ABSTRACT

1.0 GC-MS

1.1 INTRODUCTIONGas chromatography (GC) or gas-liquid chromatography (GLC) is one of the types of gas chromatographic analysis. The other type is gas-solid chromatography (GSC) which based on a solid stationary phase. The retention of analytes occurs by the means of physical adsorption. While in GC, the partition of analyte occurs between a gaseous mobile phase and liquid stationary phase, on the surface of an inert solid packing or on the walls of the columns.In 1941, Martin and Synge suggested the concept of GLC. They were also responsible for the liquid-liquid partition chromatography. This technique was used as a tool in laboratory practices. In 1955, the first GLC apparatus was commercialized into the market. The application of the GLC technique has been phenomenal. Since then, GLC technology has also grow and consequently led to many advanced GC instruments in this modern day.In general, the significant components in GC instrument are;i) Stationary phase – area of dispersion of analytesii) Mobile phase – liquid, superficial fluid, or in this case gas that carry

analytes to stationary phaseiii) Sample injection compartment – the port to introduce the sample into the

systemiv) Column – a device to which the stationary phase is fixed in.v) Detector – system that identifies, records or indicates the variables

changed in its environment.

1.2 THEORY

Figure 1.0 showed a schematic diagram of the basic component of a typical Gas

Chromatography Instrumentation. The instruments in GC-MS system are basically

similar to any other GC system. What differ the GC-MS system to the other GC

system is the detector used in a GC-MS. As the name proposed, a GC-MS system

must be connected to the mass spectrometer to function as the detector of the

system. The instrumentation of a GC-MS includes;

1.2.1 Carrier gas system;

In GC, the carrier gas system consists of the mobile-phase gas, which

makes it to be known as carrier gas. The carrier gas used in GC must

be chemically inert. In a common practice, helium gas is usually used.

Despite that, other gases like argon, nitrogen and hydrogen are also

used. These gases are readily contained in pressurized tank.

In order to control the flow rates of the carrier gas, the system had to

have the pressure regulator, gauges and the flow meters. Normally, the

flow rates are controlled in two stages. The first stage is at the gas

cylinder, while the other one is at the chromatograph. For packed

columns, the flow rates usually set to the range of 25 – 150 mL/min,

while for the open tubular capillary columns, the flow rates are usually

set to 1 – 25 ml/min.

In general, if the inlet pressure remains constant, the flow rates are

assumed to remain constant. The inlet pressures are usually set to be at

the range of 10 – 50 psi above room pressure. As for the gas flow rates

at the column, they can be established using a rotometer at the column

head. However, a more accurate device to perform this is the simple

soap-bubble meter. While nowadays, as the technology advances,

many gas chromatographs are equipped with electronic flow meters

which are can be regulated through a computer to maintain the flow

rate at the desired level.

1.2.2 Sample injection system;

The sample port, located at the head of the column, needs to be pre-

heated before introducing the samples into it. Typically, it is heated to

a temperature of about 50OC higher than the boiling point of the least

volatile component of the sample.

Samples are injected to the heated sample port through a rubber or

silicon diaphragm or septum. This is done by using calibrated

microsyringes. The sample is usually introduced into the

chromatographer in the form of a ‘plug’ of vapour. It is important that

the sample is of the form that is suitable for the GC system in order to

achieve high column efficiency, as the slow injection or oversize

samples could lead to a band spreading and poor resolution. Sample

sizes range from few tenths of a microliter to 20µL for ordinary packed

analytical columns, while the capillary columns require samples to be

smaller by a factor of 100 or more.

In most of advanced gas chromatographs, they are readily equipped

with the Auto-injectors together with the automatic sampling trays.

These improvements in GC provide convenience for sample injection

as they help to obtain a more precise volume of injected samples

compared to the manually sample injection using the syringe

1.2.3 Column;

a) Column types:

There are two types of columns used in GC; packed columns and

open tubular columns or also known as capillary columns. Open

tubular columns offer a higher efficiency than the packed columns.

Hence, capillary columns are widely used nowadays.

Open Tubular Columns

Two types of open tubular columns are;

Wall-coated open tubular (WCOT) – capillary tubes coated with a

thin layer of stationary phase.

