Analytical Chemistry Part 2

INSTRU TOPIC

Basis of Separation: size

Separation Technique:

Filtration

Dialysis

Size-exclusion Chromatography

Basis of Separation: Mass or Density

Separation Technique:

Centrifugation

Basis of Separation: Change in Physical State

Separation Technique:

Distillation

Sublimation

Recrystallization

Basis of Separation: Change in Chemical State

Separation Technique:

Precipitation

Electrodeposition

Volatilization

Basis of Separation: Partitioning between phases

Separation Technique:

Extraction

Chromatography

Gas Chrom

What classification of chrom if:

Stationary phase: liquid film or solid adsorbent on support

Column type: capillary or packed

Analyte: volatile, thermally stable compounds

liquid chrom

What classification of chrom if:

Stationary phase: solid of liquid bonded to solid support

Column type: packed

Analyte: nonvolatile, thermally labile compounds

Supercritical fluid chrom

What classification of chrom if:

Stationary phase: solid of liquid bonded to solid support

Column type: capillary or packed

Analyte: Moderately volatile or thermally sensitive compounds

Paper Chromatography

What type of planar chrom?

SP (more polar): Thin layer of water absorbed on the paper surface

MP (less polar): Solvent/ Solvent Mixture

Separation Principle: Primarily partition chromatography between water absorbed on paper and solvent

TLC Chromatography

What type of planar chrom?

SP (more polar): Thin layer of adsorbent like silica gel or alumina coated on glass/plastic/metal plate

MP (less polar): Solvent/ Solvent Mixture

Separation Principle: Primarily adsorption chromatography between sample and adsorbent layer

Nonpolar

MP is nonpolar

SP is polar

Which travels farther in a planar chrom?

polar or nonpolar

↑Rf = Nonpolar

In planar chrom:

↑Rf =

Gas-Liquid Chrom

What type of GC?

Stationary: Liquid coated on solid support

Separation technique: Partitioning

Gas-solid chrom

Stationary:Solid adsorbent

Separation technique: Adsorption

Split injection

sample injection for GC if concentration of sample is pure or high

Splitless injection

sample injection for GC for trace analysis

Carrier Gas System

Gas chrom component:

delivers an inert gas to carry the analytes through the column

Flow controller/ pressure regulator

Gas chrom component:

Maintains consistent carrier gas flows and pressure for accurate analysis

Sample Injector

Gas chrom component:

Introduce the sample into the GC; typically a heated port that instantly vaporizes the liquid

column

Gas chrom component:

Contains the stationary phase where the separation of compound occurs

Oven

Gas chrom component:

Houses the column and controls its temperature to optimize the separation

detector

Gas chrom component:

Identifies compounds as they elute from the column by responding to specific properties

Data system

Gas chrom component:

Software/Hardware that records and interprets detector signals into chromatograms

Packed Column

Type of GC column:

Length: Shorter 0.5-5 m

Resolution: Moderate

Best for gases and large sample loads

Capillary/Open Tubular Column

Type of GC column:

Length: Longer 5 - 100 m, 30 m as common

Resolution: High

Best for complex mixtures and trace analysisG

FLAME IONIZATION DETECTOR (FID)

GC detector:

Universal for organic compounds

Application: hydrocarbons, alcohols, solvent

THERMAL CONDUCTIVITY DETECTOR (TCD)

GC detector:

Universal for organic and inorganic compounds

Application: permanent gases, general use

hint: least sensitive detector

ELECTRON CAPTURE DETECTOR (ECD)

GC detector:

Highly selective for halogenated and nitro groups

Application: Pesticides, PCBs, refrigerants

NITROGEN PHOSPHORUS (NPD) / THERMIONIC SPECIFIC DETECTOR (TSD)

GC detector:

Selective for Nitrogen and Phosphorus containing compounds

Application: Drug residue, pesticides, explosives

MASS SPECTROMETER (MS)

GC detector:

