PHAR2202 Chemical Analysis Notes

The Language of Analytical Chemistry

• Reporting analytical results.

• Important aspects of analytical methods.

• Calibrating instrument response (Exercise 1).

Molecular Spectroscopy

• UV/Vis absorbance.

• Fluorescence (Experiment 2).

Atomic Spectroscopy

• Emission

• Absorbance (Experiment 2 and 3).

Matter and Energy: Atomic Structure

• Many objects emit light over a continuous range of wavelengths.

• Atoms, even hydrogen, do not.

• H atom spectrum consists of discrete “lines.”

• The H atom emits and absorbs at specific wavelengths.

• First observed in 1885 (the Balmer series).

• Atomic lines are spectrally narrow compared to molecular transitions.

• Emission spectrum and Absorption spectrum

Quantum Theory and Atomic Structure

• Niels Bohr proposed that the energy of the H atom electron is quantized.

• The electron is “trapped” in stationary orbits or “orbitals” around the nucleus.

• Discrete spectral lines arise from the electron “hopping” between orbitals.

• The energy of the light emitted (hv) can be calculated from the difference between the orbital energies.

• hv = E3-E1

Ground vs. Excited States

• Absorption: An electron transitions from a lower energy level (Ei) to a higher energy level (Ej) by absorbing a photon with energy ΔE = hv = Ej - Ei.

• Emission: An electron transitions from a higher energy level (Ej) to a lower energy level (Ei) by emitting a photon with energy ΔE = hv = Ei - Ej.

• A similar principle applies to other atoms.

• Electrons occupy a series of “orbitals”.

• Orbitals are filled with electrons according to a set of rules.

• Electrons in atoms can be promoted to an excited state from the ground state by absorbing light.

• Electrons in atoms can relax to the ground state from an excited state by emitting light.

• The energies at which these transitions occur for each element produce the element’s line spectrum, which is a unique fingerprint for that element.

Atomic Spectrophotometry

• By detecting or measuring absorption or emission from an atomized sample at a wavelength corresponding to a transition in the line spectrum for a particular element, we can determine whether that element is present in the sample, and in what quantity.

Atoms in Pharmaceuticals: Elemental Impurities

• Elemental impurities have several sources (e.g., catalyst residues in API synthesis).

• Some elements are of more concern than others due to toxicity and bio-accumulation.

• ICH classifies these according to risk profile.

ICH Permitted Concentrations

• Depend on the route of administration (Oral, Parenteral, Inhalation).

• For the oral route, when no more than 10 g of drug per day:

  • Class 1:

    • < 3 mg/g

    • Cd, As, Pb, Hg

  • Class 2A:

    • < 20 mg/g

    • Co, V, Ni

  • Class 2B:

    • < 30 mg/g

    • Tl, Au, Pd, Ir, Os, Rh, Ru, Se, Ag, Pt

  • Class 3:

    • < 1100 mg/g

    • Li, Sb, Ba, Mo, Cu, Sn, Cr

Atomic Spectra

• Several types of spectroscopy are possible:

  • Atomic Emission

  • Atomic Absorption

  • Atomic Fluorescence

Technique: Atomic Emission Spectroscopy

• Electrons in atoms can relax to the ground state from an excited state by emitting light.

• In atomic emission spectroscopy, the source of excitation is thermal energy (e.g., flame, furnace, plasma).

• Flames are typically low-temperature (2,000 – 3,000 K), so excitation is limited for many elements, which leads to simple spectra and few emission lines.

• Light emitted from the flame is sent through a polychromator, and specific wavelengths are detected.

Emission & Temperature

• In atomic spectroscopy, temperature determines how much sample is atomized and the populations of atoms in ground, excited, and ionized states.

• At thermal equilibrium, the relative populations of electrons in two different states in any atom (or molecule) are determined by the temperature (T), according to the Boltzmann Distribution.

• Variables:

  • N0, N* = ground, excited state populations

  • g0, g* = ground, excited state degeneracies

  • T = temperature (K)

  • ΔE = absorption energy

  • λ = absorption wavelength

  • k = 1.3806488 x 10- 23 J K- 1 (Boltzmann’s constant)

Temperature Effects

• Emission intensity is proportional to excited state populations.

