Analytical chemistry

A philosophical and historical introduction

GENERAPHARM

Some Basic Questions to be addressed in this section

1) Have Analytical Instruments improved in recent years ?

2) Do Analytical Chemists provide meaningful answers to problems ?

3) Are Analytical Chemists providing answers with better accuracy and precision compared to 60 years ago ?

HISTORY OF ANALYTICAL CHEMISTRY1950’s-70’s

• 1950’s – pH meters – single wavelength spectrophotometers– electrochemical techniques.– Lots of titrations, gravimetric analysis.

• 1960’s– Gas Chromatography– Atomic Absorption spectrophotometry

• Analysis of ppm and ppb levels of metals and organics in the environment

• 1970’s– Liquid chromatography– Mass spectrometry

• Sensitivity allowing part per trillion trace analysis.

1980’s – 1990’s

• 1980’s– Hyphenated methods

• GC-MS, LC-MS, GC-IR, etc.– Computers control instruments, manipulate data, and

run experiments– Automation– Robots conduct complete analytical schemes

• 1990’s– Sample preparation is a major focus

• Old, time consuming methods of solvent extraction are replaced with fast, automated procedures.

– LC/MS brings MS to biochemistry

Most recent advances are related to biological systems, 3-dimensional analysis and high speed throughputs

Types of analytical methods

• Gravimetric Methodsmeasure the mass of an analyte (or something chemically equivalent to the analyte)

• Titrimetric (Volumetric) Methodsmeasure the quantity of a reagent needed to completely react with the analyte

• Electroanalytical Methodsmeasure the change in the electrical potential, current, resistance or charge produced by an analyte

• Spectroscopic Methodsmeasure the interaction between electromagnetic radiation (light, UV, IR, etc.) and the analyte

These interactions can be used for both quantitative and qualitative analysis

Different methods measure different attributes

Wet chemistryClassical Analytical Methods • Gravimetric methods• Volumetric methods - Titrations with Indicators

Chemistry in solutionPrecipitationPrecipitation

Acid-base reactionsAcid-base reactions

Metal complexesMetal complexes

ElectrochemistryElectrochemistry

The Pharmacopeiastill contains

many classical methods

Gravimetric methods

• LOD• Ash values• Gravimetric assays

– Chloride by precipitation as a silver salt and then heating in a furnace to measure the silver

LOD-moisture content?

• There are a number of “methods” which are loosely termed Loss on Drying– The only absolute method is to dry to constant weight

[different conditions-110OC, vacuum etc]– Thermobalances determine the loss in weight at a

specific temperature in a specific time• This is a comparative method-is it the same as it was last

time? Different instruments-different results!

– Karl Fisher titration• A chemical titration determining water only

Even LOD can be confusing

• It is possible to have LOD higher than KF – If the test substance contains both water and organic

solvents then LOD>KF

• It is possible to have KF higher than LOD– If the test substance contains “bound” water such as

water of crystallization that is not removed during the drying process then LOD<KF

An error at this point will result in incorrect results throughout the whole analytical process because if we weigh 100mg of the substance thinking it is pureand it has 5% water then we are actually only putting95mg into the equation

Gravimetric analysis

1. Dissolve the sample

2. Add a precipitating agent

3. Collect the precipitate

4. Wash and dry the precipitate

The two measurements are both weightsand the balance is the most accurate of

all routine analytical equipments

The only errors are related to handlingand the solubility of the precipitate

Determination of Halide salts

• AgNO3 + NaX AgX + NaNO3

• The silver halide precipitate is collected and burnt in a muffle furnace giving pure silver

• From the weight of silver the amount of halide can be calculated

Solubility constants

G/100G H2OCOMPOUND

0.000003AgI

0.000014AgBr

0.00019AgCl

172AgF

The method will work for Chloride, Bromide and Iodide but not FluorideThe precipitation of the Fluoride is not sufficiently complete i.e. recovery isno good.

It is also an example of an accurate method with no selectivity, i.e. it doesnot distinguish between the halides, i.e. it can’t be used for a mixture oftwo halides.

