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PH3505- Medicinal chemistry > Lec 1- Gas Chromatography > Flashcards

Lec 1- Gas Chromatography Flashcards

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1
Q

Constituents of a gas chromatography equipment

A
  • Gas supply => Injector => Oven (column) => Detector => Recorder
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2
Q

Analysis procedures: Sample preparation

A
  • Samples (Blood urine etc) have to be subjected to clean up procedure prior to analysis
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3
Q

Analysis procedures: Derivatisation

A
  • Where considered necessary, the sample can be derivatised with a choice of special reagents to convert it into a more volatile material
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4
Q

Analysis procedures: Injection

A
  • A solution of analyte mixture (1 uL, containing perhaps several hundred ng of materia) is injected onto the GC column via a heat injection port
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5
Q

Analysis procedures: GC separation

A
  • Following volatilisation in the heated injector, the mixture is pushed by a pressurized carrier gas (Usually N2 or He) through the GC column
  • Where different compounds are separated depending on their partition between the stationary phase and the mobile carrier gas
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6
Q

Analysis procedures: Detection

A
  • A detector (such as flame ionisation detector) detach each component in the mixture as they emerged from the column
  • This information is sent to the recorder
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7
Q

Effect of changing chromatographic parameters:

Column/Oven temperature

A
  1. Increase in column/ oven temperature
  • Results in Decrease resolution
  • And Decreased retention time
  1. A decrease in Column/ Oven temperature
  • Results in Increased resolution
  • And Increased retention time
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8
Q

Effects of changing chromatographic parameters

Carrier gas flow rate

A
  • There is an optimum flow rate for each carrier gas used so is unique
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9
Q

Effects of changing chromatographic parameters

Column Length

A
  1. Increase in column length
  • Results in Increase in resolution
  • Increase in retention time
  1. Decrease in column length
  • Results in Decrease in resolution
  • Decrease in retention time
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10
Q

Effects of changing chromatographic parameters

Stationary phase loading

A
  1. Increase in stationary phase loading
  • Results in Increased resolution
  • Increased retention time
  1. Decrease in stationary phase loading
  • Results in Decreased resolution
  • Decreased retention time
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11
Q

Determination of propranolol in plasma by GC

  1. Extraction method
A
  • To the sample of 1ml plasma were added 50-100 uL of an aqueous solution of internal standard (Oxprenolol hydrochloride) and 0.1mL of 5M NaOH in a glass-stoppered 10mL centrifuge tube
  • The samples were shaken with 5Ml of toluene for 5 minutes and centrifuged at 3000 rpm for 15 minutes
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12
Q

Determination of propranolol in plasma by GC- Extraction

  1. Why was NaOH added
  2. What is the purpose of the centrifuge
  3. What types of materials being removed from the above procedure
A
  • Q1) Why was NaOH added?
    • To ensure that both the drug and IS are un the unionised form. Only unionised drug and IS will partition into the organic layer
  • Q2) What is the purpose of centrifuge?
    • To get rid of emulsion to ensure that no aqueous phase is carried through to the next stage
  • Q3) What type of materials being removed from the above procedure?
    • Acidic materials were removed
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13
Q

Back extraction method

  • The initial toluene phase from the single extraction procedure (3ml) was transferred to a tube containing 1ml 0.2 N H2SO4
  • The tube was shaken for 5 minutes and centrifuged
  • The acidic aqueous phase was transferred to another tube, make it to alkaline with 5N NaOH and shaken with 3 ml of toluene
  • The toluene phase was separated and then evaporated to a small volume (50 uL) with a gentle stream of N2

Q. What is the purpose of the second procedure (back extraction)

A
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14
Q
  1. Derivatisation
  • To the 50 uL of toluene was added 25 uL of trimethylamine (1M) in toluene and 50 uL TFAA (Trifluoroacetic Anhydride)
  • The tube, after being tightly stopped, was heated for 5 min in a 50’C water bath
  • After cooling the reaction mixtures were shaken vigorously for 30 seconds with 1 mL of phosphate buffer (pH 6.0) and centrifuged
  • Part of the toluene phase (1-5 uL) was taken for electron capture analysis

Q1. Why is derivatisation necessary

Q2. What is the purpose of the addition of trimethylamine

A
  • Q1)Why is the derivatisation necessary?
    • Propranolol and oxprenolol have functional groups which are too polar (Not volatile enough) to be analysed directly by GC
  • Q2) What is the purpose of the addition of trimethylamine
    • It ensures that the amine groups in the drug and IS are not protonated. If they are protonated they will not form the TFA derivative
  • Q3) What derivatives would be formed
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15
Q
  1. Chromatography
  • A gas chromatograph equipped with 63Ni Electron-capture detector was used
  • A 150cm glass column with an internal diameter of 2mm was pack with 2% OV-17 on re-silanised CHROMOSORB W (80-100 mesh)
  • The nitrogen flow rate was 30mL/min and the column was conditioned at 250’C for 72 hours
  • The injector temperature was 230’C and the column temperature was 170’C and the detector temperature was 270’C
  • The 63Ni detector gave a linear response for quantities ranging from 15-800 pg of propranolol

