Zone, IRZ - Initial Radiation Zone, NAZ - Normal Analytical Zone.

Figure 6 The Process that takes place when a sample droplet is introduced into an ICP discharge.

While the exact mechanisms for excitation and ionization in the ICP are not yet fully understood, it is believed that most of the excitation and ionization in the ICP takes place as a result of collisions of analyte atoms with energetic electrons. There is also some speculation about the role of argon ions in these processes. In any case, the chief analytical advantage of the ICP over other emission sources are derived from the ICP’s ability to vaporize, atomize, excite, and ionize efficiently and reproducibly a wide range of elements present in many different sample types.

One of the important reasons for the superiority of the ICP over flames and furnaces for the above is in the high temperature within the plasma. While flames and furnaces have upper temperature ranges in the area of 3300 K, the gas temperature in the center of the ICP is about 6800 K. Besides improving excitation and ionization efficiencies, the higher temperature of the ICP also reduces or eliminates many of the chemical interferences found in flames and furnaces.

Other electrical discharge emission sources, such as arcs, sparks, direct current plasmas, and microwave induced plasmas, also have high temperature and thus may be as efficient at excitation and ionization as the ICP. However, it is largely the ICP’s combination of stability and freedom from sample matrix interferences that makes the ICP a better source for atomic emission spectrometry than these other electrical discharge sources.

An important feature of the ICP that is not common to most other emission sources is that since the sample aerosol is introduced through the center of the ICP, it can be surrounded by the high temperature plasma for a comparatively long time, approximately 2 milliseconds. It is this long residence time of the analyte particles in the center of the plasma that is largely responsible for the lack of matrix interferences in the ICP. In addition, because the aerosol is in the center of the discharge and the energy-supplying load coil surrounds the outside of the plasma, the aerosol does not interfere with the transfer of the energy from the load coil to the discharge. In some other sources, the sample travels around the outside of the discharge where it does not experience uniform high temperature for as long. In the arcs and sparks, the sample may commingle with the entire electrical discharge and interfere with the production and sustainment of the discharge. These situations lead to the higher levels of matrix effects and poorer stability that are often characteristic of non-ICP discharges.

2.3.2 Detection of Emission

In ICP-OES, the light emitted by the excited atoms and ions in the plasma is measured to obtain information about the sample. Because the excited species in the plasma emit light at several different wavelengths, the emission from the plasma is polychromatic. This polychromatic radiation must be separated into individual wavelengths so the emission from each excited species can be identified and its intensity can be measured without interference from emission at other wavelengths. The separation of light according to wavelength is generally done using a monochromator, which is used to measure light at one wavelength at a time, or a polychromator, which can be used to measure light at several different wavelengths at once. The actual detection of the light, once it has been separated from other wavelengths, is done using a photosensitive detector such as a photo-multiplier tube (PMT) or advanced detector techniques such as a charge-injection device (CID) or a charge-coupled device (CCD).

2.3.3 Extraction of Information

Extracting qualitative and quantitative information about a sample using ICP-OES is generally straightforward. Obtaining qualitative information, i.e., what elements are present in the sample, involves identifying the presence of emission at the wavelengths characteristic of the elements of interest. In general, at least three spectral lines of the element are examined to be sure that the observed emission can be indeed classified as that be- longing to the element of inter- est. Occasional spectral line interferences from other elements may make one uncertain about the presence of an element in the plasma. Fortunately, the relatively large number of emission lines available for most elements allows one to overcome such interferences by choosing between several different emission lines for the element of interest
Obtaining quantitative information, i.e., how much of an element is in the sample, can be accomplished using plots of emission intensity versus concentration called calibration curves (Figure 8.). Solutions with known concentrations of the elements of interest, called standard solutions, are introduced into the ICP and the intensity of the characteristic emission for

Figure 7 Temperature regions of a typical ICP discharge.

.

each element, or analyte, is measured. These intensities can then be plotted against the concentrations of the standards to form a calibration curve for each element. When the emission intensity from an analyte is measured, the intensity is checked against that element’s calibration curve to determine the concentration corresponding to that intensity.

Figure 8 Calibration curve used for ICP-OES.

The computers and software used with ICP-OES instruments represent these calibration curves mathematically within the computer’s memory. Thus, it is not necessary for the analyst to construct these curves manually for quantitation of the elements in the sample. Because calibration curves are generally linear over four to six orders of magnitude in ICP-OES, it is usually necessary to measure only one or two standard solutions, plus a blank solution, to calibrate the ICP instrument. In contrast to ICP-OES, arc and spark sources require five or more standards per element because of nonlinear calibration curves. The nonlinearity in these sources is a direct result of self-absorption which is the process by which some of the emitted radiation of the analyte is absorbed by ground state atoms in the plasma. In conventional ICPs, nonlinearity in the calibration curves is usually only observed for high analyte concentrations; i.e., greater than 5 to 6 orders of magnitude above the detection limit.

2.3.4 ICP INSTRUMENTATION

In inductively coupled plasma-optical emission spectrometry, the sample is usually transported into the instrument as a stream of liquid sample. Inside the instrument, the liquid is converted into an aerosol through a process known as nebulization. The sample aerosol is then transported to the plasma where it is desolvated, vaporized, atomized, and excited and/or ionized by the plasma. The excited atoms and ions emit their characteristic radiation which is collected by a device that sorts the radiation by wavelength. The radiation is detected and turned into electronic signals that are converted into concentration information for the analyst. A representation of the layout of a typical ICP-OES instrument is shown in Figure 9.

Figure 9 Major components and layout of a typical ICP-OES instrument.

2.3.4.1. SAMPLE INTRODUCTION

Auto Sampler

The most widely used accessory for ICP-OES instruments is the autosampler. Typical autosamplers have a capacity of almost 100 samples while some larger models can hold 300 - 500 samples. The main function of the autosampler is to free the analyst from having to switch the sample input tubing manually from one solution to the next. Ideally, the analyst should be able to load the autosampler with standards and samples, start the analysis, walk away from the instrument, and return to find the analysis completed. This is called unattended operation and has particular relevance to laboratories with high sample throughput requirements.

PUMPS

Most nebulizers require that the solution be pumped into the nebulizer. With a pumped solution, the flow rate of the solution into the nebulizer is fixed and is not as dependent on solution parameters such as viscosity and surface tension. The controlled flow rate of liquid also allows for more rapid washout of the nebulizer and spray chamber

Peristaltic pumps, such as the one shown in Figure 10, are almost exclusively the pumps of choice for ICP-OES applications. These pumps utilize a series of rollers that push the sample solution through the tubing using a process known as peristalsis. The pump itself does not come in contact with the solution, only with the tubing that carries the solution from the sample vessel to the nebulizer. Thus, the potential for contamination of the solution that may exist with other types of pumps is not a concern.

Peristaltic pump tubing is the one part of an ICP system that usually requires frequent replacement. The analyst should check the pump tubing daily for wear, which is generally indicated by permanent depressions in the tubing that can be felt by running one’s fingers over the tubing

Figure 10 Peristaltic pump used for ICP-OES.
NEBULIZER:

Nebulizers are devices that convert a liquid into an aerosol that can be transported to the plasma. The nebulization process is one of the critical steps in ICP-OES. The ideal sample introduction system would be one that delivers all of the sample to the plasma in a form that the plasma could reproducibly desolvate, vaporize, atomize and ionize, and excite. Because only small droplets are useful in the ICP, the ability to produce small droplets for a wide variety of samples largely determines the utility of a nebulizer for ICP-OES.

Figure 11: Concentric nebulizer used for ICP-OES

SPRAY CHAMBER

Once the sample aerosol is created by the nebulizer, it must be transported to the torch so it can be injected into the plasma. Because only very small droplets in the aerosol are suitable for injection into the plasma, a spray chamber is placed between the nebulizer and the torch. The primary function of the spray chamber is to remove large droplets from the aerosol. A secondary purpose of the spray chamber is to smooth out pulses that occur during nebulization, often due to pumping of the solution.

In general, spray chambers for the ICP are designed to allow droplets with diameters of about 10 m or smaller to pass to the plasma. With typical nebulizers, this droplet range constitutes about 1 - 5% of the sample that is introduced to the nebulizer. The remaining 95 - 99% of the sample is drained into a waste container. The material from which a spray chamber is constructed can be an important characteristic of a spray chamber. Spray chambers made from corrosion-resistant materials allow the analyst to introduce samples containing hydrofluoric acid which could damage glass spray chambers.

Figure 12: Cyclonic Spray Chamber used in ICP-OES.

