1. GC – Mass Spectrometry (GC-MS)
GC-MS is a sophisticated instrumental technique that produces,
separates, and detects ion in the gas phase.
Today, relatively inexpensive compact benchtop system are available
and widely used in laboratories.

2. GC – Mass Spectrometry (GC-MS)
Mass spectroscopy is used to determine the molecular formula of the unknown compound.
Mass spectroscopy data that provides structural information tends to be unreliable and thus will only be used to verify a possible structure or in the event that the other spectral techniques are unsuccessful.

3. Principle of GC-MS
Block diagram of mass spectroscopy

4. The inlet transfers the sample into the vacuum of the mass spectrometer. In the source region, neutral sample molecules are ionized and then accelerated into the mass analyzer.
The mass analyzer is the heart of the mass spectrometer. This section separates ions, either in space or in time, according to their mass to charge ratio.
After the ions are separated, they are detected and the signal is transferred to a data system for analysis.
All mass spectrometers also have a vacuum system to maintain the low pressure, which is also called high vacuum, required for operation.
High vacuum minimizes ion-molecule reactions, scattering, and neutralization of the ions.
In some experiments, the pressure in the source region or a part of the mass spectrometer is intentionally increased to study these ion-molecule reactions. Under normal operation, however, any collisions will interfere with the analysis.

5. Ionization Source

6. Electron Impact (EI)
Electron Ionization (EI) is the most common ionization technique used for mass spectrometry. EI works well for many gas phase molecules, but it does have some limitations.
Although the mass spectra are very reproducible and are widely used for spectral libraries, EI causes extensive fragmentation so that the molecular ion is not observed for many compounds. Fragmentation is useful because it provides structural information for interpreting unknown spectra.

7. Electron Ionization Source

8. Electron Ionization Process
A) Ionizing electron approaches the electron cloud of a molecule
B)Electron cloud distorted by ionizing electron
C) Electron cloud further distorted by ionizing electron

9. D) Ionizing electron passes by the molecule
E) Electron cloud of molecule ejecting an electron
F) Molecular ion and ejected electron.

10. Chemical Ionization
Chemical Ionization (CI) is a “soft” ionization technique that produces ions with little excess energy. As a result, less fragmentation is observed in the mass spectrum.
Since this increases the abundance of the molecular ion, the technique is complimentary to 70 eV EI.
CI is often used to verify the molecular mass of an unknown. Only slight modifications of an EI source region are required for CI experiments.

11. In Chemical Ionization the source is enclosed in a small cell with openings for the electron beam, the reagent gas and the sample.
The reagent gas is added to this cell at approximately 10 Pa (0.1 torr) pressure.
This is higher than the 10-3 Pa (10-5 torr) pressure typical for a mass spectrometer source. At 10-3 Pa the mean free path between collisions is approximately 2 meters and ion-molecule reactions are unlikely.
In the CI source, however, the mean free path between collisions is only 10-4 meters and analyte molecules undergo many collisions with the reagent gas.
The reagent gas in the CI source is ionized with an electron beam to produce a cloud of ions. The reagent gas ions in this cloud react and produce adduct ions like CH5+ ,which are excellent proton donors.

12. When analyte molecules (M) are introduced to a source region with this cloud of ions, the reagent gas ions donate a proton to the analyte molecule and produce MH+ ions.
The energetic of the proton transfer is controlled by using different reagent gases.
The most common reagent gases are methane, isobutane and ammonia. Methane is the strongest proton donor commonly used with a proton affinity (PA) of 5.7 eV. For softer ionization, isobutane (PA 8.5 eV) and ammonia (PA 9.0 eV) are frequently used.
Acid base chemistry is frequently used to describe the chemical ionization reactions. The reagent gas must be a strong enough Brønsted acid to transfer a proton to the analyte.
Fragmentation is minimized in CI by reducing the amount of excess energy produced by the reaction. Because the adduct ions have little excess energy and are relatively stable, CI is very useful for molecular mass determination.

