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Soil Testing


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Soil Testing

  1. 1. Certificate Course in Agricultural BiotechnologyBIOFERTILIZER TECHNOLOGY Institute team: Dr. B.K.Datta Dr. R.Datta Dr. S.K.Das Dr. S.K.Si Mr. S. Sahoo Mr. D. Biswas Mr. S. Giri Mr. T. Nayek Expert Consultants: Dr. P.K. Singh, IRAI, New Delhi Dr. O.P. Rupela, ICRISAT, Hyderabad Dr. R.K.Basak, BCKV, Mohanpur, W.B Dr. D.J. Bagyaraj, Univ. of Agric. Sciences, Bangalore Dr. R. Kale, Univ. of Agric. Sciences, Bangalore Dr. Sunil Pabbi, IARI, New Delhi Dr. Aloke Adholeya, TERI, New Delhi Vivekananda Institute of Biotechnology
  2. 2. First published in July, 2004by Vivekananda Institute of BiotechnologyNimpith, South 24 Parganas, West Bengal, IndiaDesigned by Tellywallah, CalcuttaPrinted by Swapna Printing Works Private Limited, CalcuttaAll rights reserved.c Vivekananda Institute of Biotechnology, 2004This manual is sold subject to the condition that it shall not, by way of trade or otherwise,be lent, resold, hired out or otherwise circulated without the publishers prior consentin any form of binding or cover other than that in which it is published.
  3. 3. ForewordFifty years back a monk, Swami Buddhanandji, was deeply inspired by SwamiVivekananda’s ideals. He started a different kind of journey of life and a seedof development was sown at Nimpith. Late Swami Buddhanandaji establishedthis Ashram. Vivekananda Institute of Biotechnology is a branch of that tree. TheInstitute initiated its activities in 1991. Its aim is to develop an advanced,functional, research as well as a resource center for the people of the Sunderbans.One of the chosen fields is Agricultural Biotechnology. For the last few years theInstitute has been implementing programmes on entrepreneurship development,in this field, in rural areas. The present manual is one of such activity to supportthis programme, which is the need of the hour. The Department of Science and Technology, Govt of India and UnitedNations Development Programme have come forward benevolently in bringingout this manual. It is a result of the combined efforts of scientists at our Instituteand of other National Institutes and Universities. Senior scientists like Dr.P.K.Singh, Dr. O.P.Rupela, Dr. Radha Kale, Dr. Ranjan Basak, Dr. Sunil Pabbi,Dr. D.J. Bhagyaraj and Dr. Alok Adholeya have enriched this effort by theirvaluable guidance and by sharing their experience . Shri Gautam Bose, Shri AmitKumar and Shri Pradip Nair of Tellywallah have worked hard and extensively tomake it in this present form. We hope that this great effort will be used by the rural agro-biotechnologists,whose services, we believe, will bring a new dawn to rural India.Nimpith Swami SadanandaJuly 2004 Chairman
  4. 4. PrefaceIn today’s world, technology is moving very fast, in certain sectors at a fasterpace. Biotechnology is such an area. The rural India which is depending onagriculture for its day to day life provides an immense market for new technologies.The only condition is the proper training and marketing. The present manual is the outcome of the project ‘Technical HumanResource Development – Vocational Training For Employment Generation’supported by UNDP & DST, Govt. of India. The objective of the programme isto develop human resources through competency based training in innovativeareas for the production and service sectors in new, high technology areas basedon market needs. Agricultural biotechnology is the area in which we have worked on underthis project. This manual is first of its kind in this series. It deals with BiofertilizerTechnology which has six modules - Soil testing and fertilizer recommendation,Production and Application of Blue-green algae, Azolla, Microbial inoculants,Vesicular Arbuscular Mycorrhiza and Vermicompost. The target are the rural youth, who have passed their 10th std. It isdesigned and presented in such a way that a complicated subject like soil testingor microbial inoculant production becomes an easily adaptable skill, a demystificationof technology indeed. All the techniques mentioned here are of world standardbut no doubt many other options can also be opted for, for instance, in thesection of microbial inoculant production only the use of vessel is mentioned inthis manual though the use of other types of fermentors or shakers are alsopossible. This effort is the result of hard work of a team of our Institute, the samewas complemented by experts of other organizations, which are of world repute.Mr. Khudiram Sardar, Mr. Diwakar Haldar, , Mr.Tarun Das, Mr. Deepankar Haldar,Mr.Tapan Haldar and Mr. Shubankar Malik have helped while filming the experimentsfor this manual. Shri Gautam Bose , Shri Amit Kumar, Shri Pradip Nair and Shri ParthaBhattacharya of Tellywallah gave their best to make this dream a reality byfilming, designing, adding inputs and finally printing the manual.Nimpith Dr. B.K. DattaJuly 2004 Principal Scientist, VIB
  5. 5. Using this manual This manual has been written to complement classroom lectures pertaining to the Biofertilizer Technology Section of the “Certificate Course in Agricultural Biotechnology” taught at the Vivekananda Institute of Biotechnology — VIB. However, it may also be read by anyone with school level knowledge of Science who is interested in setting up a soil testing lab or a microbial inoculant production facility. We have departed from traditional styles of writing Course material. Instead of dry and forbidding lists of procedures and equations, the manual tries to expose the student to the various aspects of the three disciplines that this course straddles — Microbiology, Agricultural Science and Chemistry. Of course, one cannot entirely avoid equations and procedures! But we felt it is possible to present these topics in a friendlier manner. Our approach to making the subject “friendlier” and also maintaining sufficient scientific detail was two-pronged. We’ve split the material being discussed into two parts — quite literally. The odd-numbered pages in this manual contain descriptions of experiments and processes in a step-by-step manner, devoid of detailed explanations — rather like a traditional lab manual which expects students to follow the steps described bothering to evoke an interest in the topic at hand. The objective of the VIB course was the opposite. As scientists we are amazed and intrigued by our respective fields of study. We wanted the reader to feel this amazement as well; to undestand that science is an exciting subject! To help us out, we enlisted the help of Shubham, an inquisitive, imaginary friend, who can’t stop asking questions. Shubham can be found loitering around on the even-numbered pages — asking questions about the text on the facing page. The even-numbered pages also contain supplementary information about the topic at hand. Definitions, little bits of trivia, brief forays into the history of science, suggestions for further reading, tips on how to simplify a process and so on may also be found on even numbered pages. The course material was meant to enable an interested student set up his own Soil Testing Centre and an Inoculant Production Centre. Which is why the material in this manual is presented in a “modular” fashion — typical of industrial processes. Therefore, operations like autoclaving, working in a Laminar Flow Cabinet and using a Fermentor have been dealt with in a separate section. The step-by-step description concentrates on following the process being discussed segment-wise. The line at the bottom of each odd-numbered page is a “station map”. This helps the students to see the “whole railway line” and on which station he stands — this is typical of any industrial process where one goes from A (A test tube of bacteria) to B (application of the inoculant in the field) and the various stages that must be crossed to do so.