Support-coated open tubular (SCOT) – the inner surface of the

capillary is lined with a thin film of support material, diatomaceous

earth as such.

SCOT has a greater sample capacity than WCOT, but in efficiency

aspect, WCOT is more efficient than SCOT. The commonly used

capillary columns are fused-silica wall-coated open tubular

columns (FSWC), which have thinner wall and strengthen by an

outside protective polyimide coating. These columns also offer

flexibility in which they are able to bend into coils with a few

inches of diameter.

b) Stationary Phase:

It is often crucial to decide which immobilized liquid phase to be

used in the GC. Stationary phase can be chosen based on the

properties of; lowing volatility, thermal stability, chemical

inertness and solvent characteristics. The guidelines to select the

best stationary phase can be obtained from literature review,

internet search, prior experience or advice from the vendor of

chromatographic equipment and supplies. The most widely used

stationary phase are polydimethyl siloxane, polyethylene glycol

and 5% Phenyl-polydimetyl siloxane. Their applications differ

from each other, depending on the types of sample analysed.

c) Column configuration and column oven temperature:

The column is ordinarily housed with a thermostatted oven in order

to control the temperature of the column, since the column

temperature in one of the important variables to obtain a precise

work. Based on previous studies, the temperature that set to be

equal or a few degrees above the average boiling point of a sample

led to an acceptable result. Thus, the optimal temperature of the

column is set based on the boiling point of the sample and the

separation degree required.

1.2.4 Detector;

There are many types of detector available to be coupled as detection

system to the GC. They depend on the types of analysis to be

conducted. Examples of the detectors are flame ionization detectors,

thermal conductivity detectors, electron-capture detectors, and atomic

emission detectors. While in a GC-MS system, as the name proposed,

the mass spectrometer is coupled to the GC for detection purpose.

Hence, this system able to help to identify the analytes present in a

sample, instead of only detects their appearance.

Analysis by mass spectrometer begins with the formation of gaseous

analyte ions. The mass spectra results from a mass spectrometer

depend on the method used for the ion formation. The ion-sources

commonly used in GC/MS are;

i) Electron-impact ionization:

The processes occurred in electron impact are;

a) Sample is heated to a temperature that is high enough to

produce a molecular vapour

b) The molecules formed are then ionized by bombarding

them with a beam of energetic electrons.

The energetic electrons approach very closely to the molecules.

This results in the loss of electrons from the molecules by

electrostatic repulsion. The primary product of the ion impact is

singly charged positive ions.

Though the electron-impact ionization is less efficient, but the

use of this technique is still important in many mass

spectrometers due to its convenient, high ion currents

production and sensitivity.

ii) Chemical ionization:

In chemical ionization process, ionization of gaseous sample

atoms occurred by collision of the atoms with ions produced by

electron bombardment of an excess of a reagent gas. This

process also produces singly charged positive ions. Some of the

reagents used in chemical ionization are methane, propane,

isobutene and ammonia. The use of the reagents results to a

different spectrum according to analytes of the analysis.

Modern technology has led to the designation of mass spectrometer

equipped with interchangeable ion source. Thus, it is able to do either

electron-impact ionization or chemical impact ionization. To perform

chemical impact ionization, the electron beam ionization area of the

electron-impact ionization is modified by addition to the capacity of

vacuum pump and reduction of the width of the slit to mass analyser.

These modifications lead electron beams to react with reagent

molecules nearly exclusively. Hence, due to the addition of proton in

the presence of the reagent ions, the spectra from the chemical

ionization are generally contain well-defined peaks than electron-

impact ionization spectra.

In a nutshell, to run an analysis using a GC-MS, the sample is firstly injected

into the pre-heated sample port of the system, the carrier gas will flow will

carry the analytes of the sample to the column of the gas chromatographic

system for separation, then the output from the column is fed directly into the

ionization chamber of the mass spectrometer. Finally, a schematic illustration,

which is the outcome of the analysis, is printed out of the output system.

