|Year : 2022 | Volume
| Issue : 3 | Page : 213-221
A comparative analysis of macro and micronutrient content in nutraceutical “Asparagus racemosus (Shatavari Roots)” by energy dispersive X-ray fluorescence and ion chromatography
Shalvi Agrawal, Astha Pandey
Teaching & Research Assistant, Senior Research Scholar, School of Forensic Science, National Forensic Sciences University, Gandhinagar, Gujarat, India
|Date of Submission||10-May-2021|
|Date of Decision||10-Sep-2021|
|Date of Acceptance||26-Dec-2021|
|Date of Web Publication||28-Sep-2022|
School of Forensic Science, National Forensic Sciences University, Gandhinagar - 382 007, Gujarat
Source of Support: None, Conflict of Interest: None
Introduction: Shatavari is indeed a highly rejuvenating herb for both males and females, a revitalizing tonic for most of the problems related to hormonal changes and reproductive system and is a commonly used nutraceutical. Macro and micro elemental concentration of the Ayurvedic medicinal plant, Shatavari (Asparagus racemosus), is investigated for the first time in detail, using Energy Dispersive X-Ray Fluorescence (EDXRF) technique and Ion Chromatography with Conductometric Detector (IC-CD). Methods: In the present study, root powders have been analyzed directly by EDXRF, and extracted root samples have been analyzed by IC-CD. The data of standard plant sample has been compared with ten other market samples. Results: The EDXRF results have been given for 20 elements in percentage composition for K, Ca, P, Si, S, Fe, Zn, Mn, Ti, Cu, Ba, Sr, Ni, V, Cr, Os, Ho, Br, Ge, and Rb whereas IC-CD results have been given in ppm level for six essential elements for Li, K, NH4+, Ca, and Na. Discussion: It has been found from the results that there are many essential elements that were absent in some of the commercial samples. Some samples reported very low concentration of one or the other elements that are crucial for its role in human body owing to which the nutraceutical is consumed. Conclusion: The results revealed a significant difference between the standard and the sample drug by both the methods suggesting that there is indiscriminate use of exhausted and adulterated drug in commercially available nutraceuticals. Furthermore, there is a crucial need to standardize such drugs before it reaches consumers.
Keywords: Adulteration, Asparagus racemosus, ayurvedic, energy dispersive X-ray fluorescence, herbal medicines, ion chromatography, nutraceuticals
|How to cite this article:|
Agrawal S, Pandey A. A comparative analysis of macro and micronutrient content in nutraceutical “Asparagus racemosus (Shatavari Roots)” by energy dispersive X-ray fluorescence and ion chromatography. J Ayurveda 2022;16:213-21
|How to cite this URL:|
Agrawal S, Pandey A. A comparative analysis of macro and micronutrient content in nutraceutical “Asparagus racemosus (Shatavari Roots)” by energy dispersive X-ray fluorescence and ion chromatography. J Ayurveda [serial online] 2022 [cited 2022 Nov 29];16:213-21. Available from: http://www.journayu.in/text.asp?2022/16/3/213/357292
| Introduction|| |
Our Indian history is well known for the use of several of numerous medicinal plants in the treatment of various diseases. This treatment of health-related problems using various medicinal herbs is known as Ayurveda. This paper focuses on one of the most important medicinal herbs used extensively in Ayurveda, Asparagus racemosus.
A. racemosus is also called the “Queen of herbs,” because of its properties that promote love and devotion. A. racemosus locally known as Shatavari is the most commonly used Ayurvedic rejuvinative tonic for female, similarly to Withania is used for the male. Shatavari has been found to exhibit properties such as madhurrasam, madhurvipakam, seet-veeryam, somrogam, and effective in treating chronic fever and internal heat. The use of shatavari has also been mentioned in Caraka Samhita.
