Chrom test 2

Question 1

what type of pump is used on HPLC systems?

Answer 1

Piston- single and dual types

Question 2

whare are the disadvantages of single piston pumps?

Answer 2

creates pulses which affects consistency. change in baseline. erratic flow

Question 3

What are the differences between nano, UPLC, HPLC - analytical, and LC - preparative columns?

Answer 3

difference in size. column is larger for each. so higher flowrate and pump required.
preparative= bigger column
analytical = smaller

nano: uL/min
UPLC:
HPLC

Question 4

What is the difference between a dual piston and an accumulator pump
-How many check valves are in each?

Answer 4

dual piston pump: left piston = fill
right piston = deliver
4 check valves
Accumulator-piston pump: bottom piston= fill
top piston = deliver

3 check valves

Question 5

What is a check valve?

Answer 5

one way valve that prevents the flow from going backwards

Question 6

What is a pulse dampener and what is it used for?

Answer 6

used to lower pulsations

Question 7

What happens to our chromatography if we have a leak in the system?

Answer 7

longer retention times, mp may go in wrong direction, lower back pressure

Question 8

What are four common symptoms of pump failures?

Answer 8

1) retention time fluctuation
2) noisy baseline
3) visual abnormalities
4) audible abnormalities

Question 9

What are the symptoms of air in the pump and how do we fix it (don’t need procedure)?

Answer 9

pressure not constant (you cant set pressure, you observe it)
inexplicable chromatographs
solution: prime the pump (removes air and fills line with solvent

Question 10

What are the causes of lower or no pressure and how do we solve them?

Answer 10

is the pump turned on? = turn it on
is the pump actually pumping solvent? = check waste line
is there a leak in the joints? = check for leaks
faulty transducer?

Question 11

What are the causes of higher pressure and how do we solve them?

Answer 11

clog in system? = work backwards to see what brings pressure down
this is more efficient

Question 12

What do we use pressure for?

Answer 12

to make sure that the instrument is working properly.
column packing, injection, elution

Question 13

What can cause no flow and how do we fix it?

Answer 13

is pump on?
is pressure where it should be?
is solvent below LC?
are there any leaks?

Question 14

What are the differences between isocratic and gradient methods?

Answer 14

*isocratic: can only provide one mobile phase composition throughout the run. useful for size separation
*Gradient: can pump more than one solvent at a time and blends them

Question 15

What is the difference between high pressure mixing and low pressure mixing? What are the differences between binary and quaternary systems?
o Dwell volume, number of solvents, mixers

Answer 15

high pressure mixing: 2 pumps (binary system). the speed of pump is managed by a gradient controller and the two streams are combined. between pump and column so its under pressure. (2 solvents)

low pressure mixing: one pump and a gradient proportioning valve that combines the solvent and sends to the pump. its before the pump so low pressure. (up to 4 solvents, quat)

Question 16

What is dwell volume? What is it and what is its impact?

Answer 16

how much tubing is between column and point of mixing. determines length of isocratic hold. large dwell volume = loss of resolution

Question 17

What is the flow of a manual injector in the load position and injector positions (general)?

Answer 17

Load position: mobile phase flows from pump. valves, column.

injector position: mobile phase flows from the pump, valves, loop and then column

Question 18

What type of loop does a manual injector use?

Answer 18

fixed loop

Question 19

What are the benefits and disadvantages of fixed loop and variable loop injectors?

Answer 19

fixed loop: benefits: low system volume. disadvantage = carryover.

Variable: benefits: little carryover. disadvantage: high system volume

Question 20

Why do we prefer to overload fixed loops?

Answer 20

band spreading and wider peaks

Question 21

Why can we use partial fill with autosamplers but not manual injectors?

Answer 21

autosampler can insert gaps on either side of the sample

Question 22

What determines the ideal injection volume?

Answer 22

depends on loop and column size.
should be less than 5% of column
depends on injection solvent

Question 23

How do we calculate injection volume?

Answer 23

less than 5% of column volume

Question 24

What are the two types of overloading? What are the symptoms?

Answer 24

mass loading
injection volume
symptoms: shark fin

Question 25

What is the impact of diluent on one’s chromatography? What is an ideal diluent?

Answer 25

diluent should be the same as injection solvent or weaker.
higher strength = broadened peaks

Question 26

When do we overload the column on purpose - two situations?

Answer 26

when purifying samples which elutes away from the other compounds
impurity profiling

Question 27

How does mobile phase pH impact mass loading for ionizable species?

Answer 27

reversed phase retention can be altered significantly by manipulating pH. impacts peak shape (shark fin)

Question 28

Contrast carry over and contamination. How can we tell the difference between them?

Answer 28

carryover= small amount of previous sample left in the injector which gets injected with the new sample. to tell, peaks go away with repeated blank injections

Question 29

What is longitudinal diffusion and why is it important in chromatography

Question 30

What is extra column dead volume? Where does it reside and what is its impact?

Answer 30

any volume external to the column that does not contribute to the separation process.
resides in filters, fittingsm connections, tubing.
impact= poor chromatography. too much tubing = poor peak shape. causes leaks, broadening of bands. poor efficiency

Question 31

What happens to our chromatography when we increase tubing ID and length?

Answer 31

increased tubing = poor peak shape
ID increase = dispersion and distortion of bands. broadening of peaks, less sensitive. sample must travel further through tube so more dispersion will occur (more band spreading)

Question 32

What is the impact of tubing machining on one’s chromatography?

Answer 32

symmetrical, smooth opening = better results
if not cut properly, flow path can be altered.

Question 33

Label a cross section of a connection indicating the position of the ferrule, nut, and pilot

Answer 33

nut, ferrule, pilot

Question 34

Where is a ferrule, what is its purpose?

Answer 34

ensures that everything is seated and sealed

Question 35

What is a pilot, what is its purpose? What happens if it is too long or too short?

Answer 35

pilot: length of tubing that extends from the ferrule
too long: screw cant fully sit in the fitting. liquid can leak out. lowers the pressure.
too short: a void is created in the system. affects peak shape

Question 36

Why don’t we use Teflon tape?

Answer 36

it can dissolve in the mobile phase and contaminate analysis.
it can break up and introduce particulates into the system.

Question 37

Compare void volume vs dwell volume vs ECDV

Answer 37

dwell volume: distance from head of column to point of mixing

void volume: volume between injector and detector (including column)

ECDV: distance from injector to detector, not including column

Question 38

How do we measure void volume?

Answer 38

inject compound known not be retained.
monitor for change in signal
first peak seen will represent the void.

for reverse phase, sodium nitrate can be used. very polar salt while stationary phase is non-polar.
sometimes “blips” in baseline = void.