Transcript Instrumental Analysis

Prof . Dr. Hisham Ezzat Abdellatef
Professor 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, distillation
b.) 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: spectrophotometer
Stimulus: monochromatic light energy
Analytical response: light absorption
Transducer: photocell
Data: electrical current
Data processor: current meter
Readout: 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 – m)
Relative Error (%) = 100(X – m)/m
where:
X = The experimental result
m = 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.
Illustrating the difference between “accuracy” and “precision”
Low accuracy, low precision
High accuracy, low precision
Low accuracy, high precision
High accuracy, high 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):
SD 
n
2
(
X
i

X
)
/(n  1)

i 1
Relative Standard Deviation (RSD) or
Coefficient of Variation (CV):
RSD(%)  (SD / X )100
where: n = total number of measurements
Xi = measurement made for the ith trial
X = mean result for the data sample
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 – measured signal
c – analyte concentration
Sbl – instrument signal for blank
Sensitivity:
S = mc + Sbl
calibration sensitivity = slope (m) of calibration curve.
analytical sensitivity (g) = slope (m)/standard deviation (Ss)
ability to discriminate between small
differences in analyte concentration.
Slope and reproducibility of the
calibration curve.
70
60
50
Method A
40
30
Method B
20
10
0
0
2
4
6
8
Concentration (mM)
10
12
Selectivity: degree to which the method is free from interference by other
species
the sample
70
No method is totally free from
interference from other
species.
60
50
40
Species A
Selectivity coefficient (k):
30
20
kB,A = mB/mA
Species B
10
Relative slopes of calibration
curves indicate selectivity:
0
0
2
4
6
8
Concentration (mM)
10
12
S = mA(cA + kB,Acb) + Sbl
Interested in detecting species A, but signal will be a combination of signal
from the presence of species A and species B.
Calibration methods
Basis of quantitative analysis is magnitude of measured property is
proportional to concentration of analyte
Signal α[ x ]
𝐱 =
or
Signal = m[x]+ Signal blank
𝐒𝐢𝐠𝐧𝐚𝐥 – 𝐬𝐢𝐠𝐧𝐚𝐥 𝐛𝐥𝐚𝐧𝐤
𝐦
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 limit
The 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 quantitation
LOL - 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)
𝑺𝒎− 𝑺𝒃𝒊
𝑪𝒎 =
𝒎