13. Mass Analyzer
After ions are formed in the source region they are accelerated into the mass analyzer by an electric field. The mass analyzer separates these ions according to their m/z value.
The selection of a mass analyzer depends upon the resolution, mass range, scan rate and detection limits required for an application.
Each analyzer has very different operating characteristics and the selection of an instrument involves important tradeoffs.

14. Quadrapole Mass Filter
The quadrupole mass spectrometer is the most common mass analyzer.
Its compact size, fast scan rate, high transmission efficiency, and modest vacuum requirements are ideal for small inexpensive instruments.
It ‘filter’ and only allow specific ions to pass.
Most quadrupole instruments are limited to unit m/z resolution and have a mass range of m/z 1000.
Many benchtop instruments have a mass range of m/z 500 but research instruments are available with mass range up to m/z 4000.

15. Quadrapole Mass Analyzer
Only compound with specific m/z ratio will resonate along the field (stable path)
Achieved by rapidly varying the voltage

16. Time of Flight Analyzer
The time-of-flight (TOF) mass analyzer separates ions in time as they travel down a flight tube.
This is a very simple mass spectrometer that uses fixed voltages and does not require a magnetic field. The greatest drawback is that TOF instruments have poor mass resolution, usually less than 500.
These instruments have high transmission efficiency, no upper m/z limit, very low detection limits, and fast scan rates.
For some applications these advantages outweigh the low resolution.
Recent developments in pulsed ionization techniques and new instrument designs with improved resolution have renewed interest in TOF-MS.

17. This picture shows the working principle of a linear time of flight mass spectrometer.
To allow the ions to fly through the flight path without hitting anything else, all the air molecules have been pumped out to create an ultra high vacuum.
Ions of different m/z ratio travel at different velocities.
The difference in arrival times separate two (or more) ions.

18. Graph of ion intensity versus mass-to-charge ratio (m/z)
(units daltons, Da)

19. High Performance Liquid Chromatography

20. Liquid Chromatography
Chromatography in which the mobile phase is a liquid.
The liquid used as the mobile phase is called the “eluent”.
The stationary phase is usually a solid or a liquid.
In general, it is possible to analyze any substance that can be stably dissolved in the mobile phase.
“Liquid chromatography” (LC) is chromatography in which the mobile phase is a liquid.
Stationary Phase
Usually a solid or a liquid is used as the mobile phase. (This includes the case where a substance regarded as a liquid is chemically bonded, or applied, to the surface of a solid.)
The most common form of stationary phase consists of fine particles of, for example, silica gel or resin packed into a cylindrical tube. These packed particles are called “packing material” or “packing” and the separation tube into which they are packed is called the “separation column” or simply the “column”. In day-to-day analysis work, “column” is sometimes used to refer to the stationary phase and “stationary phase” is sometimes used to refer to the column.
Mobile Phase
Various solvents are used as mobile phases. The mobile phase conveys the components of the dissolved sample through the separation field, and facilitates the repeated three-way interactions that take place between the phases and the sample, thereby leading to separation.
The solvent used for the mobile phase is called the “eluent” or “eluant”. (In LC, the term “mobile phase” is also used to refer to this solvent. In this text, however, we shall use the term “eluent”.)
Sample
In general, it is possible to analyze any substance that can be stably dissolved in the eluent. This is one advantage that LC has over GC, which cannot be used to analyze substances that do not vaporize or that are thermally decomposed easily.
The sample is generally converted to liquid form before being introduced to the system. It contains various solutes. The target substances (the analytes) are separated and detected.

