Liquid chromatography Flashcards
LC
- LC is emerging as an important separation technique in forensic toxicology and analysis of drugs
- Complements GC
- Need to know how to optimise separations
High performance liquid chromatography
- Stationary phase – solid
Contained in a column of fixed dimensions - Mobile phase – liquid
Pumped through the system at high pressure at a fixed flow rate
what is LC
- Separate non-volatile organic compounds
- Analyte in liquid solution
- Suitable for thermally labile, polar compounds
how big are the columns
- short (5-30 cm)
what does the mobile phase consist of
- phase often a combination of water and organic solvent
what is separation based on
polarity, electrical charge and molecular size
organic molecules are sorted into classes
- according to the principal functional groups each contains
for polarity
- the chromatographic retention of different kinds of molecules if largely determined by the nature and location of these functional groups
look at ppt
stationary phase- normal phase HPLC
– Based on polar silica (SiO2) – stationary phase
– Uses non-polar or less polar mobile phase
– Robust – can stand high P
– Microspheres ~3 – 10 μm
– Packed in stainless steel columns
– Less polar compounds eluted before polar ones
drawback of normal phase
very polar solvents bond to silica makes column useless
Reverse-phase HPLC (RP-HPLC) – most common
– Silica has been functionalised
– Long chain hydrocarbon bonded to silica
– C-8 or C-18 chains
– Later known as ‘octodecylsilyl’ or ODS
– Make stationary phase non-polar
– Can use mobile phases with a range of polarities
– More polar compounds eluted before less polar ones
– Can be used with a variety of compound types
– Separation depends on mobile phase composition
RP-HPLC
look on ppt at graph
mobile phase commonest solvents are
– Water - polar
– Methanol - polar
– Acetonitrile - moderately polar
– Tetrahydrofuran (THF) – moderately polar
generally mobile phases are made up of mixtures of solvents of
- different polarities
– Water with acetonitrile or THF or methanol
mixtures in mobile phase remain
- constant – isocratic
composition can change over time- gradient
- In RP-HPLC start with a less polar mix and move towards a more polar mix
- Mix may include a pH buffer (instead of water)
- Will ionise or un-ionise compounds depending on nature
Where are the reverse phase solvents are by convention installed where
- the HPLC channels A and B
‘A’ solvent
- the aqueous solvent (water or a buffer)
- generally HPLC grade water
‘B’ solvent
- the organic solvent (acetonitrile, methanol, THF).
- generally an HPLC grade organic solvent such as acetonitrile or methanol.
- Sometimes acid (0.1%) is used in each to the improve the chromatographic peak shape
what is more polar
mobile phase is more polar than stationary
mobile phase can have a range of polarities
stationary phase
hydrophobic group chemically bonded to silica
the two phases
- Analyte ‘partitions’ between the two phases depending upon its chemistry (hydrophobicity)
- Increasing the % organic in the mobile phase increases the ‘elution power’ of the mobile phase
- Retention and Selectivity are altered by changing the chemistry of the stationary phase, mobile phase and the temperature
- Most analytes tests are likely to have a weak polarity
increase the polarity of the MP (water)
- this will increasingly repel the hydrophobic (non-polar) sections of the analyte molecules into the stationary phase
- Be retained longer.
decrease the polarity of the MP (acetonitrile)
- this will increasingly repel the hydrophilic (polar) sections of the analyte molecules into the stationary phase
- the weak polar analytes will elute faster
pros
- Acetonitrile has lower viscosity - reduces back pressure and often results in slightly better peak shape
- Acetonitrile has lower UV cut-off - advantage for UV detection
- Methanol is less expensive and less toxic
- Methanol is more polar - reducing the risks of solid buffer precipitation
elution order
- The less water soluble a sample is, the more retention
retention time increases as
- the number of carbon atoms increases
what elutes more rapidly
- Branched-chain compounds elute more rapidly than straight-chain compounds (isomers) e.g. C4H10
unsaturation decreases
retention
in RP-LC
- more polar compounds eluted before less polar ones
trial and error
- Carry out separation at high %A (80%)
- This saves time vs. starting at low A
- Reduce by 5-10% A in steps to assess retention – make mobile phase less polar
- Really only works for neutral compounds
- Ionisable species need to employ pH control – a buffer as A
controlling ionisation
- Charged (ionised) compounds are more hydrophilic so more polar than when non-charged
- Need to know the pKa of a compound and pH of the mobile phase
- Adjust pH so that you get a stronger or weaker retention
the 2pH rule
look on ppt
In a RP-LC separation which of the following compounds elute first and last?