Gravimetric analysisash value

Method II (Ph. Eur. method 2.4.16)

Heat a silica or platinum crucible to red heat for 30 minutes, allow to cool in a desiccator and weigh. Unless otherwise specified in the monograph, evenly distribute 1 g of the substance being examined in the crucible, dry at 100° to 105° for 1 hour and ignite to constant weight in a muffle furnace at 575° to 625°. Allow the crucible to cool in a desiccator after each ignition. Flames should not be produced at any time during the procedure. If after prolonged ignition a carbon-free ash cannot be obtained, take up with hot water, filter through an ashless filter paper and ignite the residue and the filter paper. Combine the filtrate with the ash, carefully evaporate to dryness and ignite to constant weight.

1000.0 mg gave a residue of 100.0 mg gives an ash value of 10.0

Limit Tests for Arsenic (Ph. Eur. method 2.4.2)

Test A The apparatus (Fig. 7-1) consists of a 100-ml conical flask closed with a ground-glass stopper through which passes a glass tube about 200 mm long and 5 mm in internal diameter. The lower part of the tube is drawn to an internal diameter of 1.0 mm and 15 mm from its tip is a lateral orifice 2 to 3 mm in diameter. When the tube is in position in the stopper the lateral orifice should be at least 3 mm below the lower surface of the stopper. The upper end of the tube has a perfectly flat, ground surface at right angles to the axis of the tube. A second glass tube of the same internal diameter and 30 mm long, with a similar flat ground surface, is placed in contact with the first and is held in position by two spiral springs. Into the lower tube insert 50 to 60 mg of lead acetate cotton, loosely packed, or a small plug of cotton and a rolled piece of lead acetate paper weighing 50 to 60 mg. Between the flat surfaces of the tubes place a disc or a small square of mercury(II)bromide paper large enough to cover the orifice of the tube (15 mm × 15 mm).

In the conical flask dissolve the prescribed quantity of the substance being examined in 25 ml of water or, in the case of a solution, dilute the prescribed volume to 25 ml with water. Add 15 ml of hydrochloric acid, 0.1 ml of tin(II) chloride solution AsT and 5 ml of potassium iodide solution, allow to stand for 15 minutes and add 5 g of activated zinc. Immediately assemble the two parts of the apparatus and immerse the flask in a water bath at a temperature such that a uniform evolution of gas is maintained. After not less than 2 hours any stain produced on the mercury(II) bromide paper is not more intense than that obtained by treating 1 ml of arsenic standard solution (1 ppm As) diluted to 25 ml with water in the same manner.

The conservatism of the Ph. Eur [B.P.]

• Many pharmaceutical compounds are supplied as salts such as sulphates

• Sulphuric acid was made by the lead chamber process [burning sulphur in the presence of K/Na Nitrate] in lead chambers [the lead chamber process]

– Lead impurities antimony, arsenic, bismuth, copper, nickel, silver, tin, and zinc.

• Replaced by a contact catalyst process [originally Pt and now Vanadium] from 1930’s

Many monographs still contain tests for heavy metals,

lead and/or arsenicThe 3 pharmacopeias are changing

their testing procedures but thereis little sign of harmonization

Classification of methods

GRAVIMETRYTITRATIONS

TLC

CLASSICMETHODS

SPECTROSCOPYELECTROCHEM

GLC, HPLC

INSTRUMENTALMETHODS

DISSOLUTIONDISINTEGRATION

HARDNESSVISCOSITY

USETESTS

IR, NMR, MS, X-RAY, Thermal methods

Basic concepts

Types of analysis

QUANTITATIVEANALYSIS

QUALITATIVEANALYSIS

ABSOLUTE COMPARATIVE

Qualitative analysis is what.

Quantitative analysis is how much.

QUALITATIVE ANDQUANTITATIVE ANALYSIS

Quantitative or qualitative?

• Qualitative analysis– What compounds are present

• Answers the question is compound x present

– What is the structure of a compound• What is the chemical structure of compound x

• Quantitative analysis– How much of a specific compound is present

• How much of compound x is found in the matrix

QUALITATIVEANALYSIS

STRUCTURAL ANALYSISIDENTITY TESTS

FROM FIRSTPRINCIPLES

BY COMPARISON

Qualitative analysis

WHAT IS IT?

