liquid chromatography and mass spec

Question 1

difference of isocratic and gradient mode in LC

Answer 1

isocratic = mobile phase composition is the same every run

gradient = mobile phase changes throughout the run

Question 2

what causes separation of anaylte bt mobile and stationary phase

Answer 2

differential equilibrium aka partition coefficient

Question 3

what is another name for partition chromatography

Answer 3

liquid liquid chromatography bc both mobile and stationary are liquid

Question 4

purpose of a column in LC

Answer 4

prevent dissolution of stationary phase by covalent binding solica

Question 5

explain normal and reverse phase LC column

Answer 5

normal phase - stationary phase is more polar than mobile phase

reverse phase - mobile phase more polar than stationary

Question 6

two most common solvents use in LC

Answer 6

methanol
acetonitrile

Question 7

what is considered when choosing a solvent

Answer 7

polarity and UV cut-off

Question 8

purpose of buffers

Answer 8

control pH (selectivity)
reduce peak tailing
give well shaped peaks

Question 9

What should the buffers pH be

Answer 9

+/-1 of buffer’s pKa

Question 10

are volatile or non volatile buffers preferred in LC-MSMS

Answer 10

Volatile because non volatiles contaminate ion source

Question 11

Thoery of UV detector

Answer 11

UV and photometers with intense light sources are quantified using beers law

Question 12

theory of electrochemical/amperometric detector

Answer 12

1. effluent passes voltage electrode
2. large voltage = movement of electrons = current
3. current is proportional to concentration of analyte

Question 13

what is voidtime (to)

Answer 13

time to elute unretained substance

Question 14

what is retention time

Answer 14

time elasped from injection of sample to recording of peak

Question 15

what value is good resolution

Answer 15

1.25 or greater

Question 16

what factors affect peak retention and peak width

Answer 16

retention
- capacity factor (k)
- selectivity (a)

width
- efficiency (N)

Question 17

what does capacity factor (k) measure and how to adjust it

Answer 17

retention time - change organic solvent

Question 18

what does selectivity factor (a) measure

Answer 18

retention time of two peaks - most important factor

Question 19

what does efficiency factor (N) measure and how

Answer 19

peak width - calculate number of theoretical plates

Question 20

why is selectivity the most imporant factor for resolution

Answer 20

selectivity is a function of column packing, mobile phase, and solute chemistry

Question 21

what does a large number of theoretical plates mean

Answer 21

narrow peaks = efficient column

Question 22

what factors increases efficiency (narrower peaks)

Answer 22

well packed and lengthy column

increasing flow rate

less volume injected

Question 23

why are samples extracted before injection

Answer 23

-prolong life of column
- peaks of interest
-improve sensitivity

Question 24

what does mass spec detect

Answer 24

fragmentation patterns

Question 25

theory of ionization -electron impact- in mass spec

Answer 25

1. electron beam of 70eV from a heated filament onto ion source 2. sample loses e- (unstable) = fragmentation 3. voltage on repeller plate causes ions to be accelerated to analyzer

Question 26

what is a quadrupole

Answer 26

1. mass analyzer 2. four rods (dc and rf voltages)

Question 27

what is the path of mass spec

Answer 27

1. inlet 2. ionization (electron impact) 3. mass analyzer (quadrupole) 4. detector and data

Question 28

what is the molecular ion and base peak

Answer 28

molecular ion = largest m/z peak base = most abundant peak

Question 29

problems of gc mass spec

Answer 29

heat labile compounds; methadone acetaminophen has poor chromatographs

Question 30

full scan vs SIM (select ion monitoring)

Answer 30

full scan = every ion SIM = derivitization step for selected ions (increase sensitivity)

Question 31

what is deuterated internal standard

Answer 31

3 hydrogen replaced with deuterum for controls

Question 32

pros and cons of LC MS

Answer 32

pro - LC is good for volatile and non volatile (GC only uses volatiles) - LC works with polar analytes (GC is poor extraction with polar) con - Ion suppression with high salt

Question 33

steps in electrospray ionization

Answer 33

1. form charged droplets 2. evaporation 3. ionic repulsion 4. ion enter mass analyzer

Question 34

what happens in triple quadrupole (tandem) MS

Answer 34

Q1 = single m/z selection Q2 = fragmentation in collision cell Q3 = single m/z selection to the detector

Question 35

principle of QTOF MS

Answer 35

separation of m/z by TOF -smaller = faster - heavier = slower Q1= filter Q2= fragmentation TOF = separation of ions