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The Medicine 2 - Sem 1 > Analytical techniques > Flashcards

Analytical techniques Flashcards

1
Q

How is EMR emitted by sources classified?

A

in order of increasing wavelength

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2
Q

What does a high wavelength mean for the frequency and energy?

A

low frequency, low energy

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3
Q

What makes the light waves move?

A

the energy from the moving electric and magnetic fields which are vibrating at 90 degrees to each other

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4
Q

What are photons

A

packets or quantum of energy / particles of light

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5
Q

How is EMR produced?

A

when excited particles return to lower energy levels or ground state

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6
Q

What are the uses of spectrophotometry in BP?

A

identification of drugs / measuring reactions and ionisation / quantification of drugs

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7
Q

What are the basic components of a spectrophotometer?

A

light source / monochromator / optics / detector / display

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8
Q

What processes does spectroscopy involve?

A

absorption, emission or scattering of light

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9
Q

What is total molecule energy a sum of?

A

energy from electrons, vibrations between molecule’s own atoms and rotations of the molecule

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10
Q

What does the position and intensity of absorption depend on?

A

substituent group / degree of conjugation / nature of solvent pH

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11
Q

What is bathochromic shift?

A

red shift, the position shift of a peak/signal to a longer wavelength

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12
Q

Why do conjugated structures with double bonds alternating to single bonds cause bathochromic shift?

A

because electrons are easily delocalised in conjugated systems making energy orbitals smaller so less energy needed to excite

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13
Q

What is hypsochromic shift?

A

blue shift, shift to a shorter wavelength

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14
Q

What causes delocalization to extend?

A

benzene ring present / alternating double and single bonds / lone pairs on substituent groups with N/O/halogen

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15
Q

What is an auxochrome?

A

a functional group containing lone pair which doesn’t absorb much UV/Vis but shifts peaks of molecules attached to longer wavelength (hyperchromic shift)

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16
Q

What is a chromophore?

A

the part of a molecule that absorbs UV or visible light

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17
Q

What is the criteria for a cell to be suitable for IR spectroscopy?

A

inactive in interested wavelength / for non aq samples use KBr, NaCL, CAF2 or CsI and for aq samples use diamond, Ge, ZnS or AgCl cuvette for NIR made out of quartz

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18
Q

How are liquid and solid samples prepared to generate an IR spectrum?

A

liquid: fill cell that can be held in position of IR beam / solid sample: grind to form paste in liquid paraffin then add drop of sample between 2 circular plates

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19
Q

What is the IR spectra displayed as (axes)

A

%T vs Wavenumber

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20
Q

What are the types of bending and stretching?

A

Bending: scissoring ,rocking ,wagging & twisting / Stretching: symmetrical & asymmetrical

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21
Q

Which bonds give no IR peaks?

A

Symmetrical bonds with no dipole moment. N2 O2, Cl2 etc

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22
Q

What must a vibration cause in order to be IR active?

A

a change in dipole moment

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23
Q

What does the absorption of IR for a given bond depend on?

A

mass of atoms connected to the bond/ strength of the bond / dipole moment

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24
Q

What are the 2 types of IR spectrophotometer and the functions of their components?

A

Dispersive instrument: source – heated metal filament, cells, monochromator, detector, thermocouple & Fourier transform instrument: same principle but monochromator replaced by interferometer

