HPLC INTRO AND TROUBLESHOOTING

1. High Performance
Liquid Chromatography
NAME : SIRSHENDU ROY

2. INTRODUCTION
DEFINATION OF HPLC :
It is a chromatographic technique used to separate
components of mixture for the purpose to identify, quantify or
purify the individual components of the mixture.
This is widely used in field of biochemistry and analytical
chemistry.

3. Types of HPLC techniques
Based on mode of chromatography.
Based on principle of separation.
Based on elution technique.
Based on scale of operation.
Based on types of analysis.

4. Based on modes of chromatography
Normal Phase mode:
The stationary phase is polar in natural & the mobile phase is
non-polar.
This is not advantageous in pharmaceutical application since most
of the drug molecules are polar in nature and takes longer time to
be eluted and detected.
Reverse phase:
He stationary phase is non-polar & the mobile phase is polar
in nature.
Since most of the drugs and pharmaceuticals are polar in nature,
they are not retained for a longer time and eluted faster.

5. Based on principle of separation
Adsorption chromatography: Separation of components
takes place because of the difference in affinity of compounds
towards stationary phase.
Ion exchange chromatography: An ion is used to separate
a mixture of similar charged ions.
 Size exclusion or gel permeation chromatography: A
mixture of components with different molecular sizes are
separated by using gels which acts as sieve.

6. Based on elution technique
Isocratic separation: In this technique, the same mobile
phase combination is used throughout the process of
separation. The same polarity or elution strength is maintained
throughout the process.
Gradient separation: In this technique, a mobile phase
combination of lower polarity or elution strength is used
followed by gradually increasing the polarity or elution
strength.

7. Based on the scale of operation
Analytical HPLC: Where only analysis of the samples are
done. Recovery of the samples is not done
Preparative HPLC: Where the individual fractions of pure
compound can be collected using fraction collector. The
collector samples are reused.

8. Based on the type of analysis
Qualitative analysis: Which is used to identify the
compound, detect the impurities, to find the number of
components, etc.
Quantitative analysis: Which is done to determine the
quantity of the individual or several components in a mixture.
This can be done by comparing peak area of the standard and
sample

9. INSTRUMENTATION HPLC instrument
consists of following components:
 Pump
 Mixing unit
 Solvent degassing
 Injector
 Column
 Detectors
Application

11. Pump
The role of the pump is to force a liquid ( called the mobile
phase) through the liquid chromatography at a specific flow
rate, expressed in milliliters per min (mL/min)
Normal flow rates in HPLC are in the 1-2mL/min range.
During the chromatographic experiment, a pump can deliver
a constant mobile phase composition (isocratic) or an
increasing mobile phase composition (gradient).

12. Types of pump
Mainly three types:
Constant flow reciprocating pump
 Syringe type pump
 Pneumatic pump

13. MIXING UNIT
Mixing unit is used to mix solvents in different proportions and pass
through the column.
There are two types of mixing units.
1.they are low pressure mixing chamber which uses helium for
degassing solvents.
2.high pressure mixing chamber does not require helium for
degassing solvents
Mixing of solvent is done either with a static mixer which is packed
with beads or a dynamic mixer which uses magnetic stirrer and
operates under high pressure.

14. INJECTOR
• The injector serves to introduce the liquid sample into the
flow stear the mobile phase.
• Typical sample volumes are 5-20microliters
• The injector must also be able to withstand the high pressure
of the liquid system.
• An auto sampler is the automatic version for when the user
has many samples to analyze or when manual injection is not
practical.

15. COLUMN
It is the heart of the chromatograph Column length: varies from
5cm to 30cm Column diameter: ranges from 2mm to 50mm
Particle size: from 1µ to 20µ Particle nature: spherical, uniform
sized, porous materials are used.

16. DETECTORS
Detectors are used with high performance
liquid chromatography to detect and identify analytes in the
sample.
 UV detectors
Refractive index detector
 Flourimetric detector
Conductivity detector
Amperometric detector

17. APPLICATIONS OF HPLC
• Qualitative analysis
• Checking the impurity of a compound
• Presence of impurities
• Quantitative analysis
• Isolation and identification of drugs
• Isolation and identification of mixture of components
• Biopharmaceutical and pharmacokinetic studies
• Stability studies
• purification.