This plant belongs to the genus Asparagus which has recently moved from the subfamily Asparagae in the family Liliaceae to a newly created family Asparagaceae. It has 30–100 cm thick, short and fusiform tuberous roots. The powdered roots contain 2.95% protein, 5.44% saponins, 52.89% carbohydrate, 17.93% crude fiber, 4.18% inorganic matter, and 5% oil. The important bioactive constituents of Shatavari are a group of steroidal saponins (Shatavarin I, II, III, and IV). This plant also contains vitamins namely A, B1, B2, C, E, minerals like Mg, P, Ca, Fe, and folic acid. Its other important chemical constituents are essential oils, alkaloids, asparagine, arginine, tyrosine, flavonoids (kaempferol, quercetin, and rutin), resin, tannin, and terpenoids. Phytosteroidal saponins present in the shatavari possess anti-oxidant property, galactagogue effect,, antibacterial activity, anti-hepatotoxic activity,, anti-diabetic, immunomodulatory activity, anti-diarrheal activity, anti-dyspepsia activity, and anti-ulcer activity.,
According to FSSAI, “Nutraceuticals” can be defined as a naturally occurring chemical compounds that have a physiological benefit or that provide protection against chronic disease. Nutraceuticals can be isolated and purified from food or nonfood source and may be prepared and marketed in the form of granules, powder, tablet, capsule, liquid or gel and may be packed in sachet, ampoule, bottle, etc., and to be taken as measured unit quantities.
Schedule V of FSSAI enlists A. racemosus as a nutraceutical from plant or botanical origin and recommends its permissible daily dose. Maximum usage levels of A. racemosus per day for use as a health or food supplement (given in terms of raw herb/material) is 3–6 g for adult, half of the adult usage levels for 5–16 years of age group and 1–4th of the adult usage levels for 1–5 years of age groups in form of powder.
Due to the growing population, there is a great demand of food leading to degradation in food quality to meet the requirements on large scale. Increase in population is not only affecting food manufacturing sectors but also the medicinal sectors. Medicinal plants which are the prime source of drug preparation are now being replaced by the materials or substances having similar appearance and functional properties but are of inferior quality, cheaper, and easily available in the market. It reduces the cost of production and increases the rate of production at the same time but this ultimately affects the health of the consumer. Adulteration can be defined as any practice that involves the intentional or unintentional addition of foreign substance as a substitute to original crude drug either partially or fully which is inferior or sub-standard in therapeutic and chemical properties or addition of low grade or spoiled or exhausted or entirely a different drug morphologically similar to that of original drug adding with an intention to enhance profits.,, In adulterated drugs, the Adverse Event Reports are not due to the intended herb, but due to the presence of an unintended herb.,, Medicinal plant dealers and traders over the time have discovered many scientific methods that create adulterants of such a high quality that without detailed microscopic and chemical analysis, it becomes very difficult to trace these adulterations.,, Also for a common consumer, it is near to impossible to detect these adulterations.
The destructive use of Shatavari has led to endangering of the wild species found in India. To make up with the requirement for the treatment, many artificial drugs or plants having similar properties are added as adulterants in the herbal formulations.
In this paper, a comparison has been done between the two elemental techniques, i.e., energy dispersive X-ray fluorescence (EDXRF) and IC with conductometric detector The most important advantages of EDXRF for the quantitative and qualitative analysis are (1) simultaneous determination of many elements, (2) determination in a wide concentration range, (3) simple and fast sample preparation, and (4) much lower equipment cost than that of a conventional wavelength X-ray fluorescence spectrometer. Ion chromatography separation is based on the principle of ionic (or electrostatic) interactions between ionic and polar analytes, present in the eluent along with mobile phase ions and ionic functional groups attached to the chromatographic support column known as stationary phase. Two distinct mechanisms take place in interaction between the ions, one is the ion exchange due to competitive ionic binding or attraction and other is the ion exclusion due to repulsion between similarly charged analyte ions and the ions fixed on the chromatographic column. These two interactions along with other forces play a major role in the separation in ion chromatography. The most important advantages of ion chromatography among many are a broad range of applications, well-developed hardware, many detection options, reliability with good accuracy and precision, high selectivity, high speed, high separation efficiency, good tolerance to sample matrices, and low cost of consumables.
| Materials and Methods|| |
Standard Shatavari plant roots have been collected from Varanasi Region, U. P. with the help of local people and gardeners. The collected sample was dried and pasted in a herbarium sheet and given for authentication. The sample was authenticated at the Department of Botany, Institute of Science, BHU, Varanasi. A voucher specimen number Lilia. 2019/3 was provided for the plant sample. Besides standard sample, ten marketed samples of different brands have also been collected.