• At < 3,000 K, these are very small, particularly for higher energy excited states (shorter wavelength transitions).

• Low-temperature flames lead to low emission signals.

• Temperature fluctuations in the flame will lead to significant deviations in emission intensity because small changes in temperature lead to big changes in excited state populations.

• Atomic emission spectroscopy often requires the use of higher-temperature, more stable flames.

Wavelength and Temperature

• The table shows that as the temperature increases, the N*/No (excited state population/ground state population) ratio increases significantly, especially for shorter wavelengths.

• Wavelength of transition / nm DE / J atom- 1 N/No (when g/go = 1) 2500 K 6000 K

• 250 7.95 x 10^{-19} 1.0 x 10^{-10} 6.8 x 10^{-5}

• 500 3.97 x 10^{-19} 1.0 x 10^{-5} 8.3 x 10^{-3}

• 750 2.65 x 10^{-19} 4.6 x 10^{-4} 4.1 x 10^{-2}

Flame Emission

• Limited to metals that have high emission strength and/or longer wavelength transitions (emit enough light even when excited state populations are low).

• Examples: alkali metals and alkali earth metals.

• Instrumentation is relatively simple:

  • Photometer: measures the number of photons being emitted by the source.

  • Flame: Propane or natural gas and air.

  • Nebulizer.

  • Filter: selects emission line (wavelength); different filters for different elements.

  • Photon detector.

  • Readout circuits.

Flame Atomisation

• Atomic spectroscopy needs atoms to work.

• To atomize a sample in solution:

  • A solution of analyte is sprayed into a flame (aspirated and nebulized).

  • Several reaction pathways that can be a source of some types of interference.

• Parameters:

  • Flame temperature.

  • Height in the flame.

  • Aspiration rate.

Flame Photometry Interferences

• Ionization:

  • Alkali and alkali earth metals have low ionization energy. Example: K ⇌ K^+ + e^-.

  • Ions have different emission spectra compared to the parent atom, which reduces the atom signal.

  • The equilibrium can be shifted to the left by the addition of another readily ionized element to the sample (produces electrons).

    • Emission lines from the added metal are unlikely to interfere because AE lines are very narrow.

  • Example: strontium chloride solution is added in order to suppress the ionization of K in the BP assay of effervescent KCl tablets.

• Viscosity

  • Organic substances in a sample can either increase or decrease the rate at which it is drawn into the nebulizer compared to a standard (aqueous) solution by increasing or decreasing the viscosity.

  • Example: sucrose decreases the aspiration rate, thus giving a false low reading; ethanol increases the aspiration rate, thus giving a false high reading.

  • Use of standard addition can overcome this, as samples and standards have the same viscosity.

• Anionic interference:

  • Anions such as sulfate and phosphate form involatile salts with metal ions, which reduces the reading of the sample solution.

  • Can be removed by the addition of lanthanum chloride, which precipitates them out and replaces them with the chloride anion.

• Self Absorption:

  • Flames are hottest in their centers, but cooler outer regions may contain atoms that can absorb photons emitted by the hotter atoms.

  • This reduces the collected emitted intensity as the atom concentration increases, leading to non-linear calibration curves.

Applications of AES

• Clinical:

  • Sodium and potassium concentrations in urine.

• Pharmaceutical:

  • Quantification of alkali metals in infusion and dialysis solutions.

  • K, Na, and Ba in calcium acetate used to prepare dialysis solutions.

  • Alkali and alkali earth metals in manufacturing and formulation water.

  • Na in albumin solution and plasma protein solution.

  • Ca in adsorbed vaccines (e.g., diphtheria and tetanus).

Atomic Emission Spectroscopy at Higher Temperatures

• Excited state populations are higher at higher temperatures.

• This means more elements are capable of emission of intensity that is high enough to detect, including some non-metals. Variables:

  • Wavelength of transition / nm

  • DE / J atom- 1

  • N/No (when g/go = 1) 10 000 K

• 250 7.95 x 10^{-19} 0.003

• 500 3.97 x 10^{-19} 0.056

• 750 2.65 x 10^{-19} 0.147

Inductively Coupled Plasma (ICP)

• Intense, high radio-frequency fields (27 - 41 MHz).

