Prof . Dr. Hisham Ezzat AbdellatefProfessor of pharmaceutical analytical chemistry

Introduction to Analytical Chemistry Background

A.) ANALYTICAL CHEMISTRY: The Science of Chemical Measurements.

B.) ANALYTE: The compound or chemical species to be measured, separated or studied

C.) TYPES of ANALYTICAL METHODS:

1.) Classical Methods (Earliest Techniques)a.) Separations: precipitation, extraction, distillationb.) Qualitative: boiling points, melting points, refractive index,

color, odor, solubilities

c.) Quantitative: titrations, gravimetric analysis

2.) Instrumental Methods (~post-1930’s)a.) separations: chromatography, electrophoresis, etc.b.) Qualitative or Quantitative: spectroscopy, electrochemical

methods, mass spectrometry, NMR, radiochemical methods, etc.

CHOOSING AN ANALYTICAL METHOD What Factors to Consider:

What are the advantages or disadvantages of the technique versus other methods?

How reproducible and accurate is the technique?

How much or how little sample is required?

How much or how little analyte can be detected?

What types of samples can the method be used with?

Will other components of the sample cause interference?

Other factors: speed, convenience, cost, availability, skill required.

How Do We Answer or Address These Questions?

Types of Instrumental Methods: Example methods

Radiation emission Emission spectroscopy, fluorescence,phosphorescence, luminescence

Radiation absorption Absorption spectroscopy spectrophotometry, photometry, nuclear magnetic resonance NMR

Electrical potential Potentiometry

Electrical charge Coulometry

Electrical current Voltammetry - amperometry, polarography

Electrical resistance Conductometry

Thermal Thermal gravimetry, calorimetry

Example:Spectrophotometry

Instrument: spectrophotometerStimulus: monochromatic light energyAnalytical response: light absorption

Transducer: photocellData: electrical current

Data processor: current meterReadout: meter scale

Performance Characteristics: Figures of Merit:

How to choose an analytical method? How good is measurement?

How reproducible? - Precision

How close to true value? - Accuracy

How small a difference can be measured? - Sensitivity

What range of amounts? - Dynamic Range

How much interference? – Selectivity

CHARACTERISTICS OF AN ANALYTICAL METHODS Accuracy: The degree to which an experimental result

approaches the true or accepted answer.

Ways to Describe Accuracy:

Error: An experimental measure of accuracy. The difference between the result obtained by a method and the true or accepted value.

Absolute Error = (X – )

Relative Error (%) = 100(X – )/

where: X = The experimental result = The true result

All Methods, except counting, contain errors – don’t know “true” value

CHARACTERISTICS OF AN ANALYTICAL METHODS Precision: The reproducibility of results. The degree to which an

experimental result varies from one determination to the next.

Low accuracy, low precision Low accuracy, high precision

High accuracy, low precision High accuracy, high precision

Illustrating the difference between “accuracy” and “precision”

Accuracy vs Precision

CHARACTERISTICS OF AN ANALYTICAL METHODS Ways to Describe Precision:

Range: the high to low values measured in a repeat series of experiments.

Standard Deviation: describes the distribution of the measured results about the mean or average value.

Absolute Standard Deviation (SD):

Relative Standard Deviation (RSD) or Coefficient of Variation (CV):

where: n = total number of measurements Xi = measurement made for the ith trial = mean result for the data sample

n

i

i nXXSD1

2 )1/()(

100)/((%) XSDRSD

X

CHARACTERISTICS OF AN ANALYTICAL METHODS Response: The way in which the result or signal of a method

varies with the amount of compound or property being measured.

Ways to Describe Response:

Calibration Curve: A plot of the result or signal vs. the known amount of a known compound or property (standard) being measured.

Calibration expression is Absorbance = slope [Analyte (ppm)] + intercept

CHARACTERISTICS OF AN ANALYTICAL METHODS Parameters used to Describe a Calibration Curve: S = mc + Sbl

S – measured signal c – analyte concentration Sbl – instrument signal for blank

Sensitivity: calibration sensitivity = slope (m) of calibration curve.analytical sensitivity () = slope (m)/standard deviation (Ss)

ability to discriminate between small differences in analyte concentration. Slope and reproducibility of the calibration curve.

0

10

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0 2 4 6 8 10 12

Concentration (mM)

Method A

Method B

Selectivity: degree to which the method is free from interference by other species

the sample

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Concentration (mM)

Species A

Species B

No method is totally free frominterference from other species.

Selectivity coefficient (k):

kB,A = mB/mA

Relative slopes of calibration curves indicate selectivity:

S = mA(cA + kB,Acb) + Sbl

Interested in detecting species A, but signal will be a combination of signalfrom the presence of species A and species B.

Calibration methodsBasis of quantitative analysis is magnitude of measured property is proportional to concentration of analyte

Signal α[ x ] or Signal = m[x]+ Signal blank

ሾ𝐱ሿ= 𝐒𝐢𝐠𝐧𝐚𝐥 –𝐬𝐢𝐠𝐧𝐚𝐥 𝐛𝐥𝐚𝐧𝐤𝐦

Detection Limit: The smallest [analyte] that can be determined with statistical confidence. analytical signal = 2 or 3 times std. dev. of blank measurement

(approx. equal to the peak-peak noise level).

Calculation of detection limitThe minimum detectable analytical signal (Sm) is given by:Sm = Sbl + k(stdbl); for detection use k =3

Dynamic Range: linear region of calibration curve where the lower limit is ten times the standard deviation of the blank.LOQ - limit of quantitationLOL - limit of linearity

Concentration (mM)

To Experimentally Determine

Perform 20 – 30 blank measurements over an extended period of time.

Treat the resulting data statistically to obtain Sbl (mean blank signal) and stdbl (std. dev. of blank signals). Use these to obtain Sm value.

Using slope (m) from calibration curve. Detection limit (Cm) is calculated by: (Rearranged from Sm = mc + Sbl)

𝑪𝒎 = 𝑺𝒎− 𝑺𝒃𝒊𝒎