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Analytical Chemistry > Liquid chromatography > Flashcards

Liquid chromatography Flashcards

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1
Q

LC

A
  • LC is emerging as an important separation technique in forensic toxicology and analysis of drugs
  • Complements GC
  • Need to know how to optimise separations
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2
Q

High performance liquid chromatography

A
  • 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
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3
Q

what is LC

A
  • Separate non-volatile organic compounds
  • Analyte in liquid solution
  • Suitable for thermally labile, polar compounds
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4
Q

how big are the columns

A
  • short (5-30 cm)
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5
Q

what does the mobile phase consist of

A
  • phase often a combination of water and organic solvent
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6
Q

what is separation based on

A

polarity, electrical charge and molecular size

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7
Q

organic molecules are sorted into classes

A
  • according to the principal functional groups each contains
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8
Q

for polarity

A
  • the chromatographic retention of different kinds of molecules if largely determined by the nature and location of these functional groups

look at ppt

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9
Q

stationary phase- normal phase HPLC

A

– 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

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10
Q

drawback of normal phase

A

very polar solvents bond to silica makes column useless

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11
Q

Reverse-phase HPLC (RP-HPLC) – most common

A

– 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

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12
Q

RP-HPLC

A

look on ppt at graph

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13
Q

mobile phase commonest solvents are

A

– Water - polar
– Methanol - polar
– Acetonitrile - moderately polar
– Tetrahydrofuran (THF) – moderately polar

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14
Q

generally mobile phases are made up of mixtures of solvents of

A
  • different polarities

– Water with acetonitrile or THF or methanol

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15
Q

mixtures in mobile phase remain

A
  • constant – isocratic
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16
Q

composition can change over time- gradient

A
  • 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
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17
Q

Where are the reverse phase solvents are by convention installed where

A
  • the HPLC channels A and B
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18
Q

‘A’ solvent

A
  • the aqueous solvent (water or a buffer)

- generally HPLC grade water

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19
Q

‘B’ solvent

A
  • 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
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20
Q

what is more polar

A

mobile phase is more polar than stationary

mobile phase can have a range of polarities

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21
Q

stationary phase

A

hydrophobic group chemically bonded to silica

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22
Q

the two phases

A
  • 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
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23
Q

increase the polarity of the MP (water)

A
  • this will increasingly repel the hydrophobic (non-polar) sections of the analyte molecules into the stationary phase
  • Be retained longer.
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24
Q

decrease the polarity of the MP (acetonitrile)

A
  • this will increasingly repel the hydrophilic (polar) sections of the analyte molecules into the stationary phase
  • the weak polar analytes will elute faster
25
Q

pros

A
  • 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
26
Q

elution order

A
  • The less water soluble a sample is, the more retention
27
Q

retention time increases as

A
  • the number of carbon atoms increases
28
Q

what elutes more rapidly

A
  • Branched-chain compounds elute more rapidly than straight-chain compounds (isomers) e.g. C4H10
29
Q

unsaturation decreases

A

retention

30
Q

in RP-LC

A
  • more polar compounds eluted before less polar ones
31
Q

trial and error

A
  • 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
32
Q

controlling ionisation

A
  • 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
33
Q

the 2pH rule

A

look on ppt

34
Q

In a RP-LC separation which of the following compounds elute first and last?

A
  • 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

35
Q

HPLC instrument

A

look at ppt for diagram

36
Q

HPLC injector

A

look at ppt

37
Q

injector

A

heavy duty valve with internal tubing that allows free flow of
mobile phase at all times but operates in two modes

38
Q

LOAD

A

where mobile phase flows directly onto column BUT allows sample
loop to be filled from external syringe (loop, 20 – 100 μL)

39
Q

INJECT

A

directs flow through sample loop and pushes sample onto column.

40
Q

calibration graphs

A

look at ppt

41
Q

detectors

A
  • Sensitive, stable, appropriate to compounds

- Detect and measure change in a parameter – converts to electrical signal

42
Q

most common type of detector

A

UV

Only useful for compounds that absorb in the UV

43
Q

Other detectors

A
  • Refractive index

- conductivity

44
Q

UV/Vis detections

A
  • 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
45
Q

Lamber- Beer’s Law

A

Concentration of analyte is proportional to the intensity of transmitted light – detected by a photodiode

46
Q

Lamber- Beer’s Law equation

A

look at power point

A= e b c
A = absorbance
e= molar extinction coefficient
b= path length (1cm)
c= concentration
47
Q

UV detector

A
  • Single beam UV spectrometer
48
Q

range of UV detector

A
  • 190 – 400 nm (deuterium lamp)
49
Q

Cons of UV detector

A
  • Only works with solvents that absorb UV
  • Usually only works at one wavelength
  • Problem when compounds have different λmax
50
Q

alternatives to UV detector

A
  • Dual wavelength

- Diode array – scans over wavelength range very rapidly

51
Q

Photodiode Array Detector (DAD)

A
  • 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
52
Q

refractive index detector

A
  • For compound with no UV abs. (sugars,
    polymers) .
  • Detects changes in RI when compound passes through. Very sensitive.
53
Q

Drawbacks of refractive index detector

A
  • 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.
54
Q

conductimetric detector

A
  • Used for detecting ions

- Ion chromatography – a special type of HPLC

55
Q

what does conductimetric detector measure

A

change in an electrical current as ions pass through

  • Response is proportional to ion concentration
  • Can’t tell which ion – that needs standards
56
Q

cons of conductimetric detector

A
  • Can’t tell which ion – that needs standards
57
Q

data collection

A
  • Either an integrator or computer program
    Takes signal, produces chromatogram and reports;
  • Retention time
  • peak area
  • Peak height
  • % areas
58
Q

GC and LC

A

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
59
Q

BOTH GC AND LC

A
  • efficient
  • selective
  • small sample size
  • can be non destructive
  • easy to adapt to quantitative analysis

Analytical Chemistry (20 decks)

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