• Generate Ar ions and electrons.

• Ion, electron, and gas atoms collisions result in a plasma.

• Temperatures: 6,000 – 10,000 K.

• Compared to a flame:

  • Twice as hot.

  • More stable.

  • Chemically inert.

  • Longer residence time.

  • A good source for emission spectroscopy.

ICP-OES: Optical Emission Spectroscopy

• Radial and Axial configurations:

  • Axial is more sensitive by a factor of about 5 due to a longer pathlength.

• Simultaneous multi-element analysis: many elements can be detected in one sample.

• Polychromator.

• CCD/CID detector.

• Element discrimination relies on spectral resolution of the spectrometer.

• Higher resolution is required than AAS, which costs more.

Internal Standards

• Atomic emission signals can vary due to:

  • sample matrix effects,

  • sample introduction,

  • atomization,

  • plasma/flame conditions,

  • source stability,

  • drift.

• Multielement capability allows the use of an internal standard to compensate for this in some cases.

• USP <730> indicates that an IS should be used.

• An internal standard must:

  • not be in the sample or standards already,

  • be stable in the sample and standard solutions,

  • behave in the plasma in the same way as the analytes,

  • have neutral or ionic emission lines.

• Common ICP-OES internal standards: Y, Sc, Co, Pt, Au, and Mn.

• An element different from the analyte is added to the samples and standards at a known concentration.

• The relative response of the detector to the analyte (X) and internal standard (S) is usually constant over a wide range of conditions:

  • [X]/[S] = f([X signal]/[S signal])

• If the signal from the analyte increases, the signal from the internal standard usually also increases by the same amount.

• As long as the concentration of the standard is known, the correct concentration of the analyte can be calculated.

ICP–MASS Spectrometry

• In an Ar plasma, analyte elements can be ionized by collisions with Ar+, excited Ar atoms, or energetic electrons.

• The plasma can be coupled to a mass spectrometer, which separates and measures ions according to their mass-to-charge ratio.

• Mass spectrometers have high sensitivity, with sub-part-per-trillion detection limits for many elements.

• Cleanliness of reagents, plasticware, and procedure is important, as dust is a major source of contamination.

ICP-MS

• Trace and ultra-trace element impurity analysis.

• Isotope distribution can provide information about impurity origin.

• Measuring m/z means isotopes can be used as internal standards (Isotope dilution).

• Polyatomic isobaric interferants may need to be dealt with (Collision cell).

Cold Vapour Atomisation

• Metals like mercury don’t need a flame to generate atoms.

• Atomic vapor can be generated at low temperature (e.g., by reduction of mercury ions and purging).

• Hydride generation:

  • Reaction of As, Sb, Bi, Ge, Sn, Pb, Se, and Te with sodium borohydride forms volatile hydrides (e.g., arsine, AsH3).

  • Swept by argon into a heated quartz cell where atomization occurs.

• In flames, the atom residence time is < 1 s. In hydride generation atomization, the quartz tube confines the atomized sample in the optical path for several seconds, resulting in higher sensitivity.

Atomic Fluorescence

• Usually uses resonant fluorescence: λ{ex} = λ{em}.

• Atomize and excite with:

  • HCL

  • Laser

  • Discharge lamp

• Wavelength selection can be simple.

• Selectivity comes from the excitation source.

• Fluorescence is more sensitive than absorption because you are observing a weak fluorescence signal above a dark background vs. looking for small differences between large amounts of light reaching the detector (e.g., 0.5 ppt for Hg).

Populations and Atomic Absorption Spectroscopy

• In low-temperature (2,000 – 3,000 K) flames, most atoms are in the ground state.

• This means that most of the atoms present are able to absorb radiation.

• In addition, natural temperature fluctuations in the flame (±10 K) do not significantly alter absorbance, as the ground state population is not significantly altered.

Atomic Absorption Spectrometer

• A light source is used to produce specific wavelengths of light, which pass through the flame.