2.3.4.2 PRODUCTION OF EMISSION

Torches

The torches contain three concentric tubes for argon flow and aerosol injection. The space between the outer tubes is kept narrow so that the gas between them emerges at high velocity. The outside chamber is also designed to make gas spiral tangentially around the chamber as it proceeds upwards. One of the functions of this gas is to keep the quartz wall of the torch cool and thus this gas flow was originally called the coolant flow or plasma flow but is now called the "outer" gas flow. For argon ICPs, the outer gas flow is usually about 7 - 15 liters per minute.

The chamber between the outer flow and the inner flow sends gas directly under the plasma toroid. This flow keeps the plasma discharge away from the intermediate and injector tubes and makes sample aerosol introduction into the plasma easier. In normal operation of the torch, this flow, formerly called the auxiliary flow but now the intermediate gas flow, is about 1.0 L/min. The intermediate flow is usually introduced to reduce carbon formation on the tip of the injector tube when organic samples are being analyzed. However, it may also improve performance with aqueous samples as well. With some torch and sample introduction

Figure 13 Demountable ICP Torch

configurations, the intermediate flow may be as high as 2 or 3 L/min or not used at all.
The gas flow that carriers the sample aerosol is injected into plasma through central tube or injector. Due to small diameter at the end of injector, the gas velocity is such that even 1L/min of argon used for nebulization can punch a hole through plasma, this flow is known as inner gas flow. The torch consists of three concentric quartz tubes sealed together.
Radio Frequency Generators

The radio frequency (RF) generator is the device that provides the power for the generation and sustainment of the plasma discharge. This power, typically ranging from about 700 to 1500 watts, is transferred to the plasma gas through a load coil surrounding the top of the torch. The load coil, which acts as an antenna to transfer the RF power to the plasma, is usually made from copper tubing and is cooled by water or gas during operation.

Most RF generators used for ICP-OES operate at a frequency between 27 and 56 MHz.

2.3.4.3. COLLECTION AND DETECTION OF EMISSION

Transfer Optics

The emission radiation from the region of the plasma known as the normal analytical zone (NAZ) is sampled for the spectrometric measurement. Until recently, the analytical zone was observed from the side of the plasma operating in a vertical orientation as shown in Figure 14. This classical approach to ICP spectroscopy is referred to as a radial or side-on viewing of the plasma. In the early 1990’s, a new "look" at the normal analytically zone of the ICP was commercialized. The plasma is rotated to a horizontal position and the zone is observed from the end of the plasma as illustrated in Figure 15. This configuration has become known as an axial or end-on viewing of the ICP. Recently, instruments that combine both radial and axial viewing, called dual view, have been introduced

Figure 14 Side-on ICP Viewing. Figure 15 End-on ICP Viewing.

No matter whether the ICP is a side-on or end-on viewing type configuration, the radiation is usually collected by a focusing optic such as a convex lens or a concave mirror. This optic then focuses the image of the plasma onto the entrance slit of the wavelength dispersing device or spectrometer. In some side-on configured instruments, the focusing optic is used in conjunction with mirrors that allow measurement of emission at different heights within the plasma. This viewing height adjustment makes the instrument more flexible, although it is not necessary for most analyses.

Wavelength Dispersive Devices:

The next step in ICP-OES is the differentiation of the emission radiation from one element from the radiation emitted by other elements and molecules. The discrimination of this emission may be done in several ways. The physical dispersion of the different wavelengths by a diffraction grating is by far the most common.

A reflection diffraction grating is simply a mirror with closely spaced lines ruled or etched into its surface. Most gratings used in ICP-OES instruments have a line, or groove, density from 600 to 4200 lines per mm. When light strikes such a grating, its diffracted at an angle that is dependent on the wavelength of the light and the line density of the grating. In general, the longer the wavelength and higher the line density, higher the angle of diffraction will be.

To separate polychromatic light predictably, the grating is incorporated in an optical instrument called a spectrometer. The function of the spectrometer is to form the light into a well-defined beam, disperse it according to wavelength with a grating, and focus the dispersed light onto an exit plane or circle. In other words, the spectrometer receives white light or polychromatic radiation and disperses it into monochromatic radiation. One or more exit slits on the exit plane or circle are then used to allow certain wavelengths to pass to the detector while blocking out other wavelengths.

The monochromatic radiation which is diffracted from the grating is composed primarily of wavelengths representative of the light emitted by a particular elemental or molecular species in the ICP.

Detectors: Once the proper emission line has been isolated by the spectrometer, the detector and its associated electronics are used to measure the intensity of the emission line. By far the most widely used detector for ICP-OES is the photomultiplier tube or PMT. The PMT is a vacuum tube that contains a photosensitive material, called the photocathode; eject electrons when it is struck by light. These ejected electrons are accelerated towards a dynode which ejects two to five secondary electrons for every one electron which strikes its surface. The secondary electrons strike another dynode, ejecting more electrons which strike yet another dynode, causing a multiplicative effect along the way. Typical PMTs contain 9 to 16 dynode stages. The final step is the collection of the secondary electrons from the last dynode by the anode. As many as 106 secondary electrons may be collected as the result of a single photon striking, the photocathode of a nine-dynode PMT. The electrical current measured at the anode is then used as a relative measure of intensity of the radiation reaching PMT.

Figure 16 Photocathode, dynode and anode layout of a photomultiplier tube.

Figure 16 shows schematically how a PMT amplifies the signal produced by a photon striking a photocathode. The major advantages of the PMT over other detection devices are that it can be used to measure light over a relatively wide wavelength range, it can amplify very weak emission levels, and range of response can be extended to over nine orders of magnitude in light intensity.

2.3.4.4. SIGNAL PROCESSING AND INSTRUMENT CONTROL

Signal Processing

The electronics used for signal processing in ICP-OES systems utilizing PMT detection are generally straightforward. Here, the electrical current measured at the anode of the PMT is converted into information that can be used by a computer or the analyst. The first step is to convert the anode current, which represents emission intensity, into a voltage signal. Since virtually all commercial ICP-OES instruments today, including advanced detector systems, utilize digital signal processing, the voltage signal is converted into digital information via an analog-to-digital, or A/D, converter. This digital information can then be used by a computer for further processing, the end result being information passed on to the host computer or to the analyst in the form of a number representing either relative emission intensity or concentration.

With PMT-base sequential ICPs, it is possible to reprocess stored spectral information, such as background intensities and intensities of spectral interferences, but this data cannot be easily reviewed and changes made at a later date without severe time penalties. On the hand, with advanced detectors, e.g., CIDs and CCDs, a "snapshot" of a wavelength region is obtained and all of the intensities within this region are converted into digital information and may be permanently stored. Thus, post processing of the data is easily accomplished. This means that if it is found 1.) that the data might be susceptible to some spectral interference that was not previously corrected for or 2.) that the background correction was performed at the wrong wavelength, then these corrections can still be applied and the analyte concentrations recalculated.

Computers and Processors

An important part of any ICP-OES instrument is the computer control incorporated into the instrument. The majority of automated functions of an ICP-OES instrument are directly controlled by an on-board computer. For the simplest instruments, the analyst interacts directly with the on-board computer through buttons or a keypad located on the instrument.
All ICP-OES instruments, however, use an external computer, interfaced to the instrument’s on-board computer, to act as an interface between the analyst and the instrument.

At the simplest level of multielement ICP-OES instrumentation, a computer is needed to handle the massive amounts of data that such an instrument generates. While virtually every commercial ICP-OES instrument available today uses some type of computer to control the spectrometer and to collect, manipulate, and report analytical data, the amount of computer control over other functions of the instrument varies widely from model to model. For many instruments, the generator controls, argon flows, viewing height, and other variable parameters are controlled manually by the user. In the most sophisticated ICP-OES instruments, every function of the instrument is automated, i.e., under the control of the computer.

In addition to the obvious benefits of using computers for data collection and reporting, automation in ICP-OES instrumentation has other important advantages. For example, when the plasma is ignited, adjustments to the input power, the matching circuitry, and the gas flows may need to be made. In the early days of ICP-OES, these adjustments were made manually, making the utility of the ICP instrument partially dependent on the manual dexterity of the operator. Manual adjustments of the RF generator and argon flows upon ignition have now been made obsolete by automatic matching networks and computerized flow controls. Additional automated functions, such as automatic control of sample selection, nebulizer washout, and operating parameters, have vastly improved the speed and quality of the analytical determinations.

Software

At least as important as the degree of automation of an ICP-OES instrument is the computer software that controls the instrument and through which the analyst communicates with the instrument. While the issue of computer software may be a highly subjective topic for many analysts, there are certain aspects of software that warrant discussion in this chapter on instrumentation.

There have been many philosophical discussions regarding the attributes of the ideal analytical instrument. Some of the attributes of the "ideal" ICP-OES instrument would be that it could prepare the standards and samples, develop the analytical method, analyze the samples, report the results, and make decisions based on those results - all from a single keystroke. The expertise and hardware required to perform all of these tasks is readily available today. However, it is the software required to coordinate these tasks which is presently unavailable.

