SAGAR SAVALE 1

Liquid chromatography and the mass spectrometry

(LC-MS)

Mr. Sagar Kishor Savale

(Department of Pharmaceutics, North Maharashtra University, college of

R.C.Patel Institute of Pharmaceutical Education and Research, Shirpur, 425405,

Dist.Dhule, Maharashtra.)

avengersagar16@gmail.com

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1. Introduction

• It is the combination of liquid chromatography and the mass spectrometry.

• Liquid chromatography-mass spectrometry (LC-MS) is an analytical chemistry

technique that combines the physical separation capabilities of liquid

chromatography with the mass analysis capabilities of mass spectrometry.

• The combination of these two powerful techniques gives the chemical analyst the

ability to analyze virtually any molecular species; including, thermally labile, non-

volatile, and high molecular weight species.

• It has been said that over 80% of known organic species are amenable to separation

with liquid chromatography.

• Mass spectrometry is capable of providing structure, molecular weight, empirical

formula, and quantitative information about a specific analytes so, LCMS in recent

years, liquid chromatography/mass spectrometry (LC/MS) has become one of the

most powerful analytical techniques for qualitative and quantitative analysis .

• AIMS - To identify the different proteins, peptides drugs in various samples also to

study the bioequivalence in future

2. Advantages of LC-MS

2.1 Provides compound identity.

2.2 Provides sensitive response to most analytes.

2.3 Provides compound class information.

2.4 Provides compound structure.

2.5 Provides sequence information.

2.6 Provides molecular weight information.

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2.7 Provides the five 5 Important Criteria

Speed, Selectivity, Specificity, Sensitivity, Low Cost

3. Sample Consideration for LC-MS

3.1 The analytes must have ionizable groups such as Amines, Carboxylic Acids, Ketones

and Aldehydes.

3.2 For best sensitivity, work at a pH where the analytes is ionized

3.3 i.e. for acid, Neutral to basic pH (7-9) and Acidic pH (3-4) for bases.

4. Parts of LC-MS

5. Flow Chart of LC-MS

Sample Column Detector Eluent

Port

Collector

Ionization Source Vacuum

Mass analyzer

Detector

Read out device

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6. Liquid Chromatography

6.1 Liquid chromatography is type of chromatography in which analytes molecule get

partitioned between moving mobile phase and stationary phase

6.2 In liquid chromatography mobile phase is always liquid while stationary phase is either

liquid or solid.

6.3 Liquid chromatography includes following chromatographic techniques:

6.3.1 Paper chromatography

6.3.2 Thin layer chromatography

6.3.3 Adsorption column chromatography

6.3.4 High performance liquid chromatography

6.3.5 Ion exchange chromatography

6.3.6 Liquid-liquid chromatography

7. Instrumentation of

1.1 Detectors

1.2 Pumps

1.3 Column heaters

1.4 Auto samplers

8. Applications of LC:

8.1 Separation

8.2 Purification

8.3 Quantification

9. Mass Spectroscopy

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9.1 Mass spectroscopy is an analytical technique used to measure the mass-to-charge-

ratio of ions it is most generally used to find the composition of a physical sample by

generating a mass spectrum representing the masses of sample components.

Sample Inlet Ionization source Mass Analyzer Detector Read out

Vacuum

9.2 The stages within the mass spectrometer are:

9.2.1 Producing ions from the sample.

9.2.2 Separating ions of differing masses.

9.2.3 Detecting the number of ions of each mass produced.

9.2.4 Collecting the data and generating the mass spectrum.

9.3 Application of MS

9.3.1 Identifying unknown compounds by the mass of the compound molecules or their

fragments. Determining the isotopic composition of elements in a compound.

9.3.2 Determining the structure of a compound by observing its fragmentation.

9.3.3 Quantifying the amount of a compound in a sample using carefully designed methods.

9.3.4 Determining other physical, chemical, or even biological properties of compounds with

a variety of other approaches.

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10. Interfaces Used In LC-MS

10.1. Direct Liquid Introduction

The first attempts to introduce a liquid into an MS using the classic electron impact

ionization (EI)/chemical ionization (CI) source were based on the simple principle that by

minimizing the amount of liquid, the vacuum system would remove the solvent leaving the

analytes in the gas phase for ionization.

Figure 1: Scheme of the DLI interface. 1 _ connection to LC column, 2 _ diaphragm 5 μm

Opening to MS, 3 _ needle valve, 4 _ cooling region, 5 _ to UV detector or waste.