  6. 6. Using this manual In sections where extra explanation on the left hand pages was unnecessary,the step-by-step description continues. Addressing the reader directly (”You could then....” “We might look atthis from another angle....”) is something most textbooks never do but we feltthere was no reason why the manual should not attempt to make the readerfeel as if he was in a classroom while reading it. The manual is accompanied by an interactive CD-ROM. This contains allthe course material viewable in a non-linear fashion. This enables a studentto quickly refer to topics without having to flip through the manual. Further, allthe “modules” that are part of the production process, were filmed during themaking of this manual and the footage, with narration, is available on the CD.This could be used in the classroom too, during, say, the first time the topicis brought up for discussion by the instructor. The glossary in this manual is available in a searchable format on theCD-ROM.The CD-ROM contains two computer programmes written specifically for SoilTesting Centres. Chemicalc and X-Base are a scientific formula calculator anda Soil Database, respectively. X-Base allows Soil Testing Centres to share theirdata over the Internet with no additional software or hardware requirementsapart from a very rudimentary computer system and a telephone line. A few of the pages also contain material that is not immediately relevantto the course (eg., a brief description of the gene cascade in R.Meliloti that leadsto the production of nitrogenase, may be found in the pages that describeRhizobia). These portions are italicised. These sections are rare and are onlyfound, if at all, in the introductory part of the section. These could, if nothingelse, be food for thought for an inquisitive reader. Biotechnology is the fastest growing scientific field around the world. Asscientists around the world learn more about the amazing internal workings ofliving things, literature — this manual included — needs to be updated with aregularity, which, to a scientist, is more exciting than monotonous. The authorswould greatly appreciate any feedback about this manual. VIB hopes that this initiative will help fuel the next agricultural revolutionin our country — one powered not just by fertilizers and technology but also bya more aware and knowledgable farmer who understands the science behindthe word biotechnology... VIB team Nimpith July 2004
  7. 7. Soil Testing - Collection and preparation of soil samplesComposite soil samples, packed for lab. analysis 1-1
  8. 8. Introduction..A bit about the Soil Collection Process The process of soil testing begins in the field — where collection of samples is done. This needs to be done methodically — to ensure that the samples taken from the field represent the soil characteristics of the entire field. The process stated here is simple and easy to follow — though it does seemelaborate when you first read about it! Locating areas to take samples... Start at the bottom -left corner of the field and walk along the path indicated by the arrows. Along the way, mark areas (with a piece of wood — or anything easy to locate) from where you will be taking soil samples. Avoid marking prohibited areas (see the next page). The marked areas are shown in Red in the figure. Notice, that the red dots along the path are not necessarily on the path itself. That’s perfect — because this is a random sample. Apparatus required Plastic bucket, spade and wooden stakes or markers. Why is it necessary to go through such an elaborate process? It’s quite simple — ideally you’d want to test all the soil in your field — but that would take a lot of time. So, it becomes necessary to take samples from different places in the field. But how do you ensure that the samples taken to the testing centre represent, more or less, the soil in the field? The answer is the reason behind the elaborate process. By taking samples from randomly selected spots, you ensure thatyour cumulative soil sample represents the soil in most areas of the field. Thezig zag walk is just to establish a technique of selecting random spots.1-2 Collection and Preparation of soil samples
  9. 9. Collecting soil samples The locations from where soil samples are taken may be marked by wooden stakes as shown. The stakes need not be numbered. The locations are chosen by walking along a zig-zag path starting at any corner of the field. These are marked by white spots in the picture. A typical location is shown in the next picture. Each collection location needs to be cleared of vegetation and dust. This is done by scraping away a very thin layer of soil.Collection Grinding Partitioning Storage Drying Mixing SievingCollection and Preparation of soil samples 1-3
  10. 10. Prohibited samples... Prohibited Samples? It’s just another thing to do with the statistics (see page 2.1). Your soil samples need to represent the average soil characteristic of the field. However, every field has areas in it that tend to distort the average characteristic because the soil there has properties very different from the rest of the soil. (It’s called deviation from the mean, by the way) So, you need to ignore these areas when collecting samples.These include— Areas near gates, farmways, buildings etc. and areas on crop hills and in rows.— Areas where organic/chemical manure is or was kept.— Areas which are permanently in the shade.Why must the pit be 6” (15 cm) deep?6” is the depth of tillage i.e., the depth up to which the root system of the croppenetrates. Since soil testing is carried out to determine the availability ofnutrients to crops, samples i.e., the furrow slices, are 6” long.Why should a PVC bucket be used?An iron bucket may have rust, which might contaminate the sample. Presenceof iron would skew the results of the organic carbon test as we shall see later.Jute or nylon bags which may have been used to store fertilizer etc. must notbe used too.PVC (or any other plastic) would not contaminate the soil. Besides, they are easyto clean.1-4 Collection and Preparation of soil samples
  11. 11. Collecting soil samples Remove a wedge-shaped lump of soil from the cleared sampling location and discard it. The resulting pit should be about 6 inches (15 cm) deep. From the two larger surfaces of the pit, remove a half inch thick slice of soil — called the furrow-slice. Thus, from each sampling location on the field, two furrow- slices are obtained. Carry these in a PVC bucket.Collection Grinding Partitioning Storage Drying Mixing SievingCollection and Preparation of soil samples 1-5
  12. 12. Collecting soil samplesAfter collection — what now?You now have a bucketful of furrow-slices. However, it would be time-consumingto test all the soil in each of the furrow-slices separately and then average theresults. Ideally, you would want to test a relatively small sample of soil which,nevertheless, represented the soil present in the entire field.This is achieved by thoroughly mixing the soil. Further, to simplify the labexperiments, the samples are ground and sieved... We’ll discuss this issue in thenext section...1-6 Collection and Preparation of soil samples
  13. 13. Collecting soil samples After all the samples are collected, they could be taken directly to the laboratory — if one is close enough. Else, the samples may be prepared on location itself, and a composite soil sample may be sent for testing. The samples are now air-dried in the shade.Collection Grinding Partitioning Storage Drying Mixing SievingCollection and Preparation of soil samples 1-7
  14. 14. Drying the soil samplesWhy are the samples dried?In dried soil, any reversible chemical reactions that usually take place in it arein equilibrium.However, drying does change the chemical constituents of soils. Ferrous ironis oxidised to ferric iron, exchangeable potassium content increases or decreasesdepending upon the soil and hydrogen ion activity changes to some extent.Therefore, concentration of ferrous iron (if required) should be determined witha field-moist soil sample. Also, concentration of exchangeable potassium andsoil pH may also be determined without drying the sample.Dried soil is easier to grind.For reasons mentioned earlier, metallic apparatus must not be used for grindingas particles might break off and contaminate the soil.Therefore, a wooden mortar and pestle must be used.1-8 Collection and Preparation of soil samples
  15. 15. Grinding the soil samples Spread a clean polythene sheet on the ground and place a wooden mortar on it. Transfer the dried soil samples to the mortar. With a wooden pestle, grind the soil to break down any aggregates. After grinding, transfer the soil to the polythene sheet and spread evenly across the surface.Collection Grinding Partitioning Storage Drying Mixing SievingCollection and Preparation of soil samples 1-9
