Assignment

work
TOPIC- Assignment on analysis of
fatty acid by gas chromatography.

SUBMITTED TO- Ms. Anita singh

(Incharge ,instrumentation lab)

SUBMITTED BY- Shivani Sharma

(Bsc.Ist yr) Microbial And food technology

1. Gas Chromatography
In early 1900s, Gas chromatography (GC) was discovered by Mikhail Semenovich Tsvett as a separation
technique to separate compounds. Gas chromatography (GC), is a common type of chromatography used
in analytical chemistry for separating and analyzing compounds that can be vaporized without
decomposition. Typical uses of GC include
1.testing the purity of a particular substance
2. separating the different components of a mixture
3. help in identifying a compound
4. can be used to prepare pure compounds from a mixture.
Gas chromatography in which the sample is dissolved and vapourized to separate analytes
consists of two phases :-

MOBILE PHASE:- which is carrier as gas, it has to be inert gas such as; helium,
nitrogen (unreactive).It is to carry the molecules of the analyte through the heated
column.
STATIONARY PHASE:- It is solid support having microscopic layer of liquid or
polymer, which is inside a piece of glass or metallic tubing called column. The stationary
phase is either a solid adsorbant, termed gas-solid chromatography (GSC), or a liquid on
an inert support, termed gas-liquid chromatography (GLC).

In this instrument the gaseous compound which is to be analyzed interact with walls of the
column (being coated with a stationary phase).Due to this arrangement each compound elute at
specific temp (different for different compound) which is being analyzed by detector and finally
comparison of retention times obtained.

PRINCIPLE:- Gas chromatography has principle same as fractional distillation. Since the
components are separated on the basis of different boiling points. The component having lower
boiling point will elute faster as compared to those having comparatively higher boiling point
and finally detected by detector. GC technique is used for small scale analyzisation .

The gaseous compounds being analyzed interact with the walls of the column, which is coated with a
stationary phase. This causes each compound to elute at a different time, known as the retention time of the
compound. The comparison of retention times is what gives GC its analytical usefulness.
Gas chromatography is also sometimes known as vapor-phase chromatography (VPC), or gasliquid
partition chromatography (GLPC). These alternative names, as well as their respective abbreviations, are
frequently used in scientific literature. Strictly speaking, GLPC is the most correct terminology, and is thus
preferred by many authors.
2.Instr um entation

a) Sample Injection
A sample port is necessary for introducing the sample at the head of the column. Modern injection
techniques often employ the use of heated sample ports through which the sample can be injected and
vaporized in a near simultaneous fashion. A calibrated microsyringe is used to deliver a sample volume in
the range of a few microliters through a rubber septum and into the vaporization chamber.

Most separations require only a small fraction of the initial sample volume and a sample splitter is used to
direct excess sample to waste. Commercial gas chromatographs often allow for both split and splitless
injections when alternating between packed columns and capillary columns. The vaporization chamber is
typically heated 50 C above the lowest boiling point of the sample and subsequently mixed with the carrier
gas to transport the sample into the column.

Figure 1. A cross-sectional view of a microflash vaporizer direct injector.

b)Carrier Gas
The carrier gas plays an important role, and varies in the GC used. Carrier gas must be dry, free of oxygen
and chemically inert mobile-phase employed in gas chromatography. Helium is most commonly used
because it is safer than, but comprable to hydrogen in efficiency, has a larger range of flow rates and is
compatible with many detectors. Nitrogen, argon, and hydrogen are also used depending upon the desired
performance and the detector being used. A carrier gas is typically required in GC system to flow through
the injector and push the gaseous components of the sample onto the GC column, which leads to the
detector.
c) Column Oven

The thermostatted oven serves to control the temperature of the column within a few tenths of a degree to
conduct precise work. The oven can be operated in two manners: isothermal programming or temperature
programming:

(i) In isothermal programming, the temperature of the column is held constant throughout the entire
separation. The optimum column temperature for isothermal operation is about the middle point of the
boiling range of the sample.

