High Performance Liquid Chromatography (HPLC) drl.pptx

HIGH
PERFORMANCE
LIQUID
CHROMATOGRAPH
Y (HPLC)
BY- PRAKHAR RAI

INTRODUCTION
 HPLC is a physical separation technique conducted in the liquid phase in which a sample is
separated into its constituent components (or analytes) by distributing between the mobile
phase (a flowing liquid) and a stationary phase (sorbents packed inside a column).
 An online detector monitors the concentration of each separated component in the column
effluent and generates a chromatogram.
 The solvent usually flows through column with the help of gravity but in HPLC technique the
solvent will be forced under high pressures up to 400 atmospheres so that sample can be
separated into different constituents with the help of difference in relative affinities.
 It is a popular analytical technique used for the separation, identification and quantification of
each constituent of mixture, and widely used in field of clinical research, biochemical
research, industrial quality control and etc.

PRINCIPLE
 The principle of HPLC is based on adsorption as well as partition chromatography and
depending on the nature of stationary phase, if stationary phase is solid principle is based on
adsorption chromatography and if stationary phase is liquid principle is based on partition
chromatography.
 It is important for determination of volatile and non volatile compounds
 It is important for determination qualitative and quantitative analysis.
 It is important for determination of Retention Time ( the time is required, after sample
injection maximum angle peak reaches to detector)

 The compounds bind at specific regions of stationary phase based on certain physical and chemical
properties . These bound molecules are then eluted with suitable buffer and the same are collected with
time.
 These are –
 polarity
 charge
 molecular weight
 Present of functional group
PRINCIPLE CONTI…

GOAL
 The goal of HPLC analysis is to separate the analyte(s) from the other components in the sample in
order to obtain accurate quantitation for each analyte. Towards this goal, several common-sense axioms
are:
 The sample must be soluble
 For separation, analytes must be retained and have differential migration in the column
 The mobile phase controls HPLC separation
 The final analyte solution should be prepared in the mobile phase

FUNDAMENTAL CONCEPTS
 Retention: The time between the sample injection point
and the analyte reaching a detector is called the retention
time (tR). The retention time of an unretained component
(often marked by the first baseline disturbance caused by
the elution of the sample solvent) is termed void time (to).
 Capacity Factor (k’): The capacity factor measures how
many times the analyte is retained relative to an unretained
component. A more fundamental term that measures the
degree of retention of the analyte is the capacity factor or
retention factor (k’).

 Void Volume (V0): Void volume is the volume of the empty column minus the volume occupied by the
solid packing materials. It is the liquid holdup volume of the column that each analyte must elute from.
Note that the void volume is equal to the void time multiplied by the flow rate (F).
V0=t0 F
 Selectivity (∝): Selectivity or separation factor (a) is a measure of differential retention of two analytes.
It is defined as the ratio of the capacity factors (k') of two peaks as shown in Figure.
FUNDAMENTAL CONCEPTS CONTI…

 Column Efficiency (tR): As readily observed in most chromatograms, peaks tend to be Gaussian in
shape and broaden with time, where Wb becomes larger with longer tR .This is caused by band-
broadening effects inside the column, and is fundamental to all chromatographic processes.
 Plate Number (N): The term, plate number (N), is a quantitative measure of the efficiency of the
column, and is related to the ratio of the retention time and the standard deviation of the peak width (σ).
Since Wb is equal to 4σ, the following equation has been derived:

 Height Equivalent of a Theoretical Plate or Plate Height: The height equivalent of a theoretical
plate (HETP or H) is equal to the length of the column (L) divided by the number of theoretical plates
(N) even though there are no discrete plates inside the HPLC column.
Height Equivalent of a Theoretical Plate, HETP (H)= L/N
 As will be shown later, HETP is determined by particle diameter of the packing material
(dp) and to a lesser extent by the flow rate (F) and column temperature (T). For a well-
packed column, the plate height is roughly equal to 2.5dp.
 Therefore, a column packed with 5micrometer particles would have a plate height of
12.5micrometer.
 A typical 15cm long column packed with 5micrometer materials should have
N=L/H=150,OOOmicrometer/ 12.5 micrometer = 12,000 plates.