- Polar compounds will come out first because the stationary phase is non polar
1) (first) benzoic acid
2) phenol benzene
3) benzene
4) (last) methyl benzene (has an aliphatic chain so makes it more non polar)
look at ppt
HPLC instrument
look at ppt for diagram
HPLC injector
look at ppt
injector
heavy duty valve with internal tubing that allows free flow of
mobile phase at all times but operates in two modes
LOAD
where mobile phase flows directly onto column BUT allows sample
loop to be filled from external syringe (loop, 20 – 100 μL)
INJECT
directs flow through sample loop and pushes sample onto column.
calibration graphs
look at ppt
detectors
- Sensitive, stable, appropriate to compounds
- Detect and measure change in a parameter – converts to electrical signal
most common type of detector
UV
Only useful for compounds that absorb in the UV
Other detectors
- Refractive index
- conductivity
UV/Vis detections
- Foundation for UV/Vis spectroscopy was laid in mid-1800s
- Lambert-Beer’s Law
- Concentration of analyte is proportional to the intensity of transmitted light – detected by a photodiode
Lamber- Beer’s Law
Concentration of analyte is proportional to the intensity of transmitted light – detected by a photodiode
Lamber- Beer’s Law equation
look at power point
A= e b c A = absorbance e= molar extinction coefficient b= path length (1cm) c= concentration
UV detector
- Single beam UV spectrometer
range of UV detector
- 190 – 400 nm (deuterium lamp)
Cons of UV detector
- Only works with solvents that absorb UV
- Usually only works at one wavelength
- Problem when compounds have different λmax
alternatives to UV detector
- Dual wavelength
- Diode array – scans over wavelength range very rapidly
Photodiode Array Detector (DAD)
- Operates over a bigger wavelength range 190-600nm
- Allows the acquisition of the entire spectra passed through
- Spectra is a 3D plot of response vs time vs wavelength
refractive index detector
- For compound with no UV abs. (sugars,
polymers) . - Detects changes in RI when compound passes through. Very sensitive.
Drawbacks of refractive index detector
- Extremely temp. sensitive – needs a controlled environment
- Sensitive to changes in mobile phase
- Cannot be used with a gradient mobile phase
- Sensitive to turbulence – needs a stable flow rate.
conductimetric detector
- Used for detecting ions
- Ion chromatography – a special type of HPLC
what does conductimetric detector measure
change in an electrical current as ions pass through
- Response is proportional to ion concentration
- Can’t tell which ion – that needs standards
cons of conductimetric detector
- Can’t tell which ion – that needs standards
data collection
- Either an integrator or computer program
Takes signal, produces chromatogram and reports; - Retention time
- peak area
- Peak height
- % areas
GC and LC
GC
- samples analysed by GC must be volatile and thermally stable
- derivatization to increase analyse volatility is possible but cumbersome and introduces possible quantitative errors
- most GC analyses are under 500 Da molecular weight for volatility purposes
- resolution unparalleled (capillary columns)
- simple and inexpensive equipment
- rapid
LC
- HPLC analysis has no volatility issues, however the analyse must be soluble in the mobile phase
- HPLC can analyse samples over a wider range of polarity and is able to analyse ionic samples
- HPLC has no real upper molecular weight limit and large proteins of many thousands of Daltons may be analysed
- thermally unstable compounds
- macromolecules
- more complex interface to MS
- applicable for inorganic ions
BOTH GC AND LC
- efficient
- selective
- small sample size
- can be non destructive
- easy to adapt to quantitative analysis
