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Analytical Chemistry > Liquid Chromatography > Flashcards

Liquid Chromatography Flashcards

1
Q

what does HPLC stand for

A

high performance liquid chromatography

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2
Q

what state is the stationary phase in HPLC

A

solid

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3
Q

where is the stationary phase contained in HPLC

A

in a column of fixed dimensions

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4
Q

what state is the mobile phase in HPLC

A

liquid

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5
Q

what happens to the mobile phase during HPLC

A

pumped through the system at high pressure at a fixed flow rate

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6
Q

what does liquid chromatography do

A

separate non-volatiles organic compounds

analyte in liquid solution

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7
Q

what is liquid chromatography suitable for

A

thermally liable, polar compounds

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8
Q

what is the mobile phase often a combination of

A

water and organic solvent

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9
Q

how big are the columns

A

short (5-30cm)

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10
Q

what is the separation based on?

A

polarity, electrical charge and molecular size

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11
Q

how are organic molecules sorted

A

into classes according to the principal functional groups each contains

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12
Q

for polarity the chromatographic retention of different kinds of molecule is determines by what

A

the nature and location of the functional groups

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13
Q

see powerpoint for

A

polar and non polar molecule diagrams

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14
Q

what are the two kind of stationary phase

A

normal phase HPLC

reverse phase HPLC

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15
Q

which stationary phase is most common

A

reverse phase HPLC

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16
Q

normal phase HPLC

A

Based on polar silica (SiO2) – stationary phase

Uses non-polar or less polar mobile phase

Robust – can stand high pressure

Microspheres – 3-10um

Packed in stainless steel columns

Less polar compounds eluted before polar ones

Drawback: very polar solvents bond to silica makes column useless

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17
Q

reverse phase HPLC

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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18
Q

see powerpoint for

A

hydrophilic and hydrophobic graph

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19
Q

what are 4 common solvents for the mobile phase

A

water - polar

methanol - polar

Acetonitrile – moderately polar

Tetrahydrofuran – moderately polar

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20
Q

what are mobile phases generally a mixture of

A

solvents of different polarities

water with acetonitrile or THF or methanol

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21
Q

what is it called when it mixtures can remain constant

A

isocratic

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22
Q

can change composition over time in mobile phase - 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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23
Q

the reverse phase solvents are by convention what

A

installed on the HPLC channels A and B

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24
Q

what is solvent A by convention

A

the aqueous solvent (water to buffer)

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25
Q

what is solvent B by convention

A

organic solvent (acetonitrile, methanol, THF)

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26
Q

A solvent is generally HPLC what

A

grade water

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27
Q

B solvent is generally HPLC what

A

grade organic solvent

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28
Q

in stationary phase the silica is bonded to what

A

hydrophobic group

29
Q

is the mobile phase or the stationary phase more polar

30
Q

increasing the % organic in the mobile phase does what

A

increases the elution power of the mobile phase

31
Q

how are retention and selectivity altered

A

by changing the chemistry of the stationary phase, mobile phase and temperature

32
Q

most analyses are likely to have what

A

a weak polarity

33
Q

RP-HPLC has a what stationary phase

34
Q

mobile phase can have a range of what

A

polarities

35
Q

what does increasing the polarity of the mobile phase do

A

increasingly repel the hydrophobic (non-polar) sections of the analyte molecules into the stationary phase

Be retained longer

36
Q

what does decreasing the polarity of the MP do

A

increasingly repel the hydrophilic (polar) sections of the analyte molecules into the stationary phase

The weak polar analytes will elute faster

37
Q

pro’s of acetonitrile

A

lower viscosity- reduces back pressure and often results in slightly better peak shape

lower UV cut-off – advantage for UV detection

38
Q

pro’s of methanol

A

less expensive and less toxic

more polar – reducing the risks of solid buffer precipitation

39
Q

elution order

A

The less water soluble a sample is, the more retention

40
Q

retention time increases as what else increases

A

number of carbon atoms

41
Q

which compounds elute more rapidly

A

branched-chain compounds

42
Q

what decreases retention

A

unsaturation

43
Q

elution in RP LC

A

more polar compounds eluted before less polar ones

44
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

Only works for neutral compounds

Ionisable species need to employ pH control – a buffer as A

45
Q

controlling ionisation

A

Charged (ionised) compounds are more hydrophilic 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

46
Q

see powerpoint for

A

diagram of 2 pH rule

47
Q

if compound has pKa of 9.6-10.2, what would the pH be when ionised

48
Q

if a compound has pKa of 9.6-10.2, what would the pH be when non-ionised

49
Q

see powerpoint for

A

diagram of HPLC

50
Q

see powerpoint for

A

example of eluting compounds

51
Q

what is the injector in HPLC

A

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

52
Q

what is the load in HPLC

A

Where mobile phase flows directly onto column but allows sample loop to be filled from external syringe (loop, 20-100uL)

see pp for diagram

53
Q

what does inject do in HPLC

A

Directs flow through sample loop and pushes sample onto column

see pp for diagram

54
Q

advantages of detectors

A

sensitive
stable
appropriate to compounds

55
Q

what do detectors do

A

Detect and measure change in parameter – converts to electrical signal

56
Q

what is the most common type of detector

A

UV

only useful for compounds that absorb in the UV

57
Q

give example of two other types of detector

A

refractive index

conductivity

58
Q

when was the foundation for UV/Vis spectroscopy laid

59
Q

Lambert-Beer’s Law

A

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

A = e b c

60
Q

A = e b c

A 
A= absorbance 
e= molar extinction coefficient 
b= path length (1cm)
C= concentration
61
Q

UV detector range

A

190-400nm (deuterium lamp)

62
Q

UV detector

A

Single beam UV spectrometer

Only works with solvents that absorb

Usually only works at one wavelength
-Problem when compounds have different λmax

63
Q

alternatives to UV detector

A

Dual wavelength

Diode array – scans over wavelength range very rapidly

64
Q

photodiode array detector

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

65
Q

refractive index detector

A

For compound with no UV abs. (sugars,
polymers).

Detects changes in RI when compound passes through. Very sensitive.

66
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

67
Q

data collection

A

Either an integrator or computer program

Takes signal, produces chromatogram and reports;

  • Retention time
  • peak area
  • Peak height
  • % areas
68
Q

see powerpoint for

A

comparisons of GC and LC

Analytical Chemistry (17 decks)

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