Highly sensitive and specific, enables structural identification

Application: complex mixtures. trace analysis

PHOTOIONIZATION DETECTOR (PID)

GC detector:

Selective for aromatics and unsaturated compounds

Application: VOCs, envi monitoring

FLAME PHOTOMETRIC DETECTOR (FPD)

GC detector:

Selective for sulfur and phosphorus compounds

Application: Petrochemicals, pesticides, sulfur analysis

• Increase carrier-gas flow rate

• low column temp

• use thinner stationary-phase films

How to GC troubleshoot?
Problem: Band broadening (longitudinal diffusion)

Effect on Chromatogram:

Wider peaks

Reduced efficiency

• Use longer column

• use smaller particle size (packed) or thinner film (capillary)

• apply temp programming

How to GC troubleshoot?
Problem: Poor resolution peak

Effect on Chromatogram:

Overlapping peaks

Analytes are difficult to separate

• Use cross-linked stationary phase

• operate within temp limits

• use guard column

How to GC troubleshoot?
Problem: Baseline noise

Effect on Chromatogram:

Fluctuating or elevated baseline

difficult peak integration

• Use deactivated or cross-linked stationary phases

• clean or replace column

How to GC troubleshoot?
Problem: Peak tailing

Effect on Chromatogram:

Asymmetric peaks with long tails

• Reduce injection volume

• Dilute sample

How to GC troubleshoot?
Problem: Peak fronting

Effect on Chromatogram:

Distorted peaks with flattened front

• Derivatize sample to increase volatility and thermal stability

How to GC troubleshoot?
Problem: Nonvolatile or thermally unstable samples

Effect on Chromatogram:

no peaks or distorted peaks

Partition

Type of LC

Separation principle: Partitioning between two immiscible phases

Stationary phase: Liquid coated on solid support

Analyte interaction: Partitioning between the two phases

Adsorption

Type of LC

Separation principle: Adsorption onto solid surface

Stationary phase: Solid adsorbent (silica, alumina)

Analyte interaction: Adsorption and desorption

Ion Exchange

Type of LC

Separation principle: Electrostatic interactions

Stationary phase: Resin with charged functional groups

Analyte interaction: Ionic attraction and repulsion

Size exclusion

Type of LC

Separation principle: Molecular size and shape

Stationary phase: Porous beads

Analyte interaction: Physical sieving through pores

Affinity

Type of LC

Separation principle: Specific binding interactions

Stationary phase: Ligand immobilized on solid support

Analyte interaction: Highly selective binding interactions

Cation Exchange Chromatography

Type of ion exchange chrom:

separates cations: metal ions, amino ions and protein

Stationary phase: SP has negatively charged groups

elutes first: negatively charged (anionic) or neutral ions

Anion Exchange Chromatography

Type of ion exchange chrom:

separates anions: DNA, RNA, protein purification

Stationary phase: SP has positively charged groups

elutes first: positively charged (cationiic) or neutral ions

Gel Filtration Chromatography

Type of size-exchange chrom:

Uses an aq. mobile phase to separate water-soluble biomolecules

For protein purification, enzyme studies

Gel Permeation Chromatography

Type of size-exchange chrom:

Uses an organic mobile phase suitable for hydrophobic or synthetic polymers

For polymer analysis, plastic and rubber industries

Solvent Reservoir

Liquid chrom component:

Holds the mobile phase

Pump

Liquid chrom component:

Delivers the mobile phase through the system at high pressure

Injector

Liquid chrom component:

Introduces the sample into the mobile phase stream

Column

Liquid chrom component:

Performs separation based on the interaction between analyte and stationary phase

Detector

Liquid chrom component:

Detects separated components as they elute from the column

Data System

Liquid chrom component:

Records and processes the detector signal

Column oven (optional)

Liquid chrom component:

Maintains constant temp for better reproducibility

Waste Collector

Liquid chrom component:

Collects waste or separated fractions in preparative HPLC

Reverse Phase Column

SP: nonpolar

MP: polar

Normal-Phase Column

SP: polar

MP: nonpolar

UV-Vis

LC detector:

Most widely used, sensitive, needs chromophores

Application: Pharmaceuticals, organics, biological samples

Photodiode Array (PDA)

LC detector:

Offers full spectra; useful for peak purity analysis

Application: Complex mixtures, peak identification

Fluorescence Detector (FLD)

LC detector:

High sensitivity and selectivity, limited to fluorescent compounds

Application: Biomolecules, pharmaceuticals and food additives

Refractive Index (RI)

LC detector:

universal detector, low sensitive, nondestructive

Application: Sugars, alcohols, lipids (non-uv absorbing)

Mass spectrometry (MS)

LC detector:

High sensitivity and selectivity, gives molecular weight and structure

Application: Metabolomics, drug analysis

Main cause: Mobile - phase mass transfer

• Use narrower columns, and smaller stationary-phase particles

Main cause: Dead volume due to extra-column flow

• Use shorter and narrower-column tubing

• Match injection volume to column dimensions

How to LC troubleshoot?
Problem: Band broadening

Effect on Chromatogram:

Wider peaks, reduced efficiency

• Use smaller particle sizes

• Increase column length

• Apply gradient elution

How to LC troubleshoot?
Problem: Poor Peak Resolution

Effect on Chromatogram:

Overlapping peaks, analytes are hard to separate

• Degas mobile phase

• Filter solvents

• Maintain constant temp

• Use guard column

How to LC troubleshoot?
Problem: Baseline noise / irreproducible results

Effect on Chromatogram:

Fluctuating or elevated baseline, difficult peak integration

• Adjust mobile-phase pH

• Use end-capped columns

How to LC troubleshoot?
Problem: Peak tailing

Effect on Chromatogram:

Asymmetric peaks with long tail

• Dilute sample

• Decrease injection volume

How to LC troubleshoot?
Problem: Peak fronting

Effect on Chromatogram:

Distorted peaks with flattened front

• Check system leaks

• Ensure proper connection

• Choose suitable detector

• Derivatize analyte

How to LC troubleshoot?
Problem: Low detector response

Effect on Chromatogram:

Very small peaks or missing peaks

Adjust the solvent strength of MP

Prev board question

During an HPLC analysis, chromatogram shows broad tailing peaks. What is the appropriate initial action?

Methods: Atomic Emission, Fluorescence

What spectrochemical method?

Type of interaction: emission

Process in which a substance releases energy, usually after excitation by an external source

Methods: Fluorescence, Phosphorescence

What spectrochemical method?

Type of interaction: luminescence

A substance absorbs light and re-emits it at a longer wavelength

Method: Raman, Rayleigh scattering

What spectrochemical method?

Type of interaction: Scattering

Light is redirected by a substance without absorption

UV-Vis, IR, NMR

What spectrochemical method?

Type of interaction: absorption

substance absorbs energy from electromagnetic radiation (EMR) and transitions to a higher energy state

Fluorescence

Nature of emission:

Immediate light emission after excitation, stops almost immediately

Phosphorescence

Nature of emission:

Delayed light emission after excitation, continues after excitation source is removed

Glow in the dark >:D

UV-Vis scpectrophotometer:

Ultraviolet and Visible regions (200-800 nm)

Range of UV-Vis

• Derivatize analyte to introduce UV-absorbing groups

How to UV-Vis troubleshoot?
Problem: Sample does not absorb in UV-Vis region (analyte has no chromophore)

Effect on Chromatogram:

No observable absorption peak

• Dilute sample to remain in linear range

How to UV-Vis troubleshoot?
Problem: Deviation from Beer’s Law (high conc, strong molecular interactions)

Effect on Chromatogram:

Nonlinear calibration

Absorbance too high

• Match reference and sample cells

• Clean cuvettes

• Consider std addition

• Replace light soure

How to UV-Vis troubleshoot?
Problem: Inaccurate readings / baseline drift

Effect on Chromatogram:

