ASSIGNMENT CHE 515

INSTUMENTAL CHEMISTRY
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
&
GAS CHROMATOGRAPHY MASS SPECTROSCOPY (GC-MS)
PREPARED BY
SITI NURAJJAR BINTI JAMI
2013 762 607
EH 2204
PREPARED FOR
DR KAMARIAH
2015

ABSTRACT

1.0GC-MS

1.1 INTRODUCTION
Gas 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 analytes
ii)
Mobile phase liquid, superficial fluid, or in this case gas that carry
analytes to stationary phase
iii)
Sample injection compartment the port to introduce the sample into the
system
iv)
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
1.2.2

rate at the desired level.

Sample injection system;
The sample port, located at the head of the column, needs to be preheated 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 20L 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
1.2.3

compared to the manually sample injection using the syringe

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 electronimpact 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 ecigarette 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
ii)

mouthpiece.
Volunteers were asked to smoke tobacco cigarette, by inspired and expired

iii)

deeply three times, then retain the breath for 20 seconds

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

iv)
v)

disposable cardboard mouthpiece.

Step ii) was repeated five times.
The sample from the Bio-VOC was transferred into a sorbent cartridge
through a screwed-in plunger. The VOCs of tobacco cigarette exhaled

vi)
vii)

breathe were accumulated in the same cartridge.

Steps from ii) through iv) were repeated for e-cigarette Type I and Type II.
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 0.5g/L to 200g/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 150 oC. 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 1m film thickness
Experimental Procedures:
i)
The sample port (thermal desorber) was pre-heated to a
ii)

temperature of 300oC.
Standard solutions were prepared from commercial solutions,

iii)

and then introduced into clean sorbent cartridges.

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

iv)

equipped with an auto-sampler.

Then, the analysis of the VOCs followed. The VOCs samples
were transferred from the cartridges to the thermal desorption

v)
vi)

instrument. Then the system was run for analysis.

The chromatographic peaks illustrations were printed out.
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 ecigarette 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 ecigarette 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 10m 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 10m. 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)
ii)
iii)
iv)
v)

Generate pressures to up to 6000psi

Produce pulse-free output
Regulate flow rates at the range of 0.1 to 1.0 mL/min
A better flow reproducibility
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 500L. 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 1L to 100L. 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 1L 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
40m. 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
10m. 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)
ii)
iii)
iv)
v)
vi)

Adequate sensitivity
Good stability and reproducibility
Linear response to solutes that extends over several orders of magnitude
Short response time independent of flow rate
Minimal internal volume to reduce zone broadening
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)
ii)

iii)
iv)
v)

To prepare the roasted ground coffees brew, 5g of the samples were firstly
weighted in a filter paper Whatman no. 4
50 mL of ultrapure water of about 92 oC 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.
To prepare the soluble coffees brew, 0.2g of the samples were weighed, and
transferred into 10mL volumetric flasks.
They were dissolved by ultrapure water at temperature of 25 oC up to the
calibration marks of the flasks.
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), 5caffeoylquinic 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

ii)

weight

by

using

zinc

acetate

solution

and

potassium

hexacyanoferrate solution.
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

iii)

solution, and 0.8mL of methanol were added into the brew solution.
The mixtures were shaken for 30 seconds, and then left to settle for 10

iv)

minutes.
The precipitate was separated from the mixtures by centrifugation. The

v)

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

vi)

samples followed.
While for the other compounds determination, the brews were treated using

vii)
viii)

0.2mL of aqueous lead acetate solution.

The analysis using HPLC systems followed.
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.

LIMITATIONSREFERENCES
http://ac.els-cdn.com/S0021967315010821/1-s2.0-S0021967315010821main.pdf?_tid=e1300718-8f58-11e5-9a8100000aab0f02&acdnat=1448004940_137b57971409e30d258f4a917d4d50b4
hplc2- http://ac.elscdn.com.ezaccess.library.uitm.edu.my/S0889157513001166/1-s2.0S0889157513001166-main.pdf?_tid=aa2d8be6-902f-11e5-abf300000aacb361&acdnat=1448097189_3cd2ffa7c20b96440fdd3aa7cab6627d