While the "ideal instrument" software is not yet a reality, the role of software in the operation of an ICP-OES instrument is still important. The objective of a good software package is not only to control the automated features of the instrument during collection of analytical data but to simplify the overall operation of the instrument. Areas in which this is important include not only running an analysis but developing analytical methods and reporting results. The methods development task involves selecting proper operating parameters for an analysis, such as wavelengths, PMT voltages, background correction points, and standards concentrations. The ability to view spectral data displayed graphically with a minimum of effort is indispensable during the selection of these parameters. 2.4 MATRIX EFFECTS

In inductively coupled plasma optical emission spectrometry (ICP-OES) maintaining accuracy and precision is generally limited by the non-spectral interferences (so-called matrix effects) that are caused by the major elements in the samples (e.g. easily ionizable elements (EIEs) such as Na, K, Li) or reagents used for sample digestion and solution storage (e.g. mineral acids such as HNO3 )

Since the first studies on ICP-OES, matrix effects have been widely investigated by several researchers to find possible explanations about their origins and their influences on analyte signal. It was reported that matrix effects cause either suppression or enhancement of the analyte signal (Brenner et al. 1997, Dubuisson et al. 1998c, Grotti et al. 2000, Stepan et al. 2001, Iglesias et al. 2004). Furthermore there is a general agreement that matrix effects are generally caused by two major factors:

i. changes in the energy transfer between the plasma and sample (during the processes of atomization, excitation and ionization) and ii. changes in the efficiency of sample aerosol formation, transport and filtration.
The studies which attempt to find possible mechanisms in order to understand the causes of EIE effects and to investigate the characterization of these effects, mainly focused on the plasma properties, such as electron temperature (Te), electron number density (ne), gas-kinetic temperature (Tg), analyte atom and ion number densities which affect the electrical and thermal conductivity, viscosity and processes occurring in the plasma, such as atomization, excitation and ionization equilibria, volatilization, collision processes, ambipolar diffusion, lateral diffusion etc.

The matrix effects (i.e. changes that occur in the behavior of the system induced by the matrix), are generally classified in two groups. The first group includes the changes related to operating conditions resulting from changes in the physical properties of the solution occurring in the sample introduction system (i.e. the reduction in the nebulizer aspiration rate when free aspiration is used, primary and tertiary aerosol drop size distributions, the modification of mass of the solution transported to the plasma, and the element concentration as a function of the drop size, change in the aerosol characteristics due to a variation of the surface tension and volatility, decreased solution uptake as a result of increased viscosity). The second group includes the effects caused by the processes that occur in the plasma (such as changes in atomization and excitation conditions).

2.4.1. Variables Affecting the Matrix Effects

The liquid sample introduction system has a major effect on the matrix interferences. This can be easily understood by taking into account that the introduction system influences the total mass of the analyte and solvent transported towards the plasma and the aerosols characteristics. The solvent injected into the plasma modifies its thermal characteristics, whereas the aerosol drop size changes the plasma location at which the drop vaporization is complete.

The other most important variables which have an influence on the matrix effects are the plasma observation height, the nebulizer gas flow rate (also injector i.d.), and the rf power.

In the Initial Radiation Zone (IRZ), elemental matrix effects are known to be strongest while in the Normal Heating Zone (NAZ) few effects exist. It was reported that studying matrix effects (acid and elemental effects) at a given observation height may result in signal variations that do not correspond to the actual situation.

It has been reported that by applying a high power (>1.2kW), a low carrier gas rate (< 0.8 ml/min) and high injector i.d. (>2 mm), robust plasma conditions (i.e. operating conditions that could allow changes in the nature or concentration of the matrix components without a significant change in the analyte signals) are achieved. It is thought that under robust plasma conditions any observed effect is mainly due to the aerosol generation and transport system, i.e. to the sample introduction system and matrix effects can be reduced by using operating conditions that lead to an efficient energy transfer between the plasma and the sample (Fernandez et al. 1994)

A decrease in the gas flow rate leads to a decrease in the solvent and matrix plasma load and also an increase in the aerosol residence time, thus increasing the efficiency of the energy transfer to the analyte. Increasing the residence time is also achieved by employing injector diameters higher than 2 mm. An increase in the rf power leads to increases in the total amount of energy available to excite the analyte. Moreover, the role of the electrons in the analyte excitation should also be considered. For a plasma operated at low rf power and high nebulizer gas flow rate, the electron density is low.

Although robust operating conditions and proper choice of observation height are said to decrease the effects caused by matrices that contain the acid and salt, these interferences can not be totally eliminated. Different strategies have been suggested to compensate for matrix effects.

2.4.2 Methods for Overcoming Matrix Effects

The methods for correcting the matrix effects should fulfill the following conditions: i. single set of reference solutions for any aqueous sample matrix should be used ii. it must be applicable to both single and multielement analysis

iii. it should be simple and applicable to a mixture of matrices and to different compounds iv. it should not require periodic calibration The use of Internal Standard can meet most, if not all of these requirements. 2.5 Use of Internal Standard with ICP-OES

The concept of measuring the ratio of signals of analyte and internal standard is well known in atomic spectroscopy where the internal standard is an added metal of known concentration and not present in the sample. Internal standard addition can be performed manually or by use of an automated flow injection technique at the nebuliser during sample introduction. The advantage of an internal standard in metal analysis is that it can be used where different samples with different viscosities require analysis against the same calibration curve to overcome matrix effects. Later, this theory was extended to correct for fluctuations in sample transport effects, instrumental drift, and electronic and plasma noise. The element selected as an internal standard must be similar in chemical behavior and excitation energy as the analytes of interest.

Signal enhancements can be achieved by several means without affecting the precision of analysis. For optimisation with respect to the higher signal response, parameters such as the power of the ICP-OES, nebuliser gas flow, burner geometry, correct peak height and correct horizontal peak position of the plasma in relation to the optics of the instrument must be studied for each analyte and sample. Increasing the nebuliser gas flow rate helps the droplet size decrease due to collisions in the spray chamber introducing higher sample efficiency to the plasma torch. However, this may be at the expense of residence time, flow mechanics and plasma temperature. These improvements in signal enhancement of the analyte will also affect the internal standard present in the same solution.

3. MATERIALS AND METHODS

Multielemental determination of nutritional elements using Inductively Coupled Plasma- Optical Emission Spectroscopy after preparation of sample using Microwave Digestion System has the following steps: * Preparation of Standards * Test Sample Preparation * Test sample Digestion * Analysis using ICP-OES
These steps are described in detail in the following sections

3.1 Preparation of Standards

3.1.1 Internal Standard Solution with Ionization Buffer:
Ionization buffer (CsCl 1000mg/ml) is mandatory to stabilize the plasma; it is particularly useful for consistent analysis of matrices with high salt content such as soups, tastemakers or inorganic raw materials. To prepare the internal standard solution: a. 12.67 g of CsCl is weighed into 1000ml volumetric flask. b. 500 ml ddH2O is added and is mixed well c. 4 ml of Indium plasma emission standard 10009 mcg/ml and 1ml of Strontium Plasma Emission standard 10000 mcg/ml is also added to the above solution. d. Carefully add 10ml HNO3, super boiled or suprapure (65% w/v). e. The volume is made up to 1L with deionized water and is mixed well. It is made sure that the volume of the internal standard solution is sufficient for the whole run analysis. A new solution of the internal standard cannot be added throughout the analysis.

