LIQUID CHROMATOGRAPHY-

MASS SPECTROSCOPY(LC-MS)

Presented by:
Name: Mohammad Altaf Khan
Roll number:155
 INTRODUCTION

• LC-MS(liquid chromatography-mass spectroscopy)

LC-MS is achieved by combination of two the techniques.
1. HPLC(high performance liquid chromatography)
It is used to separate, identify, and quantify each component in a
mixture.
2. Mass Spectroscopy
It is used to measure the mass-to-charge ratio of ions. 
• Combining HPLC and MS is difficult becasue.
HPLC MS
Liquid Phase Vaccum operation
operation.
25 to 50 degree 200 to 300 degree
celcius. celcius.
Require inorganic Require volatile
buffers. buffers.

Hence we can’t COMBINE THEM DIRECTLY.

• For combining we use INTERFACE.
(Interface means by which interaction or communication is achieved)
• Types of Interfaces.
1. Electrospray ionization (ESI)
2. Thermospray ionization(TSI)
3. Atmospheric pressure chemical ionization(APCI)
4. Atmosphheric pressure photoionization (APPI)
• Removing detectors from HPLC and using these interfaces we can combine mass
spectroscopy.
 HPLC AND MS

• HPLC
1. It has two phases, one is mobile phase and another is stationary phase.
2. Mobile phase: Is Liquid in which sample is flow. Commonly used mobile
phases are organic solvents such as acetonitrile, and methanol.
3. Stationary phase: Are the column packed with a porous particles.
• Column used for LC-MS
1. Fast LC column: 15-50mm
2. Micro LC column: 20-150mm
• HPLC can analyse analyse samples over a wide polarity range
including ioninc samples.
• HPLC samples prepared in organic solvent.
• Sample injection volume is 1-100µl is common.
• MS (Mass spectroscopy)
1. Operates under high vaccum
2. Generating ions from ionizer.
3. Analyzer (magentic sector analyzer, Quadrupole, Ion trap mass
analyzer)
• Analyses the ions into charge to mass ratio
INSTRUMENTATION

LC-MS
• HPLC
• Interface(ionization
source)
• Mass analyzer
• Detector system
• Mass spectrum
 WORKING
• ESI (Electrospray ionization)
1. ESI generates ions by spraying the sample from tip of the capillary
tube.
2. And high voltage is applied about 3 to 5kv
3. Nebulizer or heating gas outside the capillary speed up the
evaporation.
4. Solvent get evaporated from droplets, and then undergo reduction.
ESI(Electrospray ionization)
• Mass analyzer
• The ionozed sample now in mass analyzer.
• Magnetic sector mass analyzer

Ionized sample
from ESI
• Lighter ions are deflected too much and heavier ions are deflected too
little.
• Only ions that matches to the mass range reach to the detector.
• A 10‐7 vacuum is applied to the metal analyzer tube.
• Mass to charge ratio can be calculated with this formula:
m/z=B²r²/2V
m = mass of ion; B = magnetic field
z = charge of ion r = radius of circle
V = voltage
DETECTION
(Ions from mass analyzer hit detector)
• Detector such as electron multiplier
1. Ions strikes to the wall of detector
2. Electrodes are placed discrete manner
3. Ions are hitting the electrodes continuously and multiplies.
4. Mmultiplied ions are equivalent to electrical signal and get recorded.
APPLICATION AND DISADVANTAGES

• It has a wide range of apllications

1. In Proteomics: systematic analysis of protiens.
2. Pharmokinetics: To study the drug absorption, distrubution,
metabolism, and excretion.
3. Forensic science: Drug analysis, toxicology, and trace elements.
4. Agrochemistry: The determination of compounds that are present in
fertilizers, pesticides, etc.
5. It also used in cosmetic industries.
• Disadvantages
1. High cost.
2. Required an experienced technician.
 REFERENCES

1. https://www.shimadzu.com/an/service-support/technical-support/analysis-basics/fun
damental/interfaces_for_lcms.html#section1
2. https://www.shimadzu.com/an/service-support/technical-support/analysis-basics/fun
damental/mass_analyzers.html#section1
3. https://en.m.wikipedia.org/wiki/Liquid_chromatography%E2%80%93mass_spectro
metry
4. https://www.khanacademy.org/science/ap-chemistry-beta/x2eef969c74e0d802:atomi
c-structure-and-properties/x2eef969c74e0d802:mass-spectrometry-of-elements/a/isot
opes-and-mass-spectrometry
5. https://www.google.com/url?sa=t&source=web&rct=j&url=https://m.restek.com/pdf
s/GNBR1000A-UNV.pdf&ved=2ahUKEwiintDYpqzuAhUFzTgGHb3TA34QFjABe
gQIDxAH&usg=AOvVaw0UuAN5VGBoaFDr-L1hAqQg

6. https://warwick.ac.uk/fac/sci/lifesci/research/sigtraf/animations/
7. https://youtu.be/EzvQzImBuq8
THANK YOU!