Processing of standard sample
The freshly collected roots (about 500 g) were washed under running tap water to remove the adhered soil particles and other dirt. The washed roots were boiled in distilled water till the roots become soft. The boiled roots were then peeled off and dried under sun for 2–3 days. The roots can also be dried in oven at 100°C. The dried roots were then powdered and stored in an air-tight container.
Processing of marketed samples
The commercial samples were procured from online stores as well as from local markets. These were already in the form of fine powder so they were not processed further. They were also labeled and stored in air-tight containers.
Sample preparation for energy dispersive X-ray fluorescence
For EDXRF analysis, all the samples including standard drugs were used in the powder form only and no sample preparation was required further.
Sample preparation for IC
Extraction of samples
Forty gram g of each sample was taken and extracted in a mixture of distilled water and methanol taken in a ratio of 70:30 in Soxhlet Apparatus of 500 mL. Each sample was extracted for 6 h. The extracted solution would be dark brown in color. The solution was collected and excess solvent was evaporated. The final extract would be thick tar like dark brown in color. The dried extracts were stored at 4°C for further use.
Preparation of standards and samples for cation analysis in IC
10 ppm standards of calcium, lithium, potassium, sodium, and ammonium of high purity were purchased from Sigma Aldrich. All the cations standards were mixed in equal quantity and 25 mL of standard cation solution was prepared. The standard cation mixture solution was kept in IC tubes supplied by Metrohm. 0.1 g of each extract was weighed in separate tubes and 10 mL of water and methanol in the ratio of 7:3 was added to make 100 times diluted solution. The water and methanol used were of High-performance liquid chromatography grade purchased from Merck. The dissolved samples were filtered by 0.45 μ nylon filters separately and the final (diluted and filtered) samples were kept in IC tubes supplied by Metrohm. Similarly, one control was also prepared by taking 10 mL of water-methanol solvent only.
Experimental method for energy dispersive X-ray fluorescence
One gram of powdered samples was packed into a 20 mm wide polyethylene cup and then covered with 6-μm-thick polypropylene film (Mylar®). The samples were irradiated for 600s under vacuum using a “Shimadzu EDX” EDXRF system. Rh X-ray tube that was operated at 15 kV (Na to Sc) and 50 kV (Al to U) was used for the irradiation of the samples. The current was automatically adjusted (maximum of 1 mA). A 10 mm collimator was used. The detection was carried out using the Si (Li) detector cooled with liquid nitrogen.
Experimental method for IC
The cation analysis was performed on Metrohm Ion Chromatography instrument attached with an autosampler and conductivity detector. The samples prepared for IC were put in IC tubes and put on autosampler rack. The following specifications are given in [Table 1]. The data were finally reprocessed with the standard.
| Results and Discussion|| |
Energy dispersive X-ray fluorescence analysis
The EDXRF results of all shatavari samples and standard have been tabulated below in [Table 2]. Based on the data formulated in [Table 2], it can be summarized that a standard shatavari root powder must contain following essential elements like Potassium (50.156%), Calcium (30.327%), Sulfur (5.917%), Phosphorus (5.267%), Iron (3.173%), and Zinc (1.920%). Apart from these, Silicon, Manganese, Titanium, and Copper were found to be present in trace amounts. It has been found that high concentration of Potassium along with Calcium plays an important role in regulating neuromuscular activity. Potassium also helps in activating enzymes, metabolizing carbohydrates and proteins, and in regulating heartbeat. Calcium as is well known is essential for bones and it has been reported in many studies that it helps in increasing bone density in postmenopausal women when supplemented with other trace minerals. Various studies suggest that the human blood contains almost 60 minerals and these minerals are also needed in human diet regularly. The mother's milk, the most required and essential diet of newborn child also contains these 60 minerals along with nickel and vanadium. Zinc has a lot to do with the fertility of both men and women. In women, Zinc is found to be beneficial in treating infertility whereas in men, it plays an important role in improving the sperm count, motility, and morphology of sperm in sub-fertile men having idiopathic asthenozoospermia. Manganese is a good antioxidant and is essential in functioning of the central nervous system. Manganese (≈0.11 mg/g in leaf and ≈ 0.5 mg/g in root) is also known to facilitate insulin production. Mitochondrion is the storehouse for manganese and it activates enzymes and molecules involved in metabolism of fatty acids and synthesis of proteins and is also involved in neurological functions. In women, deficiency of manganese causes osteoporosis. It is critically associated with the normal functioning of thyroid glands and plays a major role in energy metabolism. Phosphorus prevents from osteoporosis in women and is necessary during pregnancy, lactation, and in post menopause, when protective effect of estrogens is depleted.,
|Table 2: Percentage composition of elements in various commercial products containing shatavari by energy dispersive X-ray fluorescence analysis|
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On comparing this data with the results of different commercial products containing shatavari, the following conclusions were made:
AR11 contains the maximum amount of potassium, i.e., 48.182%, while AR2 contains the least, i.e., 0.815% and other samples lay in between the range of AR2 and AR11.