3.1.2 Working Standard Solutions :
5 Working Standard solutions are prepared which are used for the calibration of the ICP-AES equipment which is the PERKIN ELMER OPTIMA 5300 DV with oval viewing system. To make each of these 5 standard solutions, a commercially available multi-element solution is used in different volumes along with HNO3 suprapure 65% (w/v). a. Std 1: (BLANK) 90ml ddH2O + 10ml HNO3 b. Std 2: 0.2 ml Multi-element soln. + 100ml ddH2O + 10ml HNO3 c. Std 3: 0.8 ml Multi-element soln. + 100ml ddH2O + 10ml HNO3 d. Std 4: 1.6 ml Multi-element soln. + 100ml ddH2O + 10ml HNO3 e. Std 5: 3.2 ml Multi-element soln. + 100ml ddH2O + 10ml HNO3

Elements | Concentration Range (mg/Kg)Std1 Std 2 Std 3 Std 4 Std 5 | Calcium | 0.0 | 15 | 60 | 120 | 240 | Potassium | 0.0 | 20 | 80 | 160 | 320 | Sodium | 0.0 | 10 | 40 | 80 | 160 | Magnesium | 0.0 | 5 | 20 | 40 | 80 | Phosphorous | 0.0 | 10 | 40 | 80 | 160 | Iron | 0.0 | 0.5 | 2.0 | 4.0 | 8.0 | Zinc | 0.0 | 0.2 | 0.8 | 106 | 3.2 | Copper | 0.0 | 0.1 | 0.4 | 0.8 | 1.6 | Manganese | 0.0 | 0.0025 | 0.010 | 0.020 | 0.040 |
Table 2: Concentration Range of Different elements in Calibration Standards

3.2 Preparation of the Test Sample
Preliminary treatment of heterogeneous products (lab samples) is mandatory to prepare representative test sample (e.g. a serving size or specific size according to the specifications of the concerned product like 25g for standard cereals or 50g for complete cereals) before analyzing the digested test portion by the ICP-AES. 1. For food matrices: Homogenization is done using suitable grinding equipments or by preparing slurry with water and the test sample 2. For inorganic raw material: A test sample preparation for water soluble inorganic raw material is a simple aqueous dilution with deionized water in acidic condition. A minimum dilution factor of 500 is mandatory to quantify element impurities in salt matrices. A higher dilution is necessary to measure the major ion present in the salt. For some specific mineral salts which are partly water soluble, a previously digested test portion is used which is digested by microwave digestion.
For dilution factor of 500: a. 0.2g salt is weighed into a 100ml volumetric flask. b. 10ml of deionized ddH2O and 10ml of suprapure HNO3 are added. c. Volume is made up to 100ml with ddH2O.

3.2.1 Test Portion Digestion

Microwave digestion (closed vessel) digestion is used for the digestion of the test portions. CEM MARS Xpress is the microwave that is used for microwave digestion at the NQAC. It is used because it applies mostly to food samples with high fat or carbohydrate content when using adopted digestion program. Moreover it does not require constant operator attention as only HNO3 is used in closed vessel system.

Figure 17 Microwave Digestion System

CEM MARS Xpress uses Teflon vessels for closed vessel digestion. Before usage these vessels are decontaminated to ensure that the samples are free from any contamination.

Figure 18 Teflon Vessel Figure 19 Vessel Stand or Carousel

Decontamination:

* Carefully add 5mlof 5% HNO3 (sub-boiling or Suprapur,65%) into the digestion vessel * Tightly close the digestion vessels with the Teflon caps the help of capping station. * Distribute the digestion vessels into the microwave carrousel to ensure homogeneous microwave power application on all samples * Carefully & properly fit the carrousels on the turntable & shut the microwave door properly to avoid any leakage. * Select the program “Decontamination” from the front display panel & Press “Start” button (Green) & the program will start/run * After completion of the cycle the “Cool down” program run automatically for 30 min. to cool down the samples. * Carefully remove the carrousel from the microwave. * Allow the flasks to cool down to room temp. * Wash the vessels properly in the washing machine. Step for | CEM MARS Xpress (1600 Watts max.) | 1 to 40 tubes with 5 mL HNO3 | Power (Watt) | Ramp time(min.) | Ramp tempTo °C | Hold time(min.) | 1 | 1600 (75 %) | 5 | 180 | 5 | 2 | Cool to RT | - | - | - |

Table 3: Decontamination program for CEM MARS-Xpress system using a rotor with 40 positions

Steps for Microwave Digestion

* Weigh a maximum of 0.5g test sample (0.5ml in slurry) to the nearest 0.1mg into 50ml MDS Teflon liners. * Carefully add 5ml of HNO3 (sub-boiled or suprapure, 65% w/v). * Leave the sample to stand for at least 10 minutes at ambient temperature to let the quickly formed gases escape. Then close the liners with their lids and caps. * Distribute the liners into MDS carousel to ensure homogenous microwave power application on all samples * Start the appropriate power program (as shown in the table) depending on the number of liners and MDS used. A digestion is judged complete when clear to yellow analytical solutions are produced. * Allow the liners to cool down to room temperature for 30 minutes. * Carefully unseal the caps, rotating them slowly at first to let the fumes and gases at high pressure escape and then remove the lids as well. * Transfer into 50ml volumetric flasks and make up the volume with deionized ddH2O. * Filter the digested solution using an ash less filter paper if necessary (for turbid samples), discarding the first 20ml of the filtrate and collecting the remaining filtrate for analysis. Step for | CEM MARS Xpress (1600 Watts applied) | 6 to 40 tubes | Power (Watt) | Ramp time(min.) | Ramp tempTo °C | Hold time(min.) | 1 | 1600 | 75 % | 5 | 120 | 5 | 2 | 1600 | 75 % | 5 | 200 | 25 | 3 | Cool to RT | - | - | - |

Table 4: Closed-vessel microwave digestion program for CEM MARS-Xpress system using a rotor with 40 positions

3.3 Analysis using ICP OES

Figure 20: PERKIN ELMER OPTIMA 5300DV

PERKIN ELMER OPTIMA 5300DV with dual viewing system is used for the analysis of the digested samples. Before starting the analysis, the following maintenance checks are performed

3.3.1 Peristaltic Pump Tubing

* Check for depressions or flat spots on the tubing. New pump tubing may require a short break-in period to stretch out to a consistent length. This break in period can be minimized by manually stretching the tubing a few times before it is placed on to the peristaltic pump. * Make sure that the pump tubing used is appropriate for the sample type. 3.3.2 Nebulizer * Make sure that the nebulizer is not clogged or leaking. While making visual observations of the aerosol to check for a uniform spray pattern use deionized water and wear proper eye protection.

3.3.3 Drain System

* The drain system should be filled with liquid to the level that will provide the proper backpressure for the inner or nebulizer gas flow. * Sample waste that is drained from the spray chamber should flow smoothly through the drain system. Uneven flow indicates the restriction in the drain line.

3.3.4 Torch

* Check for leaks that can be caused by cracks or other damage to the quartz tubes, O-rings or gas fittings. * Accumulated deposits on the torch should be removed. (Small depositions are normal) * When analyzing solutions containing high levels of particulates or dissolved solids, the injector may become clogged and should be removed or cleaned.

3.3.5 RF Generator

* The RF load coil should be inspected occasionally for signs of corrosion and leakage. * The high voltage wire, rods and other components of the plasma initiation system should be inspected occasionally and replaced if corroded or worn.

3.3.6 Spectrometer

* Purge windows should be regularly inspected and carefully cleaned or replaced if necessary. * The air filters of the electronic section of the spectrometer should be cleaned when needed.

3.3.7 Change of Carrier Gas Cylinders:

* The cylinder for carrier gas i.e. Argon is fixed on semiautomatic gas panel. This gas panel has four cylinders connected to it all the time. Two of them are running and the other two are standby for the changeover in case the first cylinder finishes. When the current cylinder finishes the gas supply automatically shifts to the second cylinder after we open the line valve for that cylinder. The direction of the changeover lever is also to be changed with the arrow pointing towards the cylinder running currently. Similar is true for other gas cylinders of nitrogen . * The emptied cylinder is to be replaced with a filled one as soon as possible. * For changing the cylinder people from the mechanical workshop are called . * After changing the cylinder it is necessary to check the leakage at the concerned joints. * The line valve of the filled cylinder is kept closed till not in use.

After the above checks are complete, the following procedure is followed
3.3.8 Starting the instrument:

* Turn on the gases and the cooling water supply or chiller. * Switch ‘ON’ the ICP-OES unit. * Turn on the autosampler and other accessories.

3.3.9 Starting the software:

* Switch ‘ON’ software system i.e. PC & Monitor. Enter the password. * Click the Start button. Select Programs ►PerkinElmer Win Lab 32►Win Lab32. The Win Lab 32 software starts. * Click the plasma switch to On to ignite the plasma. Check the stability of plasma through the viewing window. If the plasma is unstable, immediately click the Plasma switch to Off to turn off the plasma or press the red Emergency Plasma off button above the sample compartment. * Let the instrument warm up for 15 minutes.

3.3.10 Giving a sequence

* Click twice on Method untitled option in the tool bar and select the required method from the given list of saved methods. Click SamInfo icon in the toolbar. The sample information table appears on the screen. Enter the complete information about the samples including QCS no., autosampler location, sample weight, dilution factor and information whether it is a blank, standard or sample. Enter the batch ID and save the sequence. * Create ‘Result data sheet’ & click on ‘save results’ to store all values during calibration & analysis.

3.3.11 Calibration:

* A nine elements standard in known amounts is injected daily. This is done to check the sensitivity of the instrument and the shift in the retention times of the peaks in case it happens. QC is also injected at regular intervals in the sequence. The frequency of this injection is first in the beginning of the sequence, after calibration and then after every six samples. * The calibration is done and saved daily for every sequence. The software calculates and reports the results on the basis of calibration. * After 15 min. of turning the plasma ON start calibration procedure by clicking on “Calibrate” in the Method window. * The automatic liquid sampler will select the specified location and sample is drawn from that location with the help of sample probe. * The spectra and calibration curve for the samples are simultaneously displayed on the PC screen. * In case the QC fails after calibration, recalibrate the whole sequence.

