INTRODUCTION Analytical Chemistry – that branch of chemistry which is concerned with the theory and practice of methods used to determine the composition (quantity and nature) of matter.

Analytical Chemistry involves separating, identifying, called qualitative analysis; and determining the relative amounts (concentration) of the components in a sample of matter, called quantitative analysis.

There are several different areas of analytical chemistry, namely: environmental analysis, forensic analysis, clinical analysis, bioanalytical analysis, and pharmaceutical analysis.

This introductory analytical chemistry module is done by students pursuing different fields of study, including industrial and analytical chemistry, environmental science, forensic chemistry, pharmacy, medical technology and chemical engineering. Therefore, this module we introduce the theory and application of analytical chemistry to these various fields of study.

Analytical Chemistry would answer the question “how much dioxin is in a soil sample” or “how much hydrocortisone is in a topical preparation used for eczema treatment” or “how much mercury is in a blood sample” and an analytical chemist would have an interest in the methods and techniques involved in answering this question.

These persons would need to have a deep knowledge of wide areas in chemistry, statistics, perhaps geology, anatomy and physiology, etc.

An analytical chemist is therefore one who utilises analytical thought processes, in conjunction with specialised knowledge of analytical measurement techniques, methodology, and chemistry to experimentally characterise chemical systems in quantitative and qualitative terms. Sub-groups include the forensic analytical chemist, the pharmaceutical analytical chemist, the medical technologist and others, who could all utilise the principles and methods you will learn more about this semester.

Roles of the Analytical Chemist in Industry To the average student and more-so an outsider, the analytical chemist is one who simply receives samples, execute measurements and report results (H.A. Laitenien, Anal Chem., 1979, 51, 1601).

The analytical chemist obviously does more than that since that person is involved in method and technique development, problem solving, consultancy, and applying standard methods.

Scope and Application to Science & Technology In any industrialized society, the analytical chemist has a very vital role to perform. Most manufacturing industries rely upon both quantitative and qualitative chemical analyses to ensure that the raw materials are of acceptable specifications as well as to check the quality of the final product. A list of some of the areas where a person who is trained in analytical chemistry can be found follows.

Hospitals: That chemist can be used to assist in the diagnosis of illness and in monitoring the condition of patients (clinical chemist). For example, the presence of a measurable amount of bilirubin and more than 12 mg % of alkaline phosphatase (an enzyme) in a patient’s blood serum is indicative of an impaired liver function. Also, Na+/K+ concentration in serum can be measured by flame emission spectroscopy.

Detection and quantitation of poisonous and banned substances is also done.

Agriculture: Monitor nutrient levels in soils to aid farmers – the nature and level of fertilizer application is based on information obtained by analysis of the soil to determine its content of the essential plant nutrients, N, P, and K and of trace elements like Mn, Co, Fe which are needed for healthy plant growth. Therefore, the soil will have to be sampled and tested to determine the level of nutrient deficiency and to ensure that nutrients are added accordingly.

Also, to conduct pesticide residue analysis on crops. For example, samples of canned callaloo or ackee can be analysed for pesticide levels before export. 3

Food and Drug Administration (FDA): Chemical analysis is applied to monitor the food and drug taken to determine harmful or deleterious effect of ingredients or for quality control to ensure that ingredients are present in the amount purported on the label.

The Bureau of Standards in Jamaica has the responsibility to ensure that food and drug being imported is of acceptable quality. Their chemistry lab is equipped with many high-tech instruments that are used to carry out such analyses.

The Caribbean Regional Drug Testing Laboratory, located in Hope Gardens, undertakes analyses of drugs that have been reported in regional territories.

Research & Development: Many environmental research companies worldwide invest millions of dollars to develop and validate methods to determine varying types of analytes in different matrices.

Sports: In most if not all sporting meet that are sanctioned by the IAAF and other international bodies, athletes are selected randomly to have to their urine samples analyzed for banned substances like modafinil, nandrolone, ephedrine, stanozolol and tetrahydrogestrinone (THG).

Expensive and elaborate instruments such as HPLC, GC, GC-MS, LC-MS are used to undertake these analyses as on many occasions these drugs are masked.

Industry/Engineering: All chemical industries require raw material testing to assess the quality of the material and make informed decisions regarding the chemical processes involved in transforming the raw material into finished products.