18. what to do before start Analysis in HPLC ?

19. How to deal with solvents
• Use clean bottles only.
• Exchange water-based solvents daily.
• Select solvent volume to be used up within 1 – 2 days.
• Use only HPLC-grade solvents and water filtered through 0.2 μm
filters.
• Label bottles correctly with bottle content, and filling date / expiry date.
• Use solvent inlet filters to protect the system from incoming particles.
• Reduce risk of algae growth: use brown bottles for aqueous solvents,
avoid direct sunlight or wrap the bottles in aluminium foil.

20. Daily tasks
• Replace solvents and solvent bottles for mobile phases based on
water/buffer.
• Replace solvents and solvent bottles for organic mobile phase latest
every second day.
• Check presence of seal wash solvent.
• Purge each channel with fresh solvent at 2.5 – 3 mL/min for 5 min.
• Equilibrate your system with composition of your application for 15
min.
• Check the instrument analysis label and calibration label.

21. Weekly tasks
• Change seal wash solvent (10 % / 90 % isopropanol/water) and
bottle.
• Flush all channels with water at 2.5 – 3 mL/min for 5 min to
remove salt deposits if buffer applications were used.
• Inspect solvent filters for dirt or blockages. Clean or exchange if
no flow is coming out of the solvent line when removed from the
degasser inlet.

22. Power-up the system
• Power up the pump
Use new or different mobile phase (as required).
Purge each channel with 2.5 – 3 mL/min for 5 min. Open the purge valve or use the purge
command .
Equilibrate your system with composition of your application for 15 min. Use conditioning for
systems.
• Power up the sampler
Purge the autosampler daily, and before and after sample analysis, especially if you are using
buffers.
Set flow to required value of your application and close the purge valve.
Pump for approximately 10 min.
Use fresh needle wash and/or needle seat backflush solvents like methanol or acetonitrile and
water mixtures without buffer.
Ensure that the vials contain enough sample solution for all injections.
• Power up the detector
Warmup the lamp for at least 1 h.
For RI detectors only: flush the reference and sample side with fresh solvent used for the current
application.

23. Shut-down the system
• Long-term storage of the column
Flush the column with appropriate solvent found in the column manual.
Remove and seal column, and store according to good laboratory practice if
needed.
• Long-term shut-down of the system
Flush system with water to remove buffer.
Remove all samples from the sampler and store according to good laboratory
practice.
Use recommended solvents to store the system.
Power off the system.

24. Recommendations for pumps
• Check pumps performance on regular basis.
• Perform preventive maintenance in the recommended usage
interval.
• Prepare the pump as recommended like described in the power up
section to ensure optimal performance and best life time.
• Use the seal wash function as recommended to ensure optimal
performance and best life time, see below.

25. Recommendations for samplers
• Purge the autosampler after sample analysis.
• Always use fresh wash solvent for the needle or seal wash
function.
• Place the wash solvent reservoir for needle wash (optional: needle
seat flush) into the solvent cabinet.
• Check the drainage routing of the wash port outlet into a waste
container.
• Fill each vial with enough sample solution for all injections.
• Filter, decant, or centrifuge sample to separate from insoluble
solid.

26. Recommendations for detectors
• Warm-up the lamp at least 1 h.
• Keep environment and ambient temperature stable.
• Do not expose the detector to too much air current from the HVAC.
• Use the recommended waste lines for each detector type. Avoid pinching
the waste tube after the cell outlet.
• Ensure that the detector flow cell is bubble free.
• For RI detectors only: flush the reference and sample side with fresh
solvent used for the current application.
• Flush the flow cell after use.
• Use isopropanol to remove organic solvents & milli-q water to remove
salts.

27. Additional Information for pumps
• Purge
Fill the system with fresh or different solvent.
Remove air bubbles in tubes and pump heads.
• Condition
If micro air bubbles persist in the pump head the overall pump
performance may look correct but the pump will perform extra
work and accuracy/precision are negatively affected. To remove
the air efficiently the Condition function can be used.

28. THANK YOU