  16. 16. Mixing Why is mixing important? After all, we just ground the samples — that should have mixed everything quite well. Mixing is necessary to ensure that the composite soil sample (all the soil samples taken from different areas in the field) represents the field’s soil composition as closely as possible even in small quantities. For example, to determine the amount of phosphorus, as little as 2.5 g of soil is used in the experiment. You would have collected nearly 3 kg of soil from the field of which only about 500g of soil is sent to the soil-testing centre. Therefore, unless properly mixed, it is likely that soil from some parts of the field might not reach the testing centre. Yes, during grinding, mixing does take place — but it is random and might not be enough. Besides, like the collection stage, the mixing stage described here is to establish a process that minimises chances of statistical error.1-10 Collection and Preparation of soil samples
  17. 17. Mixing Lift one end of the sheet and fold it till the soil collects in the centre. Repeat the process with the diagonally opposite corner as shown in the picture... and then with the other two corners... This will cause the soil to collect in the centre of the sheet. This process is called Mixing and must be repeated 5 times. Cone the soil and flatten the top.Collection Grinding Partitioning Storage Drying Mixing SievingCollection and Preparation of soil samples 1-11
  18. 18. Partitioning the soil sample Partitioning? At this stage, you have about 3kg of a composite soil sample. This would be a rather unwieldy package to transport to the testing labs. Besides, the lab will need only about 300g of soil for all the tests. Here is where the purpose behind the monotonous mixing process becomes apparent. Because the sample is mixed, statistically, the average chemical composition of the field is represented by surprisingly small amounts of soil — as little as a few grams!So, you don’t need to send in all the soil you’ve collected painstakingly — youcould send in 500g which is relatively easier to transport and store.There is a catch though — selecting the final soil sample must also be done atrandom . This is fulfilled by the next Stage — Partitioning.This involves halving the mass of the soil sample in successive stages till it isabout 500g. At each stage, a random portion of soil is selected.The method described here is one of many that may be used to quarter thesoil sample. There are others, such as : The Riffle Technique and The PaperQuartering Technique.However, the method described needs no extra apparatus and is very easy.Hence its use in this manual.1-12 Collection and Preparation of soil samples
  19. 19. Partitioning the soil sample Divide the soil into 4 equal portions, as shown. Discard any two diagonally opposite portions. This process is called Partitioning.Collection Grinding Partitioning Storage Drying Mixing SievingCollection and Preparation of soil samples 1-13
  20. 20. Partitioning the soil sample1-14 Collection and Preparation of soil samples
  21. 21. Partitioning the soil sample Continue partitioning the sample till about 500g of soil remains. The amount of soil retained depends upon the number of experiments that the Soil Chemist intends to conduct. 500 g is enough if all tests are to be carried out. Transfer all the soil to a sieve.Collection Grinding Partitioning Storage Drying Mixing SievingCollection and Preparation of soil samples 1-15
  22. 22. Sieving the soil sampleWhat mesh size is to be used?A fine sieve (80 mesh) is used for determination of oxidisableorganic carbon and the elements i.e., Nitrogen, Phosphorus andPotassium.A coarse sieve (20 mesh) is used for determination of soil pHand salinity.The entire volume of the partitioned sample should pass throughthe sieve. Soil aggregates that are too large to be sieved shouldbe ground in a mortar and sieved again.1-16 Collection and Preparation of soil samples
  23. 23. Sieving the soil sample Sieve the soil. Preparation is complete. The soil particles have been ground, mixed, partitioned and sieved. Before the soil is transported to the lab, it must be stored in polythene bags.Collection Grinding Partitioning Storage Drying Mixing SievingCollection and Preparation of soil samples 1-17
  24. 24. Packing the soil sample1-18 Collection and Preparation of soil samples
  25. 25. Packing the soil sample Seal the open end of the bag with thread. Label the bag. The information required by the laboratory for testing is shown in the Information Sheet (see Page 9-28) The samples are now ready for transport. The picture shows 3 polythene bags — these are samples from adjoining fields all headed to the testing centre.Collection Grinding Partitioning Storage Drying Mixing SievingCollection and Preparation of soil samples 1-19
  26. 26. 1-20 Collection and Preparation of soil samples
  27. 27. Soil testing - Determination of soil pHpH electrodes dipped into a soil-water suspension for pH measurement 1-21
  28. 28. Introduction A bit about pH... pH is the quantitative measure of acidity or alkalinity of liquid solutions. A solution with a pH value less than 7 is considered acidic and a solution with a pH more than 7 is considered alkaline. pH 7 is considered neutral. Soil pH between 6 and 8 is safe for most crops. If the tested sample has a pH value outside this “safe range”, steps must be taken to artificially correct the problem. The acidity of a solution is directly proportional to its hydrogen ion concentration. The term pH is derived from p representing the German word potenz, ‘power’, + H, the symbol for hydrogen. pH meters are extremely sensitive instruments. They consist of one (or two) glass electrodes connected to a digital display. The pH of a solution is displayed when the electrodes of the meter are dipped in it. Soil water suspension : A suspension, as opposed to a solution, is a heterogeneous mixture, i.e., its constituents may be separated by physical means. The mixture of soil in water is therefore a suspension, not a solution. Apparatus and reagents required Buffer tablets of pH 4.0 and 7.0, a top loading balance, a 100mL beaker, a wash bottle, a glass rod and a pH meter Determination of Lime Requirement : An acidic soil is treated with Lime to increase its pH. Recommendation of liming is also done after a pH test. The difference being the addition of an extra ingredient to the soil. Take 5g of soil instead of 20g as shown here. Add 5mL of distilled water and then add 10mL of SMP Extractant Buffer. Proceed with the pH exactly as shown in the following pages. When you obtain the pH value, refer to the Lime Recommendation Table on page 9-28.1-22 Determination of soil pH
  29. 29. Preparing a soil suspension Weigh out 20g of soil. Transfer the soil to a 100mL beaker. Add 50ml of distilled water. This creates a soil-water suspension. The soil : water ratio for conducting this test should be 1 : 1.25 Stir the suspension occasionally for about half an hour or shake in a shaker for 5 minutes.Preparing the soil suspension Measuring the pH of the sample Calibrating the pH meterDetermination of soil pH 1-23
  30. 30. Using the pH meter... Calibration? Why is it necessary? Think about this — how does the meter know a solution’s pH? It doesn’t. It’s just programmed to display different pH values depending upon the voltage across its electrodes. The electrodes, though, are sensitive to a whole lot of other things — like temperature for instance. So, even though a change in room temperature will not change the pH of a solution, it will cause the electrodes to report the pH incorrectly. And this is true of any measuring instrument. We calibrate by measuring a known amount and then re-programming the meter to display that amount — this is sometimes as simple as pushing a switch. In this experiment, we calibrate the meter with 2 buffer solutions — with pH values of 7.0 and 4.0. See page 1-26 for a description of buffer solutions. First, we dip the electrodes in the pH 7.0 buffer. While we were conducting the experiment, the meter read 7.2. This was because it was set to measure correctly at a slightly lower room temperature. So, the adjustment knob was turned till the display read 7.0. Between readings, wash the electrodes with distilled water and wipe them dry with a piece of clean tissue paper. Repeat the process with a buffer solution of pH 4.0. The volumetric flask in the third picture contains the pH buffer solution. The meter might need a few minutes to “warm up”. The time varies from model to model and you should check the literature that came with your meter. Generally, “warming up” takes a few minutes. Also, it takes a few seconds for the display to stabilise after you’ve dipped the electrodes in a solution. So, wait a while before noting down a pH reading.1-24 Determination of soil pH
