Lecture 1: Basic laboratory skills and investigative approach for sample preparation

1. Lecture 1: Basic laboratory skills and investigative approach for sample preparation Ahmad Razali Ishak Dept. of Environmental Health Faculty of Health Sciences UiTM Puncak Alam

2. Learning outcomes • To be aware of safety aspect in the laboratory • To be able to record numerical data with appropriate units • To understand the importance of sample handling with respect to both solid and liquids • To be able to perform numerical exercises involving dilution factors. • To understand the concept of quality assurance • To understand the difference between accuracy and precision and be able to use them appropriately • To understand the concept of representative sampling • To understand the principle of sampling soil and sediment, water and air • To understand the concept of sample storage • To appreciate the different method available for sample preservation for metals and organics

3. Introduction • All scientific studies involve some aspect of practical work • Essential to observe and record information correctly • In environmental analysis-Not all practical work is carried out in laboratory-sampling of environmental sample (air, water and soil) • Knowledge and implementation of storage condition and containers to be used are important

4. Safety Aspect • No lab work without due regard to safety both for yourself and people around • Occupational Safety health (Classification, Packaging and labeling of Hazardous Chemical ) Regulations 1997 • Occupational Safety health (Use and standard exposure of chemicals Hazardous to Health) regulation 2000

5. Basic rules • Always wear appropriate protective clothing • Never smoke, eat or drink in laboratory • Never work alone in lab • Make yourself familiar with the fire regulation in your laboratory and building • Be aware of the accident/emergency procedures in your laboratory and building • Only use/take the minimum quantity of chemical required for your works • Use a fume cupboard for hazardous chemicals. Check that it is functioning properly before starting the work • Clear up the spillage on and around equipment and in your own workspace as they occur • Work in a logical manner • Think ahead and plan your work accordingly

6. Recording of practical results • All experiment observation and data should be recorded in the notebook-use ink –at the same time that they are made. • Key factor to remember are as follows: • Record data correctly and legibly • Include the data and title of individual experiment • Outline the purpose of the experiment • Identify and record the hazards and risk associated with the chemical/equipment being used • Refer to the method/procedure being used and/or write a brief description of method • Record the actual observation and not your own interpretation • Record numbers with correct units • Interpret data in the form of graph, spectra and etc • Record conclusions • Identify any actions for future work

7. Metric System • Most countries use the metric system for measurement • Examples: • Gasoline by liter • Body weight in kilograms • Distance in meters or kilometers • U.S. uses English system of measurement in everyday life • Examples: • Gasoline in gallons • Weight in pounds • Distance in miles

8. Metric System • English system of measurementis not accurateenough for mostscientificmeasurements • Becausemetric system is a decimal system, itcanbeused for verysmallquantitieswithaccuracy • International System of Units (SI) is a form of the metric system adopted for use by the worldwidescientificcommunity.

9. Units of Metric System • Base Units • Distance = meter (m) • Mass or Weight = gram (g) • Volume = liter (L) • Prefixes are used to indicate larger or smaller quantities of the base units above

10. Common Metric Prefixes • Kilo (k) = 1000 x base unit • Centi (c) = .01 x base unit • Milli (m) = .001 x base unit • Micro (µ) = .000001 x base unit • Nano (n) = 10 -9 x base unit • Pico (p) = 10-12 x base unit

11. Converting between English and Metric Systems • Mile x 1.6 = kilometers • Pound x 0.454 = kilograms • Quart x 0.95 = liters • Kilometer x 0.6 = miles • Meter x 3.3 = feet • Meter x 39.37 = inches • Centimeter x 0.4 = inches • Gram x .0022 = pounds • Liter x 1.06 = quarts

12. SI System (International System) • Base Units of the SI System • Length = Meter (m) • Mass = Kilogram (kg) • Time = Second (s) • Amount of Substance = Mole (mol) • Electric Current = Ampere (A) • Temperature = Kelvin (K)* • Luminous Intensity = Candela (cd) • Volume = Liter (L)** *Although Kelvin is the SI unit, Celsius (C) is used almost exclusively in the clinical laboratory. **Liter (L) was not included in the list of base units in the SI system because the liter is a unit derived from other units. However, the liter has been accepted for use in measuring volume.

