What is pump in HPLC?
Pump
is a part of hplc which push the mobile phase and spl to pass through the
column with a specific flow rate which we provide.
Types of Pump
Isocratic
Pump
Mobile
phase composition remains same throughout the analysis, we mix the mobile phase
here
Gradient
pump
Mobile phase composition changes over time, we can set and changes the polarity of mobile phase in the system software, here system mix the mobile phase according to polarity ratio
How to differentiate that it is Back or system pressure?
First
Solution is to remove Column and check if pressure of the systems become stable
it is due to back pressure if it is still high it is due to Contamination it is
system pressure washed the columns properly
Why zigzag peak appeared in Gradient HPlC?
Because
pump continuously not flow, it mix mobile phase and push so peak issue occur.
Retention Time: The time required for a sample to withstand in a column is called RT
Relative Retention Time: Ratio between peak area of interest to the peak area of reference
RRT=
RT of peak A/ RT of peak B
RRT=
5/10= 0.5 minutes
If
it is less than 1 first peak will be RRT
In HPlC Calibration which Compound is used?
Caffeine
Why caffein?
ü Cheap and easily available
ü Stable durable
ü Show 2 maxima 205,273 and one minima 245
Column is made of?
Steel
What is Stationary phase made of?
Silica
gel
Why silica gel?
stable
inert
Which column is used mostly?
- C18 mostly and C8
- C18 is hydrophobic RT more
- C8 less hydrophobic RT less
Related substance and chromatographic impurities different - why?
We
can find both Known and unknown impurities with the help of Related substance
We
can find the known impurities only with the help of chromatographic impurities
Baseline
Stable
signal obtain when Mobile phase pass through the column without spl
Why we used buffer in Mobile phase?
Bcause
to stable the pH of mobile phase
Why
we used Guard column?
To
avoid impurities from the column
What is carryover?
Small
amount of previous spl remain in the system which lead to the failure in the
results of new spl, analyt from previous effect new one
How many kind of buffer used?
Two
kind mostly
1.
phosphate buffer
2.
acetate buffer
How to store column?
Store
on the basis of storage condition, mostly methanol and Acetonitrile (organic
solvent used in reverse phase)
Water
is not used because it will lead to fungal growth
Highest
pressure range: depends on company water US base company has 5000psi
Dead
volume: injector to column inlet and outlet to detector volume is dead volume, extra
volume
Dwell
volume: in Gradient HPlC mixer to column inlet is Dwell volume
Back pressure high / causes
ü blockage or air trap
ü inline filter block
ü Column wrong or contaminated
ü Fitz block
Why area variation occur?
ü Air trap
ü spl not mix well
ü Column contaminated
Baseline are not become stable why?
Mobile phase contaminated or column is not wash or high flow rate is used, if baseline disturb in mid it mean column is weak