  31. 31. Calibration Calibrate the pH meter with any two known buffer solutions. Just prior to taking any readings, stir the soil-water suspension with a glass rod. Dip the electrodes of the pH meter into the suspension and take a reading.Preparing the soil suspension Measuring the pH of the sample Calibrating the pH meterDetermination of soil pH 1-25
  32. 32. A buffer story A bit about Buffer Solutions We know about pH and how it describes the acidity or alkalinity of a solution. Now, why is a solution acidic? Or alkaline? Modern definitions of acidity refer to the ability of the compounds in a solution to accept or release electrons — the Lewis Concept. But historically, an acid was a compound that, in solution, could release hydrogen ions into the solution, and a base was a compound which could accept hydrogen ions. Since HCl dissociates into H+ and Cl- ions, in solution, it is an acid. There are situations when we want the pH of a solution to remain constant — irrespective of change in the concentrations of its acidic or alkaline constituents. This is done by adding an acid-base pair to the solution that acts as a reservoir, or buffer. What this reservoir does is suppress or increase the dissociation of other compounds depending upon the pH of the solution. A commonly used buffer solution is the NH4Cl - NH4OH pair. These are readily soluble chemicals and keep each other’s dissociated concentrations in check — in line with the solubility product principle. The NH4Cl - NH4OH pair is an alkaline buffer and maintains the pH of the solution at around 8.5. You can read more about Buffer Solutions in books on Physical Chemistry. A thin “chemical film” is deposited on the electrodes each time a pH measurement is taken. Unless removed, this film causes the electrodes to report inaccurate values. Therefore, between readings, wash the electrodes of the pH meter with a stream of distilled water and then wipe them dry with tissue paper. When not in use keep the electrodes dipped in distilled water.1-26 Determination of soil pH
  33. 33. Meter readings... The pH of the sample tested is 7.79. The display on most pH meters takes about a minute to stabilise.Preparing the soil suspension Measuring the pH of the sample Calibrating the pH meterDetermination of soil pH 1-27
  34. 34. 1-28 Determination of soil pH
  35. 35. Soil Testing - Determination of salinityConductivity readings are taken from the supernatant liquid.. 1-29
  36. 36. Introduction A bit about salinity... The determination of the quantity of water-soluble salts is of special importance for arid, semi-arid as well as coastal areas. It helps in taking reclamation measures as well as in the selection of crops which differ in their tolerance to salts. While we could measure the concentrations of the salts by chemical analysis, it would be time-consuming, expensive — and entirely unnecessary. That’s because, we don’t need to identify all the salts lurking about — we’re only interested in the water-soluble ones. The concentration of all of these salts taken together is what matters to plants. So, we take a more practical approach to measure soil salinity — we add distilled water into the soil and stir it till the soluble salts get dissolved. Then, we measure the electrical conductivity of the water. And how does that tell us anything about salinity? Indirectly, it does — because in solution, ions are the carriers of electric charge and therefore, the electrical conductivity of a solution is directly proportional to its soluble salt concentration. The conductivity of a soil sample is measured with the help of a conductivity meter and is expressed in mmhos/cm. or, in SI units, in dS/m. You don’t even need to calculate the concentrations for the purposes of recommending fertilizers. The recommendation is based upon the conductivity measurement itself. ( See the recommendation tables). Most soil testing labs mention “Electrical Conductivity” or just “E.C.” in their reports. Apparatus and reagents required 0.01N KCl solution, a 100mL beaker and a conductivity meter.Using a conductivity meter...An electrical conductivity meter is very similar to a pH meter. It also consistsof an electrode connected to a digital display. Measurements are made by dippingthe electrode in the solution being tested.The precautions to be observed while using this instrument are the same asthose with a pH meter (see page 1-24 ).1-30 Determination of salinity
  37. 37. Calibrating the conductivity meter This is a typical Conductivity Meter. Like the pH meter, its electrode must be kept immersed in distilled water when not in use. Calibrate the meter. This is done with distilled water and a 0.1N Potassium Chloride solution. Distilled water should display 100 in the digital panel. Then, dip the electrode in a 0.1N KCl solution.Calibration of the Conductivity meter Measuring conductivity Supernatant LiquidDetermination of salinity 1-31
  38. 38. A bit about supernatant liquid What is Supernatant liquid? The liquid that floats above a suspension after it has been allowed to stand for a while is termed “Supernatant”. When measuring conductivity, the conductivity cell should remain in the Supernatant liquid and not touch the soil below as shown. So, why are we measuring the conductivity of the supernatant liquid? After all, during the pH experiment we had specified that the soil shouldbe in suspension while the reading was being taken.The supernatant liquid is a solution. The salts present in the soil dissolve in waterand dissociate into ions, which are charged particles. The concentration of solublesalts in the soil may, therefore, be calculated from the conductivity of thesupernatant liquid.Soil - water ratio? Why is that important?Soil to water ratio should be 1:2. The ratio influences the amount of salts in theextract. Some laboratories use different ratios while conducting this test. Eitherway, the ratio must (and is) always mentioned in a soil analysis report.1-32 Determination of salinity
  39. 39. Conductivity readings... Adjust the cell constant knob till the meter displays 14.1 m.mhos/cm. The soil water suspension from the pH experiment is allowed to stand till a clear supernatant liquid is obtained. After setting the range switch to maximum, the electrode is dipped in the supernatant liquid. Reduce the range setting on the meter one at a time till the most appropriate setting is found. In this case, the conductance of the soil sample is 1.20 m.mhos/cm.Calibration of the Conductivity meter Measuring conductivity Supernatant LiquidDetermination of salinity 1-33
  40. 40. 1-34 Determination of salinity
  41. 41. Soil Testing - Determination of available organic CarbonDiphenylamine indicator being added drop by drop 1-35
  42. 42. Introduction A bit about Oxidisable organic carbon... Decomposed plants and microbial residues are the constituents of organic matter. The percentage of oxidisable organic matter can be determined by multiplying its percentage of organic carbon by 1.724. Oxidisable organic carbon consists of partly decomposed residues of plants, animals and microorganisms. This constitutes most of the usable carbon present in the soil. The other forms of carbon which are present, but not useful as a source of nutrients, include inorganic carbon (such as carbonates), elemental carbon (such as coal and graphite) and completely decomposed organic carbon. For areas known to have very low organic matter content take 2g of soil in the conical flask, for peat soils, take 0.05g and for areas known to have about 1-2% of organic carbon content, take 0.5g of soil. Apparatus and reagents required 1N potassium dichromate solution, 0.5N ferrous ammonium sulphate solution, diphenylamine indicator, concentrated sulphuric acid and 85% orthophosphoric acid solution. 500mL conical flask, titration setup (50mL burette, chromyl chloride solution to clean the burette and titration stand) 10mL bulb-type pipette, chemical balance, 1000mL volumetric flask and two watch glasses. Why are two conical flasks used? This experiment is based upon the Walkley and Black method according to which soil is digested with chromic acid resulting in the oxidation of its organic content. The excess chromic acid is determined by titration with a standard ferrous ammonium sulphate solution. After titration, in the case of the soil sample, the amount of titrant consumed is obtained. The amount of titrant consumed in a blank titration (without soil) could be calculated stoichiometrically. But this would require accurate weighing of all the reagents involved in the reaction. Therefore, it is much simpler to perform a blank titration to obtain the required figures i.e. the volume of ferrous iron solution consumed.1-36 Determination of oxidisable organic carbon
