1. MAULANA ABUL KALAM AZAD UNIVERSITY OF TECHNOLOGY
CA-4 : POWERPOINT PRESENTATION
TOPIC : HPLC AND ITS RELATION WITH MASS SPECTROSCOPY.
GROUP 4:
B.SC. BIOTECHNOLOGY(4TH SEMESTER)
 DEBJANI SAHA
 DEBANSHU GHOSH
 ANKITA SHOME
 RAJDIP GHOSH
 SREEJIT DAS
 DOYEL PAL
 SALMAN HOQUE
B.SC BIOINFORMATICS(4TH SEMESTER)
 MEGHA GHOSH
 SAYARI PAL

2. INTRODUCTION
High Performance Liquid Chromatography (HPLC) is a
type of chromatography used to separate, identify,
and quantify compounds in a mixture. It is widely used
in the pharmaceutical, food, and environmental
industries. HPLC utilizes a high pressure pump to push
a mobile phase (a liquid or a gas) through a column
containing a stationary phase (a liquid or solid). The
compounds in the sample are separated based on
their interactions with the stationary and mobile
phases. The separated compounds are then detected
and quantified using a variety of detectors. This
technique is highly sensitive, reliable and reproducible,
making it one of the most widely used
chromatography techniques. The Liquid
Chromatography-Mass Spectrometry (LC-MS) is
hyphenated analytical technique which is combination
of Liquid Chromatography (LC) and Mass
Spectrometry (MS).

3. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY(HPLC)
 Aim: HPLC separates a mixture components based on their
polarity.
 Stationary phase: Dimethylpolysiloxane (Hydrophobic).
 Mobile phase: Polar solvent like borate buffer/acetic
acid(Hydrophilic).
 Principle:
1)The sample is injected into the mobile phase flow from the
pump to the separation column using a syringe.
2)Subsequently, the individual components of the sample
migrate through the column at different rates because they are
retained to a varying degree by interactions with the stationary
phase.
3) After leaving the column, the individual substances are
detected by a suitable detector and passed on as a signal to
the HPLC software on the computer.
4) At the end of this operation/run, a chromatogram in the
HPLC software on the computer is obtained.
5) The chromatogram allows the identification and
quantification of the different substances.

4.  There are three types of detector in HPLC:- (i)Refractive Index Detector
(ii) UV-Vis spectrophotometer(Scanning and variable wavelength detector).
(iii) Fluorescence Detector.
 More hydrophobicity, less polarity, retention time increases.
 Less hydrophobicity, more polar, retention time decreases.

5. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY-MASS
SPECTROSCOPY (HPLC-MS)
 High performance liquid chromatography-
mass spectrometry (HPLC-MS) is an
effective analytical technique for
determining the composition and purity of
chemicals.
 The combination of high performance
liquid chromatography (HPLC) and mass
spectrometry (MS) offers great capabilities
in physical separation and mass analysis,
providing accurate data on sample
composition.

6. OPERATING PRINCIPLE OF HPLC-MS
 In HPLC-MS, the two techniques (HPLC and MS) are connected by an interface that
transfers the separated components from the liquid chromatograph column into the
mass spectrometer ion source. An interface is needed since HPLC operates at high
pressure and the MS system has a high vacuum.
 HPLC includes a mobile phase and a stationary phase, both of which can be modified to
suit the sample matrix and the desired properties to be determined. Usually, the mobile
phase is adjusted to suit the sample, and the stationary phase is adjusted to work well
with the mobile phase. The degree of compound separation is based on the compound’s
affinity for the mobile phase.
 HPLC methods can be divided into two main categories based on the properties of the
stationary and mobile phases. A combination of polar stationary phase and non-polar
mobile phase is called "normal-phase chromatography" and the opposite of that, the
combination of non-polar stationery and polar mobile phase is called "reverse-phase
chromatography".

7. COMBINING HPLC WITH MASS SPECTROSCOPY
 Once compounds have been separated using HPLC, they are identified and their
contents are determined by mass spectrometry (MS), which creates a mass spectrum
that is unique for every compound. In mass spectrometry, the compounds and their
fragments are ionized using either electron or chemical ionization. The sample is then
accelerated through a mass analyzer, which includes either a quadrupole or an ion
trap, and the ions are identified based on their mass-to-charge (m/z) ratios.
 It is also possible to combine HPLC with detectors other than the mass spectrometer.
In HPLC-DAD analysis, for example, a diode-array detector is used instead. The choice
of the most appropriate detector depends on the analyte and the goal of the analysis.
In general, HPLC-MS is more suitable for identifying unknown components, while the
DAD-detector works well in quantifying known components.