21. Column Chromatography and Planar Chromatography
Separation column
Column Chromatography
Paper or a substrate coated with particles
Paper Chromatography
Thin Layer Chromatography (TLC)
Liquid chromatography can be categorized by shape of separation field into column-shaped and planar types.
A representative type of chromatography that uses a column-shaped field is “column chromatography”, which is performed using a separation column consisting of a cylindrical tube filled with packing material. Another type is “capillary chromatography”, which is performed using a narrow hollow tube. Unlike column chromatography, however, capillary chromatography has yet to attain general acceptance. (In the field of GC, however, capillary chromatography is a commonly used technique.)
Types of chromatography that use a planar (or plate layer) field include “thin layer chromatography”, in which the stationary phase consists of a substrate of glass or some other material to which minute particles are applied, and “paper chromatography”, in which the stationary phase consists of cellulose filter paper.
Packing material

22. Separation Process and Chromatogram for Column Chromatography
Output concentration
Time
Chromatogram
The separation process for column chromatography is shown in the above diagram.
After the eluent is allowed to flow into the top of the column, it flows down through the spaces in the packing material due to gravity and capillary action. In this state, a sample mixture is placed at the top of the column. The solutes in the sample undergo various interactions with the solid and mobile phases, splitting up into solutes that descend quickly together with the mobile phase and solutes that adsorb to the stationary phase and descend slowly, so differences in the speed of motion emerge. At the outlet, the elution of the various solutes at different times is observed.
A detector that can measure the concentrations of the solutes in the eluate is set up at the column outlet, and variations in the concentration are monitored. The graph representing the results using the horizontal axis for times and the vertical axis for solute concentrations (or more accurately, output values of detector signals proportional to solute concentrations) is called a “chromatogram”.

23. Chromatogram tR : Retention time t0 : Non-retention time A : Peak area
Intensity of detector signal
Peak
tR : Retention time h A
t0 : Non-retention time
A : Peak area
h : Peak height
Usually, during the time period in which the sample components are not eluted, a straight line running parallel to the time axis is drawn. This is called the “baseline”.
When a component is eluted, a response is obtained from the detector, and a raised section appears on the baseline. This is called a “peak”. The components in the sample are dispersed by the repeated interactions with the stationary and mobile phases, so the peaks generally take the bell-shape form of a Gaussian distribution.
The time that elapses between sample injection and the appearance of the top of the peak is called the “retention time”. If the analytical conditions are the same, the same substance always gives the same retention time. Therefore, the retention time provides a means to perform the qualitative analysis of substances.
The time taken for solutes in the sample to go straight through the column together with the mobile phase, without interacting with the stationary phase, and to be eluted is denoted as “t0”. There is no specific name for this parameter, but terms such as “non-retention time” and “hold-up time” seem to be commonly used.
Because the eluent usually passes through the column at a constant flow rate, tR and t0 are sometimes multiplied by the eluent flow rate and handled as volumes. The volume corresponding to the retention time is called the “retention volume” and is notated as VR.
The length of a straight line drawn from the top of a peak down to the baseline is called the “peak height”, and the area of the raised section above the baseline is called the “peak area”.
If the intensities of the detector signals are proportional to the concentrations or absolute quantities of the peak components, then the peak areas and heights are proportional to the concentrations of the peak components. Therefore, the peak areas and heights provide a means to perform the quantitative analysis of sample components. It is generally said that using the peak areas gives greater accuracy.
Time

24. From Liquid Chromatography to High Performance Liquid Chromatography
Higher degree of separation!  Refinement of packing material (3 to 10 µm)
Reduction of analysis time!  Delivery of eluent by pump  Demand for special equipment that can withstand high pressures
The arrival of high performance liquid chromatography!
In order to increase the separation capability of column chromatography, in addition to increasing the surface area of the stationary phase so that the interaction efficiency is increased, it is also necessary to homogenize the separation field as much as possible so that dispersion in the mobile and stationary phases is minimized. The most effective way of achieving this is to refine the packing material.
Refining the packing material, however, causes resistance to the delivery of the eluent to increase. This is similar to the way that water drains easily through sand, which has relatively large particles, whereas it does not drain easily through clay-rich soil, which has relatively fine particles.
Depending on gravity and capillary action would cause analysis to take a very long time to be completed, and the idea of delivering the eluent forcibly using a high-pressure pump was proposed. This was the start of high performance liquid chromatography.