  43. 43. Oxidising the carbon in the soil sample Take two 500mL conical flasks. Add 1g of soil to one of the flasks. Add 10mL of K2Cr2O7 to each of the flasks with a pipette.Oxidizing the carbon in the soil Titration of the soil sample suspension Blank Titration CalculationsDetermination of oxidisable organic carbon 1-37
  44. 44. Precautions... Concentrated sulphuric acid is a very corrosive chemical. It fumes in contact with moisture. Observe the following precautions when using sulphuric acid : The acid must be poured into the beaker along a glass rod or along its inner walls. DO NOT use a pipette to measure out the acid - if any of the acid gets into your mouth, there might not be enough of it left to talk about the experience! This step should ideally be carried out in an Exhaust Cabinet because the fumes are extremely corrosive as well. Don’t try to smell the fumes however tempting it might seem! What happens during the half hour? The oxidisable organic carbon in soil is oxidised by potassium dichromate 3C + 2K2Cr2O7 + 8H2SO4 = 3CO2 + 8H2O + 2K2SO4 + 2Cr2 (SO4)3 Potassium dichromate is converted to potassium sulphate and chromium sulphate. Cr6+ is reduced to Cr3+. The colour of the oxidised form of chromium(Cr6+) is yellow (or orange) and that of it’s reduced form (Cr 3+ ) is green. The volume of K2Cr2O7 solution added to the soil should be large enough so that only a small fraction of it is reduced - which is indicated by yellow (or orange) c o l o u r o f t h e r e a c t i o n m e d i u m a f t e r c o m p l e t i o n o f ox i d a t i o n . This occurs over a period of 30 minutes. Because sulphuric acid fumes, reagents might get deposited on the watch glass. Therefore, when adding water, if you see flecks of chemicals deposited on the watch glass, rinse them and allow the water to drip into the conical flasks.1-38 Determination of oxidisable organic carbon
  45. 45. Oxidising the carbon in the soil sample With a measuring cylinder, add 20 mL. of concentrated sulphuric acid to each of the flasks. Cover the flasks with watch-glasses and allow them to stand for about half an hour. Then, add about 200 mL of distilled water to each of the flasks.Oxidizing the carbon in the soil Titration of the soil sample suspension Blank Titration CalculationsDetermination of oxidisable organic carbon 1-39
  46. 46. Titration A bit about titration... Titration is a process by which the amount of an oxidisable or reducible substance in solution is determined by measuring the volume of a standard reagent required to react with it. The burette used must be cleaned with chromyl chloride prior to titration. Dirty burettes are the most common cause of errors. Carry out the blank titration first. This will give you a general idea about the volume of titrant, i.e., Ferrous ammonium sulphate that will be consumed. With this value in mind, the titration of the soil sample usually takes less time. Observe the colours that the solution assumes during the process. In the first phase, the solution is a dark burgundy. After a while, it turns violet. This indicates, approximately, the mid point of the titration. Local action is also observed at this point. The remainder of the titration needs to be carried out carefully, i.e., by agitating the contents of the flask after every 2 drops. The end-point is indicated by a sudden change of colour of the solution to viridian, or dark green. During the titration of the soil sample, all the indicative colours are more cloudy than those observed during the blank titration. This is due to suspended soil particles. Why is Orthophosphoric acid used? Orthophosphoric acid, H3PO4, is added so that the colour change at end point is clearly defined. Diphenylamine should be added just prior to titration. This is to avoid the potassium dichromate from oxidising the indicator instead of the organic content of the soil sample being tested. During titration, a small amount of diphenylamine is oxidised, however, the error is negligible.1-40 Determination of oxidisable organic carbon
  47. 47. Blank Titration Add 10mL of orthophosphoric acid to each of the flasks. Just prior to titration, add about 10 drops of Diphenylamine indicator to the flask. The solution turns a dark burgundy. The blank titration is done first. Titration is done with a 0.5N Ferrous ammonium sulphate solution.Oxidizing the carbon in the soil Titration of the soil sample suspension Blank Titration CalculationsDetermination of oxidisable organic carbon 1-41
  48. 48. Local action... And a few calculations What is local action? Local action is a phenomenon observed midway during titration. At this stage, even though the titration is not complete, a faint, localised “end point” may be observed in the solution where the titrant drops fall. To observe local action during this experiment allow a drop of titrant, i.e., Ferrous ammonium sulphate, to drop on the solution without agitating the flask as is normally done during titration.The solution in the immediate vicinity of the drop turns green momentarily. A few Calculations... The CD-ROM has a Chemical calculator that does all the work for you but since we’ve set out to understand the science behind Agricultural Biotechnology, let’s dive headfirst into yet another bout with theory — and learn a bit about Stoichiometry. The Appendix contains an article that explains why you need to add and divide all these numbers... The percentage of oxidisable organic carbon (%OC) in the soil sample is given by % O.C. = [VK x (1– VS/VB) x SK x 0.3] / W where Vk = Volume of Potassium dichromate solution VS = Volume of Ferrous iron solution consumed in titration with soil VB = Volume of Ferrous iron solution consumed in blank titration Sk = Strength of Potassium dichromate solution W = Weight of soil1-42 Determination of oxidisable organic carbon
  49. 49. Indicative colours during titration... Midway through the titration, the colour of the solution turns to clear purple. At this stage, local action may be observed. End point is indicated by a sudden change of colour to viridian, or dark green. Titrate the soil sample as well. Notice that all the indicative colours with the soil sample are cloudy. The picture shows the titrated soil sample suspension at end point.Oxidizing the carbon in the soil Titration of the soil sample suspension Blank Titration CalculationsDetermination of oxidisable organic carbon 1-43
  50. 50. 1-44 Determination of oxidisable organic carbon
  51. 51. Soil Testing - Determination of available NitrogenAmmonia bubbling up the neck of a Kjeldahl flask 1-45
  52. 52. A bit about the Experiment A bit about the Experiment Plants generally take up nitrogen as nitrate under aerobic conditions. In anaerobic situations, some crops, such as rice, can take up nitrogen as ammonium ions. Most of the nitrogen present in soil is present in complex compounds. This is considered as a potential reserve source and, as such, it may be measured to assess the nitrogen-supplying capacity of the soil. Soil testing centres do not usually conduct a separate test for determining the quantity of available nitrogen in a soil sample brought to them for testing. Instead, they calculate this quantity directly from the quantity of oxidisable organic carbon. And how exactly is that possible? The ratio of the amount of oxidisable organic carbon is proportional to the amount of nitrogen in a given area. The ratio is unique to each region. These ratios have been tabulated. In Nimpith, where VIB is located, the ratio is 1 : 5. With this value, we need only perform the organic carbon test to determine the quatities of both nitrogen and oxidisable organic carbon. Apparatus and reagents required Boric acid solution, Mixed indicator, 0.32% potassium permanganate solution, 2.5% sodium hydroxide solution, liquid paraffin. Kjeldahl flask(s), distillation setup, titration setup, 250mL conical flask and a few glass beads. Glass beads and liquid paraffin These are used to reduce frothing and the formation of bubbles in the solution when the flask is heated. The bubbles may carry soil into the delivery tube and deposit them in the conical flask connected to the other end of the tube. The presence of soil makes it hard to detect the end point when we titrate the contents of the conical flask. More on this topic later...1-46 Determination of Available Nitrogen
  53. 53. Extracting the Nitrogen as Ammonia Take 20g of soil in a Kjeldahl Flask. Add 20mL of distilled water. Coat a few glass beads in liquid paraffin and put them in the flask. Extracting the Nitrogen as Ammonia Calculations Titration of the condensateDetermination of Available Nitrogen 1-47