9. APPLICATION OF HPLC-MS
 IN FOREINSIC SCIENCE: LC-MS is used for
determination of toxicity, in drug analysis
and also in trace analysis.
 IN DOPING TEST: The LC/ESI-MS with
positive mode can be used for detection
in urine of 4-Methyl-2-hexaneamine
doping agent.
 IN PHARMACOKINETICS: LC-MS is used in
the study of absorption, metabolism, and
excretion of drugs.
 IN BIOAVAILABILITY AND
BIOEQUIVALENCE STUDY: Comparative
bioequivalence studies in which
quantitative determination of drugs or
metabolites is measured in biological
matrix, pharmacodynamics, clinical trials
and In-vitro dissolution tests.

10.  IN DETERMINATION OF MOLECULAR
WEIGHTS: LC-MS is used for determination of
molecular weights of known and unknown
compounds.
 IN DETERMINATION OF ASSAY OF DRUG AND
INTERMEDIATES: LC-MS is used in
pharmaceutical industry for determination of
assay of drug substances, drug products,
intermediates and their related compounds.
 IN AGROCHEMICAL AND PESTICIDES
INDUSTRY: It is used in determination of
different components present in the fertilizers
and pesticides.
 ENVIRONMENTAL APPLICATIONS: LC-MS is
used for detection of phenyl urea herbicides,
detection of low level of carbaryl in food.

11. SOME RESEARCH WORK ON HPLC-MS
 THE DEVELOPMENT AND USE OF HPLC-MS FOR THE STUDY OF METABONOMICS IS REVIEWED.
 TO DATE THE TECHNIQUE HAS BEEN APPLIED TO THE ANALYSIS OF URINE SAMPLES OBTAINED
FROM STUDIES IN RODENTS IN INVESTIGATIONS OF PHYSIOLOGICAL VARIATION (E.G., FACTORS
SUCH AS STRAIN, GENDER, DIURNAL VARIATION, ETC.) AND TOXICITY.
 EXAMPLES ARE PROVIDED IN THE FORM OF GRAPHS TO THE USE OF CONVENTIONAL HPLC,
CAPILLARY METHODS AND THE RECENTLY INTRODUCED HIGH-RESOLUTION SYSTEMS BASED
ON A COMBINATION OF HIGH PRESSURE AND SMALL PARTICLE SIZE (“UPLC”).
 COMPARISON IS ALSO MADE OF THE USE OF 1HNMR SPECTROSCOPY AND HPLC-MS FOR THE
ANALYSIS OF BIOFLUID SAMPLES AND THE ADVANTAGES AND LIMITATIONS OF THE TWO
APPROACHES ARE ASSESSED.
 LIKELY FUTURE DEVELOPMENTS ARE CONSIDERED.

13. CONCLUSION
High-performance liquid chromatography or commonly known as HPLC,
is an analytical technique used to separate, identify or quantify each
component in a mixture. The mixture is separated using the basic
principle of column chromatography and then identified and quantified
by spectroscopy. The LC-MS is a hyphenated technique used in
combination with separation power of HPLC with detection power of
Mass spectrometry. It is widely used in pharmaceutical, chemical, food,
agrochemical industries, environmental and forensic applications. LC-MS
is used for qualitative and quantitative determination of drug substances
and biological samples. Also it is commonly used in drug research and
quality control.

14. ACKNOWLEDGEMENT
We would like to thank Mr. Arnab Kumar Ghosh, our respected professor for
introducing us to such a nice topic and for his valuable guidance in
completing our presentation.
I would like to take this opportunity to express my gratitude to all of my
group members Debanshu Ghosh, Rajdip Ghosh, Ankita Shome, Sreejit Das,
Doyel Pal, Salman Hoque, Megha Ghosh and Sayari Pal for their valuable
comment and suggestions on this presentation. We all have equally
contributed in this PowerPoint presentation.
by Debjani Saha

15. THANK YOU