AR2 contains maximum amount of calcium, i.e., 76.671% while AR5 contains the least, i.e., 8.137%. In maximum number of samples except AR5, Calcium is found to be present in large quantities when compared with the standard sample.
Silicon is found to be present in very high concentration in AR5 and AR9, i.e., 39.837% and 33.319%, respectively, which is 20 times more than that of silicon content in the standard sample.
Essential element like Zinc is found to be absent in AR2 and AR10 while phosphorus is absent in only AR2.
Apart from essential elements, several nonessential elements such as Barium, Strontium, Holmium, Germanium, Bromine, Osmium, Chromium, Vanadium, Nickel, and Rubidium were found to be present in trace quantities, although germanium is only present in AR6. The reason for the presence of these trace elements may be the manufacturing process.
When compared with the standard data, AR11 is found to be the most resembling product containing all the essential elements in required concentration.
The IC results of all shatavari samples, control sample, and standards have been tabulated below in [Table 3]. The analysis of both mono and divalent inorganic cations simultaneously can be easily performed by IC with conductometric detector. [Figure 10],[Figure 11],[Figure 12],[Figure 13],[Figure 14],[Figure 15],[Figure 16],[Figure 17],[Figure 18],[Figure 19],[Figure 20] show the separation of cations from the root extracts of Shatavari through chromatographic column. Good selectivity was obtained using the “Metrosep C 6-250/2.0” column and “Metrosep C 6 Guard/2.0” as guard column. Elution of cations was isocratically done with 3 mM Nitric acid for Na+, NH4+, K+, Li + and Ca2+. The eluted cation peaks were identified by comparing it with the retention time of standard peaks as can be seen from the graph. The standard mixture was also injected in three different concentrations under the same experimental conditions as shown in [Figure 1],[Figure 2],[Figure 3]. For quantitative determination of cations, calibration curves were plotted using the using standard solutions as shown in [Figure 4],[Figure 5],[Figure 6],[Figure 7],[Figure 8]. Along with this, a control sample was also run under the same chromatographic conditions, result of which is shown in [Figure 9]. However, as compared to spectroscopic method, ion chromatography with conductometric detector (IC-CD) is a more reliable and valuable alternative for herbal samples,, because it offers less interference from matrix and other cations.,, The reliability of the data sets can be confirmed further by doing statistical analysis with the help of Principle component analysis as is seen from [Figure 21],[Figure 22]a,[Figure 22]b and [Figure 23] Furthermore, IC-CD has the ability to reveal in a single run the complete cation profile including NH4+., In [Table 3], the quantitative value of the identified cations in the root extract is reported. Based upon the results of Ion chromatographic analysis, Potassium was found to be the predominant element in all the shatavari samples except AR 2 and AR 5. Sodium was the major element in AR 2 and AR 5. Other cations occurred variably depending on the sample analyzed. Na + was the second most abundant cation found in samples except AR 11 in which NH4 + was the second most abundant cation. Standard of Mg2 + has not been used since the samples were root and Mg2 + is a major constituent of chlorophyll found in leaves.
