HPLC

Question 1

HPLC can be divided into the following categories:

Answer 1

• adsorption
• ion exchange
• size exclusion
• partition chromatography
  > reverse
  > normal

Question 2

the composition of mobile phase

Answer 2

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

Question 3

the choice of solvent affects ________ and ________

Answer 3

selectivity and retention

Question 4

solvents often used in the lab

Answer 4

methanol*
DMSO
ethanol
acetonitrile*
tetrahydrofuran
dioxane
isopropanol

Question 5

what are buffers used for in HPLC?

Answer 5

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

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

Question 6

criteria for choosing a detector

Answer 6

sensitivity
detectability
linearity
reproducibility
peak shape
flow and temp

Question 7

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

Answer 7

1.25

Question 8

k’

Answer 8

capacity factor
measure of degree of retention

Question 9

how is retention time easily adjusted?

Answer 9

by changing the amount of organic solvent

Question 10

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

Answer 10

10%; two-three

Question 11

isocratic mode

Answer 11

mobile phase composition remains constant throughout the chromatographic run

Question 12

gradient mode

Answer 12

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

Question 13

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

Answer 13

differential eqm

Question 14

what is HPLC?

Answer 14

separation technique

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

stationary = column
mobile phase = solvent/buffers

Question 15

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

Answer 15

normal phase

Question 16

T or F. Normal phase more popular

Answer 16

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

Question 17

the choice of solvent affectes these (2)

Answer 17

retention and selectivity
chosen based on their polarity

Question 18

solvents usually used

Answer 18

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

DMSO, ethanol, tetrahydrofuran, etc.

Question 19

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

Answer 19

increases ionization efficiency
- lower sampel viscosity means fine droplets produced

Question 20

what are buffers used for in HPLC?

Answer 20

control pH = pH affects selectivity or separation of compounds

reduce peak tailing

give well-shaped narrow peaks

Question 21

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

Answer 21

retention
ion suppressed analytes = better retention than analyzed analytes

Question 22

which buffers are preferred in LC-MS/MS

Answer 22

volatile buffers
- minimizes ion suppression and maintains sensitivity

Question 23

non-volatile buffers

Answer 23

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

Question 24

what is back pressure?

Answer 24

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

Question 25

frequently the source of elevated back pressure

Answer 25

particulates - sample - mobile phase - instrument wear and tear

Question 26

how to troubleshoot back pressure

Answer 26

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

Question 27

what are detectors responsible for?

Answer 27

detecting compounds as they elute from column

Question 28

criteria for choosing detector

Answer 28

sensitivity detectability linearity reproducibility peak shape flow and temp

Question 29

examples of detectors

Answer 29

UV fluorescence electrochemical MS etc.

Question 30

fluorescence detector theory

Answer 30

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

Question 31

amperometric detector theory

Answer 31

- 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

Question 32

t0

Answer 32

void time time required to elute non-retained substances

Question 33

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

Answer 33

retention time (tR)

Question 34

resolution

Answer 34

R degree of separation between two components by chromatography

Question 35

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

Answer 35

1.25

Question 36

resolution is controlled by factors that affect:

Answer 36

- peak retention > capacity factor [k] > selectivity [alpha] - peak width > efficiency [N]

Question 37

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

Answer 37

- first adjust the capacity factor (k), then selectivity factor (alpha) and then the efficiency factor

Question 38

k'

Answer 38

(tR-t0)/t0 measure of retention time

Question 39

alpha

Answer 39

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

Question 40

N

Answer 40

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

Question 41

measure of the degree of retention

Answer 41

k' capacity factor

Question 42

how do we adjust retention time?

Answer 42

by changing the amount of organic solvent

Question 43

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

Answer 43

T

Question 44

this is the most important factor in terms of resolution

Answer 44

selectivity factor

Question 45

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

Answer 45

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

Question 46

obtained by calculating the number of theoretical plates for a column

Answer 46

efficiency of a column (N)

Question 47

what is a theoretical plate?

Answer 47

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

Question 48

what does a large number of theoretical plates mean?

Answer 48

indicates relatively narrow peaks and thus, an efficient column

Question 49

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

Answer 49

T

Question 50

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

Answer 50

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

Question 51

how to maximize column efficiency (4)

Answer 51

- 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

Question 52

reasons for extraction of samples

Answer 52

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

Question 53

why do people prefer LC-MS/MS?

Answer 53

short run time but sensitive and specific