1. Applications of Advanced Chromatographic Techniques
Subject :- Methods in Pharmaceuticals Research (MPR -PHSCC1201)
DEPARTMENT OF PHARMACEUTICAL SCIENCES
Dr. HARISINGH GOUR VISHWAVIDYALAYA
Sagar ( M.P)- 470003, India
(A Central University )
SUBMITTED BY :-
SUMIT S. KOLTE
( M Pharm Sem I )
Phaemaceutics
SUBMITTED TO:-
PROF. ASMITA GAJBHIYE
(PROFESSOR, DOPS)
PROF. SUSHIL K. KASHAW
(PROFESSOR, DOPS)
Session 2023-2024

2. CONTENTS
 Introduction
 Advanced chromatographic Techniques ;
 LC / MS
 GC / MS
 UPLC
 OPLC
 Chiral chromatography
 Applications
 References

3. INTRODUCTION
 Chromatography:- Chromatography is a physical method of separation in which the
components to be separated are distributed between two phases, one of which is
stationary (stationary phase) while the other (the mobile phase) moves in a definite
direction.
 Mikhail Tswett’s invented chromatography in 1903 during his research on plant
pigments.

5. Advanced chromatographic Techniques
1) Liquid chromatography–Mass spectrometry (LC–MS)
o (LC–MS) is an technique that combines the physical separation capabilities of liquid
chromatography (or HPLC) with the mass analysis capabilities of mass spectrometry
(MS).

6. Applications of LC/MS
 LC/MS plays a key role in the drug discovery .
 LC-MS is frequently used in drug development because it allows quick molecular
weight confirmation and structure identification.
 LC-MS is used in proteomics as a method to detect and identify the components of a
complex mixture.
 LC-MS is also used for the analysis of natural products and the profiling of
secondary metabolites in plants.
 LC-MS include the analysis of food, pesticides, and plant phenols.
 The LC-MS technique is useful for the detection of steroid drugs in body fluids and
in profiling endogenous steroids.
 LC-MS can be used to analyse a wide range of environmental samples – from air
and soil to wastewater, sludge and drinking water

8. 2 ) Gas chromatography–Mass spectrometry (GC–MS)
 (GC/MS) combines two powerful techniques to provide the identification of
compounds with low detection limits and the potential for quantitative analysis.
 Gas Chromatography Mass Spectrometry analysis can be performed on liquids,
gases or solids.

10. Applications of GC/MS
 GC-MS is used in screening tests for the detection of several congenital metabolic
diseases. It detects trace levels of compounds present in the urine of patients with
genetic metabolic disorders
 Monitoring environmental pollutants is a major application of GC-MS.
 GC-MS is used in research and development, production, and quality control. It is
used in identification of impurities in active pharmaceutical ingredients.
 Aromatic compounds such as fatty acids, esters, aldehydes, alcohols, and terpenes
present in food and beverages can be easily analyzed using GC-MS. This technique
can also be used to detect c spoilage or contamination of food.
 GC-MS is widely used in forensic toxicology to identify poisons and steroids in
biological specimens and in anti-doping labs to detect performance enhancing drugs
such as anabolic steroids.

11.  GC-MS can be used for the bioanalysis of body fluids to detect narcotics,
barbiturates, alcohols, and drugs such as anticonvulsants, anesthetics,
antihistamines, sedative hypnotics, and anti-epileptic drugs
 Explosive detection systems in public places use GC-MS technique for the analysis
and detection of chemical warfare agents.
 Identification and quantitation of volatile and semi volatile organic compounds in
complex mixtures.
 GC-MS can be used for Petrochemical and hydrocarbon analysis.
 Also Used in Food, beverage and perfume analysis

12. 4) Ultra Performance Liquid Chromatography (UPLC)
 UPLC enhance mainly in three areas: "speed, resolution and sensitivity.
 The principles of UPLC are same principle as HPLC
 The basic difference is in designer of the column material particle size which
less than 2-um.
 The separation and quantification in UPLC is done under very high pressure (up to
100M Pa)
 Time and solvent consumption is less in UPLC
 The principle of UPLC is based on Van Deemeter equation which describes the
relationship between flow rate and column efficiency.

13. Applications of UPLC
 UPLC is widely used for analysis of natural products and herbal medicines &
provides high-quality separations And detection capabilities to identify active
compounds in highly complex samples .
 UPLC help in critical laboratory function by increasing efficiency, reducing costs,
and improving opportunities for business success.
 UPLC is used for the highly regulated, quantitative analyses performed in QA/QC
laboratories. The supply of consistent, high quality consumable products Plays an
important role in a registered analytical
 Determination of pesticides: - UPLC couples with triple Quadra tandem mass
spectroscopy will help in identification of trace level of pesticides from water
 Analysis of amino acid: - UPLC used for accurate, reliable and reproducible analysis
of amino acids in the areas of protein characterizations, cell culture monitoring and
nutritional Analysis of foods.