25. Flow Channel Diagram for High Performance Liquid Chromatograph
Pump
Sample injection unit
(injector)
Column
Column oven
(thermostatic column chamber)
Detector
Eluent
(mobile phase)
Drain
Data processor
A high performance liquid chromatograph differs from a column chromatograph in that it is subject to the following performance requirements.
Solvent Delivery Pump
A solvent delivery pump that can maintain a constant, non-pulsating flow of solvent at a high pressure against the resistance of the column is required.
Sample Injection Unit
There is a high level of pressure between the pump and the column; a device that can inject specific amounts of sample under such conditions is required.
Column
The technology for filling the column evenly with refined packing material is required. Also, a material that can withstand high pressures, such as stainless steel, is required for the housing.
Detector
Higher degrees of separation have increased the need for high-sensitivity detection, and levels of sensitivity and stability that can respond to this need are required in the detector.
Degasser

26. What’s the different between HPLC and GC?
Solvent Delivery Pump
A solvent delivery pump that can maintain a constant, non-pulsating flow of solvent at a high pressure against the resistance of the column is required.
Sample Injection Unit
There is a high level of pressure between the pump and the column; a device that can inject specific amounts of sample under such conditions is required.
Column
The technology for filling the column evenly with refined packing material is required. Also, a material that can withstand high pressures, such as stainless steel, is required for the housing.
Detector
Higher degrees of separation have increased the need for high-sensitivity detection, and levels of sensitivity and stability that can respond to this need are required in the detector.

27. Detection in HPLC *There are six major HPLC detectors:
Refractive Index (RI) Detector
Evaporative Light Scattering Detector (ELSD)
UV/VIS Absorption Detectors
The Fluorescence Detector
Electrochemical Detectors (ECDs)
Conductivity Detector
* The type of detector utilized depends on the characteristics of the analyte of interest.

28. Refractive Index Detector
Based on the principle that every transparent substance will slow the speed of light passing through it.
Results in the bending of light as it passes to another material of different density.
Refractive index = how much the light is bent
The presence of analyte molecules in the mobile phase will generally change its RI by an amount almost
linearly proportional to its
concentrations.

29. Refractive Index Detector
Affected by slight changes in mobile phase composition and temperature.
Universal-based on a property of the mobile phase
It is used for analytes which give no response with other more sensitive and selective detectors.
RI = general
responds to the presence of all solutes in the mobile phase.
Reference= mobile phase
Sample= column effluent
Detector measures the differences between the RI of the reference and the sample.

30. Evaporative Light Scattering Detector (ELSD)
Analyte particles don’t scatter light when dissolved in a liquid mobile phase.
Three steps:
1) Nebulize the mobile phase effluent into droplets.
Passes through a needle and mixes with hydrogen gas.
2) Evaporate each of these droplets.
Leaves behind a small particle of nonvolatile analyte
3) Light scattering
Sample particles pass through a cell and scatter light from a laser beam which is detected and generates a signal.

31. UV/VIS Absorption Detectors
Different compounds will absorb different amounts of light in the UV and visible regions.
A beam of UV light is shined through the analyte after it is eluted from the column.
A detector is positioned on the opposite side which can measure how much light is absorbed and transmitted.
The amount of light absorbed will depend on the amount of the compound that is passing through the beam.

32. UV/VIS Absorption Detectors
Beer-Lambert law: A=εlc,
A= absorbance, ε=molar absortivity, l=length of solution that light pass thru, c=concentration
absorbance is proportional to the compound concentration.
Fixed Wavelength: measures at one wavelength, usually 254 nm
Variable Wavelength: measures at one wavelength at a time, but can detect over a wide range of wavelengths
Diode Array Detector (DAD): measures a spectrum of wavelengths simultaneously

33. The Fluorescence Detector
Measure the ability of a compound to absorb then re- emit light at given wavelengths
Some compounds will absorb specific wavelengths of light which, raising it to a higher energy state.
When the compound returns to its ground state, it will release a specific wavelength of light which can be detected.
Not all compounds can fluoresce / more selective than UV/VIS detection.
fluorescence.jpg

34. Electrochemical Detectors (ECDs)
Used for compounds that undergo oxidation/reduction reactions.
Detector measures the current resulting from an oxidation/reduction reaction of the analyte at a suitable electrode.
Current level is directly proportional to the concentration of analyte present.
Conductivity Detector:
Records how the mobile phase conductivity changes as different sample components are eluted from the column.