• The wavelength depends on the element of interest.

• The ratio P / P0 can be correlated to the element’s concentration in the sample, typically via a linear relation.

AAS Flames

• The nature of the flame is important:

  • Fuel ‘rich’ – reducing environment.

  • Fuel ‘lean’ – hotter.

• Atom concentration depends on position in flame.

• Different flame types and positions for different elements.

• Common Fuel-Oxidant Combinations and their Temperatures (K):

  • Acetylene/Air: 2400-2700

  • Acetylene/Nitrous oxide: 2900-3100

  • Acetylene/Oxygen: 3300-3400

  • Hydrogen/Air: 2300-2400

  • Hydrogen/Oxygen: 2800-3000

  • Cyanogen/Oxygen: 4800

AAS Nebulizer Burner

• Generates a fine mist (aerosol) from the solution via the Venturi effect.

• Glass bead: Breaks up droplets.

• Baffles: Remove large droplets.

• Only 5% of the liquid leaving the nebulizer reaches the flame; the rest goes down the drain.

• Laminar flame with long pathlength, increasing sensitivity (Beer’s law).

Hollow Cathode Lamp

• Lamps rely on atomic emission.

• The source is atom sputtering by an electrical discharge.

• Usually one element per lamp.

• Some multi-element lamps are available (up to 6 metals in the cathode).

  • Electrical discharge of Argon or neon gas

Reasons HCLs Are Useful

• HCL emission is narrow band (“monochromatic”).

• All of its power falls inside the absorption line of the atom.

• Beer’s law is more likely to be obeyed.

• Atomic absorption is broader due to pressure and temperature.

• Monochromator selects the particular atomic emission line and rejects other background emission from the flame.

• It also addresses the presence of species emitting at nearby wavelengths.

• Similar resolution to UV/Vis spectrometers.

Some Pharmaceutical Applications of AAS

• Quantification of metal (residues) in drugs related to limit tests:

  • Pb and Ni in sugars.

  • Cd, Cu, Pb, and Zn in cavity wound dressings.

  • Ag in cis-platin.

  • Pd in carbenicillin.

• Assays:

  • of Ca and Mg in hemodialysis solutions.

• Zn in Zn-insulin suspension.

Electrothermal vs Flame Atomisation

• The flame is replaced with an electrically heated graphite tube.

• Ar purge prevents oxidation and formation of CN.

  • T < 2600°C
    Spectroscopy setup remains much the same as with the flame.

• Discrete amounts of sample are placed in the tube (liquid or solid). Advantages:

  • Less sample is required (10-20 µL discrete vs. 1-2 mL continuous).

  • More concentrated samples.

  • Direct solid sampling for trace-level impurities.

  • Longer residence time (several seconds vs. fractions of a second).

  • Higher atom concentration.

  • Higher sensitivity.

LODs

• Graphite furnace can be more sensitive than flame AAS or ICP-OES BUT not as sensitive as ICP-MS.
  Can do solids directly but is the slowest of those listed here.

Which Atomic Method?

Table outlining the application of each atomic spectrometry technique given:
* Objective of analysis
* Number of elements to analyze
* Concentration expected

UV/Vis OR ICP-OES?

• Amino acids tryptophan and tyrosine have aromatic rings that absorb at 280 nm.

• At this wavelength, one value for the extinction coefficient of myoglobin is 31 200 M- 1 cm- 1.

• Amino acids cysteine and methionine have sulfur atoms.

• Myoglobin has 2 methionine amino acids in its sequence.

• A solution of lyophilized myoglobin was prepared in 5 mM phosphate buffer.

• A 5 x dilution of this solution had an absorbance of 0.490 in a 1 cm cell.

• The undiluted solution was also injected into an ICP-OES, and the emission of sulfur at 180.73 nm was measured and compared to that of standard sodium sulfate solutions.

• A linear calibration curve was obtained, and the sulfur concentration in the protein solution was determined to be 5.03 mg/L.

