1.0 | 6122 LEC - Instrumental Methods of Analysis

Analytical Methods: Types (2)

1. Classical / Wet-Chemical Method

2. Instrumental Method

Classical Method: AKA

Wet-Chemical Method

Classical Method

Measurement depends on the chemical properties of the sample

Classical Method: Reagent

made to react completely with the analyte

Chemical stoichiometry

determines relationship between the measured signal and analyte concentration

Chemical stoichiometry: Signal

being recorded after reaching an endpoint

Classical Method: Measurements (3)

1. Separation

2. Qualitative analyses

3. Quantitative analyses

Classical | Separation: Types (3)

Precipitation

Extraction

Distillation

Classical | Qualitative analyses: Types (6)

Colors

boiling or melting points

Solubility

Odors

Optical activity (OA)

Refractive index (RI)

Classical | Quantitative analyses: Types (2)

Gravimetric

Volumetric

Classical Method: Characteristics (5)

1. More suitable for analysis of major constituents

2. Used to certify analytical standards

3. Generally cheaper

4. More accurate and precise

5. More robust & less susceptible to environmental fluctuations

Instrumental Method

Measurement of physical or chemical properties of the analyte

Instrumental Method: Measurements (2)

1. Separation

2. Quantitative analyses

Instrumental | Separation: Types (2)

1. LC & GC

2. electrophoretic techniques

Instrumental | Quantitative analyses: Types (4)

1. conductivity electrode potential

2. light absorption or emission

3. mass-to-charge ratio

4. fluorescence

Instrumental Method: Characteristics (6)

1. Ability to perform trace analysis

2. Most are multi-channel techniques

3. Shorter analysis time

4. Amenable to automation

5. More samples may be analyzed quickly

6. Less skill and training required

Instrumental Methods (4)

1. Spectrometric Methods

2. Chromatographic Methods

3. Electrochemical Methods

4. Miscellaneous Methods

the largest group under Instrumental Methods

Spectrometric Methods

Spectrometric

interaction of light & matter

Spectrometric Methods: Types (6)

1. Emission

2. Absorption

3. Scattering

4. Refraction

5. Diffraction

6. Rotation

Emission (3)

1. Emission spectroscopy

2. Fluorescence

3. Phosphorescence and Luminescence

Emission | Emission spectroscopy (5)

X-ray

UV

visible

electron

Auger

Emission | Phosphorescence and Luminescence (3)

X-ray

UV

visible

Absorption (3)

1. Spectrophotometry and photometry

2. Photoacoustic spectroscopy

3. Nuclear magnetic resonance and electron spin resonance spectroscopy

Absorption | Spectrophotometry and photometry (3)

X-ray

UV

IR

Scattering (3)

1. Turbidimetry

2. Nephelometry

3. Raman spectroscopy

Refraction (2)

1. Refractometry

2. Interferometry

Diffraction (2)

1. X-ray

2. Electron diffraction methods

Rotation (3)

1. Polarimetry

2. Optical rotary dispersion

3. Circular dichroism

X-ray: Spectrometric methods (4)

1. Emission: Emission spectroscopy

2. Emission: Phosphorescence & Luminescence

3. Absorption: Spectrophotometry & photometry

4. Diffraction

UV: Spectrometric methods (3)

1. Emission: Emission spectroscopy

2. Emission: Phosphorescence & Luminescence

3. Absorption: Spectrophotometry & photometry

Visible: Spectrometric methods (2)

1. Emission: Emission spectroscopy

2. Emission: Phosphorescence & Luminescence

Chromatographic Methods (2)

1. GC

2. HPLC

Electrochemical Methods (4)

1. Conductometry

2. Coulometry

3. Polarography

4. Potentiometry

Conductometry

resistance

Coulometry

charge

Polarography

current

Potentiometry

potential

Miscellaneous Methods (3)

1. Mass-to-charge ratio

2. Thermal characteristics

3. Radioactivity

Mass-to-charge ratio

Mass spectrometry

Thermal characteristics (4)

1. Thermal gravimetry and titrimetry

2. Differential scanning calorimetry

3. Differential thermal analyses

4. Thermal conductometric methods

Radioactivity

Activation and isotope dilution methods

Analytical Instruments

converts information about the physical or chemical characteristics of the analyte → information that can be manipulated and interpreted by man

General Components of Instrumental Measurement (4)

1. Signal Generator

2. Transducer or Detector

3. Signal Processor

4. Read-out Device

Signal Generator

- chemical system interacting with the stimulus from the energy source

- produces an analytical signal reflecting the presence and usually the concentration of the analyte

Signal Generator: Parts (2)

1. Energy source

2. Chemical system

energy source

light

chemical system

matter or sample

Transducer or Detector

transforms the analytical signal produced by the signal generator → electrical signal

Signal Processor

modifies and "cleans up" the electrical signal to make it more convenient to interpret

Signal Processor provides (3)

1. Attenuation: lessen the signal

2. Amplification: strengthen the signal

3. Filtering: noise removal

Read-out Device

converts the electrical signal to a form usable to the analyst

Calibration of Instrumental Methods

determines the relationship between the analytical response and the analyte concentration

Calibration of Instrumental Methods: Types (4)

1. Comparison with Standards

2. External Standard Calibration

3. Standard Addition Method

4. Internal Standard Method

Comparison with Standards: Types (2)