3.3.12 Sample analysis:

Figure 21: Screenshot of Winlab 32 Online

* After calibration stops, click Analyze samples. option to start the analysis of unknown samples and blanks. Alternatively, click on Analyze all.. for a continuous processing of standards and samples. * If analysis is to be done for selected parameters, click option Analysis ►Enable/ disable elements and select the desired elements. Click O.K. Also specify their locations by number or sequence. Then click Analyze samples… to start the analysis.

3.3.13 Shut down: * After analysis is over, flush double distilled water in place of internal standard to wash the system for 3-5 min. * Extinguish the plasma by clicking on the Plasma Off switch in the Plasma Control Window. * Shut down the software by clicking X mark in right upper side of the window. * Click ‘Start’ icon & select ‘Shut down’ & ‘Shut down the computer’. * Turn the PC & Monitor power switch ‘OFF’ * Switch off the main instrument, air compressor, chiller and the exhaust fan. * Drain water from the air compressor. * Turn off the gases.

3.4 Internal Standardization
To study the role of internal standard in removing matrix effects; routine samples of different food matrices, samples with known concentrations were analyzed using the ICP-OES, first, by using Internal Standard and then, without Internal Standard.

For the automatic addition of the Internal Standard to each and every sample, a T- connector is used and a mixture of digested sample and internal standard (1:1) reach the nebulizer.

Figure 22: The T-connector
In the Winlab 32 Online Software, internal standard wavelength corresponding to each analytical wavelength are set according to the Table ANALYTE | | INTERNAL STANDARD with Cs 10000 mg/L | Element | Wavelength | | Element | Wavelength | Conc. | | nm | | | nm | mg/L | Ca | 317.933 |  | In | 303.936 | 40 | K | 766.491 |  | Sr | 460.733 | 10 | Na | 589.592 |  | Sr | 460.733 | 10 | Mg | 285.213 |  | In | 303.936 | 40 | P | 213.618 |  | In | 303.936 | 40 | Fe | 259.940 |  | Sr | 338.071* | 10 | Zn | 213.857 |  | Sr | 338.071* | 10 | Cu | 324.754 |  | In | 303.936 | 40 | Mn | 257.610 |  | Sr | 338.071* | 10 |

Table 5. Recommended Internal Standard wavelengths corresponding to the analytical wavelengths

4. RESULTS AND DISCUSSIONS

This section is divided into 3 parts: * Calibration Standards: This part shows the effect of internal standard on Standards * Routine Samples: This part shows the results of various routine samples from different food matrices * Reference Samples or Samples with known concentrations: This part shows the results of the samples whose concentrations were known.

4.1 Calibration Standards
As mentioned in the previous sections, 5 calibration standards are used to calibrate the PERKIN ELMER OPTIMA 5300DV. To study the effect of internal standard on different food matrices, it was very important to understand its effect on the calibration standards.
The following are the Results which were observed after analyzing all the 5 calibration standards with and without Internal Standards
Note: Units of Concentration for all minerals except Mn and Cu are mg/100g. For Mn and Cu the units are μg/100g. Std | Elements | Intensity with Internal Standard | Intensity without Internal Standard | Concentration | 1 | P | 84.5 | 84.5 | 0 | | Zn | 346.1 | 346 | 0 | | Mn | 187.6 | 187.7 | 0 | | Fe | 1295.8 | 1295.9 | 0 | | Mg | -89 | -89 | 0 | | Cu | 1954.4 | 1954.3 | 0 | | Ca | 6381.2 | 6381.4 | 0 | | Na | 29463.1 | 29463.7 | 0 | | K | 12901.4 | 12901 | 0 | 2 | P | 31102.7 | 32246.9 | 10 | | Zn | 15595.1 | 16542.5 | 0.2 | | Mn | 1761.1 | 1876.7 | 0.0025 | | Fe | 52299.4 | 55484 | 0.5 | | Mg | 529242.8 | 548657.9 | 5 | | Cu | 14145.4 | 14736.3 | 0.1 | | Ca | 1287428.4 | 1334897.9 | 15 | | Na | 1182233.6 | 1254329.3 | 10 | | K | 409040.1 | 434075.1 | 20 | 3 | P | 127928.1 | 129305.4 | 40 | | Zn | 63729.8 | 66342.6 | 0.8 | | Mn | 6470.2 | 6741.6 | 0.01 | | Fe | 223455.9 | 232661.8 | 2 | | Mg | 2242195.7 | 2266320.9 | 20 | | Cu | 59981.7 | 60648.3 | 0.4 | | Ca | 5414151.8 | 5472409.7 | 60 | | Na | 4897630.9 | 5098747.9 | 40 | | K | 17211608.4 | 1792479.9 | 80 | 4 | P | 250405.5 | 257552.7 | 80 | | Zn | 125280.9 | 132217.6 | 1.6 | | Mn | 12850 | 13569.4 | 0.02 | | Fe | 4290509.6 | 452782.9 | 4 | | Mg | 4354195.4 | 4478400 | 40 | | Cu | 117850.6 | 121267.9 | 0.8 | | Ca | 10891169.1 | 11202024.8 | 120 | | Na | 9619863.8 | 10154997.4 | 80 | | K | 3370602.9 | 3557533.2 | 160 | 5 | P | 498237.7 | 502975.7 | 160 | | Zn | 240342.6 | 255248.4 | 3.2 | | Mn | 24804.9 | 26348.2 | 0.04 | | Fe | 820538.6 | 871347.1 | 8 | | Mg | 8802938.5 | 8887068.3 | 80 | | Cu | 239699.3 | 241994.2 | 1.6 | | Ca | 21690467.5 | 21894943.6 | 240 | | Na | 19529470.4 | 20737955.7 | 160 | | K | 6640389.5 | 7052061.9 | 320 |
Table 6: Results for Analysis of calibration standards with and without internal standard.

After the graphs were plotted for each element , maximum variation was observed in Zinc and Manganese.

Graph 1 Concentration vs Intensity graph or the Calibration Curve for Zinc, with and without internal standard

Graph 2 Concentration vs Intensity graph or the Calibration Curve for Manganese, with and without internal standard
4.2 Routine Samples
Routine Samples from different Food Matrices were analyzed both with and without Internal Standard. Results from different food matrices are shown below.

4.2.1 Infant Food: Infant Food | Elements | Concentration With Internal Standard | Intensity With Internal Standard | Concentration Without Internal Standard | Intensity With Internal Standard | 1 | P | 436 | 135295 | 468 | 154185.9 | | Zn | 3.89 | 30244.4 | 4.09 | 34804.3 | | Mn | 109 | 7128.3 | 115.6 | 8344.8 | | Fe | 8.41 | 92068.6 | 8.83 | 105824.5 | | Mg | 57.9 | 650027.7 | 62.1 | 741036.9 | | Cu | 340 | 3594.6 | 373 | 4590.9 | | Ca | 570 | 5000687.2 | 612 | 5700192.8 | | Na | 233 | 2966499.3 | 245 | 3410209.7 | | K | 632 | 1379194 | 663 | 1584840.5 | 2 | P | 457 | 138607 | 484 | 155876.5 | | Zn | 4.17 | 31629.3 | 4.3 | 35681.1 | | Mn | 116.5 | 7436.7 | 120.8 | 8509.7 | | Fe | 8.86 | 94673.3 | 9.12 | 106701.1 | | Mg | 60.2 | 659494.6 | 63.7 | 741895 | | Cu | 363 | 3817.4 | 391 | 4734.6 | | Ca | 607 | 5196397.5 | 643 | 5844748.6 | | Na | 254 | 3154583.1 | 261 | 3555950.7 | | K | 666 | 1420322.5 | 686 | 1600364.5 | 3 | P | 440 | 135388.1 | 460 | 150128.2 | | Zn | 3.97 | 30606.7 | 4.07 | 34303.5 | | Mn | 114 | 7387.9 | 117.4 | 8393.2 | | Fe | 8.85 | 95924.7 | 9.05 | 107414.9 | | Mg | 61.9 | 688631.9 | 64.7 | 763820.5 | | Cu | 360 | 3845 | 380 | 4649.9 | | Ca | 591 | 5132401.9 | 617 | 5692270.3 | | Na | 242 | 3051008.4 | 247 | 3416454.3 | | K | 631 | 1365754.4 | 646 | 1528695.1 | 4. | P | 472 | 150181.7 | 488 | 155622.6 | | Zn | 4.04 | 32045.4 | 4.11 | 34168.4 | | Mn | 113.9 | 7386.5 | 115.6 | 7935.1 | | Fe | 8.48 | 93570.6 | 8.62 | 99748.2 | | Mg | 59.6 | 689393.5 | 61.6 | 714392 | | Cu | 324 | 3415.2 | 334 | 3668.3 | | Ca | 613 | 5597112.3 | 633 | 5800167.2 | | Na | 237 | 3092551.9 | 241 | 3295651.3 | | K | 637 | 1410101.1 | 647 | 1502441.2 |
Table 7 Results (both with and without Internal Standard) for Infant Food Samples