(ii) Temperature programming works best only if the boiling point range of the sample is narrow. In this
method, the column temperature is either increased continuously or in steps as the separation
progresses. Rates of 5-7 C/minute are typical for temperature programming separations.

d) column

The choice of column depends on the sample and the active measured. The main chemical attribute regarded
when choosing a column is the polarity of the mixture, but functional groups can play a large part in column
selection. The polarity of the sample must closely match the polarity of the column stationary phase to
increase resolution and separation while reducing run time. The separation and run time also depends on the
film thickness (of the stationary phase), the column diameter and the column length. 2 types of column:
(i)Open tubular columns, which are also known as capillary columns. The walls of the fused-silica
columns are drawn from purified silica containing minimal metal oxides. These columns are much thinner
than glass columns. To protect the column, a polyimide coating is applied to the outside of the tubing and
bent into coils to fit inside the thermostatted oven of the gas chromatography unit. These , come in two basic
forms.

The first is a wall-coated open tubular --- WCOT column are capillary tubes that have a thin layer
of the stationary phase coated along the column walls.

The second type is a support-coated open tubular (SCOT) column. In SCOT columns, the column
walls are first coated with a thin layer (about 30 micrometers thick) of adsorbant solid, such as
diatomaceous earth. The adsorbant solid is then treated with the liquid stationary phase.

(ii)Packed columns, are made of a glass or a metal tubing which is densely packed with a solid support
like diatomaceous earth. Due to the difficulty of packing the tubing uniformly, these types of columns have
a larger diameter than open tubular columns and have a limited range of length.
e) Detection Systems
The detector is the device located at the end of the column which provides a quantitative measurement of
the components of the mixture as they elute in combination with the carrier gas.

(i)Mass Spectrometry Detectors-- In a GC/MS system, the mass spectrometer scans the masses
continuously throughout the separation. When the sample exits the chromatography column, it is passed
through a transfer line into the inlet of the mass spectrometer . The sample is then ionized and fragmented,
typically by an electron-impact ion source. During this process, the sample is bombarded by energetic
electrons which ionize the molecule by causing them to lose an electron due to electrostatic repulsion.
Further bombardment causes the ions to fragment. The ions are then passed into a mass analyzer where the
ions are sorted according to their m/z value, or mass-to-charge ratio.

(ii)Flame Ionization Detectors --FID in this common detector where electrodes are placed
adjacent to a flame fueled by hydrogen / air near the exit of the column, and when carbon containing
compounds exit the column they are pyrolyzed by the flame. This detector works only for organic /
hydrocarbon containing compounds due to the ability of the carbons to form cations and electrons upon
pyrolysis which generates a current between the electrodes. The increase in current is translated and appears
as a peak in a chromatogram. FIDs have low detection limits (a few picograms per second) but they are
unable to generate ions from carbonyl containing carbons.

(iii) Electron capture detector --ECD which uses a radioactive beta particle (electron) source to
measure the degree of electron capture. ECD are used for the detection of molecules containing
electronegative / withdrawing elements and functional groups like halogens, carbonyl, nitriles, nitro groups,
and organometalics.

(iv) Flame photometric detector --FPD which uses a photomultiplier tube to detect spectral lines of
the compounds as they are burned in a flame . Compounds eluting off the column are carried into a
hydrogen fueled flame which excites specific elements in the molecules, and the excited elements (P, S,
Halogens, Some Metals) emit light of specific characteristic wavelengths. The emitted light is filtered and
detected by a photomultiplier tube.

(v)Nitrogenphosphorus detector--NPD a form of thermionic detector where

nitrogen and phosphorus alter the work function on a specially coated bead and a resulting
current is measured.

3. Applications
Gas chromatography is a physical separation method in where volatile mixtures are separated. It can be
used in many different fields such as pharmaceuticals, cosmetics and even environmental toxins. Since the
samples have to be volatile, human breathe, blood, saliva and other secretions containing large amounts of
organic volatiles can be easily analyzed using GC.
4. Laboratory work
=>fatty acids is a carboxylic acid with a longaliphatic tail (chain), which is
either saturated or unsaturated. Most naturally occurring fatty acids have a chain of an even
number of carbon atoms, from 4 to 28. Fatty acids are usually derived
from triglycerides or phospholipids. When they are not attached to other molecules, they are
known as "free" fatty acids.

-- Fatty acids are important sources of fuel because, when metabolized, they yield large
quantities of ATP. Many cell types can use either glucose or fatty acids for this purpose. In
particular, heart and skeletal muscle prefer fatty acids. Despite long-standing assertions to the
contrary, fatty acids can be used as a source of fuel for brain cells, at least in some rodents, in
addition to glucose and ketone bodies.