1.3 SAMPLE PREPARATION

1.4 EXAMPLE OF ANALYSIS

Analysis of VOC content in the exhaled breathe of tobacco cigarette and e-

cigarette smokers using GC-MC

In a research conducted by Esther Marco and Joan O. Grimalt, GC-MS was used

to analyse the volatile organic compound content in the exhaled breath of tobacco

cigarette and electronic cigarette smokers, which included disposable e-cigarette

(Type 1) and rechargeable e-cigarette (Type 2). In their analysis, sample of

exhaled air were collected using a Bio-VOC exhaled breath sampler. This device

was developed by the UK Health and Safety Laboratory. Below is the overview of

the analysis techniques involved in sample collection for the analysis;

1.4.1 Sample Preparation Techniques:

i) Before asking volunteers to smoke, they were asked to blow at their

highest capacity into the Bio-VOC device first through a disposable

mouthpiece.

ii) Volunteers were asked to smoke tobacco cigarette, by inspired and expired

deeply three times, then retain the breath for 20 seconds

iii) Volunteers blew at their highest capacity into the Bio-VOC body through a

disposable cardboard mouthpiece.

iv) Step ii) was repeated five times.

v) The sample from the Bio-VOC was transferred into a sorbent cartridge

through a screwed-in plunger. The VOCs of tobacco cigarette exhaled

breathe were accumulated in the same cartridge.

vi) Steps from ii) through iv) were repeated for e-cigarette Type I and Type II.

vii) Sample of ambient air before and after smoking were also collected using

the same device to compare it to the analysis of exhaled breath of the

various types of cigarette.

1.4.2 Methodology

Components to be analysed: nicotine, benzene, toluene, naphthalene,

propylene glycol, glycerine, and other pollutants of general concern.

Standard solutions prepared for sample identification: 2-methylbutane,

1-pentene, cis-2-pentene, trans-2-pentene, 4-methyl-1-pentene and nine

standard solutions of organics in methanol with different concentration

ranging from 0.5µg/L to 200µg/L.

The operation of the GC-MS system;

Model:

Carrier gas: helium gas, controlled at 1.5mL/min

GC oven temperature was hold at 40oC for the first 10 minutes,

increased 5oC for every minute until it reached 150oC. After that, the

temperature was increased 15oC for every minute until the temperature

reached 210oC. This Temperature was hold for 10 minutes. While the

temperature of the transfer line between GC and MS was set to 280oC.

Columns: Capillary column with 60m length, 0.32mm internal

diameter, and 1µm film thickness

Experimental Procedures:

i) The sample port (thermal desorber) was pre-heated to a

temperature of 300oC.

ii) Standard solutions were prepared from commercial solutions,

and then introduced into clean sorbent cartridges.

iii) The standard solutions were then analysed using the GC-MS to

obtain calibration curves, by using introducing them to the

thermal desorption instrument (the sample port) which

equipped with an auto-sampler.

iv) Then, the analysis of the VOCs followed. The VOCs samples

were transferred from the cartridges to the thermal desorption

instrument. Then the system was run for analysis.

v) The chromatographic peaks illustrations were printed out.

vi) Identification of chromatographic peaks of the compounds was

based on the retention time and references to the literature of

mass spectrum

1.4.3 Results

Figure 1 and Figure 2 shows the results from the analysis of

VOC content in the exhaled breathe of tobacco cigarette and e-

cigarette smokers. These data could led to a clue of what compounds

retain in the respiratory system from smoking whether tobacco

cigarette or e-cigarette.

The ambient air of the room was compared between the

environment before smoking and after smoking in Figure 1. According

to the discussion of the results of this experiment, the exhaled breath

environment was rich in acetone and isoprene. These are the typical

compounds that can be identified in this kind of sample.

As for Figure 2, the chromatograms of the exhaled breath of the

three samples were compared to the sample of the environmental air of

the room after smoking. The chromatogram of the exhaled air from

tobacco cigarette showed a more simplified mixture of compounds,

besides lowering in the abundance of some compound as compared to

the smoke chromatogram. This indicates that most of the compounds

of the smoke were retained in the respiratory system. While for the e-

cigarette exhaled breath chromatograms, both Type 1 and Type 2

showed the absence of propylene glycol and glycerine peaks, which

indicates that these compounds were retained in the lungs.

Besides that, the chromatogram of the tobacco cigarette

exhaled breath showed a significant difference to the chromatograms

of the e-cigarette exhaled breath. On the other hand, the

chromatograms of both Type 1 and Type 2 e-cigarette exhaled breath

were almost similar. Comparing tobacco cigarette and e-cigarette

exhaled breaths, tobacco cigarette chromatograms illustrated a higher

burden of VOCs than e-cigarette.