Deviating or unstable absorbance

• Maintain optical components

• Minimize stray light

How to UV-Vis troubleshoot?
Problem: Stray light (scattered light entering detector)

Effect on Chromatogram:

Reduced peak height/ lower absorbance

• Filter or clarify sample (use sonication to disperse particles and remove trapped gases or aggregates)

How to UV-Vis troubleshoot?
Problem: Light scattering by sample (turbid or particulate sample)

Effect on Chromatogram:

Broad/ Distorted peaks; elevated baseline

• Use double-beam instrument

• Recalibrate

How to UV-Vis troubleshoot?
Problem: Instrument instability (lamp intensity fluctuations)

Effect on Chromatogram:

Fluctuating absorbance, poor reproducibility

INFRARED SPECTROSCOPY

Spectra wherein transitions of molecules between vibrational and rotational states.

Vibration or rotation must result in change in its net dipole moment

Trend in IR spec

↑frequency - lighter atoms; C-H, O-H, N-H (4000-2500 cm^-1)

↑frequency - stronger bonds; Triple bonds (2500-2000 cm^-1)

alkenes; double bond - (2000-1500 cm^-1)

fingerprint region - single bonds; C-C, C-O, C-N, C-X (1500-400 cm^-1)

Trend in IR spec

↑frequency =

↑frequency =

alkenes =

fingerprint region =

NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY

Detects absorption of radiofrequency radiation by nuclei with spin in a strong magnetic field.

Each signal corresponds to a chemically non-equivalent nucleus

Deshielded; Downfield, High frequency = (most electronegative)

(left most)

Shielded; Upfield, lower frequency = (less electronegative)

(right most)

NMR trend

Deshielded; Downfield, High frequency

(left most)

Shielded; Upfield, lower frequency

(right most)

MASS SPECTROSCOPY

Generate, separate and detect ions in the gas phase

Analytes are ionized using high-energy sources; electron bombardment to form charged species; separated according to mass-to-charge ratios (m/z)

Each signal corresponds to a chemically non-equivalent nucleus

RAMAN SPECTROSCOPY

Most light is scattered without energy change, but a small fraction exchanges energy with molecular vibrations, producing a shift in the scattered light frequency relative to the incident light.

Atomic spectroscopy

note: sa mass spec: broad band (curved peak)
sa atomic spec: sharp peak (one line)

Comprises analytical techniques used to determine the elemental composition of samples by measuring the interaction of light with atoms

involves only electronic transitions of isolated atoms, resulting in sharp line spectra

ATOMIC ABSORPTION SPECTROSCOPY

Used to determine the concentration of elements by measuring the absorption of light by free atoms in the gas phase

Nebulizaton

Which Flame atomization process in AAS?

Sample solution is converted into a fine aerosol using a nebulizer

Desolvation

Solvent evaporates, leaving a finely divided solid aerosol

Volatilization

Solid particles vaporize to from gaseous molecules

Dissociation

Molecules break apart to produce free, ground-state atoms

Ionization

A small fraction of atoms forms ions and free electrons

• Dilute samples

• Use std addition

How to AAS troubleshoot?
Problem: Nonlinear calib curve (matrix effects/ deviation from beer’s law)

Effect on Chromatogram:

Curved calib plot, inaccurate concentrations

• Apply background correction (zeeman or two-line method)

How to UV-Vis troubleshoot?
Problem: Spectral Interference

Effect on Chromatogram:

Distorted or extra peaks

• Add releasing or protecting agents

How to UV-Vis troubleshoot?
Problem: Chemical Interference

Effect on Chromatogram:

Reduced absorption (signal supression)

• Clean nebulizer

• Stabilize flame

How to UV-Vis troubleshoot?
Problem: Low precision

Effect on Chromatogram:

Irregular peak intensity

• Optimize flame temp

• Adjust burner alignment

How to UV-Vis troubleshoot?
Problem: Incomplete atomization

Effect on Chromatogram:

Weak absorption peaks