|Figure 22: (a) Principle component analysis- data variables (b) Principle component analysis-data observations|
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|Figure 23: Compliance of data variables and observations on each other based on Eigen values through PCA|
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|Table 3: Concentration of cations (in ppm) in various commercial products containing shatavari by ion chromatography analysis|
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| Conclusion|| |
The EDXRF and IC analysis show vast difference in their result. One is a powder technique whereas the other technique involves extraction and sample preparation. From the results of two analyses, the reason for the difference can be inferred as the EDXRF is a surface analysis technique and gives results for the elements present on the surface of the sample therefore the sample should be a homogenous mixture for better results whereas IC analyses the sample in dissolved and diluted form. Both the techniques are useful for elemental analysis and have their own advantages. On the one hand, EDXRF technique does not require any standard to compare with whereas IC analysis cannot be done without standards. EDXRF comprises wide range of elements for qualitative analysis but for quantitation IC is more reliable. Both the data cannot be compared against each other to much extent but if the issue of reliability comes, IC data is more reliable than EDXRF data as the instrument is more sensitive, robust, and has very high limit of detection. Results by EDXRF and IC were the same in terms of qualitative analysis though they both cannot be compared for each and every element. The daily recommended allowance of potassium and calcium according to the Indian Council of Medical Research (ICMR) (2018 guidelines) is 3750 mg and 600 mg respectively. The WHO suggests a potassium intake of at least 90 mmol/day (3510 mg/day) for adults. Standard Shatavari sample (AR1) contains potassium and calcium according to IC analysis, which is under the limit set by ICMR (3750 mg and 600 mg respectively) but other samples contain potassium and calcium either in too low concentration that suggests for their degraded quality or in too high concentration that may be due to adulteration or spiking of these elements from outside other than the natural content. The low level of nutrients from degraded or exhausted product is insufficient to cause the desired effects whereas the higher than recommended level would cause toxicity that in case of chronic consumption would result in hyperkalemia and hypercalcemia. Thus, the two studies with EDXRF and IC are helpful in qualitative and quantitative estimation of micro and macronutrients in nutraceuticals. The techniques are easy, quick, cost-effective, and reliable and can be used for vast variety of nutraceuticals.
The brand name of individual commercial samples has not been revealed as the authors do not wish to promote or downgrade any brand but want to make the readers aware of the illicit practices going on in herbal drug industry so that the consumers do not trust on any big or popular brand blindly. Furthermore, the techniques used in this work are restricted to elemental composition only and not on complete chemical fingerprinting.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Alok S, Jain SK, Verma A, Kumar M, Mahor A, Sabharwal M. Plant profile, phytochemistry and pharmacology of Asparagus racemosus (Shatavari): A review. Asian Pac J Trop Dis 2013;3:242–51.
Kumar S, Mehla RK, Dang AK. Use of shatavari (Asparagus racemosus
) as a galactopoietic and therapeutic herb – A review. Agric Rev 2008;29:132-8.
Negi JS, Singh P, Joshi GP, Rawat MS, Bisht VK. Chemical constituents of Asparagus
. Pharmacogn Rev 2010;4:215-20.
Zhu X, Zhang W, Zhao J, Wang J, Qu W. Hypolipidaemic and hepatoprotective effects of ethanolic and aqueous extracts from Asparagus officinalis
L. by-products in mice fed a high-fat diet. J Sci Food Agric 2010;90:1129-35.
Nadkarni AK. Indian Materiamedica. Bombay: Popular Book Depot; 1954. p. 153-5.
Joglekar GV, Ahuja RH, Balwani JH. Galactogogue effect of Asparagus racemosus
. Preliminary communication. Indian Med J 1967;61:165.
Mandal SC, Nandy A, Pal M, Saha BP. Evaluation of antibacterial activity of Asparagus racemosus
Wild root. Phytother Res 2000;14:118-9.
Muruganadan S, Garg H, Lal J, Chandra S, Kumar D. Studies on the immunostimulant and antihepatotoxic activities of Asparagus racemosus
root extract. J Med Arom Plant Sci 2000;22:49-52.
Fuller JH, Elford J, Goldblatt P, Adelstein AM. Diabetes mortality: New light on an underestimated public health problem. Diabetologia 1983;24:336-41.
Singh A, Sinha B. Pharmacological significance of Satavari: Queen of herbs. Int J Phytomed 2014;6:477-88.
Venkatesan N, Thiyagarajan V, Narayanan S, Arul A, Raja S, Vijaya Kumar SG, et al
. Anti-diarrhoeal potential of Asparagus racemosus
wild root extracts in laboratory animals. J Pharmacol Pharm Sci 2005;8:39-45.
Dalvi SS, Nadkarni PM, Gupta KC. Effect of Asparagus racemosus
(Shatavari) on gastric emptying time in normal healthy volunteers. J Postgrad Med 1990;36:91-4.