Testing of the product must also be conducted for quality assurance before distribution to consumers.

Forensics/Crime Scene Investigation: Analyse evidence collected at crime scenes – examples:

Forensic drug analysis utilise instrumental methods to identify illicit (illegal) drugs such as crack/cocaine, spot tests for the analysis of opiates (such as heroin and morphine), amphetamines, barbiturates, marijuana, lysergic acid diethylamide (LSD), and cocaine;

Forensic toxicology for qualitative and quantitative analysis of biological specimens for the presence of alcohol, drugs, and/or poisons and their corresponding metabolites. In 4

death investigation, the role of the toxicologist is to assist the coroners and pathologists in determining the cause and manner of death (natural, accidental, suicide, or homicide);

Trace Analysis/Arson to identify trace amounts of chemicals at crime scenes, including the analysis of gunshot and primer residue, paint, hair, and fibres.

Academia In many colleges and universities worldwide people with analytical chemistry background can be found either as researchers, lecturers, or as administrators.

Task: Think of and write down three additional examples of industries (preferably local) where chemical analysis or analytical chemistry is used. Qualitative vs. Quantitative Analysis A qualitative analysis yields information about the atomic or molecular species or the functional groups that exist in the unknown sample. Having determined the nature of a given sample, the amount of each component, or of specified components has to be determined. Thus, a quantitative method provides numerical information. The nature of the information sought may involve requirement for very detailed data, or alternatively, results of a general character may suffice. Depending on the information that is furnished, chemical analysis may be classified as: Complete Analysis: This is the determination of the amounts of all of the constituents in the sample – an assay. For example, a complete analysis of a gasoline sample would tell the % of each compounds present (hydrocarbons, tetraethyl lead, tricresyl phosphate) Partial Analysis: Deals with the determination of (a) selected constituent(s) in the sample. A partial analysis of gasoline might tell the % of aromatic hydrocarbons or the % of tetraethyl lead. Proximate (ultimate) Analysis: The amount of each element in a sample is determined with no concern as to actual compounds or ions present. For example, an ultimate analysis of gasoline would tell us its %C, %O, %H, %P and %Pb etc. 5

Major-constituent (macro) Analysis: Only the constituents that are present in high concentrations are determined. Trace Constituent Analysis: A kind of partial analysis in which specified components are determined; these are present in very minute quantities. Task: Derive your own examples for each of the types of analysis just discussed. Classification of Analytical Methods Analytical methods are often classified as being either classical or instrumental. The classification is largely historical, with classical methods preceding instrumental methods by a century or more. Classical/Non-Instrumental Methods: In the early years of chemistry, the majority of analyses were undertaken by separating the components of interest (analytes) in a sample by distillation, precipitation or extraction. For qualitative analyses, the separated components were then treated with reagents that yielded products that could be recognized by their colours, melting or boiling points, their solubilities, optical activities or refractive indices. For quantitative analyses, the amount of analyte was determined by gravimetric or titrimetric measurements. In gravimetry, the mass of the analyte or some compound produced from the analyte (e.g., precipitate) is determined. In titrimetric methods, the volume or weight of a standard reagent required to react completely with the analyte is measured. In this course we will look at four types of titrations; neutralization (acid-base), complex formation, precipitate formation and oxidation-reduction. The extent to which the classical methods are being used is decreasing with passage of time. Instrumental Methods: In the 1930's, chemists began to exploit phenomena other than those of the classical nature for solving analytical problems. Measurements of the physicochemical properties of the analyte such as electrode potential, conductivity, mass-to-charge ratio, light absorption or emission, fluorescence- began to be 6

