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Analytical Methods Exam > Gas Chromatography > Flashcards

Gas Chromatography Flashcards

1
Q

What is the definition of chromatography?

A

Differential distribution of sample components (solute) between two phases

One phase remains fixed = stationary phase
One phase is moving = mobile phase

Separation is achieved by the differences in the affinity of the solute for the mobile versus stationary phase.

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2
Q

How is a single peak defined using the capacity factor?

A

The definition of a single peak is in terms of how well a compound is retained on a column and the sharpness of the peak exiting the column.

The capacity factor is an estimate of the retention on the column – affected by type of the column and mobile phase.

On the graph there is always a small initial peak around t0, this is the void volume - peak due to minimal impurity.

                                       t(R) - t(0) Capacity factor (k) =   ————
                                             t(0)

This is the retention time corrected for void volume.

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3
Q

How is the theoretical plate number method used to determine peak sharpness?

A

Peak sharpness can be used as a quality control measure allowing you to compare different chromatographic methods.

            (     t R     )2 N = 5.4 ( ——— )
            (   w1/2   )

Where ‘N’ is an estimate of how sharpe the peak is, note:-
L
H = — = height equivalent to a theoretical plate (HETP)
N
where L is length of the column and N is the number of plates
This adds consistency between column lengths.

Diffusion also affects the resolution of peaks so should be incorporated into the definition:
H = Eddy Diffusion + longitudinal diffusion + mass transfer
A B C
(Simple diffusion - if
you’re traveling
slowly theres more
time for this. But faster
it goes, the broader the
peaks as this affects C)

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4
Q

How are multiple peaks defined in terms of resolution?

A

The resolution of two peaks is defined using the separation factor of each peak. The resolution is therefore a function of the relative retention time and peak width. Alternatively it can be expressed as:
a - 1 k’
R(s) = 1/4 ( ——- ) N^1\2 ( ——— )
a k’ + 1
(a = alpha, k’ is an average of k’1 and k’2 as they are roughly equal)

In this way, resolution is proportional to the square root of the theoretical plate count. So doubling the column length (plate count) will not double the resolution. Resolution can be altered by a, k’ or N.

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5
Q

How can resolution be altered in gas chromatography?

A

Increase column length but:-

  • Increases back pressure
  • Increased retention time - you cant put as much sample through - but you want high throughput!
  • Increased cost
  • Note again R(s)αN^1/2

Change column type

  • Packed column to capillary (cf. hplc columns)
  • Change method
  • Change column packing - reduce the liquid phase

Change mobile phase velocity, but

  • May only have small effect
  • Not always possible
  • Time factor

Temperature - affects solubility and binding of things

Summary:
Poor resolution can be improved by:
- increased volume efficiency, eg GC to capillary. This increases peak sharpness.
- altered column selectivity, this moves the peaks further away from each other by altering the RF slightly.

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6
Q

What are the effects of Eddy Diffusion minimisation?

A

Van Deemter curves can illustrate the effects of particle size in HPLC on HETP. Higher the resolution, the lower the curve

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7
Q

How is the choice of chromatographic method decided?

A

Size of the solute:

  • Small molecules (such as steroids and drugs) are often assayed with thin layer chromatography, gas chromatography or even HPLC.
  • Larger molecules such as proteins are determined by gel filtration, ion-exchange or affinity chromatography.
  • Some types of chromatography may be used for large and small molecules such as ion-exchange chromatography.
  • There are special exceptions to this when a particle packing may be used. E.g. analysis of haemoglobinopathies by LC-MS/MS.

Solubility: hydrophobic or hydrophilic?
- The basic principle of chromatographic separation is the concept of solubility (‘like dissolves like’)

pKa: acidic, basic or neutral?
- This will affect its solubility in the mobile phase and interaction with the stationary phase.

Volatility: can it be made so?
- A volatile compound is ideally suitable for methods where the mobile phase is gaseous such as gas-liquid chromatography.

Presence of closely related substances:

What is the number of samples to be analysed?

What is the cost?

Is there a suitable extraction method available?

Is there an internal standard available?

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8
Q

What is gas chromatography?

A

With gas chromatography the mobile phase is a gas and the the stationary phase is a liquid at elevated temperatures (gas liquid chromatography) or a solid (gas solid chromatography).
For this to occur the solute needs to be in the vapour phase which is achieved by heat.