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25
Why is a reference cell used?
to ensure peaks due to water or carbon dioxide in air can be cancelled out
26
What is the role of NIR in drug formulation?
quality control / particle size / blend uniformity/ identification of polymorphic drugs / determination of moisture
27
What are the uses of IR spectroscopy?
to identify drugs by matching spectrum / functional group analysis / to detect polymorphs of drugs
28
IR energy can only cause which kind of excitations?
vibrational and rotational, too low for electronic transitions
29
What is solid phase extraction used for and its advantages over LLE
to remove interferences from sample, get more reliable results and to concentrate analytes to improve sensitivity. Adv: quick, less labour, less solvent, better recovery, more selective
30
How does SPE work?
Sample filtered through sorbent particles / analytes captured from liquid matric / conc analytes eluted with solvent / eluted sample collected
31
Explain the uses of chromatography
to analyse, identify, purify and quantify
32
What are the types of chromatography available?
column, TLC, gel filtration, ion exchange, high pressure liquid chromatography & gas
33
Describe column chromatography
column either loaded dry and filled with mobile phase which is flushed through column or loaded with slurry of stationary and mobile phase together / polar at the top
34
In column chromatography, how do you determine the components?
Colour, TLC, UV and fluorometry
35
What is thin layer chromatography?
a type of chromatography that uses silica gel or alumina on a card as the medium for the stationary phase / mobile phase passes through stationary phase by capillary action. Separates molecules based on affinity for stationary and mobile phases with the most polar at the top
36
In TLC how do you detect colourless components
UV light, chemical treatment, chemical vapour. UV light (if analytes absorbs UV, gives dark spot on yellow/green background. Iodine vapour ( organic chemicals= reversible brown spots, if you spray with starch permanent spots), Potassium permanganate (for sugars and sugar like molecules), ninhydrin (purple/pink spot with amines), and alkaline tetrazolium (blue spots with corticosteroids)
37
What are the uses of TLC
select solvent system for column chromatography, quality control & purity evaluation, to follow progress of reaction, follow purification process, basic identity check on raw materials, detect impurities
38
Describe the stationary phase in gel filtration chromatography
inert, has a variety of pore sizes, eg. Dextran (sephadex), agarose (Sepharose)
39
What are the uses of gel filtration chromatography?
to find the MR of an unknown substance / to desalt or purify a sample
40
How does ion exchange chromatography work
the molecules are separated based on charge properties
41
Describe the stationary phase of ion exchange chromatography
usually cellulose or agarose resins / has either positive or negative functional groups , binds to oppositely charged ions in sample
42
Which factors affect elution?
size of charge / intensity of charge / concentration of ions
43
What are the two types of gas chromatography and what do they involve
gas solid involves adsorption of analytes & gas liquid involves partitioning of analytes
44
What are the components of a gas chromatograph?
injector / column/ oven / detectors / chart recorder or integrator
45
Describe gas chromatography and it’s principle of separation
column packed with liquid phase / sample injected, vapourised and carried by flowing gas / vapour partitions between gas and stationary liquid phases / time each component spends in column depends on vapour pressure and ability to interact with liquid phase / longer retention time = greater solubility in liquid phase
46
Why do we derivatise the sample prior to analysis?
increase volatility, reduce thermal degradation, increase detector response, decrease polarity, improve separation
47
What derivitising agents are used when preparing the sample?
sialylation, acylation, alkylation and esterification
48
What are the features of the stationary phase?
low volatility, thermally stable, chemically inert, must allow analytes to partition, coated 0.1-0.5 microns thin film
49
What are the basic components of HPLC instrumentation and their functions?
solvent mixing valve controls the proportion of each solvent used depending on gradient/ pump sets a flow rate and creates back pressure / injection valve / column separates / detector (most common UV/Vis
50
What are the mobile and stationary phases?
Mobile phase - non-polar solvent like n-hexane, heptane and chloroform. Stationary phase – silica particles
51
How is the separation performed?
column is equilibrated with starting solvent / analyte injected using injection valve / solvent composition can be changed or kept the same / analytes separate by interaction with stationary phase
52
Describe a HPLC chromatogram
displayed as absorbance Vs Time , area under peak shows the amount of analyte eluted
53
Describe the absorption process of normal phase HPLC
the analyte is retained by the interaction of its polar functional groups with stationary phase. Stationary phase usually microporous silica and mobile is a non-polar solvent initially
54
Describe reverse phase HPLC
most common, stationary phase non polar and mobile phase polar, analytes interact with the surface by partitioning. Elute in order of decreasing polarity
55
How can you improve resolution/performance?
longer column length, slower flow rate, smaller stationary phase particle size, less steep gradient
56
How does a spectrophotometer work?
directs beams of light of different wavelengths through a solution of sample and measures the fraction of the light transmitted at each wavelength. Spectra is displayed as Absorbance Vs wavelength
57
Describe the criteria for cells/ cuvettes used
2 clear and 2 frosted sides for UV and 4 clear for fluorescence, made of plastic/glass for visible region and quartz for UV region, cuvettes handled from frosted side and not filled completely
58
What does difference spectrophotometry measure?
absorbance in sample before and after adjusting a certain condition
59
What is a jablonski diagram and how are the states arranged?
An energy diagram that describes the process of photon emission. The states are arranged vertically by increasing energy and grouped horizontally by spin multiplicity
60
When does fluorescence occur?
when high energy electron returns to ground state by releasing energy in photon (S1 to S0)
61
What are the components of a spectrofluorometer and how does it function
light source, excitation monochromator, a cell an emission monochromator and a detector. 1. Light passes through excitation monochromator which transmits specific wavelength to excite analyte 2. Then passes through sample in cuvette and excites analyte 3. After excitation, analyte relaxes and emits light at an emission wavelength longer than excitation wavelength 4. Emitted light passes through emission monochromator at right angle o excitation light 5. It minimises light scatter and screens emission light before reaching detector
62
Describe some uses of spectrofluorometry
– to measure the fluorescence signature of an analyte in a sample based on its specific excitation and emission wavelengths, used in biofuel analysis, plasma monitoring, polymer analysis and quality control
63
Describe some advantages and limitations of spectrofluorometry
adv: very sensitive, inexpensive, higher selectivity than UV-vis. Limitations: not all drugs fluorescent, use of standards required in pharm analysis, changes in conditions affect fluorescent particles
64
Why is fluorescence more sensitive than UV-Vis?
Process is repeatable and emission is at a higher wavelength than absorption so background signal is low at detection wavelength thus better S/N ratio
65
What are the factors affecting fluorescence intensity?
inner filter effect, pH, temperature, viscosity, presence of oxygen, photobleaching, structural effects and solvent
66
Describe the inner filter and concentration effect
at high drug conc, fluorescence intensity reaches plateau and decreases because of inner filter effects, and so molecule absorbs fluorescence emitted by excited molecules
67
Describe the pH effect
fluorescent drugs with ionisable groups sensitive to pH
68
Describe the effect of viscosity and temp
they have opposite effects. Free moving molecules means more chance of collisions causing energy loss or deactivation of excited molecules so lower fluorescence.
69
Describe the effect of the presence of oxygen
high concentration quenches fluorescence through collisions. Copper ions quench fluorescein by complexation (static quenching)
70
Describe the effect of photobleaching
too high incidence radiation can irreversibly degrade fluorophore and cause loss of fluorescence
71
Describe the structural effects
rigidity increases fluorescence / substituents that affect resonance stability alter fluorescence / electron withdrawing groups have lower fluorescence and vice versa
72
Describe the effects of solvents
polar fluorophores are sensitive so interaction with solvent molecules can offer non-radiative pathway for energy loss

The Medicine 2 - Sem 1 (13 decks)

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