Liquid Chromatography and Mass spectroscopy

(LC-MS)

Presented by: Group # 4

 2017(F)-MS-CH-07
 2017(F)-MS-CH-26
 2017(F)-MS-CH-28
 2017(F)-MS-CH-37
 2017(F)-MS-CH-42
 2017(F)-MS-CH-45

Table of contents
 Liquid chromatography
 General scheme
 Chromatographic column
a. Stationary phase
b. Mobile phase
 Theoretical background
 Liquid chromatography-Mass Spectroscopy
 General scheme for LC-MS
 Sample Types
 Selectivity and Sensitivity
 Mass spectroscopy
 Ionization techniques
 Applications
 References
Liquid chromatography

Liquid chromatography is a technique used to separate a sample into its individual parts. This separation occurs based on the
interactions of the sample with the mobile and stationary phases.

General scheme

Components within a mixture are separated in a column based on each component's affinity for the mobile phase. So, if the
components are of different polarities and a mobile phase of a distinct polarity is passed through the column, one component will
migrate through the column faster than the other. Because molecules of the same compound will generally move in groups, the
compounds are separated into distinct bands within the column. If the components being separated are colored, their corresponding
bands can be seen. Otherwise, the presence of the bands are detected using other instrumental analysis techniques such as UV-VIS
spectroscopy.

Column Chromatography

The stationary phase in column chromatography is most typically a fine adsorbent solid; a solid that is able hold onto gas or liquid
particles on its outer surface. The column typically used in column chromatography looks similar to a Pasteur pipette (Pasteur pipettes
are used as columns in small scale column chromatography). The narrow exit of the column is first plugged with glass wool or a porous
plate in order to support the column packing material and keep it from escaping the tube. Then the adsorbent solid (usually silica) is
tightly packed into the glass tube to make the separating column. The packing of the stationary phase into the glass column must be
done carefully to create a uniform distribution of material. A uniform distribution of adsorbent is important to minimize the presence
of air bubbles and/or channels within the column. To finish preparing the column, the solvent to be used as the mobile phase is passed
through the dry column. Then the column is said to be "wetted" and the column must remain wet throughout the entire experiment.
Once the column is correctly prepared, the sample to be separated is placed at the top of the wet column.

Components

Chromatography is effective because different components within a mixture are attracted to the adsorbent surface of the stationary
phase with varying degrees depending on each components polarity and its unique structural characteristics, and also its interaction
with the mobile phase. The separation that is achieved using column chromatography is based on factors that are associated with the
sample. So, a component that is more attracted to the stationary phase will migrate down the separating column at a slower rate than
a component that has a higher affinity for the mobile phase. Also, the efficacy of the separation is dependent on the nature of the
adsorbent solid used and the polarity of the mobile phase solvent.

a) Stationary Phase: (Adsorbent material) Adsorbent material can be chosen based on particle size and activity of the solid. The
activity of the adsorbent is represented by its activity grade, which is a measure of an adsorbent's attraction for solutes in the
sample solution. The solids with the highest activity grading are those that are completely anhydrous. Silica gel and alumina
are among the most popular adsorbents used. Alumina caters well to samples that that require specific conditions to
adequately separate. However, the use of non-neutral stationary phases should be done with great caution, an increase or
decrease of pH in the alumina stationary phase may allow chemical reactions within the components of the mixture. Silica
gel, however, is less active than alumina and can generally be used as an all-around adsorbent for most components in
solution. Silica is also preferred because of its high sample capacity, making it one of the most popular adsorbent materials.

b) Mobile Phase: The proper mobile phase must also be chosen for the best separation of the components in an unknown
mixture. This eluent will be chosen based on its polarity relative to the sample and the stationary phase. With a strong polar
adsorbent stationary phase like alumina, a polar solvent used as the mobile phase will be adsorbed by the stationary phase,
which may displace molecules of sample in the mixture and may cause the sample components to elute vary quickly. This will
provide little separation of the sample, so it is best to start elution with a solvent of lower polarity to elute the components
that are weakly adsorbed to the stationary phase first.
Theoretical background

It is very important to understand the theoretical background of LC, before moving towards MS.

Band broadening

Band-broadening is a general term used to describe the overall dispersion or widening of a sample peak as it passes through a
separation system. It in Chromatography is a result of several effects. These includes diffusion processes, and transfer of solutes
between the mobile and stationary phases, etc. These effects are discussed below.

The Rate theory

According to rate theory the following Processes (i.e., parameters) Effect Band-Broadening:

1. Diffusion: (longitudinal) HL
2. Flow and Diffusion in mobile phase (Eddy or multi-path diffusion) HE
3. Resistance to mass transfer(stationary phase to mobile phase ) HS,(mobile phase to stationary phase ) HM
Sum of all these parameters is equal to the total height required for separation.