LIMITATIONS

HPLC

INTRODUCTION

High-performance liquid chromatography, HPLC is a term that can be used interchangeably

with LC, liquid chromatography. The term was used to distinguish the method it represents

with the classic gravity-flow liquid chromatography method. The general instrumentation

components consisted in LC is basically similar to the GC, but they differ in the types of the

components used, which will later be explained.

Liquid chromatography historically began with the use of glass column with diameters of 10

– 50mm. The stationary phase consisted of solid particles, packed with 50 – 500cm length.

The particles were coated with an adsorbed liquid. During that time, several modifications to

the size of the particles were made to establish a reasonable flow rates however failed,

because they led to longer time consumption. Then, attempts to speed up the partition process

was also made by putting on pressures, but led the increase of the plate height, which was

inefficient.

Liquid chromatography started to develop when scientists realized that the performance of

the column can be boosted by decreasing the size of the particles of packings. Then, in late

1960s, the technology to produce packings with particles of diameter of 3 – 10µm was

developed. The instrumentation of LC became more sophisticated in contrast to the

traditional gravity-flow liquid chromatography, as the development of the packing particles

required the operation of the system to be performed at high pressures.

LC is popularly used in analytical separation techniques due to its advantages. These

advantages include; higher sensitivity, adaptable to accurate quantitative determinations, easy

to handle and suitable for separation of non-volatile species. On top of those, the widespread

of the instrument is applicable to substances that are important in the industry, many fields of

science and the public. The substances include; hydrocarbon, drugs, proteins, carbohydrates,

pesticides, steroids, antibitics and various inorganic substances.

THEORY

The typical components of instruments in LC are shown in Figure 1. In modern LC, pressures

are pumped at several hundred atmospheres. This is required in order to achieve reasonable

flow rates for packing of 3 – 10µm. Hence, the HPLC equipment is more complex and costly

compared to other types of chromatographic instruments. The instrumentations of a general

HPLC are elaborated below.

Mobile phase reservoirs and solvent treatment system:

In a modern LC apparatus, it may consist of single or multiple glass reservoirs. The reservoirs

are contained with 500mL or more liquid solvents. Presence of dissolved gases and dust from

liquid solvents could interfere the flow rates and band spreading, as well as the detector

performance. Thus, some reservoirs have a built in sparging system, as shown in the figure.

The sparger functions as dissolved gasses remover by the means of fine bubbles of inert gas

that is insoluble in mobile phase. Other systems that may be built in the degasser are vacuum

pumping system, a distillation system, a device for heating and stirring, or as shown in the

figure, the inlet filter. It functions as a filtration to the dust and particulate matter in the

solvents which can cause damage to the pumping system and injection system, and could also

clog the column. In addition, partitioning valves are also equipped into a modern LC

instruments to help the partitioning of the solvent introduced in HPLC at ratios that can be

varied continuously.

Pumping system:

Pumps are required in LC to;

i) Generate pressures to up to 6000psi

ii) Produce pulse-free output

iii) Regulate flow rates at the range of 0.1 to 1.0 mL/min

iv) A better flow reproducibility

v) Control corrosion by solvents.

Though pump used to increase the pressures in LC, but these high pressure liquids do not

cause any hazardous explosion, since liquids are not very compressible. The results of these

high pressures may only lead to component rupture, and consequently cause solvent leakage.

Instead, this leakage may constitute to a fire and may be hazardous to the environment.

The widely used pumps types in LC are the screw-driven syringe and the reciprocating pump.

In most commercial LC instruments, they are equipped with a computerized system in which

they are more convenient in controlling the measurement of the flow rate.

Sample-injection system

Sample introduced into the column packing is required to be at a very small volume. The

volume range may be of a few tenths of microliter to of about 500µL. This helps to reduce

the limiting factor in LC precision, which is the reproducibility with which sample can be

introduced into the column.

The common conservative way to introduce the sample into LC is by using sampling loops.