employed for quantitative analysis of a variety of analytes. Additionally, highly efficient chromatographic separation techniques began to replace distillation, extraction, and precipitation for the separation of components of complex mixtures prior to their qualitative and quantitative determination. These newer methods are referred to as Instrumental methods of analysis. Instrumental methods are usually much faster than classical, they usually require small sample size, and can measure very low concentrations (sensitive). Can be very expensive. However, classical methods are not obsolete and have advantages: 1. Apparatus required is cheap and readily available in all laboratories while many instruments are very expensive and is used if numerous samples are to be analyzed. 2. With instrumental methods, it is necessary to calibrate instrument against a reference material while classical methods are absolute and usually require no calibration. 3. If a fast, non-routine analysis is required with low precision, then it is often simpler to use a classical method. STEPS IN A QUANTITATIVE ANALYSIS Chemical analysis involves the determination of a material’s composition, qualitatively and or quantitatively. Experienced analytical chemists therefore have to use a systematic approach to obtain the required information from samples. This approach consists of a series and sequence of steps and accompanying thought processes. Most of these steps play a crucial role in a successful analysis. See Fig 1.2 of text. 1. DEFINITION OF PROBLEM: Before starting work on the sample, the chemist should inquire why work is being done (purpose) and what decisions will be taken depending on the results obtained. For example “Is this water safe to drink?” The general questions must be translated into specific questions that can be answered through chemical analysis, for example “What metals and organics are present and in what concentration?” Task: Suggest five reasons for carrying out an analysis and the uses to which the results might be put? List the consequence(s) of making an error in each. 7

2. METHOD CHOICE: The goal of every chemical analysis is to obtain the required information within a period of time acceptable to the customer. The analyst, therefore, must know what information is needed to solve the problem. Suitable methods are published in the scientific literature, e.g. U.S. Pharmacoepia, British Pharmacoepia, The Analyst, AOAC International, Journal of Chromatography. These methods have been validated (tried, tested, and proven). If no method is readily available for that particular analyte, the analyst will have to develop an original procedure but it too will have to be validated. Having established the purpose of an analysis and developed or searched the literature for suitable methods, the chemist must decide which analytical procedure to employ. Several factors will influence the method chosen. The order of importance will depend on the purpose or nature of the problem and will have to be ranked by the parties involved. 1. Cost of analyses – batch or single samples

2. Limit of detection – how minute a concentration can be accurately analyzed

3. Accuracy – how close to the true value the experimental value is

4. Precision – how close to each other are replicate measurements

5. Speed of analysis – minutes, hours, or days?

6. Equipment required – can be very specialized like HPLC, LC-MS, GC-MS, NAA

7. Sample size – how many samples, how tiny is sample

8. Safety – are chemicals safe to handle and dispose

9. Sample matrix – other than the analyte, what else makes up sample

10. Dynamic range – see text

11. Sensitivity – see text

12. Robustness – see text

13. Selectivity – see text

Cost: Most analytical chemists and clients are concerned with the cost of an analysis. Analysis of a single sample is normally charged at a higher rate than a larger batch. Additionally, certain techniques like GC-MS or HPLC are expensive; classical techniques are cheapest.

Limit of Detection (LOD) is especially important in trace analysis. LOD is the lowest concentration of an analyte that the analytical procedure can reliably detect (American Chemical Society). This has to be determined by repeated measurements of a blank test 8

solution. Measurements done at such low levels immediately eliminate most classical methods. It is the analyte concentration whose signal is equivalent to the average blank signal plus 3 standard deviations:

LOD of the signal = Sb + 3b LOD can also be determined from the standard deviation of a sample’s response: LOD = 3.3s/m where m = sensitivity or slope of calibration curve Ideally, measurements should be made above the limit of quantification (LOQ): LOQ = Sb + 10b (Refer to the text: Quantitative Chemical Analysis by Daniel Harris)

Accuracy refers to the closeness of a measured value to the true value. The demands of accuracy frequently dictate the procedure chosen for an analysis. Additionally, high degree of accuracy requires greater time to complete an analysis. For some analysis a high degree of accuracy is unnecessary. For example, if the maximum permissible level of a contaminant is 50 ppb and a sample is found to contain 10 ppb, it does not matter if the analysis is in error by 100% as the level of contamination is still below the permitted maximum.

Precision refers to the closeness of a set of values. Sometimes high precision may or may not be required. If the analyst is merely trying to establish whether the concentration of a trace element falls within 5-30 ppm, a high degree of precision may not be necessary unless the result obtained lies close to the margin.

Speed: If a large number of samples have to be analysed, a method that is rapid is to be preferred so that data can be acquired quickly and with minimum effort and cost. 9

Equipment: If the analysis requires the use of an instrument that is not available, say a LC/MS or NAA, the job will have to be contracted out to specialist laboratories.