  54. 54. Extracting the Nitrogen as Ammonia A bit about Kjeldahl and the flask he invented... A Danish chemist called J.G.C.T. Kjeldahl came up with the brilliant idea of estimating nitrogen concentrations in organic substances by distilling it out as ammonia — which can be easily assayed. For boiling the organic substance, he made a round-bottomed glass flask with a long neck. A special heat-resistant glass is used which does not crack when heated to high temperatures and is expposed to relatively cooler liquids at the same time. The entire assembly is called a Kjeldahl setup or unit and the flask also bears its inventor’s name.1-48 Determination of Available Nitrogen
  55. 55. Extracting the Nitrogen as Ammonia Then add 100mL each of 0.32% potassium permanganate and 2.5% sodium hydroxide solutions. Heat the flask to about 80oC on an electric heater. Ammonia is evolved. The gas escapes into the delivery tube attached to the Kjeldahl flask. Extracting the Nitrogen as Ammonia Calculations Titration of the condensateDetermination of Available Nitrogen 1-49
  56. 56. Extracting the Nitrogen as Ammonia Why do we use mixed indicator? In this test the pH changes at two distinct points. The first is when the ammonia is absorbed by the boric acid and the solution changes from bright pink to green. The second occurs during the titration of the solution with sulphuric acid. The solution then changes back to pink. These changes occur at different pH values and a single indicator is not sufficient since indicators exhibit a colour shift only in a small pH range. Thus, we need two indicators which will show us both these changes. Hence a mixed indicator — which is a mixture of Methyl Red, Bromocresol green and Ethanol — is used in this experiment.1-50 Determination of Available Nitrogen
  57. 57. Titration of the condensate The evolved gases condense and are collected in a conical flask containing Boric Acid solution and Mixed Indicator. The Ammonia is absorbed by the acid — indicated by a change in colour of the solution to green. Continue boiling the contents of the Kjeldahl flask till about 100mL of distillate is collected. Titrate the distillate with 0.02N sulphuric acid. Extracting the Nitrogen as Ammonia Calculations Titration of the condensateDetermination of Available Nitrogen 1-51
  58. 58. Titration of the condensate My end point is brown! You did not pour in enough paraffin. Or, perhaps, you didnt use enough glass beads. These ingredients are added to reduce the surface tension of the solution in the Kjeldahl flasks. This greatly reduces bubble formation... The bubbles often carry small amounts of soil and deposit it in the conical flask. This “muddies” the indicative colours during titration and hence the end point appears brownish... A few calculations Substitute the observed volume, V, of sulphuric acid consumed in the following equation to calculate the amount of available nitrogen (in kg per hectare) of the soil sample — V X 31.36 kg/Ha1-52 Determination of Available Nitrogen
  59. 59. Titration of the condensate... and Calculations Local action is observed distinctly during titration. End-point is indicated by a change in colour from green to a brownish-pink. Note the value of sulphuric acid consumed. Carry out a blank titration — with the contents of the conical flask corresponding to the Kjeldahl flask without soil. Extracting the Nitrogen as Ammonia Calculations Titration of the condensateDetermination of Available Nitrogen 1-53
  60. 60. Titration of the condensate... and Calculations1-54 Determination of Available Nitrogen
  61. 61. Soil Testing - Determination of available PotassiumFlame view - the orange-red colour indicates the presence of potassium 1-55
  62. 62. A bit about Potassium A bit about potassium In soil, potassium may be found in four compound forms - Water soluble, Exchangeable, Fixed and Lattice-bound. Of these, plants are interested only in the first two since they cannot assimilate potassium when it is present in the last two types of compounds. Potassium is the most abundant meta-cation in plant cells. Oddly though, soil humus furnishes very little potassium during decomposition. Also, it occurs in plants only as a mobile, soluble ion, K+, rather than as an integral part of any specific compound - but, it is known to affect important aspects of a plants life such as cell division, formation of carbohydrates, translocation of sugars and resistance of the plant to certain diseases. Over 60 enzyme actions are known to require potassium for activation. Which is why it forms the “K” part of the NPK trio - the three major important elements that plants require for proper growth. Incidentally, the “K” comes from “Kalium” which is what potassium used to be called. And a bit about the experiment In the next experiment, when testing for phosphorus, we will learn about a technique called curve fitting. This experiment also uses the same principle but the curve fitting itself is done electronically by the machine itself. So what machine are we talking about? It’s called a Flame Photometer. It consists of two parts — the gas compressor (that’s the first picture on the right) and the Aspirator/Measurement unit (the second picture). The principle on which this gizmo operates is that every element, when burnt in a flame, emits energy in a set series of wavelengths. Simply put, each element burns with a different colour. Further, the intensity of colour is directly proportional to the concentration of the element. So, by measuring the intensity of colour of flame aspirated with the sample, and comparing it with a known set of colour intensities, the photometer c a n d e t e r m i n e t h e c o n c e n t ra t i o n o f p o t a s s i u m i n t h e s a m p l e . The gas compressor regulates the flow of LPG to the Photometer. The gas burns with a nearly colourless (or faint blue) flame. The soil sample extract is then sucked into the flame in minute quantites. This causes the water to vapourise instantly and the compounds in it burn in the flame. The colour of the flame changes depending upon the elements present in the extract. This is detected by electronic sensors which calculate the intensity of the colour. Potassium burns with an orange-yellow flame. Apparatus and reagents list is on page 1-601-56 Determination of available Potassium
  63. 63. Calibrating the photometer Set the Compressor to supply gas at a pressure of 0.45kg/cm2. Ignite the flame and then calibrate the photometer with solutions whose potassium concentrations are known. The calibration must be done with 4 standard solutions.Calibrating the Flame Photometer Measuring the concentration Extracting the Potassium from the sample CalculationsDetermination of available Potassium 1-57
  64. 64. Calibrating a Flame PhotometerEnter “Calibration” mode We’re working with pretty The smart meter nowusing the control panel. high concentrations of realises that it needs toThe buttons you have to Potassium here. plot a Standard Curve. Sopress will vary for different you need to tell it howmodels. Some meters are designed many standard samples for micro-analysis — like you’re going to use to plotUsually, you’ll see a the one here. In our case, the curve. We’ll use 4numbered list of options. we need to tell it to expect samples. Most meters areIn this case, we press “5” Potassium in high good enough to accuratelyon the numeric keypad to concentrations. “fit a curve” with thisenter Calibration mode. number of samples.It now wants to know the Sampling time! Aspirate The flame colour changesconcentrations, in ppm, of each of the stock solutions immediately to a brightthe standard solutions one by one — in the order orange-yellow. Thewe’re going to use. Here, in which you keyed them change in intensity of thewe use solutions with 100, into the meter. We typed colour will be barely75, 50 and 25 ppm in the concentrations in noticable to the nakedconcentrations. descending order (100,75, eye. The meter however, 50,25), so, we’ll have to can distinguish each a s p i ra t e t h e 1 0 0 p p m colour precisely. solution first.1-58 Determination of available Potassium