13. SI System (International System) • Non-SI units accepted in the clinical laboratory • Minutes (min) • Hours (hr) • Days (d) • Liter (L) • Pressure (mm Hg) • Enzyme Activity (IU) – International Unit

14. Exercises • 0.82 ppm = mg/l? • 1.21 ug/L = ? ppm • 2.32 mg/L = ? ug/ml

15. ppm = parts per million • PPM is a term used in chemistry to denote a very, very low concentration of a solution. One gram in 1000 ml is 1000 ppm and one thousandth of a gram (0.001g) in 1000 ml is one ppm. • One thousandth of a milligram is one gram and 1000 ml is one liter, so that 1 ppm = 1 mg per liter = mg/Liter. • PPM is derived from the fact that the density of water is taken as 1kg/L = 1,000,000 mg/L, and 1mg/L is 1mg/1,000,000mg or one part in one million. • OBSERVE THE FOLLOWING UNITS1 ppm = 1mg/l = 1ug /ml = 1000ug/L ppm = ug/g =ug/ml = ng/mg = pg/ug = 10 -6ppm = mg/litres of water 1 gram pure element disolved in 1000ml = 1000 ppm PPB = Parts per billion = ug/L = ng/g = ng/ml = pg/mg = 10 -9

16. Sample Handling- Liquids • The main vessels used for measuring out liquids in env. analysis can be sub divided into Quantitative works and Qualitative work. • Volumetric flask, burettes, pipettes, syringe-Quantitative • Beaker, conical flask, measuring cylinders, test tubes and Pasteur's pipettes- Qualitative • Nature of vessel may be important is some instances. Some plasticizers are known to leach from plastic vessels, especially in the presence of organic solvent. • In organic analysis, contamination risk is evident from glass vessels that may not be cleaned effectively- e.g. metal ions can adsorb to glass and then leach into solution under acidic condition---causing contamination • This can be remedied by cleaning the glassware prior to use by soaking for 24 h in 10% nitric acid solution followed by rinsing with deionized water 3 times.

17. Sample handling- Solid • The main vessel used for weighing in env analysis – weighing bottle, plastic weighing dishes or weighing boat, filter paper • Use 4 decimal place balance and tranfer a soluble solid directly into a volumetric flask • If solid not tatally soluble, transfer solid to a beaker, add suitable solvent • May be necessarry to heat the solution to achieve complete dissolution • Quantitatively transfer the cooled solution to the volumetric flask and make up the graduation mark with the solvent

18. Preparing solution for Quantitative analysis • Solution are usually in term of their molar conc. E.g. mol/l or mass conc. E.g. g/l • Both of these refer to the amount per unit volume • Important to used highest purity grade of chemicals for preparation of chemicals for quantitative analysis

19. Making up 1000 ppm solutions • From the pure metal : • weigh out accurately 1.000g of metal, dissolve in 1 : 1 conc. nitric or hydrochloric acid, and make up to the mark in 1 liter volume deionised water. • From a salt of the metal : e.g. Make a 1000 ppm standard of Na using the salt NaCl.FW of salt = 58.44g.At. wt. of Na = 231g Na in relation to FW of salt = 58.44 / 23 = 2.542g. Hence, weigh out 2.542g NaCl and dissolve in 1 liter volume to make a 1000 ppm Na standard. • From an acidic radical of the salt : e.g. Make a 1000 ppm phosphate standard using the salt KH2PO4FW of salt = 136.09FW of radical PO4 = 951g PO4 in relation to FW of salt = 136.09 / 95 = 1.432g.Hence, weigh out 1.432g KH2PO4 and dissolve in 1 liter volume to make a 1000 ppm PO4 standard.

20. Cont..Test. • Consider the preparation of a 1000ppm solution of lead from its metal salt, Pb(NO3)2 • Note the molecular weight of Pb(NO3)2 – 331.2, the atomic weigh of Lead = 207.19

21. Cont.. • Note the molecular weight of Pb(NO3)2 – 331.2, the atomic weigh of Lead = 207.19 • 331.2/207.19 = 1.5985 g of Pb(NO3)2 in 1 liter • Therefore dissolve 1.5985 g of Pb(NO3)2 in 1% HNO3 and dilute to 1 liter 1 % HNO3 will give you 100 ppm of Lead

22. Dilutions • Dilution = making weaker solutions from stronger ones Example: Making orange juice from frozen concentrate. You mix one can of frozen orange juice with three (3) cans of water.

23. Cont.. • Dilutions are expressed as the volume of the solution being diluted per the total final volume of the dilution In the orange juice example on the previous slide, the dilution would be expressed as 1/4, for one can of O.J. to a TOTAL of four cans of diluted O.J. When saying the dilution, you would say, in the O.J. example: “one in four”.

24. Calculation of dilution factor • An accurately weighed (2.1270 g) soil sample is digested in 25 ml of concentrated nitric acid, cooled and then quantitatively transfer to 100ml volumetriv flask and made up to the mark with distilled water. • This solution is then diluted by taking 10 ml of the solution and transfering further 100ml volumetric flask. • What is the dilution factor??

25. 100ml/2.127g X 100ml/10ml = 470ml/g • If the solution was then analyzed and found to be within the linear portion of the graph, the value for the dilution factor would then be multiplied by the conc. from the graph producing final value representative of the element.