35. HPLC Chromatogram

36. Types of HPLC
There are numerous types of HPLC which vary in their separation chemistry.
All chromatographic modes are possible:
Ion-exchange
Size exclusion
Also can vary the stationary & mobile phases:
Normal phase HPLC
Reverse phase HPLC

37. Chromatographic Modes of HPLC
Ion exchange:
Used with ionic or ionizable samples.
Stationary phase has a charged surface.
opposite charge to the sample ions
The mobile phase = aqueous buffer
The stronger the charge on the analyte, the more it will be attracted to the stationary phase, the slower it will elute.
Size exclusion:
Sample separated based on size.
Stationary phase has specific pore sizes.
Larger molecules elute quickly.
Smaller molecules penetrate inside the pores of the stationary phase and elute later.

38. Normal Phase HPLC Stationary phase: polar, silica particles
Mobile phase: non-polar solvent or mixture of solvents
Polar compounds:
Will have a higher affinity for the polar, stationary phase
Will elute slower
Non-polar compounds:
Will have a higher affinity for the non- polar, mobile phase
Will elute faster

39. Reverse Phase HPLC Stationary phase: non-polar
Non-polar organic groups are covalently attached to the silica stationary particles.
Most common attachment is a long-chain n-C18 hydrocarbon
Octadecyl silyl group, ODS
Mobile phase: polar liquid or mixture of liquids
Polar analytes will spend more time in the polar mobile phase.
Will elute quicker than non-polar analytes
Most common type of HPLC used today.
resources/getstart/3a01.htm

40. Advantages of High Performance Liquid Chromatography
High separation capacity, enabling the batch analysis of multiple components
Superior quantitative capability and reproducibility
Moderate analytical conditions
Unlike GC, the sample does not need to be vaporized.
Generally high sensitivity
Low sample consumption
Easy preparative separation and purification of samples
HPLC is a type of separation analysis, and this is the most important aspect of this analytical technique. Even if the sample consists of a mixture, it allows the target components to be separated, detected, and quantified. It also allows simultaneous analysis of multiple components.
It could be said that HPLC is more suited to quantitative analysis than it is to qualitative analysis. Under the appropriate conditions, it is possible to attain a high level of reproducibility with a coefficient of variation not exceeding 1%.
One advantage that HPLC has over GC is that, in general, analysis is possible for any sample that can be stably dissolved in the eluent. With GC, gas is used as the mobile phase, so substances that are difficult to vaporize or that decompose easily when heated cannot be analyzed. For this reason, particularly in the fields of pharmaceutical science and biochemistry, HPLC is used much more frequently than GC.
The level of sensitivity that can be attained varies with the detector, but detection down to the µg and pg levels is usually possible and, in some cases, even smaller quantities can be detected.
The amount of sample used is very small, and is usually in the range of 1 to 100 µL.
The components contained in the sample are eluted from the column separately. So, if a non-destructive detector is used, the preparative separation and purification of specific components is possible. In fact, liquid chromatographs specially designed for preparative separation are commercially available.