-- Types of fatty acid ;

1. Unsaturated fatty acids

Unsaturated fatty acids have one or more double bonds between carbon atoms. (Pairs of carbon
atoms connected by double bonds can be saturated by adding hydrogen atoms to them,
converting the double bonds to single bonds. Therefore, the double bonds are called unsaturated.)

(i) Some fatty acids are missing one pair of hydrogen atoms in the middle of the
molecule. This gap is called an unsaturation and the fatty acid is said to
be monounsaturated because it has one gap.eg; palmitic ,oleic, myristoleic.

Eg; oils in which MUFA is present --olive oil, rapeseed oil.

(ii) Fatty acids that are missing more than one pair of hydrogen atoms are
called polyunsaturated.eg; linoleic, linolenic.

Eg;oils in which PUFA is present-- corn, soybean, cottonseed, sesame, and sunflower
oils.

2. Saturated fatty acids

Saturated fatty acids are long-chain carboxylic acids that usually have between 12 and 24 carbon atoms and
have no double bonds. Thus, saturated fatty acids are saturated with hydrogen (since double bonds reduce
the number of hydrogens on each carbon). Because saturated fatty acids have only single bonds, each carbon
atom within the chain has 2 hydrogen atoms (except for the omega carbon at the end that has 3 hydrogens).

Eg; Caprylic acid, Capric acid

In most naturally occurring unsaturated fatty acids, each double bond has three n carbon atoms after it, for
some n, and all are cis bonds. Most fatty acids in the trans configuration (trans fats) are not found in nature
and are the result of human processing (e.g., hydrogenation)
Fame mix (Fatty Acid Methyl Esters )

1)Butyric acid 36) Nervonic acid

2)Caproic acid 37) Cis-4,7,10,13,16,19-docosahexaenoic acid
3) Caprylic acid
4)Capric acid
5)Undecanoic acid
6)Lauric acid
7)Tridecanoic acid
8)Myristic acid
9)Myristoleic acid
10)Pentadecanoic acid
11)Cis-10-pentadecanoic acid
12)Palmitic acid
13)Palmitoleic acid
14)Heptadecanoic acid
15)Cis-10-heptadecanoic acid
16)Stearic acid
17)Elaidic acid
18)Oleic acid
19)Linolelaidic acid
20)Linoleic acid
21)Arachidic acid
22)Gamma lenolenic acid
23)Cis-11-eicosanoic acid
24)Linolenic acid
25)Heneicosanoic acid
26)Cis-11,14-eicosadienoic acid
27)Behanic acid
28)Cis-8,11,14-eicosatrienoic acid
29)Erucic acid
30)Cis-11,14,17-eicosatrienoic acid
31)Arachadonic acid
32)Tricosanoic acid
33)Cis-13,16-docosadienoic acid
34) Lignoceric acid
35)Cis-5,8,11,14,17-eicosapentaenoic acid
 GC Conditions
OVEN:
Initial temperature: 140C
Initial time : 5 min
Rate: 4
Final temperature: 240C
Final time: 20 min

FRONT INLET:
Mode: Splitless
Purge flow: 35.9 ml /min
Purge time: 0.00 min
Gas saver: On
Saver flow: 15ml/min
Gas type : Nitrogen

COLUMN:
Capillary column
Mode Number: Agilent 1222362 DB-23
Max temperature: 250C
Nominal length: 60 m
Nominal diameter: 250 um
Nominal film thickness: 0.25um
Initial flow: 1.2ml/min
Inlet : Front inlet
Outlet: Front detector
Outlet pressure: ambient

FRONT DETECTOR (FID):

Temperature: 250C
Hydrogen flow: 40ml/min
Air flow :100ml/min
Make up gas type :nitrogen
Flame : On

FRONT INJECTOR:
Sample washes: 4
Sample pumps: 6
Injection volume: 1ul
Syringe size: 10ul
Post injection solvent A washes: 3
Sample Operating Procedure

I) Chemicals-
-CH2Cl2 (HPLC grade)
-Methanol (HPLC grade)
-Dichloromethane (HPLC grade)
-KOH pellets
-HCl
-petroleum ether
-certified reference material (CRM)

II) Apparatus-
- GC-FID
- Column DB-23( 50%cynopropyl + 50%dimethyl polysiloxanes)
- GC vials
- Volumetric flasks
- Micropipette (10-100 microL)

III)Reagents: FAME CRM (Fatty acid methyl ester certified refrence material), 2N methanolic KOH,
5% methanolic HCl solution, Sample
( mixture of sunflower and mustard oil in
equal amount i.e.,50:50 ratio).

