HPLC , CONTENTS OF HPLC , PRINCIPLE OF HPLC .INSTRUMENATATION OF HPLC . FACTOR AFFECTING HPLC. ADVANTAGES . DISADVANTAGES . AND APPLICATIONS OF HPLC

1. HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY

2.  HPLC is also known as High pressure Liquid
Chromatography → It is very effective seperation
Technique. Short time required for seperation.
 HPLC uses tightly packed Columns containing small
particles of the Stationary phase (solid non-polar) The
efficiency of the HPLC Column is relatively high.
 HPLC Stationary phases typically have packed particle
diameter b/w 2 and 40 micrometers. If the particle is less
than 2 micrometers, the seperative technique is Ultra high
performance liquid chromatography (UHPLC).
 The HPLC is a method of seperation in which the
Stationary phase is contained in a column, one end of
which is attached to a source of pressurized eluent mobile
phase.

3.  In HPLC, eluent from the solvent reservoir is filtered,
Pressurized and pumped through the chromatography
Column.
 A mixture of solutes injected at the top of the Column is
seperated into components on travelling down the column
and the individual solutes are monitored by the detector and
recorded as peaks on a Chart recorder.
 The main Component of HPLC are a high pressure pump, a
column/injector system and a detector.
 In addition component, such as solvent reservoir, in line
filters, pressure guages, recorders and minors components
are required.

5.  (1) Solvent delivery system including pump.
 (2) Sample Injection system.
 (3) A chromatographic Column
 (4) A detector and Recording system

6.  The mobile phase is pumped under pressure from
reservoir and flows through the column at a
Constant rate. It is advisable to use deaerated Mobile
phase solvent mixture using a vaccum pump
(deaerated means that has no effect on Composition
of mixture).
 For Normal phase seperations, eluting power
increases with increasing polarity of solvent.
 For reserved phase seperations, eluting power
decreases with increasing solvent polarity.

7.  Another most important component of HPLC is the
pump because its performance directly affects the
retention time, reproducibility and detector
sensitivity.
 Pumpes used in HPLC
 (a) Reciprocating piston pumps/constant flow pumps.
 (b) syringe type pumps.
 (c) Constant pressure pumps/non-reciprocating
pumps.
 (d) Pneumatic pumps.

8.  It consist of a small motor driven piston which
moves rapidly back and forth in a hydraulic chamber
that may vary from 35-400 μ1 in Volume.
 on the back stroke, the seperation Column Valve is
Closed and the piston pulls in solvent from the
Mobile phase reservoir.
 on the forward stroke, the pumps pushes solvent out
to the Column from the reservoir.
 Dual type of pump system is significantly smoother
because one pump is filling while the other is in
delivery cycle.

9.  single head mechanical reciprocating pumps have
been developed because of the following important
advantage.
 (a) These pumps one the most convenient to use.
 (b) These are relatively easy to flush and rinse.
 (c) cost effective.
 (d) These are more reliable.

10.  These pumps are most suitable because these pumps
deliver only a finite volume of mobile phase before it
has to be refilled.
 Volume 250 ml to 500ml.
 The pump operates by a motorized lead screw that
delivers mobile phase to the column at a constant
rate.
 The rate of Solvent delivery is controlled by
changing the voltage on the motor.

11.  In constant pressure pumps the mobile phase is
driven through the column with the use of pressure
from a gas Cylinder.
 The valving arrangement allows the rapid refill of the
Solvent Chamber whose capacity is about 70ml.

12.  The amount of the sample to be injected into the Column
after the preparation can be determined by the components
of interest have to be detected with accuracy after
seperation of the Column.
 This amount depends on the two main factors.
 (1) The sensitivity of the detector for those components.
 (2) The extent of dilution undergone in the Column.
 The injection process is depends on the following factors
 (A) Type of the injection system used.
 (B) Connection pattern b/w the injector and the Column.
 (2) Injected volume as well as the injection time.
 The injection system should be convenient to use, able to
operate at high temperature, chemically inert with the
eluent and the sample and reproducible .

13.  (1) Fixed Volume Valve Injection→ Fixed volume valve
injection in which a fixed volume is introduced by making
use of a fixed volume loop injector.
 (2) Variable Injection valve→Variable injection Valve
which a variable volume is introduced by making use of an
injection valve.
 This type of Injectors contains a needle port which can be
closed at high pressure to insert a syringe.
 The sample is loaded at almost atmospheric pressure.
 (3) On Column Injection→This is a variable Volume is
introduced by means of a syringe through a septum.
 This is simplest form of sample Introduction

14.  Depending on particle size two methods are used for Column packing
 (1) for relativelylarge particles(>15-20 μm)
 A dry packing technique has been used which involves adding the dry
column packing material slowly either continuously or in small portion
while bouncing, tapping and rotatingthe column.
 (2) In case of small particles.
 In this a slurry packing techniqueis used.
 This technique involves coupling the Column to be packed to a reservoir,
filling the column with a supporting liquid and the reservoir with slurry of
the column packing material in a suitable supportingliquid.
 The reservoir is connected to pump and a pressure of 3000-10000 psi is
applied to the system in order to force the slurry into the analytical
column.
 During slurry packing particle fractionation should be avoided by packing
the slurry into the column at high pressure by making use of correct slurry
liquid.

15.  (A) Porous, Polymeric beds based on styrene divinyl
benzene co-polymers.
 These are used mainly for ion exchange and size exclusion
chromatography. These have been now replaced by silica based
pickings which are more efficient and mechanically more stable.
 (B) Porous Layer beds (diameter 30-55 μm) consisting of a
thin shell (1-3μm) of silica on a spherical inent core.
 After the development of totally porous microparticulate
packing, these have not been used much in HPLC.
 (C) Totally porous silica particles with narrow particle size
range (diameter < 10 μm).