Sample size: Sometimes, only a small sample size is available to the chemist for analysis, especially blood or contraband. Sample size is closely related to L.O.D. When the sample is not homogeneous, too small a sample should not be taken otherwise the portion of test sample used may not be truly representative of the bulk. Improved L.O.D. can sometimes be achieved by taking a larger mass of sample.

Safety: Techniques such as RNAA, INAA require special facilities as radiation exposure poses a threat. Some methods may require solvents that are carcinogenic or highly toxic. Alternative chemicals may have to be chosen or method changed.

Sample Matrix: The chemist should have an idea of the other species (other than the analyte) that are in a sample or the nature of the sample itself. Some methods are restricted to gaseous samples while most applies to liquids and solids. Also, the method chosen should be able to discriminate between the physical and chemical properties of the analyte and that of an interferent. In other words, only the analyte should react in accordance to the design of the method. Task: Think of additional factors that might affect choice of method and discuss them. Think of a problem, for example, a final year project in chemistry then rank five factors that you would have to consider. Justify your rank. 3. SAMPLING: This is often the most difficult aspect of an analysis. Quite often, the sampling is the limiting factor of both the accuracy and the precision of an analysis.

The major problem in sampling is to obtain a “laboratory-sized sample” that is representative of the larger whole. It is essential therefore that the laboratory sample accurately represents the composition of the bulk material.

Since an analysis can be only as good as the sample, an accurate analysis of a poor sample is largely a waste of time and money.

Therefore, the two goals of sampling methods are to ensure that samples are representative of the material being analysed, and that samples analysed in the laboratory are 10

homogeneous. The more representative and homogeneous the samples are, the smaller the sampling error will be.

When you are sampling a homogenous material, few problems are presented, since by definition, the composition is uniform throughout. However, if a heterogeneous material is the subject of analysis, it is necessary to combine several portions of the material to ensure that a representative sample is selected.

The sampling procedure can involve a number of stages prior to the analysis of the material. Most of the time, bulk materials are heterogeneous e.g., foodstuff and sediments. They can contain particles of different composition which are not uniformly distributed within the material. If this is the case, a number of increments are randomly taken from points in the bulk material, thus each part has an equal chance of being selected. These increments are then combined to form a gross or primary sample. This gross sample, if large, is further divided to produce a sub-sample or secondary sample.

In order to ensure that a representative sample is collected, a sampling plan must be established.

o This is a well defined procedure that states the number and amount of samples to be taken from the bulk, the sampling location, sampling method, timing and frequency of sampling.

The sampling method depends largely on the type and characteristics of the sample and where it is to be taken from:

o Simple random sampling - Any technique that assures that object in the bulk has an equal probability of being selected into the sample.

o Stratified sampling - The bulk is first divided into subgroups using various strata (e.g. soil type, drug category, etc.) and proportionate numbers of cases are randomly selected from within each strata.

o Systematic sampling - Selecting every kth case from a list of cases, i.e. a sampling frame.

o Quota sampling - The same idea as stratified sampling, but proportionate numbers of cases are selected from within each strata but by some method other than simple random sampling.

4. PREPARATION OF A LABORATORY SAMPLE: If a sample is not representative of the material to be analysed, the results of the analysis cannot accurately reflect the content of the material.

The sub-sample, if large and bulky, may require treatment, example reduction in particle size through grinding or thorough mixing, before the analytical sample can be obtained. The analytical sample, however, should retain the same composition of the gross sample. The 11

error introduced through the sampling step can largely be minimized, however, it increases with:

a. decreasing subsample size

b. increasing particle size

c. decreasing percentage of particles with analyte.

Many of the problems occurring during sampling arise from the physical nature of the materials to be studied. Although gases and liquids can, and do, present difficulties, the greatest problems of adequate sampling undoubtedly arise with solids.

For sampling of gases and liquid samples, refer to text.

SOLIDS It is with solids that real difficulties over homogeneity arise. Even materials that superficially have every appearance of being homogeneous in fact may have localized concentrations of impurities and vary in composition.

The procedure adopted to obtain as representative a sample as possible will depend on the type of solid. The process is of great importance since, if it is not satisfactorily done, the labour and time spent in making a careful analysis of the sample may be completed wasted. If the sample is homogeneous, sampling is simple. If, however, the material is heterogeneous, sampling must be carried out with care.

