This document provides an overview of the key components and functioning of high performance liquid chromatography (HPLC). It describes the main units including pumps, mixing chambers, injectors, columns, detectors, and recorders/integrators. It explains how each component works and its role in the HPLC separation process. Some examples of applications of HPLC are also listed such as quantitative and qualitative analysis of compounds, isolation of drugs, stability studies, and more.

1. M.PRASAD NAIDU
Msc Medical Biochemistry,
Ph.D Research scholar.

2.  Pumps – solvent delivery system
 Mixing unit, gradient controller & solvent
degassing
 Injector – manual or auto injectors
 Guard column
 Analytical columns
 Detectors
 Recorders & integrators

3.  Solvents or mobile phases must be passed via column @ high pressure
(1000 – 3000psi)
 b/cos the particle size of stationary phase is few µ (5-10 µ)
 Diff types of pumps available
 Mechanical pumps operate with constant flow rate & uses sapphire
piston
 This type of pump is used in analytical scale
 Pneumatic pumps operate with constant pressure & use highly
compressed gas
 The solvents used must be of high purity preferably HPLC grade &
filtered through 0.45 µ filter
 Check valves: to control the flow rate of solvent & back pressure
 Pulse dampeners: to dampen ( make slightly wet) the pulses

4. 2 types of mixing units
Low pressure mixing chamber which uses He
for degassing solvents
High pressure mixing chamber does not
require He for degassing solvents
Mixing of solvents is done either by
Static mixer: packed with beads
Dynamic mixer : uses a magnetic stirrer &
operates under high pressure

5.  In an isocratic separation, mobile phase is of
same polarity throughout the process
 In gradient elution tech, the polarity of the
solvent is gradually increased & hence the
solvent composition has to be changed.
 Hence a gradient controller is used when two
or more solvent pumps are used for such
separations

6.  Several gases are soluble in org solvents
 When solvents are pumped under high pressure, gas bubbles
are formed – interfere the separation process
 3 methods
 1.Vacuum filtration: which can remove the air bubbles. But is
not always reliable & complete
 2. He purging: by passing He through the solvent.Very
effective but He is expensive
 3. Ultrasonication: by using ultrasonicator, which converts
ultra high frequency to mechanical vibrations – this causes
the removal of air bubbles

7.  1. Septum injectors: injecting through a rubber septum. Not
common – has to withstand high pressure
 2. Stop flow (online): in which the flow of mobile phase is
stopped for a while & the sample is injected through a valve
device
 3. Rheodyne injector ( loop valve type): the most popular
injector.
 This has a fixed volume loop like 20 µl or 50 µl or more
 Injector has 2 modes
 1. load position: when sample is loaded in the loop
 2. Inject mode: when the sample is injected

8.  It has a very small quantity of adsorbent &
improves the life of the analytical column
 Also acts a a prefilter to remove particulate
matter, if any & other material
 GC has the same material as that of analytical
column
 GC does not contribute to any separation

9.  The most imp part of the HPLC tech which decides the
efficiency of separation
 Column material: Stainless steel ( mostly used), glass,
polyethylene and PEEK (poly ethylene ether ketone) (latest)
 Column length: 5 cm to 30cm
 Column diameter: 2mm to 50 mm
 Particle size: 1 µ to 20 µ
 Particle nature: spherical, uniform , porous materials are
used
 Surface area: I gm of stationary phase provides surface area
ranging from 100-860 sq.m ( an aveg of 400sq.m)

10.  The functional group present in stationary phase
depends on the type of chromatographic separation
 In normal phase mode – contains silanol groups (
hydroxy group)
 In reverse phase mode –
 C18 – Octa Decyl Silane (ODS) column
 C8 – Octyl column
 C4 – Butyl column
 CN – Nitrile column
 NH2 – Amino column

11.  1. UV- detector: used based upon the light absorption characteristics of
the sample
 2 types of this detector are available
 a) fixed wavelength detector: which operates at 254 nm where most drug
compounds absorb
 b) variable wavelength detector: which can be operated from 190nm to
600nm
 2. Refractive index detector: non-specific or universal detector – not
much used- low sensitivity and specificity
 3. Flourimetric detector: more specificity & sensitivity
 Demerit: some compounds are not fluorescent
 4. Conductivity detector:
 5. Amperometric detector:
 6. Photodiode array detector( PDA detector)

12.  Recorders are used to record the responses
obtained from detectors after amplification
 They record the baseline & all the peaks obtained
with respect to time (Rt)
 But the area of the individual peaks cannot be
known
 Integrators: improved version of recorders with data
processing capabilities
 Rt, height and width of peaks, peak area, % area
 Integrators provide more information on peaks than
recorders

13.  Qualitative analysis
 Checking the purity of a compound
 Presence of impurities
 Quantitative analysis
 Multicomponent analysis or determination of mixture of drugs
 Isolation & identification of drugs
 Isolation and identification of mixture of compounds
 Biopharmaceutical & pharmacokinetic studies
 Stability studies
 Purification of compounds
 Environmental applications
 Forensic applications
 Biochemical separations
 Biotech, food analysis