 Resolution (Rs): Resolution is the degree of separation of two adjacent analyte peaks, and is defined as
the difference in retention times of the two peaks divided by the average peak width. Most often, a
target value of Rs>2.0 is desirable because such a condition leads to more robust separation and
quantitation.

 Tailing Factor (Tf):
 Under ideal conditions, chromatographic peaks will have Gaussian peak shapes with perfect
symmetry. In reality, most peaks are either slightly fronting or tailing.
 Tailing factors for most peaks should fall between 0.9 and 1.4, with a value of 1.0 indicating a
perfectly symmetrical peak.
 Peak tailing is typically caused by adsorption or other strong interactions of the analyte with the
stationary phase while peak fronting can be caused by column overloading, chemical reaction or
isomerization during the chromatographic process.

TYPES OF HPLC
Depending on the substrate used i.e. stationary phase used, the HPLC is divided into following types:
 Normal Phase HPLC- In this method the separation is based on polarity. The stationary phase is polar,
mostly silica is used and the non-polar phase used is hexane, chloroform and diethyl ether. The polar
samples are retained on column.
 Reverse Phase HPLC- It is reverse to normal phase HPLC. The mobile phase is polar and the
stationary phase is non polar or hydrophobic. The more is the non-polar nature the more it will be retained.
 Size-exclusion HPLC- The column will be incorporating with precisely controlled substrate
molecules. Based on the difference in molecular sizes the separation of constituents will occur.
 Ion-exchange HPLC- The stationary phase is having ionically charged surface opposite to the sample
charge. The mobile phase used is aqueous buffer which will control pH and ionic strength.

Parts of HPLC instruments:
1. Solvent storage bottle
2. Gradient controller and mixing unit
3. De-gassing of solvents
4. Pump
5. Pressure gauge
6. Pre-column
7. Sample introduction system
8. Column
9. Detector
10. Recorder
INSTRUMENTATION CONTI…

 SOLVENT/ MOBILE PHASE RESERVOIRS:
The contents of mobile phase are present in glass container. In HPLC the mobile phase or solvent is a
mixture of polar and non-polar liquid components. Depending on the composition of sample, the polar
and non-polar solvents will be varied.
 DEGASSING & FILTRATION OF MOBILE PHASE:
• In many cases, aqueous solvents & some organic solvents are degassed prior to use.
• Degassing is done to prevent formation of gas bubbles in the pump or detector (Mobile phases are
degassed by stirring of the mobile phase under vacuum, sonication or sparing with helium gas).
• The mobile phase are filtered to remove particulate matter that may clog the system.
 Tubing:
Should be inert, have ability to withstand pressure and able to carry sufficient volume

 Pump:
 The solvents or mobile phase must be passed through a column at high pressures at up to 6000 psi(lb/in²) or
414 bar.
 As the particle size of stationary phase is smaller (5 to 10µ) the resistance to the flow of solvent will be high.
 That is, smaller the particle size of the stationary phase the greater is the resistance to the flow of solvents,
Hence high pressure is recommended.
 Pulse free output & all materials in the pump should be chemically resistant to solvents.
 Flow rates ranging from 0.1 to 10 mL/min.
 Pumps should be capable of taking the solvent from a single reservoir or more than one reservoir containing
different solvents simultaneously.
 Sample Injector:
 The injector devices are available either for manual or auto injection of the sample. An injector for a HPLC
framework should give infusion of the fluid specimen inside the scope of 0.1 mL to 100 mL of volume with
high reproducibility and under high pressure (up to 4000 psi).

 COLUMN:
1. Precolumn
 It contains a packing chemically identical to that in analytical column.
 Mainly used to remove the impurities from the solvent and thus prevents contamination of the analytical
column, it can protect analytical column.
 It is also called as guard column or protective column.
 it is having large particle size.
 It is having short length of 2 to 10 cm, so does not affect separation.
2. Analytical column
 The success or failure of analysis depends upon choice of column.
 Actual separation is carried out here.
 Stainless –steel tube
 size – length -25 to 100 cm