  65. 65. Calibrating a Flame PhotometerAfter each sample is The flame becomes After aspirating distilledaspirated, the meter colourless when distilled water, repeat with the nextdemands a “washing” with water is aspirated. It might Standard sample. Notedistilled water — just like also be a faint blue colour. that the display in thethe E.C. meter and the pH The picture here has been picture reads STD4, orm e t e r. N o t i c e h o w deliberately modified to Standard Sample No. 4.different measuring exaggerate the colour ofapparatus all have the the flame — so that you This was taken when we’dsame operating principles. can easily compare the already aspirated the firstIt’s really quite simple — flame colours with and three samples. Now, weno magic, just science! without the sample. aspirate the 4th sample.Between each sample, the Calibration is over. Now, Shubham, it seems, hasmeter will ask you to the meter is ready to test no question to ask on thisaspirate distilled water. the soil sample extract - topic and wants to get onAfter all the 4 samples are we don’t know the with the experiment!aspirated, the meter concentration of Potassiumsounds a satisfied beep in this. The meter willand tells you happily that analyse the colour of thecalibration is over. flame, plot its density on the standard curve that it’s drawn for itself and tell us t h e c o n c e n t ra t i o n o f Potassium. Calibrating the Flame Photometer Measuring the concentration Extracting the Potassium from the sample Calculations Determination of available Potassium 1-59
  66. 66. Extracting the potassium from the soil sample Ammonium Acetate? Why is this used? Like in the Phosphorus experiment, we need to find a way to extract the Exchangeable Potassium from the soil sample. That’s what Ammonium acetate is used for. The ratio of soil : Ammonium acetate should be 1:5. That is, if you used 5.0g of soil, take 25mL of ammonium acetate. Ammonium acetate dissociates to yield ammonium ions CH3COONH4- CH3COO- + NH4+ The NH4+ ions replace the K+ ions, held on exchange sites of soil colloids. As a result, K+ ions are released into solution. Perfect for our purpose! The chemical equation above has two arrows pointing in both directions. This indicates a reversible reaction — i.e. one that occurs simultaneously in both directions. However, each reversible reaction has an equilibrium point at which the rates of both the forward and backward reactions remain constant. Chemistry can be a really exciting subject! You can find out more about reversible equations in any textbook on Physical Chemistry. See the Appendix for a list. Apparatus and reagents required 1N ammonium acetate solution of pH 7.0, 1000ppm potassium solution of pH 7.0. 10mL pipette, 150mL conical flask with a rubber stopper, 50mL volumetric flask, 100mL measuring cylinder, a funnel, Whatman no. 42 filter paper and a flame photometer. This experiment is shown using a direct read-out electronic flame photmeter.1-60 Determination of available Potassium
  67. 67. Extracting the potassium from the soil sample Take 5g of soil in a 150mL conical flask. Add 25mL of 1N Ammonium Acetate. The pH of the solution should be 7.0. Cork the flask and agitate its contents for about 30 minutes. This can be done with a mechanical shaker.Calibrating the Flame Photometer Measuring the concentration Extracting the Potassium from the sample CalculationsDetermination of available Potassium 1-61
  68. 68. Extracting the potassium from the soil sample Whatman No. 42... Whatman No. 1... Who is this man called What? It’s a brand name. “Whatman”, the company, makes filter paper — and a lot of other paper products used in Chemical analysis. The paper is graded according to its relative porosity. Hence, No. 1, No. 42 etc. An interesting feature of these papers is that they are “ashless”. This means that you can burn them and they do not leave behind a residue. This property is useful in a lot of experiments — such as gravimetric analysis, in which the filter paper is burned after it is used for filtration thereby leaving all the precipitate behind for weighing. Neat! Also, whoever started up the company was probably called “Whatman”... If that helps at all....1-62 Determination of available Potassium
  69. 69. Extracting the potassium from the soil sample Filter the suspension through Whatman No. 42 paper. The filtrate is used for determining Potassium concentration. Transfer the filtrate to beakers for use with the Flame Photometer. Aspirate the filtrate (the soil sample extract).Calibrating the Flame Photometer Measuring the concentration Extracting the Potassium from the sample CalculationsDetermination of available Potassium 1-63
  70. 70. Calculations... The results and some calculations.... Easy as pie! The smart Flame Photometer tells you the concentration of Potassium in the extract after politely asking you to wait for a while. In our case, the concentration of Potassium was 22.3. The K 2 O content of the soil is calculated using this formula - K20 (in kg/hectare)= [ 2 X CK ppm X Ve ] / Ws where CK ppm = Concentration of the Potassium in ppm obtained from the Photometer Ve = Volume of Ammonium Acetate used Ws = Weight of soil taken, in grams You can use Chemi-Calc, the calculator on the CD-ROM, to do the calculations. Or, if you want to show off a bit as well (like the Photometer did), use this trick Multiply the CK ppm amount by 10! Remember that the ratio of soil to extractant used should be 1 : 5. Which means that if you followed the steps, you would have taken 5g of soil and 25mL of Ammonium acetate. Those values then cancel out to give 5 in the numerator part of the equation. Multiply that by 2(also in the numerator) and you get [ CK ppm X 10] But beware, the trick works ONLY if you measured out the Ammonium acetate and the soil carefully to at least a couple of decimal places. So 25.01mL and 5.02g of soil is fine. But 25.5 mL and 5.2g of soil means your experiment will be approximately OK but you cannot show off with the calculation trick! The formula gives you the amount of K2O present in the soil (in kg per hectare). To find the amount of Potassium multiply the result from the calculation above by 0.83. However, this is not necessary for recommendation since we are interested in the K2O amount — Why? Because that’s the compound that Fertilizers Companies refer to! They aren’t very smart, are they?1-64 Determination of available Potassium
  71. 71. Measuring the concentration of Potassium in the soil sample Presence of potassium is indicated if the flame changes to a yellow-orange colour. The minute colour differences between the colours emitted by different elements are not distinguishable by the naked eye. The flame view is provided primarily for adjusting the stability of the flame and verifying that the nozzles of the aspirator are not contaminated by residues from previous experiments. The concentration of Potassium, in ppm, is displayed on the screen after a few seconds.Most soil testing labs are not equipped with direct-display Photometers like theone used here. Older equipment requires the user to plot a Standard Curvemanually on Graph Paper. If such equipment is used, the process described inSection 7 (the procedure used to determine the amount of Phosphorus) isapplicable here as well.Calibrating the Flame Photometer Measuring the concentration Extracting the Potassium from the sample CalculationsDetermination of available Potassium 1-65
  72. 72. 1-66 Determination of available Potassium
  73. 73. Soil Testing - Determination of available PhosphorusThe blue colour indicates presence of phosphorus 1-67
  74. 74. Preparing a Standard Curve A bit about available Phosphorus... Phosphorus occurs in soil in both organic and inorganic forms, most of which is not easily available to plants. A portion of the total Phosphorus is absorbed by plants during their growth in the form of H2PO4= . This is what we refer to as available phosphorus. What is a Standard Curve and what is it’s use? Simply put, it is a graphical means of determining unknowns that are variables of a linear equation. The catch is, that this is an equation of the form y = n x, where n might vary randomly as x varies. So how is that linear, you may ask. It is, approximately, because if we define n as n + e, then we find that e is a very small positive or negative number. The easier method to solve the problem, is to plot, on graph paper, a few (x,y) pairs and draw ONE line — the Standard Curve — connecting as many points as possible. If e was a relatively large number then we would have no option but to resort to esoteric mathematical tools like regression analysis because then the equation would cease to be approximately linear. But because e is a small number, we would find that most points are either on — or very close to — the straight line drawn and points are scattered almost equally on either side of the line. Then, to find the value of y for any given x (or vice versa) all you need to do is find the corresponding point on the line. Thus, we’ve found the solution to a linear equation by graphical means. Let’s call it the Graph Technique. Sounds difficult? It isn’t. As an exercise, try plotting the following value pairs on a sheet of graph paper (x,y) = (0 , 0) , (1.1 , 1) , (4.9 , 5) , ( 6.2 , 6) , (7.3 , 7) and (11 , 11). Draw a line that joins the points - you will find that the points are scattered to either side of the straight line joining (0 , 0) and (11 , 11). Using this line, find out the value of y when x is 8. You get y = 8. Now, the actual value of y might not be 8 exactly, but the graph shows that it would be pretty close to 8, if not exactly 8. In most cases, as in our present experiment, the small error is negligible. Apparatus and reagents required Olsens extractant or Bray and Kurtz No. 1 extractant, Standard 100ppm phosphate solution, ammonium molybdate reagent (containing antimony potassium tartrate and ascorbic acid), 2,4-dinitrophenol, P-free charcoal. Eight 25mL volumetric flasks, 10mL graduated pipette, 10mL measuring cylinder, a funnel, graph paper, Whatman No. 42 filter paper and a colorimeter.1-68 Determination of available Phosphorus