] [Full text]
Singh KP, Singh RH. Clinical trial on Shatavari (Asparagus racemosus
Wild.) in duodenal ulcer disease. J Res Ay Sid 1986;7:91-100.
Mangal A, Panda D, Sharma MC. Peptic ulcer healing properties of Shatavari (Asparagus racemosus
Wild). Int J Tradit Knowl 2006;5:227-8.
Tewari NN. Some crude drugs: Source, substitute and adulterant with special reference to KTM crude drug market. Sachitra Ayurveda 1991;44:284-90.
Vasudevan NK, Yoganarasimhan KR, Murthy K, Shantha TR. Studies on some south Indian market samples of Ayurvedic drugs. Anc Sci Life 1983;3:60-6.
Bisset WG. Herbal Drugs and Phytopharmaceuticals. London: CRC Press; 1984.
Sunita G. Substitute and Adulterant Plants. New Delhi: Periodical Experts Book Agency; 1992.
Uniyal MR, Joshi GC. Historical view of the basic principles of the identification of controversial drugs, problems and suggestions. Sachitra Ayurved 1993;45:531-6.
Sarin Y. Illustrated Manual of Herbal Drugs used in Ayurveda. New Delhi: CSIR & ICMR; 1996.
Saraswathy A. Adulterants and substitutes in Ayurveda. Sachitra Ayurved 2001;54:63-6.
Gupta AK. Quality Standards of Indian Medicinal Plants. Vol. 1. New Delhi: ICMR; 2003.
De Smet PA, Keller K, Hansel R, Chandler RF. Adverse Effects of Herbal Drugs. Vol. 1. Heidelberg: Springer Verlag; 1992.
Ekinci N, Ekinci R, Polat R, Budak G. Analysis of trace elements in medicinal plants with energy dispersive X-ray fluorescence. J Radioanal Nucl Chem 2004;260:127-31.
WHO. Guideline: Potassium Intake for Adults and Children. Geneva: World Health Organization (WHO); 2012.
Tapiero H, Tew KD. Trace elements in human physiology and pathology: Zinc and metallothioneins. Biomed Pharmacother 2003;57:399-411.
Zabłocka-Słowińska K, Grajeta H. The role of manganese in etiopathogenesis and prevention of selected diseases. Postepy Hig Med Dosw (Online) 2012;66:549-53.
Cashman KD. Calcium intake, calcium bioavailability and bone health. Br J Nutr 2002;87 Suppl 2:S169-77.
Kumar SS, Sudarshan M, Chakraborty A. The energy dispersive X-ray fluorescence spectroscopic study of Solanum rubrum
mill: A potential ayurvedic traditional medicinal plant. Indian J Biochem Biophys 2018;55:280-5.
Buldini PL, Cavalli S, Mevoli A. Sample pre-treatment by UV photolysis for the ion chromatographic analysis of plant material. J Chromatogr A 1996;739:167-73.
Goyal SS. Applications of column liquid chromatography to inorganic analysis in agricultural research. J Chromatogr A 1997;789:519-27.
Dionex Corporation. Determination of Organic and Inorganic Anions in Fermentation Broths. Application Note 123. Sunnyvale: Dionex; 1998.
Basta NT, Tabatabai MA. Determination of total potassium, sodium, calcium, and magnesium in plant materials by ion chromatography. Soil Sci Soc Am J 1985;49:76-81.
Morawski J, Alden P, Sims A. Analysis of cationic nutrients from foods by ion chromatography. J Chromatogr 1993;640:359-64.
Marchetto A, Mosello R, Tartari GA, Mumtau H, Bianchi M, Geiss H, et al
. Precision of ion chromatographic analysis compared with that of other analytical techniques through inter-comparison exercises. J Chromatogr A 1995;706:13-9.
Raj PS, Abbas NM. Suppressed ion chromatographic determination of lithium, sodium, ammonium and potassium concentrations in sub-surface brines. J Chroma-togr A 1996;733:93-9.
Thomas DH, Rey M, Jackson PE. Determination of inorganic cations and ammonium in environmental waters by ion chromatography with a high-capacity cation-exchange column. J Chromatogr A 2002;956:181-6.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13], [Figure 14], [Figure 15], [Figure 16], [Figure 17], [Figure 18], [Figure 19], [Figure 20], [Figure 21], [Figure 22], [Figure 23]
[Table 1], [Table 2], [Table 3]