IR, NMR, MSFunctional analysis

CHN, X-RAY

M.Pt, UV, IRCo-chromatography

HISTORICAL

• Wet chemistry techniques were used to collect information:– Functional group analysis

• i.e. ketones react with 2,4-DNP

– Degradation and identity of the pieces followed by theoretically reassembling the pieces

• Amino acid sequencing for protein analysis• Side chain oxidation of steroids

Most wet methods have been replaced by spectroscopic methods

• These are related to the relationship between the atoms that are arranged to make the molecule

• They may demonstrate spacial-sterochemical interrelationships.– UV– IR – NMR– MS

Specific properties

HOHO

HO

Level of detail

The Pharmacopeial identity test

fingerprints

Identical IR meansthe compounds areidentical

Different IR’s can begenerated by the samecompound-some [notall] polymorphs have different fingerprints

Absolute methods

• These methods refer to an inherent property of a molecule without reference to another substance or standard [pure] material

• These properties may be specific to the molecule or they may be common to a number of compounds

• There are methods which are qualitative-that is they demonstrate the presence of a compound

• There are methods that are quantitative, that is they relate to the amount [mass] of material present.

Quantitative analysis

Specificity

• May be applied to both qualitative and quantitative methods

• The infrared spectrum of a compound is like a fingerprint – it absolutely identifies the compound– No two compounds or two people have the

same fingerprint– This means that it is a highly specific method

Medium specificity

• Melting point v personal characteristics– A specific compound will have a specific melting

point, i.e. Paracetamol melts at 169OC– A person has a specific height, color of eyes etc.

• These are not unique attributes, other compounds may melt at 169OC in the same way as 1.95 m does not identify a person

• A combination of medium specific properties increases the likelihood of correct identity– Height, hair colour, eye colour etc.

Partial identities

• All Frenchmen speak French– Paracetamol melts at 169OC

• Not all people who speak French are Frenchman– Not all compounds that melt at 169OC are

paracetamol

Specificity v selectivity

• Often used interchangeably or incorrectly, even in official documents

• If the method responds to only one single analyte then this method is selective– Selective ion monitoring– Ion selective electrode– pH meter [H+

• If the method responds to a number of analytes but allows the differentiation between them, the method is specific– Chromatographic methods

USP definition

• ..selectivity of an analytical method as its ability to measure accurately an analyte in the presence of interference such as impurities, synthetic precursors, degradation products, excipients that

That is…not every compound that exists in the world

“might be expected to be present

in the sample matrix”

The significance [the corollary]

• The selectivity of a method is only valid for an analyte in the specific matrix– Change the matrix and the selectivity

must be revalidated• Change the formulation• Change the synthetic route

Measuring Vitamin C

Every method will be different-it may be sample preparation or the actual analytical method itself

An anomaly for generics

• We are required to analyse “brand” samples– For example, when we submit a generic product for

biostudy we are required to submit the brand sample together with a “certificate of analysis”

• We do not have the quantitative information on the brand nor the brands validated method

We use the method developed for our

formulation and assume that it is valid for the brand

However-ID tests for finished dosage forms take into account that the brand and a generic will have a different composition

Dosage forms

Dosage forms cannot use absolute identification such as IR because the other materials present have their own IR i.e. the matrix interferes

It would be like trying to identify a suspect from a fingerprint superimposed with several other peoples prints

The more non absolute methods used the better probability

Height alone rules out some people,Height, hair colour, eye colour togetherincreases the chance of identification

Specificity

• There are different degrees of sensitivity to the molecular structure, that is there are high resolution techniques and low resolution techniques,

• UV is a low resolution technique• IR is highly specific• Mass spec

– Low resolution = 46– High resolution =46.0418

C=12N=14H=1O=16How many combinationscould have a MW of 46?

EFFECT OF RESOLUTION ONSPECIFICITY

high reslow resformula

46.053046NH2CH2NH2

45.992946NO2

46.005446HCOOH

46.041846C2H5OH

CH3CH2OH

OR

CH3OCH3

SPECIFICITY v PRECISION

• In many analytical methods we have a trade off between the two– Gravimetric assays and titrations are precise

but not specific– HPLC is specific but not precise!!!!

