HPLC And GC Flashcards
Out of UV/Vs, Fluorescence and MS analytical techniques:
Which is the most sensitive? State sensitivities of each.
MS is the most sensitive (to 1 picogram)
Fluorescence at 3 pigograms
Then UV/Vis is the least selective at 5ng
Out of UV/Vs, Fluorescence and MS analytical techniques:
Which is/are flow sensitive?
MS is flow sensitive
UV/Vis and Fluorescence techniques are not flow sensitive
Out of UV/Vs, Fluorescence and MS analytical techniques:
Which are temperature sensitive?
None
Describe the absorption process of normal phase chromatography
The analyte (solute) is retained by the interaction of the polar functional group with the polar groups on the surface of the stationary phase
In normal phase chromatography, What is the stationary phase? Give examples. What is the particle size of it?
Polar.
Majority use microporous silicia, e.g. Unmodified silicia or chemically modified (with polar groups)
E.g.
Cyanopropyl or diol
Particle size of silicia Si-OH: 2-10um regular/irregular
In normal phase chromatography, what is the mobile phase? Give examples
Non-polar solvent E.g. N-hexane Dichloromethane Isopropanol
When choosing a detector for HPLC, you can have UV/Vis, Fluorescence, MS and what else to detect compounds?
Give the response, sensitivity, and state whether it is flow sensitive and temperature sensitive
Refractive index.
Universal response
Sensitive to 4mg
It is flow sensitive and temperature sensitive
On a print out of a HPLC chromatogram, what do the following signify?
1) Area under the peak
2) Retention time
Area under the peak reflects the amount of the particular analyte used (most accurate as takes care of fluctuations in baseline)
The retention time is the time at which a certain analyte elutes. These are characteristic of each analyte.
Briefly describe the instrumentation of HPLC
Solvents Solvent mixing valve Pump Injection valve Column Detector
In HPLC, what is the flow rate of the pump normally set at?1
1ml/min
In HPLC, what are the typical dimensions of the column?
<25cm x 0.5cm
In HPLC, how are solvents prepared?
Filtered via 0.4um pore.
Degassed to avoid air bubbles which can interfere with analysis and detection. They do this by giving noise and peaks.
In HPLC, how would you control the proportions of each solvent used?
You would use the mixing valve to control this.
From the mixing valve you can control the proportion of each solvent used depending on gradient
How would you control the pump in HPLC instrumentation?
What is produced and to what degree?
Set at flow rate (typically 1ml/min) to deliver mobile phase. This creates back pressure typically at 1000 pounds/square inch (maximum is 5000psi)
What is HPLC? What does it do?
High Performance Liquid Chromatography
Widely used method of analysis for quantification of drugs. Involves chromatographic separation.
Describe how HPLC works
Uses a mobile phase flowing over a stationary phase (fine silicia particles) packed in a column (stainless steel).
The injection of sample and the flow (pumping) of liquid mobile phase are precisely controlled by instrumentation
In HPLC, how is separation performed?
- Column equilibrated with starting solvent
- Analyte sample injected using injection valve (e.g. 20uL)
- Analytes are separated by interaction with the stationary phase packed inside the column.
- Detection (e.g. UV) is achieved as analytes pass through the detector, each analyte gives a distinct peak
In HPLC, what is the gradient method?T
When you change the solvent composition (via the solvent mixing valve) with time
In HPLC, what is the point of the solvent mixing valve?
There are 2 solvents and the solvent mixing valve can mix them in the proportions desired by the user
In HPLC, what is the isocratic method?
Whereby the solvent composition is kept constant
When is reverse phase HPLC used?
Most drugs are moderately polar and not suitable for normal phase HPLC, thus reverse phase HPLC is the most common method of separation and analysis in pharmaceutical sciences
Which is more commonly used: HPLC or Reverse phase HPLC?
Reverse Phase HPLC
Why is normal phase HPLC not good for analysing polar compounds?
Water soluble analytes are retained too strongly by the polar stationary phase
What is normal phase HPLC useful for?
Separation of analytes with low polarity and high solubility in low-polarity solvents such as hexane.
E.g.