Q. Why use ECD and what polarity is OV-17

Q. Why was the chromosorb re-silanized and what is the significance of conditioning the column

A
  • Q1) Why use ECD?
    • Very high sensitivity, 1pg (picogram)= 10-12 g
  • Q2) What polarity is OV 17?
    • Medium polarity (In between OV1 (Non-polar) and Carbowax 20 (polar)
    • The higher the number after the OV, the more polar the stationary phase
  • Q3) Why was the Chromosorb re-silanized?
    • To remove the polar silanol (Si-OH) groups on the surface of the support, which can lead to tailing of the analyte peaks
  • Q4) What is the significance of conditioning the column
    • In order to get a stable baseline. If column bleed is not both at a minimum and very steady trace analysis is impossible
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16
Q

Calibration curve

  • 1 mL control plasma samples were spiked with propranolol hydrochloride following the single extraction (5-15 ng/mL) procedure or the back extraction procedure (0.4-5 ng/mL) using oxprenolol hydrochloride as an internal standard
  • Peak area ratio’s of propranolol-oxprenolol were measured and plotted as a function of propranolol concentration
A
17
Q

Analysis of the samples

Q. is oxprenolol an ideal I.S.

A
  • It is good because there are 2 clear and defined peak
  • It would be an improvement if the IS had a longer retention time (closer to the drug)
  • Errors involved in measuring the peak heights or areas on solvent tail could be significant
18
Q

Steps involved in chromatographic analysis of drugs GC

A
  • Sample preparation
    • Concentration
    • Removing interfering substances
  • Derivatisation
  • Construction of calibration curve (or single point calibration)
  • Analysis of samples
19
Q

One point calibration

The BP format for assays run 3 solutions

A
  • The BP format for assays: Run 3 solutions
  1. A calibration standard containing the pure standard and an internal standard
  2. An extract of sample without internal standartd (to check for possible interference materials)
  3. An extract of sample containing the same amount of IS as solution 1
20
Q

Solutions question

A
21
Q

Gas chromatograph- Mass spectrometry

A
  • MAIN USE: Particularly suitable for the analysis of mixtures of volatile and relatively low molecular mass compounds (<800) such as relatively non-polar drugs, hydrocarbons, fragrances and essential oils
  • The GC/MS instrument is made up of 2 parts
  1. Gas chromatography: separates the chemical mixture into pulses of pure chemicals
  2. Mass spectrometer: Identifies and quantifies the chemicals as they emerge from the end of the GC column
22
Q

A GC/MS analysis will usually consist of the following steps

A
  • Sample preparation: Sample (Blood, plasma, urine, etc) have to be subjected to a “clean-up” procedure prior to analysis
  • Derivatisation: where considered necessary, the sample can be derivatised with a choice of special reagents to convert it into a move volatile material
  • Injection: A solution of the analyte mixture (1 uL, containing perhaps several hundred ng of material) is injected on to the GC column via a heated injection port
  • GC separation: Following volatilisation in the heated injector, the mixture is pushed by a pressurized carrier gas (usually He) through the GC column, Where different compounds are separated depending on their partition between the stationary liquid and the mobile carrier gas
23
Q

A GC/MS analysis will usually consist of the following steps (2)

A
  • Ionisation: After passing through the GC, the chemical pulses continue to the MS, where the molecules are ionised, which cause them to break into pieces and turn into positively charged particles called ions
  • Detection: A detector counts the number of ions with a specific mass. This information is sent to a computer and a mass spectrum is created
  • Recording: The data from the mass spectrometer is sent to a computer and plotted on a graph called a Mass Spectrum
24
Q

The major problem in GC/MS analysis

A
  • With interfacing, these 2 instruments are the pressures at that they operate
  • The carrier gas pressure inside the GC column >1 atmosphere
  • The operating pressure inside the MS: Almost in vacuum
25
Q

How to solve the problem

  1. Using a jet separator
A
  • If using packed columns (flow rate is about 30 ml/min) to remove as much of the carrier gas as possible with a jet separator
  • The carrier gas and separated material are passing across the narrow gap between the 2 jets
  • Highly diffusible carrier gas (Lighter) is largely removed whereas the heavier analyte molecules crossed the gap without being vented
26
Q

How to solve the problem

  1. Using capillary column
A
  • If capillary columns are used instead of packed columns
  • The problem of removing carrier gas no longer exist since GC capillary columns provide a flow rate of 0.5-2 ml/min, which can be directly introduced into the MS without losing vacuum
  • Once the interface problem solved, the analysis can be carried out as a normal GLC and MS
27
Q

Use of computer (data base) in GC/MS

A
  • The MS can scan a complete mass plectrum every 2 seconds and each scan produces a complete mass spectru, 30 scan/min
  • In the analysis of a complex mixture the GC analysis time could be up to 25 minutes
  • This would generate 750 scans (Spectra)
  • It will take 4 weeks to manually work out the mass spectra
  • The use of data systems (computers) with combined GC/MS (GC/MS/DC) has made drug analysis much more efficent
  • All the data that is generated can be store on disc and can be examined later by the data system

PH3505- Medicinal chemistry (29 decks)

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