Good analytical practice would be to prove specificity by one Method and then assay by a precise method

The B.P. used this approach – prove the compounds identity and “purity” by TLC and then assay by titration.

Do not try and argue this withthe regulatory authorities

If it isn’t HPLC it is not kosher!

PRECISION

GRAVIMETRYVOLUMETRIC

UV/VIS

GLCHPLCTLC

SPECIFICITY

GRAVIMETRYVOLUMETRIC

UV/VIS GLCHPLCTLC

Categories of Assays

Analytical methods for determination of product characteristics i.e. dissolution drug release

TYPE III

Identification testsTYPE IV

Analytical method for determination of impurities in API’s or degradation compounds in finished products. These include quantitative assays and limit tests

TYPE II

Analytical methods for quantitation of bulk drug substances or active ingredients in finished products

TYPE I

DEFINITIONCATEGORY

TLC

• Selectivity but no precision• However it is a valuable identity method

and it can be considered one of the earliest hybrid methods linking two processes– Either visual [colours] or chemical [spray

reactions]

Instrumentation

There has been a revolution in Analytical Instrumentation

• New Instruments• Computing power and improved quality

of information through computing • More detailed analyses can be done• Detection of ultratrace amounts• Faster methods of sample preparation

Equipment

Analyticalbalances

pH Meter

Five generations of equipment

Instruments for Analysis

Structure of analytical detection-human

SUNLIGHT

EYEOPTICALNERVE

Analyticalsignal

BRAIN

Electricalsignal

VISUALCOLOUR

RESPONSE

Outputsignal

RECORD IN LAB

NOTEBOOK

Instruments for Analysis

Components of a typical instrument

Signalgenerator

Inputtransducer

ordetector

Analyticalsignal

Signalprocessor

Electricalor

mechanicalinputsignal

12.301

Outputsignal

Meteror

Scale

Recorder

Digitalunit

The analyst as an observer

Colour change of indicator

AND THE PHARMACOPIASARE STILL FULL

OF THESE METHODS

Many tests rely on the analysts observation

• Tubes used for comparative tests (Ph. Eur. text 2.1.5) – Unless otherwise prescribed, tubes used for comparative tests are

matched tubes of colourless glass with a uniform internal diameter of 16 mm. The base is transparent and flat. A column of the liquid is examined down the vertical axis of the tube against a white background or, if necessary, against a black background. The examination is carried out in diffused light.

• Limit Test for Phosphates (Ph. Eur. method 2.4.11) – To 100 ml of the solution prepared and, if necessary, neutralised as

prescribed add 4 ml of sulphomolybdic reagent R3, shake, add 0.1 ml of tin(II) chloride solution R1, allow to stand for 10 minutes and examine 20 ml of the resulting solution. Any colour produced is not more intense than that produced in 20 ml of a solution obtained by treating a mixture of 2 ml of phosphate standard solution (5 ppm PO4) and 98 ml of water in the same manner.

Evaluation of colour is frequently used in Pharmacopoeias

• Colour measurement is in fact very complex because colour perception depends on: – the light source, – the object– the observer = eye + brain …complex instruments

• Current visual examination depends on the human observer

• Alternative instrumental measurement should be more objective for Ph. Eur. and for harmonization

Current Pharmacopeial Test Methods for Colour

• Ph. Eur. – 2.2.2. : Degree of coloration of liquids, visual examination,

mainly for slightly coloured solutions.– 2.2.25. : UV/VIS Absorption. For coloured solutions, maximum

absorbance limit

• USP:– <631>: Color and Achromacity / Physical test (matching fluids)– <1061>: Color-instrumental measurement / General information– <851>: Spectrophotometry and light-scattering / Physical test

• JP: – <70> general chapter (matching fluids)

Visual and instrumental correlation

• Instrumental measurement is allowed but correlation between devices is almost impossible.

