ICP-OES: An Advance Tool in Biological Research

The metal contamination in soil water and air has become a serious concern to the health, farming and food safety [1]. Therefore, metal analysis in plants, animals and microorganisms is essential for eco-toxicological studies. Several diagnostic and therapeutic studies of health issues for instance decline in the immunological effi ciency, cardiac disorders, fetal abnormalities, gastrointestinal cancer, redox reactions; cellular energetic and abnormal neurological activity patterns are also required to quantify the accumulation of trace metal in biological tissues [2]. The study of metals is an essential part of research in genetics, environmental toxicology, bioremediation, host-parasite relationship, nanotechnology, microbiology, structural biology, cytology, physiology, biomedicines and clinical biology. The interdisciplinary intrusion of metal study in biological science demands to build up more advanced, effi cient and sophisticated analytical tools and techniques of metal analysis. A combination of biochemical and spectroscopic techniques has been used to study the mechanism of metal metabolism in biological material. In biological science laboratories ICP-MS, ICP-OES (Inductively Coupled Plasma Optical Emission Spectroscopy), Flame Atomic Emission, Flame Atomic Absorption, Graphite Furnace Atomic Absorption and Atomic Absorption (cold vapor/ hydride generation) spectrometry techniques have been used for quantitative and qualitative analysis of metals. The selection of method for metal analysis depends on the range of element concentration in the sample. The ICP-MS is suitable if the range is part per trillion (ppt), furnace–AAS is used if the range is part per billion (ppb) for less than 5 elements as low dissolved solids in a sample. The fl ame-AAS is a good choice for analysis of fewer than 5 elements of part per million (ppm) and ICP-OES is selected for ppb as well as ppm high dissolved solid more than 5 multi-elements in a sample. Among them, ICP-OES is the most sophisticated multi-element analysis technique. The technique is widely used in offi cial testing of drinking water, trace elements attached with proteins, food, fi eld soil, soil-sediments, fossil fuel, bio-fuel and medicines in national and international testing laboratories [3]. A vast literature is available on the role of ICP-OES in soil, sediments, oil and other non-biological sample analysis but the application of the instrument in biological research warrant more critical analysis and compilation of research reports. The present review is an up-to-date collection of reports showing metal analysis in biological samples by ICP-OES.


Introduction
The metal contamination in soil water and air has become a serious concern to the health, farming and food safety [1]. Therefore, metal analysis in plants, animals and microorganisms is essential for eco-toxicological studies.
Several diagnostic and therapeutic studies of health issues for instance decline in the immunological effi ciency, cardiac disorders, fetal abnormalities, gastrointestinal cancer, redox reactions; cellular energetic and abnormal neurological activity patterns are also required to quantify the accumulation of trace metal in biological tissues [2]. The study of metals is an essential part of research in genetics, environmental toxicology, bioremediation, host-parasite relationship, nanotechnology, microbiology, structural biology, cytology, physiology, Furnace Atomic Absorption and Atomic Absorption (cold vapor/ hydride generation) spectrometry techniques have been used for quantitative and qualitative analysis of metals. The selection of method for metal analysis depends on the range of element concentration in the sample. The ICP-MS is suitable if the range is part per trillion (ppt), furnace-AAS is used if the range is part per billion (ppb) for less than 5 elements as low dissolved solids in a sample. The fl ame-AAS is a good choice for analysis of fewer than 5 elements of part per million (ppm) and ICP-OES is selected for ppb as well as ppm high dissolved solid more than 5 multi-elements in a sample. Among them, ICP-OES is the most sophisticated multi-element analysis technique. The technique is widely used in offi cial testing of drinking water, trace elements attached with proteins, food, fi eld soil, soil-sediments, fossil fuel, bio-fuel and medicines in national and international testing laboratories [3]. A vast literature is available on the role of ICP-OES in soil, sediments, oil and other non-biological sample analysis but the application of the instrument in biological research warrant more critical analysis and compilation of research reports. The present review is an up-to-date collection of reports showing metal analysis in biological samples by ICP-OES. uses the emission spectra of sample molecules to identify and quantify the elements present. The instrument has two parts one is inductively couple plasma and another one is an optical emission spectrometer . The major components of the ICP-OES   instrument are sampler, pump, nebulizer, spray chamber, ICP torch, monochromator /polychromator, and detector arranged in a defi nite order.
The argon gas is supplied to the torch tube and simultaneously an electromagnetic fi eld is generated by high frequency electric current in work coil positioned on the pointed end of the torch tube. The strong magnetic fi eld induces the ionization of argon in torch tube. The ionized gas produced plasma with high electron density, temperature (10,000K) and energy. The newly produced bulk of the energy is used for excitation and emission of molecules in a test sample. The plasma provides a higher degree of sensitivity and stability by restrained formation of stable complex compounds that leads to simultaneous excitation of many elements.
The pretreated or digested liquid sample is introduced with a peristaltic pump into the nebulizer and then into the torch. The nebulizer turns the sample into tiny droplets like a fi ne mist. The larger droplets of the sample are going down in the drain but fi ne droplets are directed into the high plasma.
The sample is introduced into the plasma in an atomized state through the narrow tube in the center of the torch tube.
The sample goes to the plasma and hot plasma atomizes the molecules and excites them to a higher state. When the excited atoms return to low energy position, emission rays (spectrum rays) are released and the emission rays that correspond to the photon wavelength or emission lines are specifi c for each metal.
The light is transferred to high resolution no purge seal optical system or spectrometer (OES). A radiofrequency generator (RF) produced an oscillation current in an induction coil located in the plasma tube. The tube develops an oscillating magnetic fi eld in the sample ions during exposure to plasma gas. The prisms separate the light into the specifi c wavelength for the elements to be measured. This wavelength tracks the detector ray and the light intensity for each different wavelength is quantifi ed by sophisticated analytical software that can convert them into concentration units [4]. The software also provides calibration, accuracy and precision limits after internal calculations. Thus ICP-OES is used to rapid and simultaneous analysis of elements present in the samples.