  75. 75. Preparing a Standard Curve Take eight 25 mL volumetric flasks and add 1mL, 2mL, 3mL, 4mL, 5mL and 10mL of 2 ppm Phosphate solution to six of them. Leave two flasks blank. Add 5mL of a 2ppm Standard Phosphate solution to one of the empty flasks. Add 5mL of Olsens Reagent to the same flask.Preparing a Standard Curve Measuring the concentration of Phosphorus Extracting Phosphorus from the soil sample CalculationsDetermination of available Phosphorus 1-69
  76. 76. Preparing a Standard Curve A step-by-step description of the process... First we need to plot a Standard Curve. In this test, it is plotted to determine, approximately, the relationship between the intensity of colour and the needle deflection of the Colorimeter (the reading on the scale) — the assumption being that the relationship would be linear. It is linear, by the way... Which makes this an ideal candidate for using the Graph Technique described on page no. 1-68. Now, we need a few stock solutions of Phosphorus to plot the Standard Curve. We take 6 such solutions. Then, we need to find out how much the Colorimeter would read when the solutions are placed in it. At this stage two problems crop up — (1) The Phosphorus in the soil is present primarily as a Phosphate of Calcium, Iron and Aluminium — which are all colourless. So the Phosphorus needs to be extracted AND (2) the new compound, in solution, must have a colour whose intensity varies linearly with respect to its concentration. The extraction part is done by adding either Olsen’s extractant which is a 0.5M NaHCO3 solution or by Bray and Kurtz No.1 Extractant. Ammonium molybdate reagent or "Reagent B" (which is a cocktail of Ammonium Molybdate, Tartarate and ascorbic acid) is used to obtain a heteropoly complex called phosphomolybdic acid, which is reduced, partially, by the ascorbic to give a blue coloured solution. The amount of the complex produced is directly proportional to the Phosphorus concentration. Problem solved! The two flasks to which we did NOT add any stock solution are used to simplify a technical hitch (see page 1-78) when Olsens extractant is used. The Flask with Olsens reagent described in the facing page will be used to carry out a mini-titration to determine the amount of acid required to lower the solutions pH to 3. The other flask will be used as a "blank". This lets us know if any of the chemicals we are using contain Phosphorus as an impurity. Shubham will be lurking on these pages to elaborate as we discuss the steps of the experiment!1-70 Determination of available Phosphorus
  77. 77. Preparing a Standard Curve Add two drops of 2,4-dinitrophenol indicator to the flask. The solution turns yellow. Then, with a graduated pipette or dropper, add 2.5M Sulphuric acid to the flask till the yellow colour is discharged. This indicates that the pH of the solution is 3. Note down the volume of acid consumed. In our test, 0.4mL of acid was required to lower the pH when 5mL of Olsens reagent was added. This flask is not needed any more and may be removed from the work area. This leaves 7 flasks — 6 with standard phosphate solutions and one empty flask for the blank test.Preparing a Standard Curve Measuring the concentration of Phosphorus Extracting Phosphorus from the soil sample CalculationsDetermination of available Phosphorus 1-71
  78. 78. Preparing a Standard Curve More on the pH value The value obtained in the previous step — 0.4mL of acid — pertained to 5mL of Olsens extractant. So, if we use, say, 50mL of the reagent, we need to add 4mL of 2.5M sulphuric acid to adjust the pH of the solution. Since weve standardised the procedure (always using 5mL of filtrate and so on) the value obtained earlier — 0.4mL — is used throughout.1-72 Determination of available Phosphorus
  79. 79. Preparing a Standard Curve We now have seven flasks left. To each of the flasks, add 5mL of Olsens reagent. Add exactly 0.4mL of 2.5M Sulphuric Acid to each of the flasks to adjust the pH of the solution to 3. Add approximately 10mL of distilled water to each of the flasks. Then, add 4mL of Reagent B — the Ammonium Molybdate reagent mixture - to each of the flasks. The picture shows the reagent being pipetted into the flask containing 10mL of the Standard Phosphate solution.Preparing a Standard Curve Measuring the concentration of Phosphorus Extracting Phosphorus from the soil sample CalculationsDetermination of available Phosphorus 1-73
  80. 80. Using a colorimeter Using the Colorimeter Colorimeters are usually analogue — measurements are indicated by the deflection of a needle over a semicircular scale — and they look very ancient in the laboratory, surrounded by digital equipment. However, they are very precise instruments as well! Colorimeters have a light-proof slot where the sample to be tested is inserted. If the sample is opaque, a full-scale deflection is observed, whereas a transparent sample does not cause any deflection of the needle. Calibration The zero and full scale deflections are set by “measuring” a sample of distilled water and a black, opaque cylinder (that comes as a standard accessory with the meter and is designed specifically for this purpose.) Turn the adjustment knob to set the needle to the zero when calibrating with distilled water. With the opaque cylinder in the metering slot, turn the adjustment knob so that the needle deflects all the way and stops in front of the “infinity” mark. More on the Standard Curve... After you’ve measured the optical densities of all the six solutions, plot them on a sheet of graph paper. The concentrations of Phosphorus in each of the flasks, in parts per million, go on the x-axis, while the corresponding optical densities go on the y-axis. Draw a straight line that connects most of the dots. That’s your Standard Curve! By the way, you might be wondering why it’s called the Standard Curve when it is a straight line. Well, it would be a curve if the equation were not linear. For instance, if the relationship between y and x were quadratic ( y = nx2 + c) then the graph would be a curve. Besides, (and this might sound silly) mathematically, a straight line is also a curve!1-74 Determination of available Phosphorus
  81. 81. Preparing a Standard Curve Add distilled water to each of the flasks till the volume of solution in them is exactly 25mL The 25mL value is to be maintained for all solutions. The value determines the concentration of the phosphate solution. Remember that ppm is a measure of concentration. Allow the flasks to stand for 10 minutes. The solutions assume a blue tinge. Measure the optical densities of all the solutions using a colorimeter. The blank sample should register a "zero" deflection while the sample with 10mL of the standard phosphate solution should register the highest optical density.Preparing a Standard Curve Measuring the concentration of Phosphorus Extracting Phosphorus from the soil sample CalculationsDetermination of available Phosphorus 1-75
  82. 82. Phosphorus free or not... On to the extraction We have the Standard Curve and now we proceed to extracting the Phosphorus from the soil sample. This is the part that you will be doing more often since a Standard Curve only needs to be prepared once a day. Remember to note down the volume of acid used to adjust the pH of Olsens Reagent. In our case, the volume is 0.4mL. Olsen’s Method This method is used to extract Phosphorus in soils with pH above 6. The Bray and Kurtz Method is employed to extract Phosphorus from soils with pH below 6. The reagent used for extraction, in this case is a solution of 0.03 N Ammonium fluoride in 0.025 N HCl. Olsens reagent is added at 1 : 20 ratio to the soil. Thus, when 2.5g of soil are taken for extraction, 50mL of Olsens Reagent is to be used. Bray and Kurtz Reagent is added at a 1 : 10 ratio. Usually you will find yourself using Olsen’s Method more often. The steps followed for employing both the methods are identical except for the ratio mentioned above. Preparation of the Standard Curve should also be done with the same reagent used for extraction.1-76 Determination of available Phosphorus