 Internal diameter – 2 to 4.6 mm
 Column is filled with small particles 5 – 10 micron. The solid support can be silica gel, alumina.
 The separation is result of different components adhering to or diffusion into the packing particles when the mobile
phase is forced through column. C8 and C18 columns are considered as examples of reversed phase liquid
chromatography (RP).
 The stationary phase here is seen as a thin film of non-polar liquid phase that has been designed to be chemically
similar to an inert material (Silica gel particles).
 The non-polar layer is chemically linked to the silica particles surface by reaction with the polar silanol groups on
the stationary phase surface and so rendering them less polar or non-polar.
 The difference between the two columns will be in the length of the carbon-chain attached to the silica surface.
 Accordingly C8 HPLC columns have packing material composed of silica particles attached to C8 carbon units.
 Categorically both are reversed phase but C18 columns will definitely be more "hydrophobic rather than the C8
columns.

 DETECTORS: An HPLC detector is often a modified spectrophotometer equipped with a small flow
cell, which monitors the concentration (or mass) of eluting analytes. The function of the detector used
in HPLC is to monitor the mobile phase it emerges from column.

 UV/VIS ABSORBANCE DETECTORS: The UV/Vis
absorbance detector monitors the absorption of UV or
visible lights by analytes in the HPLC eluent. They are
prevalent because most pharmaceuticals have
absorbance in this region. A typical UV/Vis detector
consists of a deuterium source, and a monochromator to
focus the light through a small flow cell.
 Specifications: Sensitivity, Linearity and Band
dispersion.

 PHOTODIODE ARRAY (PDA) DETECTORS: A PDA detector provides UV spectra of eluting
peaks in addition to monitoring the absorbance of the HPLC eluent like the UV/Vis absorbance
detector. It is the preferred detector for testing impurities and for method development. PDA facilitates
peak identification during methods development and peak purity evaluation during method validation.
Detector sensitivity was an issue in earlier models but has improved significantly (more than ten-fold)
in recent years.
 Fluorescence (FI): A fluorescence detector monitors the emitted fluorescent light of the HPLC eluent.
It is selective and extremely sensitive (pg to fg) to highly fluorescent compounds. Its application is
limited since few pharmaceutical ingredients have strong innate fluorescence. A fluorescence detector
can be a regular fluorescence spectrophotometer fitted with a small flow cell.

 Refractive Index (RI): An RI detector measures the difference in RI between the sample cell
containing the eluting analyte and the reference cell (containing pure eluent). It offers universal
detection but has lower sensitivity (0.01-0.1~tg) than UV/Vis absorbance detection and is more prone
to temperature and flow changes. RI detection is used for components of low chromophoric activities
such as sugars, triglycerides, organic acids, pharmaceutical excipients and polymers.
 Evaporative Light Scattering Detector (ELSD): An ELSD reduces the HPLC eluent into a particle
stream and measures the scattered radiation. It offers universal detection for non-volatiles or semi-
volatiles and has higher sensitivity than the RI detector (low ng) in addition to being compatible with
gradient analysis. An ELSD consists of a nebulizer equipped with a constant temperature drift tube
where a counter-current of heated air or nitrogen reduces the HPLC eluent into a fine stream of analyte
particles. A laser or a polychromatic beam intersects the particle stream and the scattered radiation is
amplified by a photomultiplier.

 Electrochemical Detector (ECD): An ECD measures the current generated by electroactive analytes
in the HPLC eluent between electrodes in the flow cell. It offers sensitive detection (pg-levels) of
catecholamines, neurotransmitters, sugars, glycoproteins, and compounds containing phenolic,
hydroxyl, amino, diazo or nitro functional groups.
 Conductivity: A conductivity detector measures the electrical conductivity of the HPLC eluent stream
and is amenable to low-level determination (ppm-ppb levels) of ionic components such as anions,
metals, organic acids and surfactants.

 Radioactivity: A radioactivity detector is used to measure radioactivity in the HPLC eluent
using a flow cell. The detection principle is based on liquid scintillation technology to detect
phosphors caused by the radiation, though a solid-state scintillator is often used around the
flow cell.
 Mass Spectrometry (MS): LC/MS is the ultimate analytical technique, which combines the versatility
of HPLC with the identification power of MS. The weak link in LC/MS has always been the interface
which connects the liquid stream at atmospheric pressure to the high vacuum present inside the mass
spectrometer.