41. Fields in Which High Performance Liquid Chromatography Is Used
Food products
Vitamins, food additives, sugars, organic acids, amino acids, etc.
Environmental samples
Inorganic ions
Hazardous organic substances, etc.
Organic industrial products
Synthetic polymers, additives, surfactants, etc.
Biogenic substances
Sugars, lipids, nucleic acids, amino acids, proteins, peptides, steroids, amines, etc.
Medical products
Drugs, antibiotics, etc.
HPLC is currently being used in a broad range of fields. In particular, in the field of biochemistry, it is widely used as an indispensable analytical technique.
From the perspective of an analytical instrument manufacturer, we observe that the industry that purchases the highest number of high performance liquid chromatographs is the pharmaceutical industry. It is said that the number of deliveries for this industry accounts for about 40% of the total. Although the number of deliveries to quality control departments is particularly high, it is also quite high for drug discovery and R&D departments.

42. HPLC Applications
Can be used to isolated and purify compounds for further use.
Can be used to identify the presence of specific compounds in a sample.
Can be used to determine the concentration of a specific compound in a sample.
Can be used to perform chemical separations
Enantiomers (mirror image molecular structure)
Biomolecules

43. *HPLC has an vast amount of current & future applications*
HPLC Applications
*HPLC has an vast amount of current & future applications*
Some uses include:
Forensics: analysis of explosives, drugs, fibers, etc.
Proteomics: can be used to separate and purify protein samples
Can separate & purify other biomolecules such as: carbohydrates, lipids, nucleic acids, pigments, proteins, steroids
Study of disease: can be used to measure the presence & abundance of specific biomolecules correlating to disease manifestation.
Pharmaceutical Research: all areas including early identification of clinically relevant molecules to large-scale processing and purification.

44. Electrophoresis

45. Electrophoresis
Electrophoresis is a separations technique that is based on the mobility of ions in an electric field.
Positively charged ions migrate towards a negative electrode and negatively-charged ions migrate toward a positive electrode.
For safety reasons one electrode is usually at ground and the other is biased positively or negatively.
Ions have different migration rates depending on their total charge, size, and shape, and can therefore be separated.

46. Electrophoresis
An electrode apparatus consists of a high-voltage supply, electrodes, buffer, and a support for the buffer such as filter paper, cellulose acetate strips, polyacrylamide gel, or a capillary tube.
Open capillary tubes are used for many types of samples and the other supports are usually used for biological samples such as protein mixtures or DNA fragments.
After a separation is completed the support is stained to visualize the separated components.
Resolution can be greatly improved using isoelectric focusing. In this technique the support gel maintains a pH gradient. As a protein migrates down the gel, it reaches a pH that is equal to its isoelectric point. At this pH the protein is natural and no longer migrates, i.e, it is focused into a sharp band on the gel.

47. Capillary Electrophoresis
Schematic of capillary electrophoresis

48. How Does CE Work?
Capillaries are typically of 50 µm inner diameter and 0.5 to 1 m in length. The applied potential is 20 to 30 kV.
Due to electro osmotic flow, all sample components migrate towards the negative electrode. A small volume of sample (10 nL) is injected at the positive end of the capillary and the separated components are detected near the negative end of the capillary.
CE detection is similar to detectors in HPLC and include absorbance, fluorescence, electrochemical, and mass spectrometry.
The capillary can also be filled with a gel, which eliminates the electro osmotic flow. Separation is accomplished as in conventional gel electrophoresis but the capillary allows higher resolution, greater sensitivity, and on-line detection.

49. (sodium dodecyl sulfate polyacrylamide gel electrophoresis)
well
Sample
(blue)
Gel Electrophoresis
(sodium dodecyl sulfate polyacrylamide gel electrophoresis)
Is a vertical gel electropheresis
To separate proteins according to their size

51. How Does GE Work?
Electrophoresis refers to the electromotive force (EMF) that is used to move the molecules through the gel matrix.
By placing the molecules in wells in the gel and applying an electric field, the molecules will move through the matrix at different rates, determined largely by their mass
Molecules move toward the (negatively charged) cathode if positively charged or toward the (positively charged) anode if negatively charged.
Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel.
This phenomenon is called sieving.

52. Gel staining
immersed in solution e.g ethidium bromide

53. Result Sample 1 Sample 2 Sample 3 Bigger protein Smaller protein
reference
marker

54. Thank you…