Hence the parts of the instrument are:-

  • carrier gas - mobile phase
  • injection - heated to vapourise
  • column - 20-25m
  • detector - MS aspect
  • data interpretation
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9
Q

What is required to perform gas chromatography?

A

A constant flow of carrier gas (mobile phase)
Ability to introduce sample vapours into flowing gas stream
Maintenance of column at the appropriate temperature
Detection of sample components as they elute from the column - MS?
Provision of a reasonable signal proportional to the amount of each component.

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10
Q

What are the different column types that can be used for GC?

A

Packed:
- uncoated solid particles: Uniform spherical particles - used by
forensics for alcohol analysis. Solely
absorption mechanism.
- solid particle support + liquid coating: Clinical use. Particles
coated by a liquid phase - only liquid at high
temp. Partition and absorption
mechanisms.

Capillary:
- wall coated open tubular column: stationary liquid phase + column wall
- porous layer open tubular column: stationary phase solid particles
- solid coated open tubular column: solid support + stationary liquid
phase coating
Advantages of capillary: no particles, so effectively 0 Eddy diffusion = very high plate counts. But columns have to be very long (~25 m).

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11
Q

What is the nature of packed columns?

A

Solid Support - usually silica (e.g. chromosorb), occasionally activated (by stemto give high loading rate) carbon (e.g. carbopack). Criteria is than it must have a large, inert surface area.
May be used uncoated such as for analysis of alcohols (e.g. Tenax GC) but has a liquid layer for most applications.
Liquid coating - >200 available, only criteria is they behave as a liquid at elevated temperatures hence most are gums and greases.

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12
Q

What is the nature of capillary columns?

A

WCOT columns are common in clinical labs. Used to be made from glass but now from fused silica with a polyimide coating for increased strength – much stronger and easier to work with.
Coating are non-polar, intermediate polarity or strongly polar.
The film thickness is determined by the balance of sample loading (injection volume) versus resolution.

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13
Q

What is involved in the injection phase of GC?

A

Injection onto packed columns is easier due to column size but not so easy with narrow bore capillary column. Example is a split/splitless injection

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14
Q

What carrier gasses are commonly used?

A

N2, He and H2

Each affects a Van Deemter plot differently, with N2 giving the lowest Hmin, thus highest resolution, but the highest height equivalent to a theoretical plate due to increased Cu

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15
Q

What is the detectors are available for use in GC?

A

Commonest detector is Flame Ionisation Detector, which has broad specificity. Burning of the column produces electrons which you can measure. This is very versatile.

Nitrogen Phosporous Detector (NPD) - similar to FID but effluent to heated alkali earth enhances the sensitivity to nitrogen and phosporous containing compounds.

Electron Capture Detector - radioactive, with increased sensitivity to electron capturing molecule such as the halogen.

Mass spectroscopy

Thermal Conductivity Detector (TCD) - little used now

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16
Q

How is MS exploited as a detector for GC?

A

GC coupled to MS must be performed at higher vacuum, meaning it is more sensitive as this prevents all air from getting in.

GC is coupled to MS by:
Capillary - introduction directly into ion source, since flow rates from column are typically 0.5 - 1.0ml/min the MS vacuum pumps can cope with this.
Packed - now rarely used with MS since high flow rates from column (10 - 40ml/min) require removal of carrier gas prior to entry into ion source.

17
Q

What are the possible ion sources for GCMS?

A

Electron Impact
- use electron beam from a heated filament (-70eV) to charge and fragment the compound (ESI). M + e- —> M^+ (parent ion) —> free radicals.

Chemical Ionisation:
- use secondary ionisation that gives gentler fragmentation.
- Primary Ion Formation:
CH4 + e- —> CH4+ + 2e-
- Secondary Reagent Ions:
CH4 + CH4+ —> CH5+ + CH3
CH4 + CH3+ —> C2H5+ + H2
- Product Ion Formation:
M + CH5+ —> CH4 + [M + H] + (protonation)
AH + CH3+ —> CH4 + A+ (H− abstraction)
M + CH5+ —> [M+ CH5] + (adduct formation)
A + CH4+ —> CH4 + A+ (charge exchange)

Electron impact (smaller fragments) vs chemical ionisation (cleaner with less interference)

18
Q

How are mass filters utilised in GC?