10.2 Moving belt/wire interface

The moving-belt interface separates the condensed liquid-phase side of the LC from the high

vacuum of the MS and uses a belt to transport the analytes from one to the other. The mobile

phase of the LC is deposited on a band and evaporated. The analytes remain on the continuously

cycling belt and are transported from atmospheric pressure into the vacuum of the ion source

through two differentially pumped vacuum locks. A heater in the ion source evaporates the

sample from the belt allowing MS analysis.

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Figure 2: Schematic showing the principal components of a moving-belt interface

10.3 Thermo spray Interface

As the name thermo spray implies, heating the liquid flow leaving an LC system creates a

spray of superheated mist containing small liquid droplets.

The most successful method involves directing the liquid flow through an electrically heated

capillary, which can be directly introduced into the MS ion source. The droplets are further

vaporized as they collide against the walls of the heated ion source.

Figure 3: Thermo spray interface.

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10.4 Particle Beam Interface (MAGIC)

MAGIC, an acronym for monodisperse aerosol generation interface for chromatography. The

LC eluent is forced through a small nebulizer using a He gas flow to form a stream of uniform

droplets. These droplets move through a desolvation chamber and evaporate to a solid particle.

These particles are separated from the gas and transported into the MS source using a

differentially pumped momentum separator.

10.5 Atmospheric pressure chemical ionization

10.5.1 In APCI, the LC eluent is sprayed through a heated (typically 250°C – 400°C)

vaporizer at atmospheric pressure. The heat vaporizes the liquid.

10.5.2 The resulting gas-phase solvent molecules are ionized by electrons discharged from

a corona needle.

10.5.3 The solvent ions then transfer charge to the analyze molecules through chemical

reactions (chemical ionization).

10.5.4 The analytes ions pass through a capillary sampling orifice into the mass analyzer.

10.5.5 APCI is applicable to a wide range of polar and non polar molecules.

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10.5.6 It rarely results in multiple charging so it is typically used for molecules less than

1,500 u.

10.5.7 Because it involves high temperatures, APCI is less well-suited than electrospray for

analysis of large biomolecules that may be thermally unstable.

10.5.8 APCI is used with normal-phase chromatography more often than electrospray is

because the analytes are usually non polar.

Figure 5 Atmospheric pressure chemical ionization

10.6. Atmospheric pressure photoionization

10.6.1 As in APCI, a vaporizer converts the LC eluent to the gas phase.

10.6.2 A discharge lamp generates photons in a narrow range of ionization energies.

10.6.3 The range of energies is carefully chosen to ionize as many analyte molecules as

possible while minimizing the ionization of solvent molecules.

10.6.4 The resulting ions pass through a capillary sampling orifice into the mass analyzer.

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Figure 6 Atmospheric pressure photoionization

10.7 Electrospray Ionization (ESI)

The analytes solution flow passes through the electrospray needle that has a high potential

difference (with respect to the counter electrode) applied to it. This forces the spraying of

charged droplets from the needle with a surface charge of the same polarity to the charge on

the needle. The droplets are repelled from the needle towards the source sampling cone on the

counter electrode. As the droplets traverse the space between the needle tip and the cone and

solvent evaporation occurs.

Figure 7 Electrospray Ionization (ESI)

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11. Applications

LC-MS USED IN FOLLOWING AREAS

11.1 Drug Discovery

11.2 Clinical Analysis.

11.3 Proteomics

11.4 Forensic Chemistry

11.5 Drug Metabolism study

11.6 Environmental chemistry

11.7 Diagnostic studies.

11.8 Molecular Weight Determination

11.9 Determination Of Molecular Weight Of Green Florescent Protein

11.10 Structural Determination

11.11 Pharmaceutical Applications

11.12 Identification Of Bile Acid Metabolites

11.13 Clinical Applications

11.14 Biochemical Genetics

11.15 Therapeutic Drug Monitoring

12. Conclusion

The study has been shown that the LC-MS is the valuable tool for analysis of various

biological samples.

The LCMS provides accuracy, specificity, selectivity & rapid less time consuming.

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Instrumentation of LC-MS is quite complicated but very efficient for their accuracy,

sensitivity.

It is mostly useful in pharmaceutical industries finds application in pharmacokinetic

study, proteomics, drug development, radio pharmaceutics and clinical analysis & in

toxicological studies.

13. Future Scope

LC-MS is the technique which have application in future in bioequivalence study.

It can be use for blood analysis, for detection of drugs whose identity is not known till

now.