IV)Procedure:

Preperation of standard (FAME CRM) :Transfer 1ml of FAME CRM in 10 ml of

volumetric flask and make up the volume with dichloromethane.

Preperation of reagent

(1) 2N methanolic KOH

Take 11.2g KOH pellets in 100 ml volumetric flask with methanol and make up the volume
with this.
(2) 5% methanolic HCl solution
Take 20ml of methanol in 100ml volumetric flask .Add 14.2 ml HCl and make up the volume
with methanol

Preparation of sample
(1) Weigh 100mg of sample in a test tube
(2) Add 200 microL of 2N methanolic KOH solution
(3) Heat it for 10 min at 50C on water bath with frequently shaking
(4) Cool it for 10 min
(5) Add 1ml of 5% of methanolic HCl solution
(6) Heat it for 10 min at 70C on water bath with frequent shaking
(7) Cool it for 10 min
(8) Add 2ml of petroleum ether and shake for 2 min
(9) Saperate out the upper layer & transfer it in GC vials
(10) Inject the sample from GC vials GC at following conditions
 V)Result - We have taken a sample that is a mixture of sunflower and mustard oil (50:50).As per
codex limit the range or limit of specific methyl esters are as follows:

CODEX LIMIT

Fatty acid methyl esters sunflowerseed oil mustard oil

C6:0 Methyl caproate ND ND

C8:0 Methyl caproate ND ND
C10:0 Methyl decanoate ND ND
C12:0 Methyl dodecanoate ND-0.1 ND
C14:0 Methyl myristate ND-0.2 ND-1.0
C16:0 Methyl palmitate 5.0-7.6 0.5-4.5
C16:1 (cis-9) Methyl palmitoleate ND-0.3 ND-0.5
C17:0 Methyl heptadecanoate ND-0.2 ND
C17:1 (cis-10) Methyl heptadecenoate ND-0.1 ND
C18:0 Methyl stearate 2.7-6.5 0.5-2.0
C18:1 (cis-9) Methyl oleate 14.0-39.5 8.0-23.0
C18:2 (all-cis-9,12) Methyl linoleate 48.3-74.0 10.0-24.0
C18:3 (all-cis-6,9,12) Methyl linolenate ND-0.3 6.0-18.0
C20:0 Methyl arachidate 0.1-0.5 ND-1.5
C20:1 (cis-11) Methyl eicosenoate ND-0.3 5.0-13.0
C20:2 (all-cis-11,14,) Methyl eicosadienoate ND ND-1.0
C22:0 Methyl behenate 0.3-1.5 0.2-2.5
C22:1 (cis-13) Methyl erucate ND-0.3 22.0-50.0
C22:2 (all-cis-13,16) Methyl docosadienoate ND-0.3 ND-0.1
C24:0 Methyl lignocerate ND-0.5 ND-0.5
C24:1 (cis-15) Methyl nervonate ND 0.5-2.5

=>in results we found that there is gradual increase in

- Palmitic
- Oleic
- Linoleic
=>whereas decrease in some methyl esters %age, those are
- Erucic
- Linolenic
- Ecosenoic
- Behenic
Precautions
1. Handle the chemicals carefully
2. blank peaks during integration should be deleted.
3. equipments should be well caliberated
4. all the volume make ups for the procedure should be done correctly.

Reference
1. http://en.wikipedia.org/wiki/Gas-liquid_chromatography
2. http://www.wfu.edu/chem/courses/organic/GC/index.html
3. http://www.chromatography-online.org/3/contents.html
4. http://www.energy.psu.edu/sp/facilities/images/tcd.jp
5. http://www.chem.agilent.com/Library/Support/Documents/a16122.pdf
6. http://www.separationprocesses.com/Absorption/GA_Chp04b.htm