HPLC/High Performance Liquid Chromatography/High Pressure Liquid Chromatography/Working&Instrumental

This video describes the principle of HPLC. The various types of mechanism that could be used with HPLC is listed. It is used to separate various components present in the mixture by the difference in the affinity of each component with the stationary phase and mobile phase. HPLC video explains the various types of columns and different types of chromatographic mechanism it can follow for each column. The instrumentation and working of HPLC, The different types of columns used are explained. The different types of detectors used in HPLC is discussed in detail. The advantages and disadvantages of this method is used. The various applications are explained.
High performance liquid chromatography is a technique used to separate the components/compounds present in a mixture based On the distribution of the components between the stationary phase and the mobile phase.
It is used to separate the liquid samples and solid samples dissolved in suitable solvents.
It is not suitable for gaseous samples.
All mechanisms of chromatography can be adopted by choosing an appropriate column
Adsorption chromatography
Normal phase chromatography
Reverse phase chromatography
Size exclusion chromatography (SEC) / Gel permeation chromatography (GPC)
Ion-exchange chromatography
Affinity chromatography
Mobile phase in HPLC
Solvent or eluent reservoirs
One reservoir - isocratic elution
Two or more reservoirs - gradient elution
moves from the reservoir to the mixing chamber regulated by the solvent proportionality valve
along with injected sample, the mobile phase is passed through the stationary phase at a constant flow rate/constant pressure.
Mixing chamber
mixes the solvents coming from the reservoirs
Mobile phase delivery system in HPLC
solvent reservoirs
solvent degassing unit
micro-filter
microprocessor - mobile phase
mixing chamber
controlled high pressure pump
High pressure pump in HPLC
Isocratic elution - One reservoir and one pump
Mixture of two solvents - binary system
Mixture of three solvents - ternary system
Mixture of four solvents - quaternary system
Gradient elution - Two or more reservoirs and independent pump for each reservoir
Single pump in conjunction with solvent proportionality valve
Sample injection port/valve injectori in HPLC - sample is injected using microsyringe into the injection port as a sharp pulse
Stationary phase in HPLC
Stationary phase is packed in the column uniformly at a very high density - increases resistance to flow of mobile phase - requires high pressure pump
It results in small plate height - large number of theoretical plates per unit length - gives rise to high separation efficiency
Adsorption chromatography and Normal phase chromatography - small rigid uniform particles of silica or alumina
Reverse phase chromatography - surface modified silica particles
Size exclusion chromatography/ Gel permeation chromatography - rigid porous polymeric beads (polystyrene)
Ion-exchange chromatography - ion-exchange resins based on synthetic or natural polymers
Affinity chromatography - consists of specific ligands coupled or immobilised on beads of agarose or other suitable solvents
Column in HPLC
Analytical Column - stainless steel or glass column containing packed bed of stationary phase- 25 cm long & 3-4mm diameter
Guard column/pre-column - placed between the sample injector and analytical column - to protect the main column from damage.
Detectors in HPLC
Solvent property detector - detects the change in the property of the mobile phase such as refractive index or conductance - refractive index detector or conductance meter
Solute property detector - detects the property of the solute - UV/Visible detector,
Photodiode array detector, fluorescence detector or electrochemical detector
Refractive index detector (RID) - used for SEC/GPC/samples without UV/visible absorption
No deflection of the light beam when the pure mobile phase passes through both the reference compartment and sample compartment.
Deflection of the light beam proportional to the amount of the solute component, when the pure mobile phase passes through the reference compartment and mobile phase mixed with solute through the sample compartment.
Conductance meter detector for HPLC- used for ion-exchange chromatography
UV/Visible/Photodiode array detector for HPLC (absorbance detectors) - samples with UV/Visible absorption
Data processor - It processes the signals coming the detector and sends to the recorder.
Recorder - Records the chromatogram by plotting the time against the detector signal response.
Advantages of HPLC
High speed compared to column and TLC- within 10 to 30 minutes.Accurate and highly reproducible.Quantitative sample recovery.Fully automated operation.High sensitivity
Disadvantages of HPLC
No universal detector.Less separation efficiency
Applications of HPLC

[@supriyahs2347]

One of the best lecture on HPLC with detailed explaination. Thank u.

[@RevathiPurushothaman]

It's my pleasure. Thank you.

[@supunkahawaththa6812]

Mam, how can i get your lecture slides if available

[@RevathiPurushothaman]

It will be available in my blog.

[@supunkahawaththa6812]

@@RevathiPurushothaman Thank You Very much

[@venkatramanan3068]

Vibrant voice.... Makes everything goes into my mind Mam. Thank you mam.....

[@RevathiPurushothaman]

Thank you very much.

[@ramneetkaurlotey1899]

Extremely useful videos

[@RevathiPurushothaman]

Thanks for your compliments.

[@sanafatima8002]

Too much fast

[@RevathiPurushothaman]

Usually I have heard people telling that I am a bit slow. This video alone was a little fast because the content was heavy. Otherwise the video will become too longer. You can always go back and see repeatedly. My other videos are not this fast.

[@amrithanagalingam1994]

Adjust playback speed

[@nandamuriganesh1709]

Nice

[@RevathiPurushothaman]

Thank you.

[@theextrovertedintrovert_am]

Hi Revathi. Is there a good textbook you can recommend that goes into the theory, instrumentation and applications of different analytical techniques. Thanks in advance.

[@RevathiPurushothaman]

Analytical chemistry by Gary Christian, Instrumental methods by Willard, Merritt,

[@theextrovertedintrovert_am]

@@RevathiPurushothaman Thank you very much🌸