Biological sample preparation
The analysis of water, oil or non-living samples is differed from biological sample due to presence of organic and inorganic substances. The removal of organic matter and extraction of pure metal from a crude sample is known as the process of sample preparation. A fi ne homogenous solution of a sample is essential to eliminate interference and chocking of the nebulizer. Prior to sample preparation, the glassware is washed with non-ionic detergents (Tween or Triton), socked in 10% HNO 3 for 24 hours and subsequent oven-dried (750 o C) to circumvent ionic contamination during analysis. The procedure of sample preparation purely depends on the nature of the source material. The biological samples have a high amount of organic content, salts or complex structures that can obstruct metal analysis so, acid digestion is used for the extraction of pure metals from the cell and cellular structure [5]. Three basic steps for sample preparation for ICP-OES analysis are digestion, evaporation and extraction. Table 1 illustrates the different methods of sample preparation reported for specifi c biological samples. Nitric acid (HNO 3 ) is used for acid digestion because hydrochloric (HCl) or sulfuric acid (H 2 SO 4 ) can develop chlorides and sulfates of metal ions [5]. Acid digestion with sample-specifi c thermal treatment or sonication is used for the isolation of metal elements form organic substances. Digestion of biological samples with individual acid or combination of HCl and HNO 3 or HNO 3 and HF, with an oxidizing agent (hydrogen peroxide), is suggested by various researchers [6]. HF is specifi c for the total dissolution of silicon-rich plant material [7]. Researcher [8] has used strong alkaline substances (pH  [6]. Enzymatic pretreatment for the hydrolysis of algal samples is also suggested by [9].

Preparation of instrument
Prior to the sample introduction, the instrument is

Digestion and Evaporation
Take a 50 ml digestion bottle with a screw. Put 0.5 g dried plant sample with concentrated HNO 3 (8ml) to destroy organic substances and keep it for 12 hours at ±25˚C in a fume hood. After the addition of 2 ml H 2 O 2 digest the mixture at 95-110 ˚C on a hot plate. After evaporation at high temperature, the remaining dry mixture is left in the digestion bottle. Extraction: Add 5 ml of deionized water in dry material and keep it for heating till yellow fumes are formed. Again add 5 ml deionized water and maintain it to cool at room temperature. Filter the extract with Whatman no 2-fi lter paper. Makeup 50 ml volume of the extract with 0.1 M HNO3. The sample is ready for metal analysis. [10] Plants

Digestion and Extraction
The 0.5 g dried sample mixed with 10 ml of HNO 3 -HCl-H 2 O 2 (8:1:1, v/v/v) in a digestion bottle and place it in the heating element at 120 o C till the solid is completely converted in a fi ne solution. Make up the fi nal volume (50 ml) of a fi ne extract with ultra-pure water. Samples can be stored in polyethylene containers. [11] Antarctic macro algae

Digestion and Extraction
Take a 0.5 g oven-dried sample (80°C) with 2 ml H 2 O 2 , 8 ml HNO 3 and 2 ml HF and left the mixture for 24 hours. Add 2 ml HNO 3 and 2 ml HClO 4 and set it for microwave digestion cycles (250W-600 W).

Extraction
Digests are allowed to evaporate on hot plate. Add 2.5 ml of HNO 3 and 47.5 ml deionized water to the residue and store the clear solution for ICP-OES. [7]

Fucus and Sargasso
Digestion and Evaporation Take 0.2 g of grounded algae with pepsin (prepared in 1% NaCl at pH1.0). Place the mixture on sonicator for 30 min at 37 °C.