4.2.2 Coffee: Matrix:Coffee | Elements | Concentration With Internal Standard | Intensity With Internal Standard | Concentration Without Internal Standard | Intensity With Internal Standard | 1 | Zn | 1.66 | 13437.8 | 1.67 | 14895.6 | | Mn | 109.8 | 6437.5 | 110.4 | 7422.4 | | Fe | 1.26 | 13491.6 | 1.31 | 15174.2 | | Cu | 34 | 133.2 | 53 | 362.6 | | Na | 276 | 3579261.1 | 290 | 3952480.8 | | K | 715 | 1604979.5 | 753 | 1771233 | 2 | Na | 245 | 3160237.4 | 257 | 3499114.1 | 3 | Na | 265 | 3420014.4 | 277 | 3774678.7 | 4 | Zn | 0.17 | 801.8 | 0.25 | 941.4 | | Cu | 38 | 138.1 | 22 | 803.6 | 5 | Zn | 0.21 | 1117.3 | 0.29 | 1241.5 | | Fe | 1.53 | 15923.2 | 1.89 | 20021.4 | | Cu | 40 | 174.6 | 22 | 816.7 | 6 | Na | 28 | 358592.1 | 32 | 435725 | | Cu | 5 | 275.3 | 4 | 288 | 7 | Cu | 19 | 502.1 | 49 | 999.7 | 8 | Cu | 15 | 441.2 | 41 | 888.3 |
Table 8 Results (both with and without Internal Standard) for Coffee Samples

4.2.3 Water: Water | Elements | Concentration With Internal Standard | Intensity With Internal Standard | Concentration Without Internal Standard | Intensity With Internal Standard | 1 | Zn | 0.03 | 19140.4 | 0.05 | 41515 | | Cu | 1 | 1798.8 | 1 | 2057 | 2 | Zn | 0.01 | 4532.2 | 0.01 | 10488 | | Cu | 0 | 241.7 | 0 | 283 | 3 | Zn | 0.02 | 18194 | 0.05 | 38142.2 | | Cu | 0 | 422.1 | 0 | 454 | 4 | Zn | 0.13 | 99145 | 0.23 | 183796.6 | | Cu | 0 | 267 | 0 | 300 | 5 | Zn | 0.01 | 5319 | 0 | 1965 | | Cu | 0 | 395 | 0 | 241.6 |
Table 9 Results (both with and without Internal Standard) for Water Samples

4.2.4 Milk: MILK | Elements | Concentration With Internal Standard | Intensity With Internal Standard | Concentration Without Internal Standard | Intensity With Internal Standard | 1 | Zn | 0.42 | 6416.2 | 0.43 | 6881.9 | | Cu | 1 | 359.2 | 3 | 499.8 | 2 | Zn | 0.46 | 7135.5 | 0.47 | 7663.1 | | Cu | -1 | 270 | 2 | 493.3 | 3 | Zn | 0.37 | 5723.9 | 0.37 | 6214.6 | | Cu | -3 | 124 | 0 | 411.2 | 4 | Zn | 0.7 | 10864.2 | 0.71 | 11754.4 | | Cu | -1 | 299.8 | 3 | 513.5 | 5 | Zn | 3.32 | 49829.8 | 3.31 | 53185.5 | | Cu | 21 | 981.3 | 26 | 1224.9 | 6 | Zn | 0.4 | 6386.9 | 0.4 | 6988.6 | | Cu | 4 | 462.7 | 7 | 639.7 |
Table 10 Results (both with and without Internal Standard) for Milk Samples

4.2.5 Flavors: Flavors | Elements | Concentration With Internal Standard | Intensity With Internal Standard | Concentration Without Internal Standard | Intensity With Internal Standard | 1. | Zn | 0.06 | 1732.4 | 0.07 | 1915.9 | | Cu | 4 | 187.5 | 10 | 532.4 | 2. | Zn | 0.16 | 1482.8 | 0.18 | 1814.4 | | Cu | -3 | -6.7 | 3 | 157 | 3. | Zn | 0.1 | -137 | 0.12 | -49 | | Cu | 20 | 708 | 30 | 931.7 | 4. | Zn | 1.21 | 7531.4 | 1.23 | 8127.6 | | Cu | 73 | 1500.5 | 94 | 1919.6 | 5. | Zn | 1.38 | 8617.2 | 1.42 | 9532.5 | | Cu | 540 | 8566.1 | 569 | 9258.1 | 6. | Zn | 0.88 | 6041 | 0.94 | 6999.1 | | Cu | 1495 | 19836.8 | 1658 | 22089.6 |
Table 11 Results (both with and without Internal Standard) for Flavor Samples

4.2.6 Chocolates: Chocolates | Elements | Concentration With Internal Standard | Intensity With Internal Standard | Concentration Without Internal Standard | Intensity With Internal Standard | 1. | Zn | 1.2 | 9150.7 | 1.22 | 9828.8 | | Fe | 2.66 | 29450.6 | 2.77 | 31513 | | Cu | 211 | 3644.3 | 225 | 4041.1 | | Ca | 221 | 2000680.4 | 229 | 2123772.8 | | Na | 94 | 1229914.1 | 96 | 1312818.2 | 2. | K | 351 | 775027.8 | 352 | 826908.6 | | Zn | 1.43 | 11046.4 | 1.44 | 11893.2 | | Fe | 2.89 | 32137.9 | 2.9 | 34497.6 | | Cu | 302 | 5116.2 | 327 | 5720.2 | | Ca | 245 | 233315.7 | 258 | 2403622.5 | | Na | 93 | 1233907 | 94 | 1321942.1 | 3. | K | 409 | 910623.8 | 410 | 974707.5 | | Zn | 1.17 | 8113.5 | 1.24 | 9117.2 | | Cu | 219 | 3665.5 | 264 | 4419.5 | | Ca | 162 | 1330974.2 | 179 | 1480127.6 | | Na | 95 | 1231731.2 | 101 | 1382846.9 | 4. | P | 209 | 67055.6 | 214 | 71326.4 | | Zn | 0.7 | 6897.3 | 0.89 | 7658.4 | | Mn | 32.8 | 2077.7 | 32.9 | 2274.8 | | Fe | 0.63 | 5794.1 | 0.63 | 6429.8 | | Mg | 31.6 | 356698 | 32.3 | 379546 | | Cu | 683 | 10442.9 | 706 | 11427.5 | | Ca | 468 | 4338808.9 | 479 | 4616597 | | Na | 107 | 1340783.3 | 107 | 1465168.3 | | K | 263 | 591061.5 | 280 | 642572 | 5. | P | 159 | 50924.9 | 164 | 54481.8 | | Zn | 0.63 | 6352.6 | 0.82 | 7052 | | Mn | 485.2 | 31399.6 | 480.5 | 33895.7 | | Fe | 2.31 | 24400.2 | 2.29 | 26484.4 | | Mg | 46.1 | 520866.8 | 47.5 | 557324.6 | | Cu | 126 | 1548.2 | 138 | 2007.6 | | Ca | 160 | 1461418.2 | 164 | 1565597.9 | | Na | 109 | 1355433.7 | 108 | 1477533.4 | 6. | K | 283 | 631863.9 | 298 | 685277.3 | | Na | 118 | 1404239 | 124 | 1502402.8 | 7. | Na | 113 | 1340190.2 | 117 | 1420713.4 |
Table 12 Results (both with and without Internal Standard) for Chocolate Samples

4.2.7 Oil: OIL | Elements | Concentration With Internal Standard | Intensity With Internal Standard | Concentration Without Internal Standard | Intensity With Internal Standard | 1. | Zn | 0.47 | 2611.6 | 0.46 | 2689.8 | | Cu | 33 | 547.1 | 46 | 724.4 | 2. | Zn | 1.05 | 4250.6 | 1.07 | 4750.8 | | Cu | 75 | 467.1 | 101 | 840.7 | 3. | Zn | 2.11 | 8058.6 | 2.05 | 8367.1 | | Cu | -37 | -854.7 | -36 | -792.1 | 4. | Zn | -0.57 | -1811.5 | -0.49 | -1789.9 | | Cu | -249 | -2309 | -225 | -2231.5 | 5. | Zn | -0.5 | -1518 | -0.42 | -1485.1 | | Cu | -261 | -2388.2 | -239 | -2334 |
Table 13 Results (both with and without Internal Standard) for Oil Samples