H=Total height= HL+ HE+ HS+ HM

1) Diffusion: (longitudinal) HL

2Dm
𝐻𝐿 = 𝛆𝐩
𝑢(1+ )
𝛆𝐞

εp: Intraparticle porosity , εe: Interparticle porosity ,Dm: Solute diffusion coefficient in mobile phase, u: Linear velocity of flow

Larger the Dm, larger the HL, larger the u, smaller the HL.
 3) Resistance to mass transfer(HR)

 x=1 for gasses and x=1/3 for liquids

 CS + CM=C

Total height=H = Au1/3+B/u+Cu

u=Velocity of mobile phase, B=Longitudinal diffusion,C=Resistance to mass transfer, A=Eddy diffusion

Liquid chromatography–mass spectrometry (LC-MS)

 Liquid chromatography–mass spectrometry is an analytical chemistry technique that combines the physical separation
capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry (MS).
 Coupled chromatography - MS systems are popular in chemical analysis because the individual capabilities of each technique
are enhanced synergistically.
 While liquid chromatography separates mixtures with multiple components, mass spectrometry provides structural identity
of the individual components with high molecular specificity and detection sensitivity.
LC-MS Interface

 LC-MS system contains an interface that efficiently transfers the separated components from the LC column into the MS ion
source. The interface is necessary because the LC and MS devices are fundamentally incompatible.
 While the mobile phase in a LC system is a pressurized liquid, the MS analyzers commonly operate under vacuum (around
10−6 torr). Thus, it is not possible to directly pump the eluate from the LC column into the MS source.
 As a requirement, the interface should not interfere with the ionizing efficiency and vacuum conditions of the MS system.
 Most extensively applied LC-MS interfaces are based on atmospheric pressure ionization (API) strategies like electrospray
ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photo-ionization (APPI).
General scheme for LC-MS

Liquid chromatography (LC) separates the sample components and then introduces them to the mass spectrometer (MS). The MS
creates and detects charged ions. The LC/MS data may be used to provide information about the molecular weight, structure, identity
and quantity of specific sample components.

Sample Types

LC/MS systems facilitate the analysis of samples that traditionally have been difficult to analyze. LC/MS significantly expands the
effective analytical use of mass spectrometry to a much larger number of organic compounds. Sample types range from small
pharmaceutical compounds to large proteins. LC/MS is suitable for the analysis of large, polar, ionic, thermally unstable and non-
volatile compounds.

Selectivity and Sensitivity

A mass spectrometer combined with a liquid chromatograph can detect masses characteristic of a compound or of a class of
compounds. The system can selectively detect compounds of interest in a complex matrix, thus making it easy to find and identify
suspected impurities at trace levels. When configured to simultaneously detect a range of masses (and depending on the compound)
LC/MS sensitivity can be comparable to that provided by a diode-array detector (DAD). Far greater sensitivity is possible when the
LC/MS is configured to detect only those masses characteristic of the compounds being monitored.

Mass spectroscopy
The following is the schematic of MS:

Description
Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio (m/z) of charged particles (ions). Although
there are many different kinds of mass spectrometers, all of them make use of electric or magnetic fields to manipulate the motion of
ions produced from an analyte of interest and determine their m/z.The basic components of a mass spectrometer are the ion source,
the mass analyzer, the detector, and the data and vacuum systems.The ion source is where the components of a sample introduced
in a MS system are ionized by means of electron beams, photon beams (UV lights), laser beams or corona discharge. In the case of
electrospray ionization, the ion source moves ions that exist in liquid solution into the gas phase. The ion source converts and
fragments the neutral sample molecules into gas-phase ions that are sent to the mass analyzer. While the mass analyzer applies the
electric and magnetic fields to sort the ions by their masses, the detector measures and amplifies the ion current to calculate the
abundances of each mass-resolved ion. In order to generate a mass spectrum that a human eye can easily recognize, the data system
records, processes, stores, and displays data in a computer.
Ionization techniques
1) Electrospray ionization (ESI)
This Ion source/ interface can be used for the analysis of moderately polar molecules (e.g., metabolites and peptides). The liquid
eluate coming out of the LC column is pumped through a metal capillary kept at 3 to 5 kV. The liquid is nebulized at the tip of the
capillary and a fine spray of charged droplets is formed. To avoid contamination, this capillary is usually perpendicularly located at
the inlet of the MS system. The heat created by the electric potential is used to rapidly evaporate the droplets in an atmosphere of
dry nitrogen. Later, the ionized analytes are transferred into the high vacuum chamber of the MS as the charged ions flow through a
series of small apertures with the aid of focusing voltages. Positively and negatively charged ions can be detected and it is possible to
switch between the negative and positive modes of operation. Most ions produced in the ESI interface are multiply charged. The use
of 1–3 mm ID microbore columns is recommended for LC-MS systems using electrospray ionization (ESI) interfaces because optimal
operation is achieved with flow rates in the 50-200 μl/min range.
2) Atmospheric pressure chemical ionization (APCI)
The APCI ion source/ interface can be used to analyze small, neutral, relatively non-polar, and thermally stable molecules (e.g.,
steroids, lipids, and fat soluble vitamins). These compounds are not well ionized using ESI. In addition, APCI can also handle mobile
phase streams containing buffering agents. The liquid from the LC system is pumped through a capillary and there is also
nebulization at the tip, where a corona discharge takes place. First, the ionizing gas surrounding the interface and the mobile phase
solvent are subject to chemical ionization at the ion source. Later, these ions react with the analyte and transfer their charge. The
sample ions then pass through small orifice skimmers by means of or ion-focusing lenses. Once inside the high vacuum region, the
ions are subject to mass analysis. This interface can be operated in positive and negative charge modes and singly-charged ions are
mainly produced. APCI ion source can also handle flow rates between 500 and 2000 μl/min and it can be directly connected to
conventional 4.6 mm ID columns.
3) Atmospheric pressure photo-ionization (APPI)
APPI is another LC-MS ion source/ interface for the analysis of neutral compounds that cannot be ionized using ESI.This interface is
similar to the APCI ion source, but instead of a corona discharge, the ionization occurs by using photons coming from a discharge
lamp. In the direct-APPI mode, singly charged analyte molecular ions are formed by absorption of a photon and ejection of an
electron. In the dopant-APPI mode, an easily ionizable compound (Dopant) is added to the mobile phase or the nebulizing gas to
promote a reaction of charge-exchange between the dopant molecular ion and the analyte. The ionized sample is later transferred
to the mass analyzer at high vacuum as it passes through small orifice skimmers.
Charge to mass ratio
Charge to mass ratio plays a vital role for the separation.