Lipids, oils, phospholipids, prostaglandins (those which are fairly lipophilic)
Describe the stationary and non-stationary phases of RP HPLC
And how they separate compounds
Stationary phase: non-polar
Mobile phase: Polar
The analytes interact with the surface by portioning
Popular bonded stationary phases for RP HPLC
Non-polar:
C18 (eg. Si-[CH2]-CH3)
C8
C4
How is the stationary phase of RP HPLC made? What is it made of?
Silicia is derivatised by colvently binding n-alkyl chains. The stationary phase can be made less poalr by using more alkyl groups e.g. C8 signifies an octyl chain and C18 an octyldecyl chain
Order of elution in RP HPLC
Most polar elutes first, followed by others in order of decreasing polairity
How does RP HPLC work? For what molecular weight range is it suitable for?
Molecules <3000 molecular weight
RP HPLC works by partitioning of the hydrophobic portion of the drug molecules into the bonded (e.g. N-alkyl) stationary phase.
What forces are involved in RP HPLC when the hydrophobic portion of the drug is partitioned?
van der Waals forces
In RP HPLC, when and how does elution occur?
Molecules remain associated with the stationary phase until the concentration of the organic modifier is high enough to break off interactions.
At this stage, it partitions into the mobile phase and elutes.
Analyte elution order in normal phase HPLC
In increasing polarity i.e. Least polar first
in normal phase HPLC, what would the effect of decreasing solvent polarity do?
The solvent polarity is already low, so making it even less polar would increase the retention time, resulting in slower elution
This would happen as the stationary phase would have a stronger affinity to the stationary phase, thus taking longer to elute
in normal phase HPLC, what would the effect be of decreasing the stationary phase polarity?
It will decrease retention time (sample will elute faster) as the substance will have a higher affinity to the mobile phase and therefore elute quicker
In Reverse Phase HPLC, what are the polarities of the stationary and mobile phases?
Stationary phase is non-polar
Mobile phase/solvent is polar
In Reverse Phase HPLC, what is the analyte elution order?
In order of decreasing polarity (most polar first)
In Reverse Phase HPLC, what would the effect of decreasing the polarity of the solvent cause?
The solvent in RP HPLC is of high polarity.
The stationary phase is of low polarity
Decreasing the polarity of the solvent will decrease the affinity of the sample going in the system and therefore it will increase retention time and elute slower.
In Reverse Phase HPLC, what would the effect of decreasing the polarity of the stationary phase be?
In RP HPLC:
The mobile phase/solvent is polar
the stationary phase is non-polar
Therefore reducing the polarity of the stationary phase even further will reduce the affinity of the solvent to the stationary phase.
This will cause an increased retention time and slower elution
How would you improve performance of HPLC?
- make the column longer
- change the pH
- decrease the stationary phase particle size (Even more)
What kind of gradient in HPLC will give better resolution?
A less steep gradient will give better resolution, but avoid peak broadening
In HPLC, on the results, what does an unsymmetrical peak mean? What will it affect?
The column may be aged.
This will affect the calculation when area under the curve is measured.
How is quantitive analysis of HPLC measured?
What does one require?
What are the two standard methods?
It is measured by measuring peak heights and areas. You require calibration and standards. The two standard methods are: -External standard method -Internal standard method
In quantitive analysis of HPLC, how do you use the external standard method? When is this method used?
- concentrations calculated from the ratio of sample peak areas to the corresponding standard peak areas.
- usually used in drug manufacture and in stability testing
In quantitive analysis of HPLC, which method is the most precise?
The internal standard method as less chance of error from reproducibility and sample preparation concerns
In quantitive analysis of HPLC, when is the internal standard method used?
Often used in pharmacokinetics where trace levels of drugs or metabolites are present in a biological matrix.
What are the two types of gas chromatography?
GSC - Gas-solid chromatography - involves adsorption of analytes
GLC - Gas-liquid chromatography - involves partitioning of analytes
What is the most common form of GC? What is the instrument called?
GLC - Gas liquid chromatography
Gas Chromatograph
Describe how GLC works
Gas-liquid chromatography works by partition of molecules between gas (mobile phase) and liquid-coated stationary phase
In GLC. What kind of gases are used in the mobile phase?
What other regulators are used?
Inert/unreactive gas to transport molecules e.g. He, Ar, N2, CO2, H2
Pressure regulators control the gas flow 10-50psi
Psi = pounds/square inch
In GLC, what is the range for which gas flow rates can be controlled within?
1-150ml/ml
Which is more sensitive, HPLC or GC?