16.  The detector for an HPLC is the component that emits a
response due to the eluting sample compound and
subsequently signals a peak on the chromatogram.
 It is positioned immediately posterior to the stationary phase
in order to detect the compounds as they elute from the
Column.
 The detector for HPLC consists of a photometric detector
fitted with a low volume flow cell.

17.  (1) Bulk property Detectors. which compare an overall
changes in a physical property of the mobile phase with and
without an eluting solute.
 Ex-Refractive index and Conductivity detectors.
 (2) solute property Detectors → which respond to a physical
property of a solute which is not exhibited by the pure mobile
phase.
 such type of detectors is about 1000 times more sensitive,
giving a detectable signal for a few monograms of sample.
 Ultraviolet, visible adsorption, fluorescence and
electrochemical detectors.

18.  Radiochemical detection involves the use of radio
Labeled materical, usually tritium (3H) or Carbon-
14 (14C). It operates by detection of fluorescence
associated with beta-particle ionization and it is
most popular in metabolite research .

19.  (A) Retention Time.
 (B) Adjusted retention Time.
 (C) Retention factor.
 (D) Column efficiency.
 (E) Asymmetry peaks.
 (F) Resolution.

20.  Difference in time b/w the sample injection and
appearance of peak maxima.It is denoted by tR
 Different compounds have different retention
times.
 Retention time may vary depending on
 (i) Pressure used.
 (ii) Nature of the stationary phase.
 (iii) Composition of the mobile phase.
 (iv) Temperature of the column.

21.  measurement b/w retention time (tR) and unretained
time(to)
 t’R = tR-to
 Ex-Retention time of compound(tR)=5min
 unretained time (to)=1min
 Adjusted retention time (t'R) = tR- to
 =5-1
 = 4 min.

22.  Ratio of adjusted retention time (t'R) and unretained
time (to)
 k = tR- to/to
 Ex- (ART) t'R = 8 min, to = 2 min.
 Retention factor (k) = 8-2/2 = 6/2=3 min.
 Higher the k value, greater the resolution.

23.  -It is also known as plate count.
 Retention time ↑- Peak width ↓- efficiency ↑
 High equivalent theoritical plate (HETP)
 HETP=L N = Length of the Column no. of
theortical plates
 HETP ↑- Column efficiency ↑

24.  Ideal chromatographic peaks are symmetrical, if
asymmetrical peak are obtained then it is said to be
inappropriate.
 There are two types of Asymmetrical peaks-
 (1) Fronting- is due to more active adsorptive site in
the Stationary phase.
 (2) Tailing- due to saturation of stationary phase.

26.  It is defined as the difference in retention times b/w
the two components divided by the combined width
of elution peaks
 Rs = 2 (tR(B)-tR(A)/Wa +Wb
 Ideal resolution value should be greater than
2.

27.  Resolution is affected by three important parameters
(1) Selectivity (seperation factor)
 (2) Efficiency
 (3) Retention (capacity factor)
 Current FDA Values for the validation of chromato
graphic methods.

28.  (1) All type of biological molecules have been purified.
 (2) Reverse phase HPLC is useful for seperation of polar
compounds such as drugs and their metabolites, peptides,
vitamins,polyphenols and steroids.
 (3) It is work widely used in clinical and pharmaceutical
as it is possibly to apply biological fluids such as serum
and urine directly to the Column.
 (4) In seperation of oligopeptides and proteins.
 (5) seperation of high polar compounds such as amino
acids, organic acids etc.

29.  Tablet dissolution study armaceutical dosages form.
Of armaceutical dosages forms.
 Shelf-life determinations of parmaceutical products•
 Identification of active ingredients of dosage forms
 Pharmaceutical quality control.
 (7)Forensics.
 Quantification of the drug in biological samples.
 Identification of anabolic steroids in serum, urine,
sweat, and hair Forensic analysis of textile dyes.
 Determination of cocaine and metabolites in blood

30.  Quantification of ions in human urine Analysis of
antibiotics in blood plasma.
 Estimation of bilirubin and bilivirdin in blood plasma in
case of hepatic disorders.
 Detection of endogenous neuropeptides in extracellular
fluids of brain.
 (9).Food and Flavor.
 Ensuring the quality of soft drink and drinking water.
 Analysis of beer.
 Sugar analysis in fruit juices.
 Analysis of polycyclic compounds in vegetables.
 Trace analysis of military high explosives in
agricultural crops.

31.  (A) High speed.
 (B) High resolution .
 (C) High Senstivity.
 (D) Automatic .
 (E)sample is recovered completely.

32.  costly
 complex to operate and doesn’t work for all samples.
 Solvents consuming.

33.  D Muralidhara Rao, AVN Swamy, D Dharaneeswara Reddy,
"Instrumental methods of Analysis", CBS Publishers and
distributers. P.no-271-277.
 ROBERT D. BRAUN- Text book of “Introduction to
Instrumental Analysis”,2nd Edition.
 GURDEEP R. CHATWAL, SHAMK. ANAND- Text book of
“Instrumental method of chemical anlysis”, Himalaya
publishing house.
 MALVIYA R. BANSAL V, PAL O.P. AND SHARMA P.K.-
“Departmennt of pharmaceutical pharmaceutical technology”,
MIET Meerut, Article in journal of global pharma technology.
June2010.

34.  F. JAMES HOLLER, DOUGLAS A. SKOOG-Text
book of “Principle of Intrumental analysis”,PP.931-
933.

35. THANKYOU
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