Apha (Hazen) Color Scale ColorQuest® XT Spectrophotometer Self-Contained Transmission Color

Measurement System

1st Generation

manual & visual interaction between instrument and analyst– examples:

• burettes• mechanical balances• colorimeter

First-generation Systems

AnalystInstrument

orProcess

Manual

manipulation

Direct

results

Second-generation Systems• analyst interacts with knobs & switches &

results are displayed on analog meters &/or strip chart recorders– Examples:

• Beckman DU, Spectronic 20

Second-generation Systems

AnalystInstrument

orProcess

Manual

manipulation

Direct

results

OutputDevice

Indirect

results

Third-generation Systems

Analyst Instrumentor

Process

Directresults

ControlComputer(External)

OutputDevice

OperatingInstructions

RawDataProcessed

Data

Indirectresults

microcomputers interfaced to existing instruments to log & process data

Fourth-generation Systems

AnalystInstrument

orProcess

ControlComputer

OutputDevice

Commands

OperatingInstructions

Processedoutputsignals

Output signals

Indirectresults

miniaturized computers integral component ( microprocessor)

Fifth-generation Systems

Analyst

Instrument or Process

User InterfaceExplanation(Knowledge)

User-levelcommands

Integrated Computers

Control Diagnostics

Intelligent System

OutputDevice

DataProcessing

Raw data

Processed data(Information)

High-levelcommands

Machine-level commands

EFFICIENCY

• SPEED– Sample preparation techniques– Chromatographic efficiency

• AUTOMATION– Off line– On line– In line

• HYPHENATED TECHNIQUES

SPEED

• Sample preparation is an area of dramatic expansion– Ultrasonics– Solid phase extraction– Microwave

AUTOMATION

Samples are made in same room as analytical equipment

hplc

Small number of samples and no automation

Two modern variants

SINK

SINKsamples

hplc

hplc

Gla

sswa

re c

up

bo

ard

s, reag

en

ts etc

hplc

hplchplc

sink

sink

Do chemists provide meaningful answers

• The development of analytical techniques provides improved information– Gas chromatography was available for some time but

the existing detection systems were not specific or highly sensitive

– The development of the Electron Capture Detector [1957] had an increased sensitivity for organochlorine pesticides of ~ 1,000

– DDT 0.04 ppm in fat [MDL]

Until this analytical procedure was available the accumulation of these compounds in the food

chain due to fat retention was unknown

Introduction to Analytical Chemistry Are Chemists providing answers with

better accuracy and precision compared to years ago ?

• New Instruments are not the complete solution

• The accuracy of analysis is dependent on the nature and complexity of the material– Errors of 1-2% are

excellent– Errors of 5-10% are

common– Higher errors observed for

trace amountsFe Mn K

05

101520253035404550

Fe Mn K

Glass

Bronze

Bauxite

The Analysis of Complex Materials

Introduction to Analytical Chemistry The Analysis of Complex Materials

• Precision for most Analyses has not improved over the years due to:-neglect of sample preparation, and

• poor knowledge of chemical interferences

1931 1951 1972 19740

0.1

0.2

0.3

0.4

0.5

1931 1951 1972 1974

Silica

Precision % sd

Year

Has analytical chemistry improved in the last 50 years

SENSITIVITY SELECTIVITY PRECISIONEFFICIENCY

Different methods provide a range of precision, sensitivity, selectivity, and speed capabilities.

Development trends in pharmaceutical analysis: its effect on analyst skills

High level of Manual dexterity

Less reliance onmanual dexterity

but more theoretical knowledge

Specialization inSpecific techniques“the specific expert”

The developerThe validatorThe technician

The General Analytical Problem

Select sample

Extract analyte(s) from matrix

Detect, identify andquantify analytes

Determine reliability andsignificance of results

Separate analytes

Selecting an Analytical Method

1. What accuracy and precision are required?2. How much sample is available?3. What is the concentration range of the analyte?4. What components of the sample will cause

interference?5. What are the physical and chemical properties of the

sample matrix?6. How many samples are to be analyzed?

Defining the problem:

Other Characteristics to be considered in Method of Choice

1. Speed2. Ease and Convenience3. Cost and availability of instrument4. Per-sample cost