14.  Dissolution Testing: - In sustained-release dosage formulations, testing higher
potency drugs is particularly important. The dissolution profile is used to
demonstrate reliability and Batch-to-batch uniformity of the active ingredient. UPLC
provides precise and reliable automated online sample acquisition. It automates
dissolution testing, from pill drop to test start.
 Bio analysis / Bioequivalence Studies: - The sensitivity and selectivity of
UPLC/MS/MS at low detection levels generates accurate and reliable data that can
be used for a variety of Different purposes, including statistical pharmacokinetics
analysis.
 ADME (Absorption, Distribution, Metabolism, and Excretion) Screening: - The
high resolution of UPLC enables accurate detection and integration of peaks in
complex matrices and extra sensitivity allows peak detection for samples generated
by lower concentration incubations and sample pooling. These are important for
automated generic methods as they reduce failed sample analyses and save time.
 UPLC fingerprint can be used for the identification of magnolia officinalis cortex.

15. 4) Overpressure layer chromatography (OPLC)
 Over pressured layer chromatography (OPLC) is a modified planar chromatographic
technique, in which the vapor phase is eliminated by covering the sorbent layer with
an elastic membrane under variable external pressure,
 And the mobile phase is forced through the sorbent layer with a pump.
 The method is particularly advantageous over thin-layer chromatography (TLC),
 A capillary-controlled system, in that better resolution and more rapid separation
can be obtained.

16. Applications of OPLC
 Determination of impurities in pharmaceutical products and reaction mixtures
 Extraction of compounds of pharmacological interest from natural products
 Isolation of metabolites in biological fluids
 Purification of reaction mixtures to extract the compound of interest for additional
studies (e.g. for NMR or MS)
 Separation of oligomers of natural (e.g. peptides) and synthetic polymers (e.g.
polystyrene)
 Determination of toxins in foodstuffs (e.g. aflatoxins in wheat)

17. 5) Chiral chromatography
 Chiral chromatography refers to the separation of optical isomers, or enantiomers.
 Chiral: A molecule is chiral if it is not superimposable on its mirror image.
 Most chiral molecules can be identified by their lack of a plane of symmetry or
center of symmetry.
 Enantiomers or Optical isomers: Two mirror images of chiral molecules.
 Chiral chromatography refers to the separation of enantiomers using a chiral HPLC
column, an HPLC column packed with chiral stationary phases.
 Approximately 60% of Pharmaceutical Drugs are chiral.

18. Applications of Chiral chromatography
 Chiral chromatography is widely used in the pharmaceutical industry particularly
for drug discovery and confirmation of enantiomeric drug purity, food science, and
environmental analysis.
 The process have been applied to a wide variety of natural products such as nucleic
acid, urine, serum, carbohydrates, lipids, amino acids, bile acids, and manufactured
products such as pharmaceuticals, pesticides, herbicides, surfactants and
antioxidants.
 Mainly used for the separation of barbiturates.
 It is used in industry primarily for separation of chiral molecules like Serine,
Soman, Glyceraldehyde's, Phosphorus, Sulfarmetal , Cobalt , Enkephalins.
 This technique is more important in case of drugs, because many drugs shows
different activities with different enantiomer.
 Resolution of Single enantiomers simplify the human toxicology profile and can
have numerous benefits such as reduced side effects and/or reduced dosage
requirements.

19. References
 Braun R., Introduction To Instrumental Analysis, Second Edition, PharmaMed Press,
Hyderabad, Page no. 251-270.
 Chatwal G.R., Anand S.K., Instrumental method of Chemical Analysis, Himalaya
Publishing House, Fifth Edition-2012, New Delhi, Page no. 420-449.
 Instrumental Methods Of Chemical Analysis By Willard
 Ultra Performance Liquid Chromatography (UPLC) A Review Taleuzzaman M, Ali
S, Gilani SJ, Imam SS and Hafeez http://www.waters.com/waters/en.IN/UPLC
 Schurig, V., Chiral separations using gas chromatography. TrAC, TrendsAnal.
Chem. 2002, 21 (9/10), 647-661.
 Raffaelli A, Saba A. Atmospheric pressure photoionization mass spectrometry. Mass
Spectrum Rev. 2003;22:318-31
 Google Images