A

Magnet filters varying magnetic field will deflect differing ions according to the relationship:

M/2 = H^2R^2/2V
Where M is mass, H is magnetic field strength, R is radius of flight tube and V is accelerating voltage putting the ions into magnetic field.

The application of a magnetic field (H(0)) bends the path of the charged fragments. Lighter molecular fragments collide into inner wall of flight tube and heavier molecular fragments collide into outer wall of flight tube.

These type of filters are only found in more expensive, high resolution instruments which often involve dual magnetic filters. The working range can be as high as 100,000amu and can differentiate masses 0.001amu differences.

19
Q

What are ion traps?

A

Trap ions by RF potential which is varied to eject ions from a central space in filter. Some potential for concentration of ions to give increased sensitivity. They are a type of mass filter.

Range: 650-1,000amu
Scan time: 0.3-2s

20
Q

What are quadruple filters?

A

Commonly found on bench top instruments - four poles, two RF change and two DC change - oscillating electrostatic field causes particle to take very contorted pathway but reproducible. The balance of RF and DC at any point in the scan will determine which size of mass fragment collides with the detector. Another type of mass filter.

21
Q

How is extraction performed during sample preparation for GC?

A

Preparation of samples prior to chromatography involves addition of internal standard extraction which may be followed by derivatisation.

Extraction is required to –
Removal of protein.
Removal from binding proteins.
Change sample matrix to one more compatible with type of chromatography.
Concentrate sample.
Removal of interfering compounds (rather than relying on Ch)

22
Q

List the different extraction methods used for sample preparation.

A

Extraction methods:

  • None, occasionally with HPLC but never with GC.
  • Protein precipitation (leaves a dirty extract = noisy report) - MeCN or MeOH
  • Liquid/liquid extraction – sample buffered (“like dissolves like”) and solvent.
  • Solid phase extraction (gaining popularity)
  • ion-exchange - Both anion and cation exchangers exist, plus the possibility of multiple interaction – co-polymer phases
  • Miscellaneous methods – dialysis (ASTED), head space analysis, column switching (clean up on one column, concentrate on another) etc.
23
Q

Why is solid phase extraction beneficial?

A

SPE is useful for molecules that are difficult to extract, fore example a zwitterion such as benzoyl ecgonine, a metabolite of cocaine that is not amenable to liquid/liquid extraction. This has contrasting pH preferences and both polar and hydrophobic regions, with this method bridging of the two is possible.
Much greater sensitivity in SPE. Higher extraction rate means you can perform more regularly. Previous technique took weeks and only measured cocaine levels from the past 12 hours. This only measures fro past 12 hours but can be performed every 2 days -if someone is still using they wont be able to stay ff for this long.

24
Q

How is derivatisation used?

A

In GC there are many compounds which will not run; there are numerous reasons for this:

  • Insufficiently volatile
  • Thermally labile - eg Valium - breaks down at high temps
  • Poor interaction with stationary phase (or too great)
  • Peak trailing

Hence need to alter properties of compound:

  • Increase volatility and decrease polarity of compound.
  • Reduce thermal degradation (e.g. benzodiazepines group).
  • Increase compatibility with detector – enhance sensitivity and/or spectral library match.
  • Enhance separation of components e.g. resolve co-eluting peaks, separation of stereoisomers
Or to summarise:
To get a peak
To improve a peak
To move a peak
To identify a peak.
25
Q

How is derivatisation used to produce peaks?

A

SILYLATION

Increases the compatibility with the detector
Using TMS

26
Q

How is derivatisation used to move a peak?

A

ACETYLATION

Creates additional peaks to improve resolution.
For example, codeine and dihydracodiene co-elute. If you acetylate the OH groups you can separate peaks

27
Q

What is the use of internal standard in GC?

A

Addition of a compound prior to extraction and possible derivatisation is mandatory if quantitation is required and highly desirable even if only simple detection is required –

  • Correction for variation in extraction
  • Correction for variation in derivatisation
  • Correction for variation on column

An internal standard should be structurally similar to compound of interest (indicated by similar retention) but must be able to differentiate between internal standard and compound of interest by either retention time or detector response (mass spec. detectors).
Closest to ideal are reiterated internal standards for mass spec. (isotope dilution) which are becoming increasing available.

In GC an internal standard is very important because the column can get so dirty, leading to a loss in sensitivity.

Analytical Methods Exam (32 decks)

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