They are integrated as a part of the LC instrument and the loops can also be interchangeable

depending on the sizes of the sample. These loops provide choices for sample sizes ranging

from 1µL to 100µL. On the other hand, most LC instruments in this modern day are equipped

with auto-injectors system. The system has sampling loops and syringe pump which allow

injection volumes to be ranging from less than 1µL to more than 1mL. While some systems

allow for controlling of the environment temperature of the sampling port and some may be

programmable to allow for unattended injections.

Columns:

There are several types of LC columns invented. These columns differ in terms of physical

constructions, sizes, packing and costs. Usually, they are built from smooth-bore stainless

steel tubing, sometimes heavy-walled glass tubing and polymer tubing, and they even built

from stainless steel, lined with glass or polymer. Common types of columns are; analytical

columns and guard columns.

Column temperature control:

Some applications of LC do not require a close control of temperature. They operated at room

temperature. But temperature regulation is often necessary to obtain better chromatograms.

Most advanced instruments now are equipped with heaters to regulate the temperature of the

column. Besides that, some columns may also be fitted with water jacket, which functioning

as the temperature regulator.

Column packing:

In HPLC, there are two well-known types of column packing used;

Pellicular particle – spherical, nonporous, glass or polymer beads with typical diameters of 30

– 40µm. There was a thin, porous layer deposited on the surface of the beads. This layer may

be made from silica, alumina, or an ion-exchange resin.

Porous particle – consists of porous microparticles with diameters ranging between 3 –

10µm. The particles are made from alumina, ion-exchange resin, or the most commonly used;

silica. The silica particle often coated with thin organic films. The films are chemically or

physically bonded to the surface.

Detectors

The ideal properties of detector used in LC include;

i) Adequate sensitivity

ii) Good stability and reproducibility

iii) Linear response to solutes that extends over several orders of magnitude

iv) Short response time independent of flow rate

v) Minimal internal volume to reduce zone broadening

vi) Compatible with liquid flow.

The detectors commonly used in HPLC are; UV-Visible absorption detectors, fluorescence

detectors, refractive-index detectors, and mass spectrometric detectors.

In general, the analysis run through the HPLC is quite similar to the analysis by GC system.

The sample is firstly sample port of the system, the mobile phase will carry the analytes of

the sample to the column of the HPLC system for separation, then the output from the

column is fed into the detector device. Finally, a schematic illustration, which is the outcome

of the analysis, is printed out of the output system.

SAMPLE PREPARATION

A research of identification and quantification of bioactive compounds in coffee brews was conducted by Naira Poerner Rodrigues and Neura Bragagnolo. High Performance Liquid Chromatography (HPLC) was involved in the methodology of the analysis.

There were several types of samples to be identified and quantified in the analysis. The samples were acquired from the local market in the city of Campina, San Paulo, Brazil. The samples were also produced of different manufacturers. As described in their packaging, the samples consisted of different degree of identity and quality, which made them to be classified into gourmet, traditional and supreme. Besides that, their degree of roasting also ranged between light to dark. The types of the samples include; 7 regular roasted ground coffees (marked as RR), 3 decaffeinated roasted ground coffees (DR), 2 regular soluble coffees (RS), and 2 decaffeinated soluble coffees (DS). The roasted ground coffees and the soluble coffees were brewed and prepared by the ratio of coffee to water as recommended in the packaging.

Sample Preparation Techniques:

i) To prepare the roasted ground coffees brew, 5g of the samples were firstly weighted in a filter paper Whatman no. 4

ii) 50 mL of ultrapure water of about 92oC was slowly poured through the centre of the filterpaper. And then, the solution was transferred into volumetric flasks. The final volume of the brew of roasted ground coffees varied from 34-37mL.

iii) To prepare the soluble coffees brew, 0.2g of the samples were weighed, and transferred into 10mL volumetric flasks.

iv) They were dissolved by ultrapure water at temperature of 25oC up to the calibration marks of the flasks.

v) The brews of the samples were immediately frozen by liquid nitrogen. And then, they were stored in a freezer at the temperature of -80oC until analysis.

EXAMPLE OF ANALYSIS

Analysis of bioactive compounds in coffee brews by HPLC

The sample preparation method was listed in the sample preparation part, section berapa

point berapa.

METHODOLOGY

The analysis procedures were quiet complex, thus, this part is only focusing on the

methodology on using the HPLC. Link to the full version of the article can be followed in the

references.