Extraction
Centrifuge the mixture at 3000 rpm for 15 min. Take supernatant and made up the fi nal volume with deionized water. [9] Fungi

Digestion and Evaporation
Fungi samples dried at 110 o C for 90 hours and ground it with the mill. Take a 0.5 gm sample with HNO3 and keep them in the microwave for digestion.

Extraction:
The digest is fi ltered with fi lter paper and the fi nal volume (15 ml) is made with deionized water. [12] Mushroom Digestion A 0.31 gm mushroom sample is mixed with phosphoric acid and digested in an ultrasonic bath.

Extraction:
Filter the sample and make a fi nal volume of 15.0 ml with deionized water. [12] Algae Digestion A 50mg of the dried sample (at 85°C for 4h) is mixed into a vial having a mixture of 25% TMAH (1ml) and 0.2 mol EDTA. After 10 minutes continuous shaking vials are capped and placed in a thermostat oven set at 120 0C. Add 2 ml of water to the remaining solution and set the pH 8-9 with HCl. Extraction Add water to a fi nal volume up to 5 ml. Centrifuge the solution at 1500 rpm for 3 min and the supernatant is used for metal analysis by ICP-OES. [8]

Bacteria
The metal-rich bacterial samples are centrifuged at 7500 rpm for 10 minutes and the supernatant is directly used for metal analysis. [13] Microbial paleontological study Pork liver, Bovine liver, Bovine muscle Take 100 mg animal tissue sample and add 25% TMAH and 2.5 ml distilled water. Place it on an ultrasonic bath for half-hour than in microwave for 30 minutes. After cooling the digest fi nal volume (20 ml) is made-up by deionized water.

Introduction of prepared samples
The prepared liquid samples are taken up by sampler and

Application of ICP-OES reported in biological research
Metal analysis by ICP-OES is a rising trend in biological research. Table 2

Advantages and limitations Of ICP-OES
The ICP-OES and ICP-MS are the two most advanced metal analysis techniques frequently used in biological sample analysis from last decay [39]. A comparative study of ICP-MS and ICP-OES is essential to establish the requirement, condition and signifi cance of a method to obtain desired results [40]. Table 3   Blood and Urine Ba [29] Procambarus clarkii Cd, Cu, Fe, Ni, Pb, Zn [30] Pork liver, bovine liver, bovine muscle Si [16] Pepperbush, chlorella, hair, mussel, tea leaves, Sargasso, rice fl our, and bovine liver  ICP-OES is used to detect 73 elements of periodic table except radioactive elements, and halogen groups.

2
The metal detection limit is parts per trillion (ppt) The lower limit of element concentration in parts per billion (ppb) 3 The tolerance for total dissolved solid is about 0.2% therefore it has low regulatory limits.
The tolerance for high total dissolved solids (TDS) or suspended solids is up to 30% with high regulatory limits.

4
A limited range of samples can be analyzed by ICP-MS. Useful for analysis of drinking water and less complex components of laboratory samples.
Multi-element analysis in a wide range of samples from soil, polluted water, soil waste, drinking water to complex biological samples can be done with ICP-OES 5 It has dynamic linear range 10 8 so the sample dilution is essential before the analysis with ICP-MS.
Sample dilution is not required because it has 10 6 dynamic linear ranges for the detection of the multi-element present with a wide concentration difference. 6 An ICP-MS system is costly due to the high consumption of argon. ICP-OES is cheaper due to the lesser requirement of argon for study.

7
The installing, operating and maintaining cost of ICP-MS system is higher than ICP-OES. ICP-OES is two to three times cheaper than ICP-MS system 8 Effi ciently remove polyatomic spectral interferences using collision cell technology Spectral interference is removed by using Fast Automated Curve-fi tting Technique 9 Rapid semi-quantitative analysis is done by a separate method for high precision.
Rapid semi-quantitative analysis is done by using the echellogram which allows users to view the relative intensity of all peaks.

10
Sample enter in the instrument for ionization and passing of ions through plasma. Therefore, there are chances for sample deposition.
Only photons are measured from the sample after passing through the plasma. Therefore, the system is more stable than ICP-MS.

11
Each time new internal standards and new calibration curve required for accurate results.
The ICP-OES is a stable system therefore, various types of semi quantities analysis may be done using previously stored calibration curves. High detection limit, inter-element interference, cost of equipment, and lab setups with expert technical staff are some limitations but ICP-OES is the most promising technology for metal detection in biological researches.

Conclusion
Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES Analysis) is a trace-level, elemental analysis technique that uses the emission spectra of a sample to identify and quantify the elements present. The major limitation is inter-element interference which is overcome by high resolutions chip and mathematical manipulation in data.
Competitively low cost, simple operation, high stability, easy method development, capacity to evaluate refractory element, high throughput, high sensitivity and dealing with large samples per day and per run make it an excellent selection for biological laboratories.