• UV/Vis: 0.490/31200 = 1.57 x 10^{-5} M x 5 = 78.5 mM

• ICP: 5.03 mg/L = 5.03/32.065 mM / 2 = 78.4 mM
  Compare The Two Methods
  Both methods rely on the protein being pure:

  • No extraneous absorbance at 280 nm

  • No extraneous S

• Literature values for the extinction coefficient vary, requiring calibration?

• The solution is injected directly into the ICP without digestion – plasma effects?

• The emission wavelength is in the vacuum-UV region – requires atmosphere control.

• ICP is destructive – you won’t ever get your sample back.
  *“The main advantages of protein determination by ICP-AES of sulfur, besides the high selectivity and multielement capability of atomic spectrometry in general, are the generality and rapidity of the method and the ease of sample preparation. The method is applicable to any protein having a known content of sulfur……A promising application of the method is the rapid and precise determination of metal binding stoichiometries in metalloproteins by determining both protein and metals for a single sample by a single method.”

What About the Rest of the EM Spectrum?

• We’ve focused on spectroscopy in the UV and visible parts of the spectrum, but other types of spectroscopy are also useful in drug development and formulation.

  • Radio Frequencies

    • Nuclear Magnetic Resonance

      • Molecular structure

      • Drug interactions

  • Microwave

    • Rotational spectroscopy

    • Molecular structure

  • THz spectroscopy

    • Low-frequency vibrational spectroscopy

    • Tablet coatings / ingredient polymorphs

  • Infrared & Raman

    • Molecular vibrations

    • Identification and purity

    • Pill testing

• Explain how internal standards are used in inductively coupled plasma emission spectroscopy.

• Distinguish between the atomic spectroscopy techniques: atomic absorption, atomic emission and atomic fluorescence.

• Describe some of the effects the atom source (flame, plasma, furnace) can have on the spectroscopic signal. In atomic spectroscopy, the atom source (flame, plasma, furnace) significantly influences the spectroscopic signal. * **Flame:** * Temperature affects atomization and excitation. * Lower temperatures limit excitation, resulting in simpler spectra but lower emission signals. * Temperature fluctuations can cause deviations in emission intensity. * **Plasma:** * Provides higher temperatures (6,000 – 10,000 K), leading to more efficient atomization and excitation. * Chemically inert and more stable compared to flames. * Results in longer residence time and is suitable for emission spectroscopy. * **Furnace:** * Graphite furnace in electrothermal atomization offers longer residence times and higher atom concentrations. * Requires less sample and suitable for direct solid sampling. * Ar purge prevents oxidation and CN formation.

• Outline the significance of elemental spectroscopy to pharmaceutical analysis.

Elemental spectroscopy is significant to pharmaceutical analysis due to its ability to:

1. Detect elemental impurities that originate from various sources like catalyst residues during API synthesis.

2. Quantify elemental impurities, which is crucial because some elements can be toxic and bio-accumulative, necessitating their control as per ICH guidelines.

3. Determine whether specific elements are present in a sample and in what quantity by measuring absorption or emission from an atomized sample.

4. Apply different atomic spectroscopy techniques such as Atomic Emission, Atomic Absorption, and Atomic Fluorescence depending on the analytical requirements.

Internal standards are used in Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) to correct for signal variations caused by matrix effects, sample introduction, atomization, plasma/flame conditions, source stability, and drift. An element not originally in the sample is added at a known concentration to both the samples and standards. The instrument response to the analyte and the internal standard should remain constant over a range of conditions, allowing for the accurate calculation of the analyte concentration even if signal intensities fluctuate. Common internal standards include Y, Sc, Co, Pt, Au, and Mn.

Atomic Absorption Spectroscopy (AAS) measures the amount of light absorbed by atoms in a sample. A light source emits specific wavelengths, and the absorption is correlated to the element's concentration. Atomic Emission Spectroscopy (AES) involves exciting atoms in a sample using thermal energy (e.g., flame, plasma), causing them to emit light. The emitted light is then analyzed. Atomic Fluorescence Spectroscopy (AFS) atomizes and excites a sample with a light source (e.g., laser), and selectivity comes from the excitation source. Fluorescence is observed against a dark background, making it highly sensitive.