Components to be analysed: caffeine, chlorogenic acids (CGA) and derivatives, trigonelline,

nicotinic acids, 5-hydroxymethylfurfural, theobromine, theophylline.

Standard solutions prepared for sample identification: standards of caffeine (99% purity), 5-

caffeoylquinic acid (95% purity), caffeic acid (98% purity), p-coumaric acid (98% purity),

ferulic acid (98% purity), trigonelline hydrochloride (98% purity), nicotinic acid (99.5%

purity), 5-hydroxymethylfurfural (99% purity), theobromine (99% purity), theophylline (99%

purity).

The operation of the HPLC system;

Model: Shimadzu HPLC, equipped with binary pump, degasser with helium, and automatic

injection system. For quantitative analysis, the HPLC system was coupled to a diode array

detector (DAD), while for compound identification purpose, it was coupled with the mass

spectrometer (MS).

Mobile phase: mixture of 80% (v/v) 10mM citric acid (pH 2), 20% (v/v) methanol and 100%

(v/v) methanol for caffeine and CGA and its derivatives analysis. While for the other

compounds analysis, the mobile phase includes; 0.5% (v/v) acetic acid (pH 3) and methanol.

Columns: For caffeine and CGA and its derivative analysis, ODS-C18 Shim-pack column of

5m, 240mm X 4.6mm inner diameter was used. While the other analysis, the same type of

column was also used.

Experimental Procedures:

i) The coffee brews were treated firstly to precipitate the compounds of higher

molecular weight by using zinc acetate solution and potassium

hexacyanoferrate solution.

ii) For caffeine, and CGA and its derivatives determination, 1 mL of each coffee

brews were transferred into test tubes, and then added with 2mL of ultrapure

water. Next, 0.1mL of zinc acetate solution and potassium hexacyanoferrate

solution, and 0.8mL of methanol were added into the brew solution.

iii) The mixtures were shaken for 30 seconds, and then left to settle for 10

minutes.

iv) The precipitate was separated from the mixtures by centrifugation. The

supernatants were collected, and then filtered using 0.45µm membrane filters.

v) The analysis was carried out using HPLC-DAD and HPLC-DAD-MS. Before

analysing the samples, standard solutions were analysed in the systems by

introducing them to the auto-sampler of the system. Then, analysis of the

samples followed.

vi) While for the other compounds determination, the brews were treated using

0.2mL of aqueous lead acetate solution.

vii) The analysis using HPLC systems followed.

viii) The mass spectra illustrations were printed out and discussed quantitative, and

qualitatively by comparing the spectra to the literature.

RESULTS

Identification of caffeine and CGA and derivatives:

Identification of trigonelline, nicotinic acids, 5-HMF, theobromine and theophylline.

Figure 1 shows the chromatogram for the analysis of caffeine and CGA and derivatives.

Based on what had been reported in the analysis, besides caffeine, there were 26 derivatives

of hydroxycinnamic. They were classified into 4 groups; CGA (peaks 1, 2, 4, 5, 7, 11, 12, 18,

19, 20, 21, 22, 23, 25, 26), chlorogenic acid lactones (peaks 10, 13, 15, 17), cinnamoyl-amino

acid conjugates (peaks 24, 27) and free cinnamic acid (peak 8).

By comparison to the UV-visible and mass spectra of authentic standards, the compounds of

trigonelline, nicotinic acids, 5-HMF, theobromine and theophylline were identified in the

chromatogram in Figure 2.

The quantification was also done to find the amount of bioactive compounds content in the

coffee brews, and the results were summarized as in Table 1.

LIMITATIONS

REFERENCES

http://ac.els-cdn.com/S0021967315010821/1-s2.0-S0021967315010821-main.pdf?_tid=e1300718-8f58-11e5-9a81-00000aab0f02&acdnat=1448004940_137b57971409e30d258f4a917d4d50b4

hplc2- http://ac.els-cdn.com.ezaccess.library.uitm.edu.my/S0889157513001166/1-s2.0-S0889157513001166-main.pdf?_tid=aa2d8be6-902f-11e5-abf3-00000aacb361&acdnat=1448097189_3cd2ffa7c20b96440fdd3aa7cab6627d