ANALYSIS OF PHARMACEUTICAL COMPOUND BY USING
HPLC
 Various steps are involved:
1. Nature of analyte / Physicochemical properties of drug molecule.
2. System suitability of the instrument.
3. Selection of chromatographic conditions.
4. Developing the approach of analysis.
5. Sample preparation
6. Method optimization
 Nature of analyte / Physicochemical properties of drug molecule:
Before performing HPLC of any drug molecules, we take some important parameters into consideration related to
the nature of analyte:
1. pH and pKa
2. polarity
3. thermostability
4. solubility

 System suitability test is an essential part of HPLC method. It is used to verify that the chromatographic system is
suitable for the intended analysis. That is to ensure that the complete testing system including instruments, electronics,
reagents, column & analyst is suitable for intended application.
Four parameters are:
1. Theoretical Plate Count
2. Peak Symmetry
3. Peak Width
4. Resolution
HPLC CONTI…

 Selection of chromatographic conditions and approach of analysis:
1. Stationary phase: HPLC systems consisting of polar stationary phases and non-polar mobile phases are described
as normal phase chromatography; those with non-polar stationary phases and polar mobile phases are called
reversed-phase chromatography.
2. Mobile phase: The choice of mobile phases is based on the desired retention behaviour and the physicochemical
properties of the analyte as well as the type of detector chosen.
a. For normal-phase HPLC using unmodified stationary phases lipophilic solvents should be employed.
b. The presence of water in the mobile phase must be avoided as this will reduce the efficiency of the stationary
phase. In reversed-phase HPLC aqueous mobile phases, with and without organic modifiers, are used.
c. The mobile phase should be filtered through suitable membrane-type filters to remove particles or
undissolved material. Multicomponent mobile phases should be prepared by measuring the required volumes
(unless masses are specified) of the individual components, followed by manual or mechanical mixing.
Alternatively, the solvents may be delivered by the individual pumps or proportioning valves of the liquid
chromatograph and mixed according to the desired proportion. Solvents are normally degassed by sparging
with helium or by means of sonification before pumping to avoid the formation of gas bubbles in the detector
cell.
d. Mobile phase should not be monophasic. Binary mobile phase is the most appropriate to use. If ternary and
quaternary mobile phase is used it should be justified of why multiple solvent is used.
HPLC CONTI…

4. Flow rate: Flow rate decide the volume of mobile phase passing through the column in unit time. When
there is an increase of the flow rate the peak will come near to zero as there will be faster elution but if there
is and decrease of flow rate the peak will move away. Flow rate of mobile phase is to be 1ml/min, 2ml/min
3ml/min etc.
5. Retention time: Retention time (RT) is a measure of the time taken for a solute to pass through a
chromatography column. It is calculated as the time from injection to detection. Retention time should be
less than 10.
6. Run time: The time during a chromatographic separation is indicated as the run-time. The
total time necessary for completing a chromatographic separation is slightly longer than the retention time of
the last peak in the chromatogram. This time is sometimes referred to as total runtime, or length of the
chromatogram. First, make sure that there are no other peaks eluting after your peak of interest. If there are
later eluting peaks, you will need to extend your run time to encompass those peaks. If you cut your run
time short and there are late eluting peaks, you run the risk of peaks wrapping around into your next
injection.
7. λmax: For HPLC we first separate and then analyze the sample peak using a DAD (aka: PDA) to find
the maximum peak wavelength in which we can reliably use to detect the sample apex. This is called
the lambda max wavelength, "λmax". Each compound will have one or more.
HPLC CONTI…

 Sample preparation:
 The drug substance being analyzed should be stable in solution (diluent). During initial method
development, preparations of the solutions in amber flasks should be performed until it is determined that
the active component is stable at room temperature and does not degrade under normal laboratory
conditions.
 The sample solution should be filtered; the use of a 0.22 or 0.45 μm pore-size filter is generally
recommended for removal of particulates. Filtration is a preventive maintenance tool for HPLC analyses.
Sample preparation is a critical step of method development that the analyst must investigate. The
effectiveness of the syringe filters is largely determined by their ability to remove contaminants/insoluble
components without leaching undesirable artifacts (i.e., extractables) into the filtrate. If any additional
peaks are observed in the filtered samples, then the diluent must be filtered to determine if a leachable
component is coming from the syringe filter housing/filter.
 After the peak is obtained the calibration curve is to be plotted taking concentration on x axis and AUC on
y axis, respectively and the required value of y=mx+c and r2 is to be acquired.
Where, y= response function m= slope x= concentration c= intercept r2= regression coefficient
HPLC CONTI…