4.2.8 Culinary: Culinary | Elements | Concentration With Internal Standard | Intensity With Internal Standard | Concentration Without Internal Standard | Intensity With Internal Standard | 1. | Fe | 134.6 | 754875.9 | 144.8 | 850753.8 | 2. | Fe | 135.8 | 746771.8 | 142 | 818034.4 | 3. | Fe | 136.9 | 779061.4 | 143.5 | 856011.1 | 4. | Fe | 139.1 | 779059.8 | 147.2 | 863722.4 | 5. | Fe | 138.1 | 753228.2 | 145.1 | 829357 | 6. | Fe | 136.4 | 754456.6 | 142.2 | 824254.4 | 7. | Fe | 135.3 | 750930.8 | 142 | 825508.1 | 8. | Fe | 124 | 691987.7 | 132 | 772399.7 | 9. | Fe | 129.4 | 705284.8 | 135.7 | 775143.9 | 10. | Fe | 132.7 | 739037.8 | 140.2 | 818069.8 | 11. | Fe | 141.7 | 771859.7 | 146.3 | 835454.7 | 12. | Fe | 134.4 | 744101.4 | 142.6 | 827090.5 | 13. | Fe | 139.9 | 770715 | 149.5 | 862885.7 | 14 | Fe | 143.2 | 804210.8 | 145.5 | 856205.3 | 15. | Fe | 143.7 | 801127.2 | 150.8 | 880947.8 | 16. | Fe | 161.5 | 925615.6 | 167.2 | 1004244 | 17. | Fe | 128.5 | 710769.5 | 133.8 | 775406.3 |
Table 14 Results (both with and without Internal Standard) for Culinary Samples

4.2.9 Discussion:
After a thorough study of the above results, the following observations are made: i. There is a lot of variation in the concentration of minerals when the internal standard is not used. ii. In some matrices e.g. Water, little difference was observed between the concentrations with internal standard and without internal standard. This is because the viscosity of water is low, and hence the matrix effects are reduced iii. In some matrices, e.g. Chocolates and Culinary the variation between the results of samples with internal standard and without internal standard is high. This is because of high viscosity and salt content of the samples.

4.3 Reference Samples:

To understand the effect of the internal standard in a better way, Reference Samples or the samples with known mineral concentration were also analyzed. After analysis, the reproducibility and repeatability of the results was calculated, which is shown in the calculation sheets below

Sample | Elements | Concentration With Internal Standard | Intensity With Internal Standard | Concentration Without Internal Standard | Intensity With Internal Standard | BFF 1 | P | 321 | 103400.9 | 336 | 114201 | | Zn | 2.32 | 19649.4 | 2.13 | 18194 | | Mn | 561.2 | 37527.2 | 590.4 | 41140.1 | | Fe | 7.25 | 84838.6 | 7.63 | 92630.1 | | Mg | 49.6 | 559431.9 | 52 | 618273.2 | | Cu | 116 | 1428.8 | 126 | 1756 | | Ca | 392 | 3595138.9 | 410 | 3975944.9 | | Na | 107 | 1342462.1 | 108 | 1465895 | | K | 520 | 1175322.4 | 539 | 1280650.7 | BFF 2 | P | 317 | 100165.5 | 334 | 111570.5 | | Zn | 2.34 | 20422.3 | 2.41 | 20265.8 | | Mn | 562.7 | 36929.4 | 596.1 | 40775.6 | | Fe | 7.47 | 85756.6 | 7.91 | 94286.9 | | Mg | 49.6 | 548245.4 | 52.3 | 611107.5 | | Cu | 121 | 1474.6 | 133 | 1836.3 | | Ca | 394 | 3551772.9 | 416 | 3961937 | | Na | 109 | 1336587.1 | 111 | 1469587.7 | | K | 513 | 1337091.6 | 534 | 1247296.9 | DDP 2/11_1 | P | 384 | 121727.8 | 419 | 140429.1 | | Zn | 6.94 | 56851.1 | 7.52 | 63912.1 | | Mn | 28.2 | 880.5 | 33.7 | 1372.8 | | Fe | 8.61 | 99465.8 | 9.34 | 112009.6 | | Mg | 55 | 61159.6 | 64.2 | 725611 | | Cu | 437 | 6369.7 | 486 | 7608.9 | | Ca | 540 | 4902288.9 | 591 | 5662865 | | Na | 175 | 2215471.3 | 185 | 2493544 | | K | 642 | 1431965.1 | 687 | 1608637 | DDP 2/11_2 | P | 385 | 122685.8 | 419 | 141197.7 | | Zn | 7.04 | 57995.6 | 7.54 | 64356 | | Mn | 28.9 | 939.5 | 33.7 | 1381.1 | | Fe | 8.77 | 101823.4 | 9.39 | 113162 | | Mg | 54.4 | 607351.8 | 59.3 | 699603 | | Cu | 432 | 6326.4 | 479 | 7537.8 | | Ca | 546 | 4978194.1 | 595 | 5737230.8 | | Na | 176 | 2241281.8 | 183 | 2489153.8 | | K | 648 | 1453512.3 | 685 | 1612031 | DDP 3/11_1 | P | 348 | 111137.8 | 374 | 120014.8 | | Zn | 6.11 | 48533.4 | 6.32 | 52613.1 | | Mn | 80.8 | 5255.6 | 83.6 | 5778.1 | | Fe | 8.63 | 95600 | 8.92 | 103653.7 | | Mg | 57 | 662049 | 64.4 | 724995.3 | | Cu | 393 | 4538 | 426 | 5185.3 | | Ca | 582 | 5336981 | 626 | 5763511.9 | | Na | 181 | 2371039 | 187 | 2570413 | | K | 495 | 1101779.9 | 512 | 1193786 | DDP 3/11_2 | P | 345 | 110161.4 | 376 | 120594.4 | | Zn | 5.97 | 47360.6 | 6.3 | 52431.9 | | Mn | 81 | 5265.6 | 85.9 | 5932.1 | | Fe | 8.24 | 91267.5 | 8.7 | 101057.6 | | Mg | 55.7 | 696643 | 60.8 | 707959.5 | | Cu | 372 | 4210 | 412 | 4947.2 | | Ca | 582 | 5344915 | 636 | 5851531.4 | | Na | 174 | 2281912 | 184 | 2526368 | | K | 502 | 1191873.9 | 363 | 849044 | DDP 3/11_3 | P | 337 | 102293 | 346 | 111465 | | Zn | 5.97 | 45176 | 5.98 | 49552 | | Mn | 76.2 | 4866.1 | 76.9 | 5435 | | Fe | 8.19 | 87692 | 8.21 | 96159 | | Mg | 56.1 | 615925.5 | 57.6 | 671327 | | Cu | 391 | 4246.7 | 403 | 4945.4 | | Ca | 561 | 4804490 | 575 | 5235876.4 | | Na | 178 | 2208699 | 178 | 2423166 | | K | 505 | 1078670 | 506 | 1182778 | DDP 3/11_4 | P | 336 | 102921.7 | 347 | 112900 | | Zn | 6 | 45819 | 5.97 | 49928 | | Mn | 78.2 | 5039 | 78.4 | 5583 | | Fe | 8.31 | 89721 | 8.27 | 97756 | | Mg | 56 | 620441.6 | 57.9 | 680774 | | Cu | 387 | 4246.8 | 403 | 5002 | | Ca | 552 | 4766769 | 570 | 5229616 | | Na | 184 | 2308868 | 183 | 2516679 | | K | 510 | 1097772.6 | 507 | 195877 |
Table 7 Results (both with and without Internal Standard) for Reference Samples

4.3.1 Calculation Sheets
These are the sheets that are prepared to check the Trueness and Precision of the Results. Precision is checked by checking Repeatability, which is the measured by comparison of the observed results and the actual results (as the actual results of these samples are already known)
Trueness is checked by checking Reproducibility, which is measured by comparison of the results of same sample, which is analyzed twice.
In these calculation sheets, the following abbreviations are used:
Ref Value: The actual concentration
R1: observed concentration of first sample
R2: observed concentration of second sample
CV%: Limits for repeatability
R%: Limits for reproducibility
Remarks 1: if OK: Results are within the Repeatability limits If CHECK: Results are NOT within the Repeatability limits
Remarks 2: if OK: Results are within the Reproducibility limits If CHECK: Results are NOT within the Reproducibility limits

