Quality control – Sec 3 – Summary by Menna Hazem From Dr Salma Abdo and Norhan Alaa Powerpoint

• Importance of spectroscopy in analytical chemistry

Definitions 1. Qualitative analysis
The study of the interaction between
Spectroscopy 2. Quantitative analysis
light components and matter
a plot of absorption intensity • Types of spectrum
Spectrum
(absorbance a) against λ . line spectrum band spectrum
a plot of solute concentration as given occur with atomic occurs with molecules
Chromatogram by detector response versus elution spectra such as sodium due to the presence of
time or elution volume. metal which have sharp different vibrational and
• chromo= colour line of λ at 589nm. rotational sub-levels.
• phore= carrier
Chromophore • Classification Of Chromatographic Methods
a covalent unsaturated group that can
According To Classification
absorb uv-vis light. (ex. c=c, c=o. c=n)
• Liquid Chromatography
a saturated group which when
(Liquid-liquid, Liquid-solid)
attached to a chromophore, alters the Mobile Phase
Auxochrome • Gas Chromatography
wavelength and/or the intensity of
(Gas-liquid, Gas-solid)
Imax. (ex. -OH, -Br,-NH2, ....)
• Column
The wavelength corresponding to Apparatus
λ max • Planner
maximum absorbance • Adsorption
Mechanism of
Bathochromic separation • Partition
change of spectral band position
Shift (retention • Ion Exchange (IEC)
to a longer wavelength mechanism)
(Red Shift) • Size Exclusion (SEC)
Hypsochromic Phase Normal Reversed
change of spectral band position
Shift Mode Of
to a shorter wavelength Polar Stationary Mobile
(Blue Shift) Operation
Non- Mobile Stationary
Hyperchromic increase in the intensity polar
Effect of absorption molecule
Hypochromic decrease in the intensity • Factors effecting the absorption spectra
Effect of absorption molecule 1. pH
a physical method of separation, in 2. Solvent polarity
which the components (of mixture) to 3. Sample concentration.
be separated are distributed between 4. Temperature
two phases.
Chromatography • Factors affecting retardation factor
1. stationary bed of large surface area
(stationary phase), 1. Absorbent
2. The solvent system
2. the other one percolate through the
3. Amount of material spotted
stationary phase ( mobile phase)
4. Temperature.
form of liquid chromatography used to
HPLC separate compounds that are • The spectrophotometer consists of:
dissolved in solution. 1. light source
Thin layer a technique used to isolate non- 2. wavelength selector (monochromator
chromatography volatile mixtures. ( prism or grating))
It's the time between injection and 3. cuvette
maximum peak height. The degree of 4. detector
Retention time Tr 5. recorder
retention is specific for each
component

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Quality control – Sec 3 – Summary by Menna Hazem From Dr Salma Abdo and Norhan Alaa Powerpoint
•
1.
HPLC instruments consist of
reservoir of mobile phase
MCQ
2. pump • The stationary phase may be either a liquid or
3. injector solid
4. separation column
• The mobile phase may be either a liquid or a gas
5. detector.
• HPLC is characterized by the use of high
• TLC Advantages pressure to push a mobile phase solution
1. one of the fastest through a column of stationary phase allowing
2. least expensive separation of complex mixtures with high
3. Simplest resolution. Compounds are separated by
4. easiest chromatography technique. injecting a sample mixture onto the column.
• The different component in the mixture pass
• HPLC Advantages through the column at differentiates due to
1. High speed differences in their partition behavior between
2. High resolution the mobile phase and the stationary phase.
3. High sensitivity • The mobile phase must be degassed to
4. Re-usable column eliminate the formation of air bubbles.
5. No destruction of the components • The TLC experiment is conducted on a sheet of
6. The instrumentation are automatic, aluminium foil, plastic, or glass which is
computerized coated with a thin layer of adsorbent material.
7. Sample is recovered completely • The material usually used is aluminium oxide,
8. Quantitative work is more easily and most cellulose, or silica gel.
sensitive • TLC is based on the principle of separation
through adsorption type. The separation relies
• HPLC Disadvantages
on the relative empathy of compounds towards
1. Costly
the mobile phase and stationary phase.
2. complex to operate
• On completion of the separation, each
3. doesn't work for all samples.
component appears as spots separated
4. Need a skill to run the instruments
vertically. Each spot has a retention factor (re)
5. Solvents consuming
• Higher the Rf value, lesser the polarity of the
• HPLC Applications substance. Lower the rf value higher is the
1. Pharmaceuticals industry polarity of the substance.
2. Analysis of natural contamination • Like other chromatographic techniques, thin-
3. Forensic test layer chromatography (TLC) depends on the
4. Clinical test separation principle.
5. Food and essence manufacture • The separation relies on the relative affinity of
compounds towards both the phases. The
• TLC applications compounds in the mobile phase move over the
1. The qualitative testing of various medicines surface of the stationary phase. The movement
such as sedatives, local anesthetics, occurs in such a way that the compounds which
anticonvulsant tranquilizers, analgesics, have a higher affinity to the stationary phase
antihistamines, steroids, hypnotics move slowly while the other compounds travel
2. biochemical analysis such as separation or fast. Therefore, the separation of the mixture is
isolation of biochemical metabolites from its attained. On completion of the separation
blood plasma, urine, body fluids, serum. process, the individual components from the
3. identify natural products like essential oils or mixture appear as spots at respective levels on
volatile oil, fixed oil, glycosides, waxes, the plates. Their character and nature are
alkaloids. identified by suitable detection techniques.
4. separating multicomponent pharmaceutical
formulations.
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Quality control – Sec 3 – Summary by Menna Hazem From Dr Salma Abdo and Norhan Alaa Powerpoint

Diagrams Questions

A=abc
A= Absorbance of unknown from table
a = slope = ∆Y / ∆X
b = 1 (always)
A
c = ab

HPLC

LAWS
LAMBERT'S LAW 𝐼
LOG 𝐼0 = KB
𝑇
BEER'S LAW. 𝐼
LOG 𝐼0 = KC
𝑇
BEER'S A = abc
LAMBERT'S LAW
Rf dist. Travelled by sample / dist.
Travelled by solvent

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