The principle of the sampling of material in bulk, say a truckload of dirt taken from a garage site for lead analysis, is to select a large number of portions in a systematic manner from different parts of the bulk and then combine them.

This large sample is mechanically crushed and then shoveled into a conical pile. Every shovelful must fall upon the apex of the cone and the analyst must walk around the cone as he shovels; this ensures a comparatively even distribution (see below).

The top of the cone is then flattened out and divided into quarters. Opposite quarters of the pile are then removed, mixed to form a smaller conical pile and then quartered again- coning and quartering.

This process is repeated, further crushing being carried out if necessary, until a sample of suitable weight (e.g. <300 g) is obtained.

If the original pile is so large that sampling cannot be done on the total – example a shipload of bauxite, portions can be removed from a vibrating conveyor belt at regular, frequent intervals throughout a cross-section.

The exact procedures for sampling many types of material vary and are specified by various professional societies. 13

In the sugar industry, for example, a core sampler burrows through the truck load of sugar cane and 'digs' out a sample. For more information, contact the sugar industry authority, SIA.

For primary samples that can be counted, e.g. a bottle of tablets, the number that is taken is either the square root of the total objects or three times the cube root. 5. DEFINE REPLICATE SAMPLES: Most chemical analyses are performed on replicate samples whose masses or volumes have been either accurately weighed on an analytical balance or measured with a pipette, buret, or volumetric flask.

Analyses are ordinarily carried out in triplicate ( on three samples simultaneously), or at least in duplicates.

Obtaining replicate data on samples usually improves the precision of the analyses.

6. DISSOLVING THE SAMPLE: Most methods, particularly non-instrumental ones, are designed for use with liquid solutions of the sample, partly because solutions are homogeneous and easy to handle.

As a rule, it is best to dissolve the entire sample using the mildest conditions. A solvent should be chosen that will not interfere in the subsequent steps of analysis.

Depending on the sample, the most suitable solvents are water, organic solvents, and mineral acids.

Several sources of error are encountered in this step and limits accuracy.

7. ELIMINATING INTERFERENCE: The ideal quantitative method for a substance would be a specific method- one that would measure the analyte accurately in the presence of any matrix.

Unfortunately, only a very few are specific while most are only selective (pg. 5).

An interference in a chemical analysis arrises whenever a species in the sample matrix either produces a signal that is indistinguishable from that of the analyte or, adds to the analyte’s signal.

There are three general ways to eliminate interferences; very careful selection of the method, separating (precipitation, ion-exchange, extraction, chromatography) the interfering species from the analyte, using a masking agent – a reagent that chemically binds an interferent to convert it to a non-interfering form.

For example, Ca2+ in tablets can be measured with EDTA. However, Al3+ interferes as it too reacts with EDTA. It can be masked by treating the sample with excess fluoride, F-.

Al3+ + EDTA4- AlEDTA- vs. Ca2+ + EDTA4- CaEDTA2- 14

To mask Al3+, the fluoride ion forms the stable complex ion, AlF63-. This complex will not react with EDTA. AlF63- + EDTA4- no reaction 8. CALIBRATION AND MEASUREMENT: This is the step in which the quantity of the substance being determined is actually determined.

All analytical results depend on a final measurement X of a physical property of the analyte. This property must vary in a known and reproducible way with the concentration CA of the analyte often, the physical property measured is directly proportional to the concentration.

CA = k . X where k = proportionality constant The process of determining k is called calibration or more accurately, calibration is the process of determining the relationship between a measured signal and the concentration of analyte that generates the signal.

The measurement step usually requires the adjustment of several conditions. For example, procedure might require adjustment of pH within a certain range for color to be developed for spectrophotometry or for extraction to take place.

Also, the dilution of the sample solution must be such that the species to be measured are in the proper concentration range.

Sometimes, the oxidation state of certain elements has to be adjusted before a titration is done or other quantitative measurement (ion-exchange).

9. CALCULATING AND EVALUATING THE RESULTS: The measurement step provides the data needed to calculate the quantity of each species in the sample. The proper use of significant figures is important.

When the results have been calculated, it is then the task of the analyst to indicate how reliable they probably are.

This is done by considering the inherent accuracy of the method used and by subjecting the numerical data to statistical treatment.

In the next unit we will extensively evaluate the reliability of results