 Procedure:
1. To equilibrate the column allow the mobile phase to flow through the chromatographic system until the baseline
and the retention times of the substances to be analysed are stable at the flow rate specified in the individual
monograph (usually about 30 minutes).
2. Prepare the prescribed test and reference solutions as directed.
3. Inject the prescribed reference solution and, if necessary, adjust the detector and/or recorder response to produce
an adequate peak size.
4. For chart recorders and integrators this should be at least 50% of the full-scale deflection of the principal peak in
the chromatograms obtained with the reference solution. Ensure that the criteria for system suitability are met.
5. The reference solution should be injected at the start, at regular intervals during and at the end of a series of assays
(e.g. every 2–4 samples). Both the reference and the test solutions should be injected in duplicate.
6. In determining the component composition of a complex mixture, a "normalization" procedure, based on the
calculation of individual peak areas as a percentage of the total area of all the peaks may be used where the
relative response factors of the individual components are similar and it has been demonstrated that the signals
(responses) of the principal (major) and the minor peaks are within the linear range of the detector.
7. The response factor is relative, being the response of the equal mass of one substance relative to that of another
according to the conditions described in the test.
HPLC CONTI…

APPLICATION OF HPLC
The HPLC has several applications in the fields of pharmacy, forensic, environment and clinical. It also helps in the
separation and purification of compound.
1. Pharmaceutical Applications: The pharmaceutical applications include controlling of drug stability, dissolution
studies and quality control.
2. Environmental Applications: Monitoring of pollutants and detecting components of drinking water.
3. Forensic Applications: Analysis of textile dyes, quantification of drugs and steroids in biological samples.
4. Food and Flavour Applications: Sugar analysis in fruit juices, detecting polycyclic compounds in vegetables,
analysis of preservatives.
5. Clinical Applications: Detecting endogenous neuropeptides, analysis of biological samples like blood and urine.

REFERENCES
1. Dong W. Michael and Ahuja Satinder, Handbook of pharmaceutical analysis by HPLC, Separation science
and technology, Published by ELSEVIER Academic Press, first Edition 2005, volume 1, Page No. 20-43.
2. Yandamuri Narayudu, K R Srinivas Nagabattula, Kurra Subrahmanya Swamy, Batthula Sahana, L P S
Nainesha Allada and Bandam Poojitha. Comparative Study of New Trends in HPLC: A Review. Int. J.
Pharm. Sci. Rev. Res., 23(2), Nov – Dec 2013; 10, 52-57.
3. Thammana Mukthi, “A Review on High Performance Liquid Chromatography (HPLC)” published in
research & review: Journal of Pharmaceutical Analysis, RRJPA | Volume 5 | Issue 2 | July – September,
2016.
4. Douglas A. Skoog, F. James Holler, Stanley R. Crouch, Principle of instrumental analysis, Published by
Belmont C.A. USA, 6th edition in 2007, Page no.746-756.
5. https://www.differencebetween.com/difference-between-isocratic-and-gradient-elution/ ( Accessed on
25/02/2022)
6. https://pediaa.com/difference-between-preparative-and-analytical-chromatography/ ( Accessed on
25/02/2022)

6. Bhardwaj k. Santosh, Dwivedi k., Agarwal D.D., A Review: HPLC Method Development and
Validation, Published in International Journal of Analytical and Bioanalytical Chemistry, 20
November 2015, 5(4), 76-81.
7. Patil Pallavi N. HPLC method development - a review, Published in SGVU Journal of Pharmaceutical
Research & Education, 2017, 2(1), 243-260
8. https://apps.who.int/phint/pdf/b/7.1.14.4.1.14.4-High-performance-liquid-chromatography.pdf
(Accessed on 25/02/2022)
REFERENCES

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