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Analytical Chemistry > Liquid Chromatography > Flashcards

Liquid Chromatography Flashcards

(68 cards)

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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
what is solvent B by convention
organic solvent (acetonitrile, methanol, THF)
26
A solvent is generally HPLC what
grade water
27
B solvent is generally HPLC what
grade organic solvent
28
in stationary phase the silica is bonded to what
hydrophobic group
29
is the mobile phase or the stationary phase more polar
mobile
30
increasing the % organic in the mobile phase does what
increases the elution power of the mobile phase
31
how are retention and selectivity altered
by changing the chemistry of the stationary phase, mobile phase and temperature
32
most analyses are likely to have what
a weak polarity
33
RP-HPLC has a what stationary phase
non-polar
34
mobile phase can have a range of what
polarities
35
what does increasing the polarity of the mobile phase do
increasingly repel the hydrophobic (non-polar) sections of the analyte molecules into the stationary phase Be retained longer
36
what does decreasing the polarity of the MP do
increasingly repel the hydrophilic (polar) sections of the analyte molecules into the stationary phase The weak polar analytes will elute faster
37
pro's of acetonitrile
lower viscosity- reduces back pressure and often results in slightly better peak shape lower UV cut-off – advantage for UV detection
38
pro's of methanol
less expensive and less toxic more polar – reducing the risks of solid buffer precipitation
39
elution order
The less water soluble a sample is, the more retention
40
retention time increases as what else increases
number of carbon atoms
41
which compounds elute more rapidly
branched-chain compounds
42
what decreases retention
unsaturation
43
elution in RP LC
more polar compounds eluted before less polar ones
44
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 Only works for neutral compounds Ionisable species need to employ pH control – a buffer as A
45
controlling ionisation
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
see powerpoint for
diagram of 2 pH rule
47
if compound has pKa of 9.6-10.2, what would the pH be when ionised
12.2
48
if a compound has pKa of 9.6-10.2, what would the pH be when non-ionised
7.6
49
see powerpoint for
diagram of HPLC
50
see powerpoint for
example of eluting compounds
51
what is the injector in HPLC
Heavy duty valve with internal tubing that allows free flow of mobile phase as all times but operates in two modes
52
what is the load in HPLC
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
what does inject do in HPLC
Directs flow through sample loop and pushes sample onto column see pp for diagram
54
advantages of detectors
sensitive stable appropriate to compounds
55
what do detectors do
Detect and measure change in parameter – converts to electrical signal
56
what is the most common type of detector
UV only useful for compounds that absorb in the UV
57
give example of two other types of detector
refractive index | conductivity
58
when was the foundation for UV/Vis spectroscopy laid
mid-1800s
59
Lambert-Beer's Law
Concentration of analyte is proportional to the intensity of transmitted light – detected by a photodiode A = e b c
60
A = e b c
``` A= absorbance e= molar extinction coefficient b= path length (1cm) C= concentration ```
61
UV detector range
190-400nm (deuterium lamp)
62
UV detector
Single beam UV spectrometer Only works with solvents that absorb Usually only works at one wavelength -Problem when compounds have different λmax
63
alternatives to UV detector
Dual wavelength Diode array – scans over wavelength range very rapidly
64
photodiode array detector
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
refractive index detector
For compound with no UV abs. (sugars, polymers). Detects changes in RI when compound passes through. Very sensitive.
66
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
67
data collection
Either an integrator or computer program Takes signal, produces chromatogram and reports; - Retention time - peak area - Peak height - % areas
68
see powerpoint for
comparisons of GC and LC

Analytical Chemistry (17 decks)

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