4.3.1.1 BFF (With Internal Standard):

Parameter | Ref Value | CV% | r% | R1 | R2 | Average | Diff % 1 | Remarks 1 | Diff % 2 | Remarks 2 | | | | | | | | | | | | Zinc | 2.49 | 7.0% | 5.0% | 2.32 | 2.34 | 2.33 | 6.4% | OK | 0.9% | OK | | | | | | | | | | | | Manganese | 580 | 15.0% | 10.0% | 561.2 | 562.7 | 562.0 | 3.1% | OK | 0.3% | OK | | | | | | | | | | | | Iron | 7.87 | 8.0% | 6.0% | 7.25 | 7.47 | 7.36 | 6.5% | OK | 3.0% | OK | | | | | | | | | | | | Magnesium | 49.3 | 6.0% | 4.0% | 49.6 | 49.6 | 49.6 | 0.6% | OK | 0.0% | OK | | | | | | | | | | Copper | 0.12 | 12.0% | 10.0% | 0.116 | 0.121 | 0.12 | 1.3% | OK | 4.2% | OK | | | | | | | | | | | | Calcium | 403 | 6.0% | 5.0% | 392 | 394 | 393 | 2.5% | OK | 0.5% | OK | | | | | | | | | | | | Sodium | 108 | 8.0% | 5.0% | 107 | 109 | 108 | 0.0% | OK | 1.9% | OK | | | | | | | | | | | | Phosphorus | 319 | 6.0% | 5.0% | 321 | 317 | 319 | 0.0% | OK | 1.3% | OK | | | | | | | | | | | | Potassium | 530 | 6.0% | 4.0% | 520 | 513 | 517 | 2.5% | OK | 1.4% | OK |

Calculation Sheet 1: Showing the checks for Repeatability and Reproducibility for BFF1 and BFF2 with Internal Standard

4.3.1.2 BFF (Without Internal Standard)

Parameter | Ref Value | CV% | r% | R1 | R2 | Average | Diff % | Remarks | Diff % | Remarks | | | | | | | | | | | | Zinc | 2.49 | 7.0% | 5.0% | 2.41 | 2.13 | 2.27 | 8.8% | CHECK | 12.3% | CHECK | | | | | | | | Manganese | 580 | 15.0% | 10.0% | 596.1 | 590.4 | 593.3 | 2.3% | OK | 1.0% | OK | Iron | 7.87 | 8.0% | 6.0% | 7.91 | 7.63 | 7.77 | 1.3% | OK | 3.6% | OK | | | | | | | | | | | | Magnesium | 49.3 | 6.0% | 4.0% | 52.3 | 52 | 52.2 | 5.8% | OK | 0.6% | OK | | | | | | | | | | | | Copper | 0.12 | 12.0% | 10.0% | 0.133 | 0.126 | 0.13 | 7.9% | OK | 5.4% | OK | | | | | | | | | | | | Calcium | 403 | 6.0% | 5.0% | 410 | 416 | 413 | 2.5% | OK | 1.5% | OK | | | | | | | | | | | | Sodium | 108 | 8.0% | 5.0% | 111 | 108 | 110 | 1.4% | OK | 2.7% | OK | | | | | | | | | | | | Phosphorus | 319 | 6.0% | 5.0% | 336 | 334 | 335 | 5.0% | OK | 0.6% | OK | | | | | | | | | | | | Potassium | 530 | 6.0% | 4.0% | 539 | 534 | 537 | 1.2% | OK | 0.9% | OK |
Calculation Sheet 2: Showing the checks for Repeatability and Reproducibility for BFF1 and BFF2 without Internal Standard

4.3.1.3 DDP 2/11 (With Internal Standard)

Parameter | Ref Value | CV% | r% | R1 | R2 | Average | Diff % 1 | Remarks 1 | Diff % 2 | Remarks 2 | | | | | | | | | | | | Zinc | 6.88 | 7.0% | 5.0% | 6.94 | 7.04 | 6.99 | 1.6% | OK | 1.4% | OK | | | | | | | | | | | | Manganese | 26.9 | 15.0% | 10.0% | 28.2 | 28.9 | 28.6 | 6.1% | OK | 2.5% | OK | | | | | | | | | | | | Iron | 8.94 | 8.0% | 6.0% | 8.61 | 8.77 | 8.69 | 2.8% | OK | 1.8% | OK | | | | | | | | | | | | Magnesium | 54.9 | 6.0% | 4.0% | 55.0 | 54.4 | 54.7 | 0.4% | OK | 1.1% | OK | | | | | | | | | | | | Copper | 0.47 | 12.0% | 10.0% | 0.437 | 0.432 | 0.43 | 7.6% | OK | 1.2% | OK | | | | | | | | | | | | Calcium | 576 | 6.0% | 5.0% | 540 | 546 | 543 | 5.7% | OK | 1.1% | OK | | | | | | | | | | | | Sodium | 185 | 8.0% | 5.0% | 175 | 176 | 176 | 5.1% | OK | 0.6% | OK | | | | | | | | | | | | Phosphorus | 392 | 6.0% | 5.0% | 384 | 385 | 385 | 1.9% | OK | 0.3% | OK | | | | | | | | | | | | Potassium | 652 | 6.0% | 4.0% | 642 | 642 | 642 | 1.5% | OK | 0.0% | OK |
Calculation Sheet 3: Showing the checks for Repeatability and Reproducibility for DDP2/11_1 and DDP2/11_2 with Internal Standard

4.3.1.4 DDP 2/11 (Without Internal Standard)

Parameter | Ref Value | CV% | r% | R1 | R2 | Average | Diff % | Remarks | Diff % | Remarks | | | | | | | | | | | | Zinc | 6.88 | 7.0% | 5.0% | 7.54 | 7.52 | 7.53 | 9.4% | CHECK | 0.3% | OK | | | | | | | | Manganese | 26.9 | 15.0% | 10.0% | 33.7 | 33.7 | 33.7 | 25.3% | CHECK | 0.0% | OK | | | | | | | | | | | | Iron | 8.94 | 8.0% | 6.0% | 9.39 | 9.34 | 9.37 | 4.8% | OK | 0.5% | OK | | | | | | | | | | | | Magnesium | 54.9 | 6.0% | 4.0% | 59.3 | 64.2 | 59.3 | 8.0% | CHECK | 7.9% | CHECK | | | | | | | | | | | | Copper | 0.47 | 12.0% | 10.0% | 0.47 | 0.48 | 0.48 | 1.1% | OK | 2.1% | OK | | | | | | | | | | | | Calcium | 576 | 6.0% | 5.0% | 595 | 591 | 593 | 3.0% | OK | 0.7% | OK | | | | | | | | | | | | Sodium | 185 | 8.0% | 5.0% | 183 | 185 | 184 | 0.5% | OK | 1.1% | OK | | | | | | | | | | | | Phosphorus | 392 | 6.0% | 5.0% | 419 | 419 | 419 | 6.9% | CHECK | 0.0% | OK | | | | | | | | | | | | Potassium | 652 | 6.0% | 4.0% | 685 | 642 | 664 | 1.8% | OK | 6.5% | CHECK |
Calculation Sheet 4: Showing the checks for Repeatability and Reproducibility for DDP2/11_1 and DDP2/11_2 without Internal Standard

4.3.2 Discussion:
The above calculation sheets show that i. In the samples with internal standard, the results are comparable to the reference values and also the results are reproduced in two same samples. Both Trueness and Precision are observed in these samples ii. In the samples without internal standard, the results are not comparable to the reference samples in some of the cases and are not reproduced in two same samples in most of the cases. These samples fail to give true and precise results.

The above difference is explained by the fact that in the absence of internal standard, the results are deviated due to the viscosity of the samples which leads to increased or decreased sample intake by the tubings (matrix effects) due to which the amount of sample reaching the plasma for detection varies and hence wrong results are produced by the ICP OES. In the samples with internal standard, this deviation is avoided as the internal standard compensates the matrix effect.

CONCLUSION ICP-OES is one of the most appropriate techniques for elemental analysis with some important features. However, its potentially excellent analytical characteristics are degraded due to the presence of matrix effects. When robust plasma conditions are used, these effects are reduced but not totally eliminated; therefore, generally different methods such as internal standardization may be applied to compensate for these effects.

The choice of appropriate internal standard is very important since the success of internal standardization highly depends on the similarity between the analyte and internal standard.

In this project, it was seen that the results of samples without the internal standard vary a lot and the effect is different in different food matrices. In water samples, less variation was observed as the viscosity effect is low in this case and also because water samples have low salt content. However, in culinary samples, the variation vas very high, especially in the case of Mn, Zn and Cu. This is because the culinary samples are made with many different ingredients, due to which these samples have high viscosity and salt content which effects the sample intake quantity by the tubings and hence, the results are high/low.

In the reference samples, it was observed that the samples with internal standard give true and precise results whereas the samples without internal standard fail to do so due to non-compensation of the matrix effects.

Also the use of internal standard stabilizes the plasma as it contains the ionization buffer Cesium Chloride. This property also helped the samples with internal standard to reproduce the results in same samples.

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