Since e, B, V are constants, therefore we can say that

m=kr2 or m ∝ r2
The ions of larger mass will fall at greater distances which becomes the basis for separation.

Peaks
The following peaks are obtained when the sample of chlorine is analyzed.
Peaks are normally not of the type just shown in the figure above. These peaks are broadened by several effects. These includes
diffusion processes, and transfer of solutes between the mobile and stationary phases, etc. Mostly we get the following peaks.

 The above figure shows the concentration of different molecules in the Rat Blood.
Several terms like Resolution (separation of peaks) and Efficiency (sharpness) are discussed with an aim to get ideal peaks.

Applications
The coupling of MS with LC systems is attractive because liquid chromatography can separate delicate and complex natural mixtures,
which chemical composition needs to be well established (e.g., biological fluids, environmental samples, and drugs.
1) Pharmacokinetics
LC-MS is widely used in the field of bioanalysis and is especially involved in pharmacokinetic studies of pharmaceuticals.
Pharmacokinetic studies are needed to determine how quickly a drug will be cleared from the body organs and the hepatic blood
flow. MS analyzers are useful in these studies because of their shorter analysis time, and higher sensitivity and specificity compared
to UV detectors commonly attached to HPLC systems. One major advantage is the use of tandem MS-MS, where the detector may
be programmed to select certain ions to fragment. The measured quantity is the sum of molecule fragments chosen by the operator.
As long as there are no interferences or ion suppression, the LC separation can be quite quick.
2) Metabolomics,
LC-MS has been useful to advance the field of plant metabolomics, which aims to study the plant system at molecular level providing
a non-biased characterization of the plant metabolome in response to its environment. LC-MS in plant metabolomics is the efficient
separation and identification of glucose, sucrose, raffinose, stachyose, and verbascose from leaf extracts of Arabidopsis thaliana.
3) Drug development
LC-MS is frequently used in drug development because it allows quick molecular weight confirmation and structure identification.
These features speed up the process of generating, testing, and validating a discovery starting from a vast array of products with
potential application. LC-MS applications for drug development are highly automated methods used for peptide
mapping, glycoprotein mapping, natural products dereplication, bioaffinity screening, in vivo drug screening, metabolic stability
screening, metabolite identification, impurity identification, quantitative bioanalysis, and quality control.
References
 Jones Jr., M. Organic Chemistry, 2nd ed. New York, NY. W. W. Norton & Company, Inc. 2000.
 Lehman, JW. Operational Organic Chemistry, 3rd ed. Upper Saddle River, NJ. Prentice Hall. 2002.
 Skoog, DA; Holler, FJ; Crouch, SR. Principles of Instrumental Analysis, 6th ed. Belmont, CA. Thomson Higher Education. 2007.
 Wade Jr., LG. Organic Chemistry, 6th ed. Upper Saddle River, NJ. Prentice Hall. 2006.
 https://chem.libretexts.org/Core/Analytical_Chemistry/Instrumental_Analysis/Chromatography/Liquid_Chromatography