GC
Name the key components of a gas chromatograph
- Injector (heated) - 0.1-10uL
- Column - inert support, range of polarity stationary phases
- Oven - RT 400 degrees Celsius
- Detectors
- Chart recorder/integrator
Name 4 different types of GC detectors
- FID (Flame ionisation detector)
- ECD (Electron capture detector)
- NPD (Nitrogen Phosphorus detecor)
- MS (Mass spectroscopy)
In GC, to what level can the oven temperature be controlled?
To a few tenths of a single degree Celsius
The oven is thermostated
In GC, the oven is usually at what temperature relative to the sample components?
Operating temperature slightly above the average boiling point of sample components to be separated.
Lower temperature means longer time for analysis as longer amount of time taken to enter the gas phase
For GC, what is needed if you have a broad range of sample boiling points?
In the oven, you can have a programmable temperature gradient.
Describe the principles of separation in GC
- The coiled column (in the oven) is packed with the liquid phase
- Sample introduced into injection block where it is vapourised and carried by flowing syringe gas stream
- sample vapour partitions between gas and stationary liquid phases
What affects the time different components spend in the column in GC?
- It depends on the vapour pressure of the components.(Low boiling point means higher vapour)
- and the ability of the components to interact with the liquid phase
What would increase the retention time in a GC sample?
Low volatility (high boiling point) = longer retention time Greater solubility in the liquid phase = longer retention time
What is the effect of gas and flow rate on GC?
Little effect
Typical temperatures in GC?
- 20-50 degrees Celsius for gases
- 200-300 degrees Celsius for steroids
Operating conditions relating to column temperature?
- at boiling point of solutes/a bit higher than boiling point
- solute needs to be in the gas/vapour phase
- heated injector helps this hapen
Name the two types of columns in GC
- Packed column
- capillary tube
Describe a packed column in GC
- contain granular solid particles (diatomaceous Earth, curushed fire brick, alumina or graphite)
- to support the stationary liquid phase
Describe the capillary tube used in GC, if it is used
And it’s dimensions
-most popular are wall-coated open tubulur (WCOT) where the stationary liquid phase is coated (chemically or as a film) on the inner wall of the tube
-the tube is 2-50m, ade of steel, copper, glass, fused silica, teflon
-coiled to about 10-30cm diametre to fit oven
Open tubular: 0.2-0.75mm diameter liquid phase coated or bonded to the inner tube
How do you tell the difference between a HPLC and a GC chromatogram? How is a GC one interpreted?
You can’t
GC chromatogram is interpreted in a similar way to HPLC for identification (area under the peak reflects the amount of component present) and quantitive analysis uses the same parameters
-each volatile component produces a peak with a characteristic retention time
What is chemical derivatisation in GC used for?
Chemically derivatised prior to analysis is necessary to:
- improve separation and reduce tailing
- increase volatility of samples
- reduce polarity of compounds
- reduce thermal degradation of samples
- increase detector response
Name some derivatising reagents used for sample preparation in GC
Methyl esters of fatty acids using methanolic HCl
Trimethylchlorositane
What ways can you derivatise a sample in preparation for GC?
- silylation
- acylation
- akylation
- esterification
What do you want from a GC stationary phase?
Thermally stable Must allow analytes to partition Chemically inert Low volatility (ideally 100 degrees over max operating temperature) Coated (0.1-0.5 microns thin film)
In these cases, what is the best stationary phase choice for GC?
1) polar analytes
2) non-polar analytes
1) polar liquid phase
2) non-polar liquid phase
In GC, how would you separate a mixture of polar compounds? What would you use for the stationary phase?
Carbowax 20M - (polyethylene glycol)
What would you use for GC separation of non-polar compounds (stationary phase) ?
-OV101 or SE-30 (polymer of methylsilicone)
-methylester of fatty acids DEGS (diethylene glycol succinate)
-Paraffins
-Hydrocarbons
-Squalene
Upper temp limit: 150 degrees Celsius
How sensitive is a FID? What is an FID? When is it used?
FID = Flame ionisation detector
Used to detect compounds during GC
It is sensitive to the ng level
How does FID work?
Burns sample with H2 from air
Current produced from ions and electrons
The number of carbons ionised is related to the current amplitude
Most drugs have carbon
Disadvantage of FID?
Not good for halogens, alcohols, amines