26. Cont.. • A waste water sample (100ml) was extracted into dicloromethane (3X 10ml) using liquid liquid extraction. The extract was then quantitatively transfer to a 50 ml volumetric flask and made up to the mark with dcm. What was the dilution factor?

27. 50ml/100ml = 0.05 • If analyzed, the value for the dilution factor, would then multiplied by the conc. from the graph.

28. Quality assurance • Getting the correct result • Involves several steps including sample collection, treatment and storage, follow by laboratory analysis. • Accuracy –closeness of determined value to its true value • Precision-closeness of determined value to each other • A determine result for the analysis of polycyclic aromatic hydrocarbon in soil could be produce precise (repeatable) but inaccurate (untrue result)

29. To achieve good accuracy and precision • Select and validate appropriate method for sample preparation • Select and validate appropriate methods for analysis • Maintain and upgrade analytical instrument • Ensure Good record keeping of methods and result • Ensure the quality of data produced • Maintain High quality of lab performance

30. Investigative approach for sample investigation

31. Sampling • Constitutes the most important aspect of env analysis. • w/o effective sampling- the subsequent data generated are worthless • Two primary types of sampling –random sampling and purposeful sampling

32. Key questions to be asked before sampling begins • Have arrangement been made to obtain samples from the site? • Is specialized sampling equipment required available? • How many sample and how many replicates are required? • What analytical method and equipment are needed? • What mass/volume of sample required for the analytical technique to be used? • What types of container are required to store the samples and do you have enough available? • Do the container require any pretreatment/cleaning prior to use and has this been carried out? • Is any sample preservation required and do you know what it is?

33. Sampling methods Random sampling • two dimensional coordinate grid is superimposed on the area to be investigate. • The selection of samples is completely “down to the luck of the draw” w/o regard to the variation of the contaminant in the soil. • The entire sample area are is not sampled but that every site on the grid has an equal chance of being selected for sampling • Ideal if the contaminant is homogeneous within the site

34. Systematic Sampling • Taking the position of the first sample at random and then taking further samples at fixed distances/direction from this.-e.g at interval of 5 m • Potential to provide more accurate result than random sampling • However, if the soil contains a periodic variation-bias samples can result. • Initial pilot study can help prevent this

35. Stratified Sampling • Commonly used in a location which is known to have contaminant heterogeneously distributed • Most common approach to sampling • The site is sub divided into smaller area-each of which is fairly homogeneous. • Each sub area is then randomly samples • The sub dividing of the site can be carried out either give equal area or be related to known features within the site.

36. Sampling soil and sediment • Soil –heterogeneous material and significant variations (chemical & physical) • The tools required for sampling include auger, a spade and trowel • For shallow-trowel is sufficient, placed in polyethene bag, sealed and clearly labeled with a permanent marker. • Deep sampling-using auger/spade by utilizing trenches, road bans or digging soil pits and exposing soil profiles.

37. General procedure for using auger • Identify the site to be sampled • Bang into the ground, using mallet, a piece of heavy-walled PVC tubing as guide for auger • Place auger in PVC tubing and turn the handle. Once the auger is filled, remove from the ground and place the collected soil in a plastic bag. • Repeat step above until the required dept has been achieved

38. Sampling water • Water sampler can be either automatic or manually operated • Automatic-commonly used in river or from point sources. Allow the collection of time average sample or precipitation • Manual-essentially open tubes of known volume(-30 L) fitted with a closure mechanism. useful when sampling in open water(oceans, sea, lakes) at specific dept.

39. Manual water sampler

40. Sampling air • Can be classified –Particulate sampling (collected on filter) and vapor/gas sampling (trapped on sorbent) • Particulate sampling-passive sampling-airborne material diffuse onto filter and retained. • Vapor sampling-actively pump through filter/sorbent and retained. • A filter simply present the physical barrier, while sorbent provide an active sites for chemical/physical retention of the material • Filter-fibre glass to cellulose fibre • Sorbent- ion exchange resin to polymeric substrates

41. Cont.. • In sorbent tube sampling, volatile and semi volatile compound are pumped from the air and trapped in the surface of sorbent. • The sorbent tube is the sealed and transport back to lab for analysis. • Desorption of volatile and semi volatile take place by the use of organic solvent (solvent extraction) or heat (thermal desorption) follow by analysis using gas chromatography

42. Different types of air sampler

43. Storage of samples • In ideal situation, sample would analysed in situ w/o the need for sampling, storage and transport to the lab • However, not ALL the sample can be analysed in situ • The need of preservation

44. The function of preservation • Retard biological action • Retard hydrolysis of chemical compoundand complexes • Reduce volatility of constituent • Reduce absorption effect

45. Preservation methods • pH control • Addition of chemical • Refrigerating • freezing

46. Examples of preservation technique for water sample

47. Thank You