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MLSCI 466 > HPLC > Flashcards

HPLC Flashcards

1
Q

HPLC can be divided into the following categories:

A
  • adsorption
  • ion exchange
  • size exclusion
  • partition chromatography
    > reverse
    > normal
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2
Q

the composition of mobile phase

A
  • solvents
  • buffers
  • mobile phase modifiers
    > ex: triethylamine (can dramatically influence retention time)
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3
Q

the choice of solvent affects ________ and ________

A

selectivity and retention

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4
Q

solvents often used in the lab

A

methanol*
DMSO
ethanol
acetonitrile*
tetrahydrofuran
dioxane
isopropanol

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5
Q

what are buffers used for in HPLC?

A
  • control pH
  • reduce peak tailing
  • give well-shaped narrow peaks

pH affects selectivity (separation of compounds relative to one another)

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6
Q

criteria for choosing a detector

A

sensitivity
detectability
linearity
reproducibility
peak shape
flow and temp

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7
Q

a resolution of _____ or greater is considered necessary for good chromatographic analyses

A

1.25

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8
Q

k’

A

capacity factor
measure of degree of retention

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9
Q

how is retention time easily adjusted?

A

by changing the amount of organic solvent

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10
Q

rule of thumb: __% change in fraction of organic solvent in water will cause ___ or ____-fold change in k’

A

10%; two-three

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11
Q

isocratic mode

A

mobile phase composition remains constant throughout the chromatographic run

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12
Q

gradient mode

A

the mobile phase composition is either changed in a stepwise or continuous fashion throughout the run

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13
Q

___________ __________ of the analytes between the mobile phase and the stationary phase of the column results in their separation

A

differential eqm

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14
Q

what is HPLC?

A

separation technique

mixtures of compounds can be resolved by exploiting differences in their chemical and physical properties

stationary = column
mobile phase = solvent/buffers

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15
Q

a partition system in which the stationary phase is more polar than the mobile phase

A

normal phase

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16
Q

T or F. Normal phase more popular

A

F! Reverse phase; more popular in biological applications b/c of polar nature of many bio compounds

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17
Q

the choice of solvent affectes these (2)

A

retention and selectivity
chosen based on their polarity

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18
Q

solvents usually used

A

methanol and acetonitrile low UV cutoff; won’t contribute to background noise

DMSO, ethanol, tetrahydrofuran, etc.

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19
Q

For LC-MS/MS how does acetonitrile compare to methanol?

A

increases ionization efficiency
- lower sampel viscosity means fine droplets produced

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20
Q

what are buffers used for in HPLC?

A

control pH = pH affects selectivity or separation of compounds

reduce peak tailing

give well-shaped narrow peaks

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21
Q

The _________ of an analyte changes rapidly when changes in pH are within +/- pH unit of the pKa of the analyte

A

retention
ion suppressed analytes = better retention than analyzed analytes

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22
Q

which buffers are preferred in LC-MS/MS

A

volatile buffers
- minimizes ion suppression and maintains sensitivity

23
Q

non-volatile buffers

A

phosphate
popular in HPLC but can lread to contam of ion source so not used in LC-MS/MS

24
Q

what is back pressure?

A

the force necessary to push a liquid mobile phase through a tightly packed bed of tiny particles
- even at low flow rates

25
Q

frequently the source of elevated back pressure

A

particulates
- sample
- mobile phase
- instrument wear and tear

26
Q

how to troubleshoot back pressure

A

start at detector and work backwards up the flow path using systematic approach

27
Q

what are detectors responsible for?

A

detecting compounds as they elute from column

28
Q

criteria for choosing detector

A

sensitivity
detectability
linearity
reproducibility
peak shape
flow and temp

29
Q

examples of detectors

A

UV
fluorescence
electrochemical
MS
etc.

30
Q

fluorescence detector theory

A

ability of molecule to emit light after excited by light radiation

since many molecules do not fluoresce, numerous methods for deriving compounds have been developed

31
Q

amperometric detector theory

A
  • electrochemical detector
  • column effluent flows past an electrode to which voltage is applied
  • voltage = large enough => analyte molecules at interface between electrode and solution can either accept e- and be reduced or give up e- to be oxidized
  • net movement of e- = current flow; current is proportional to conctn of analytes
32
Q

t0

A

void time
time required to elute non-retained substances

33
Q

the time that has elapsed from injection of sample into chrom. system to recording of peak max of the component of chromatogram

A

retention time (tR)

34
Q

resolution

A

R
degree of separation between two components by chromatography

35
Q

a resolution of _____ or greater is considered necessary for good chromatographic analyses

A

1.25

36
Q

resolution is controlled by factors that affect:

A
  • peak retention
    > capacity factor [k]
    > selectivity [alpha]
  • peak width
    > efficiency [N]
37
Q

the most cost- and time-effective approach approach to improvement of resolution

A
  • first adjust the capacity factor (k), then selectivity factor (alpha) and then the efficiency factor
38
Q

k’

A

(tR-t0)/t0
measure of retention time

39
Q

alpha

A

k’2/k1
- measure of tretention time of two peaks relative to each other

40
Q

N

A

measure of peak width
16(tR/W)^2

41
Q

measure of the degree of retention

A

k’
capacity factor

42
Q

how do we adjust retention time?

A

by changing the amount of organic solvent

43
Q

T or F. A 10% change in the fraction of organic solvent in water will cause a two- or threefold change in k’

A

T

44
Q

this is the most important factor in terms of resolution

A

selectivity factor

45
Q

because selectivity is a function of the column packing, the __________ phase and the solute chemistry can be manipulated by changing: (4)

A

mobile

  1. composition of mobile phase (organic solvent, buffer and pH)
  2. stationary phase
  3. sample chemistry through derivatization
  4. separation temp
46
Q

obtained by calculating the number of theoretical plates for a column

A

efficiency of a column (N)

47
Q

what is a theoretical plate?

A

microscopic segment of a column where a perfect eqm is assumed to exist between the solute in the mobile and stationary phase

48
Q

what does a large number of theoretical plates mean?

A

indicates relatively narrow peaks and thus, an efficient column

49
Q

T or F. The larger the N, the more efficient

A

T

50
Q

T or F. To maximize column efficiency, band broadening must be increased

A

F! DECREASED
- accomplished by using a well-packed column that contains a stationary phase packing that is small and uniform with respect to size distribution of particles

51
Q

how to maximize column efficiency (4)

A
  • decrease band broadening; well-packed column
  • increase column length
  • increasing mobile phase flow rate will also help but only to a certain pt
  • less is better with vol of injection
52
Q

reasons for extraction of samples

A

to prolong life of column
to see only peaks of interest
to improve sensitvity

53
Q

why do people prefer LC-MS/MS?

A

short run time but sensitive and specific

54
Q
A

MLSCI 466 (19 decks)

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