1. Direct Comparison

2. Titration

Direct Comparison

- property of the analyte is compared with standards

- the property being tested matches the standard

Direct Comparison: After Dilution

concentration of the analyte was then equal to the concentration of the standard after dilution

most accurate of all analytical procedures

Titration

Titration

- analyte reacts with a standardized reagent (titrant) in a reaction of known stoichiometry

- amount of the standardized reagent needed to achieve chemical equivalence can then be related to the amount of analyte present

Semi-instrumental titration

- color is no longer an indication of the end point

- uses an instrument that uses the pH level instead to signify the end point

External Standard Calibration

- series of such external standards containing the analyte in known concentrations is prepared

- response signal is obtained

Response Signal

- absorbance, peak height, peak area

- obtained as a function of known analyte concentration

- obtained for the sample

- to predict the unknown analyte concentration from the calibration curve or best-fit equation

External Standard

- to calibrate instruments and procedures

- when there are no interference effects from matrix components in the analyte solution (matrix cannot affect analyte)

- prepared separately from the sample

matrix

the sample aside from the analyte

Calibration Curve

- prepared by plotting the data or by fitting them to a suitable mathematical equation

- such as the slope-intercept form used in the method of linear least squares

Standard Addition Method

- useful for analyzing complex samples

- likelihood of matrix effects is substantial

Spiking

adding one or more increments of a standard solution to sample aliquots containing identical volumes

Standard Addition Method: Sample vs. Standard

sample: constant

standard: increasing

calibration curve: upwards slope

Internal Standard Method

plotting the ratio of the analyte signal to the internal-standard signal as a function of the analyte concentration of the standards

Internal Standard Method: Ratio

used to obtain their analyte concentration from a calibration curve

Internal Standard Method: Sample vs. Standard

sample: increasing

standard: constant

calibration curve: upwards slope

Internal Standard

- substance that is added in a constant amount

- to all samples, blanks, and calibration standards in an analysis

Internal Standard vs. External Standard

INTERNAL

- prepared with the STD (mixed with the SX)

- one soln, but will provide SX and STD

EXTERNAL

- prepared separately from the SX

- 2 solns: SX, EXTERNAL STD

Signal

analytical measurement that carries information about the analyte that is of interest to the scientist

Signal: Parts (5)

1. Absorbance

2. Peak area

3. Peak location

4. Peak height

5. Retention time

Peak area

- Chromatogram peaks

- AUC: also a peak, can give the concentration of the substance of interest

Retention time is similar to

peak location

Noise

- analytical measurement made up of unwanted extraneous information

- degrades the accuracy and precision of an analysis

- places a lower limit on the amount of analyte that can be detected

Noise: Types (2)

1. Chemical Noise

2. Instrumental Noise

Chemical Noise

- from a host of uncontrollable variables that affect the chemistry of the system being analyzed

- affects the chemical system

Chemical Noise: Factors That Affect The Sample (5)

1. undetected variations in temperature or pressure

2. fluctuations in relative humidity

3. vibrations that lead to stratification of powdered solids

4. changes in light intensity

5. laboratory fumes

Instrumental Noise

associated with each component of an instrument

Signal-to-Noise (S/N) Ratio

- equation that indicates the magnitude of an experimental effect above the effect of experimental error due to chance fluctuations

- experimental error = NOISE

- great difference between signal & noise

International Council for Harmonisation (ICH)

- an international non-profit organization

- aims to develop guidelines via a process of scientific consensus with regulatory and industry experts working together

- involves 3 countries: US, European Union, Japan

Out-of-Control Process

- a process in which variations among the observed sampling results cannot be attributed to a constant system of chance causes

- i.e., variation is NOT due to chance

Out-of-Control Process is illustrated via a

Quality Control (QC) Chart

QC Chart: Parts

1. UCL

2. Average

3. LCL

+ Zones A, B, C

Out-of-Control Process: Examples (7)

a. 1+ outside of the control limits

b. 8 consec. on one side

c. 7 consec. increasing or decreasing

d. 14 consec. up & down

e. 2/3 consec. in Zone A or beyond

f. 4/5 consec. in Zone B or beyond

g. 15 consec. in Zone C

Out of Specifications (OOS) Result

falls outside established acceptance criteria which have been established in official compendia and/or by company documentation

Out of Trend (OOT) Result

- time-dependent result

- falls outside a prediction interval or fails a statistical process control criterion

- The X-axis represents time since trend is measured based on time.

Standard

- metric, specification, gauge, statement, category or physical product sample

- against which the outputs of a process are compared and declared acceptable or unacceptable

- where the result should be compared to

Specification

- list of tests, references to analytical procedures, and appropriate acceptance criteria

- numerical limits, range, or other criteria for the test described

- Establishes the set of criteria to which a material should conform to be considered acceptable for its intended use

- A part of the standard or can be the standard itself

Specification Sheet: Parts (3)

1. Test

2. Reference

3. Acceptance criteria

System Suitability Test

used to verify that the test system will perform in accordance with the criteria set forth in the procedure

System Suitability Test: Tests are based on

the concept that the equipment, electronics, analytical operations, and samples analyzed constitute an integral system that can be evaluated as such

System Suitability Test is important in

chromatography analysis