BACHELOR OF SCIENCE ( HONS) APPLIED CHEMISTRY

(AS245)
LABORATORY REPORT
ANALYTICAL SEPARATION METHODS
CHM510
EXPERIMENT 1: GAS CHROMATOGRAPHY (GC) : OPTIMIZATION
OF FLOW RATE AND COLUMN TEMPERATURE

NAME MUHAMMAD AMIRUL AFIFI BIN MAT YAKI

STUDENT ID 2018287232
PARTNER’S NAME 1. SITI SARAH BINTI ABDUL AZIZ
2. NURHIDAYAH BINTI MOHAMAD
3. NUR AFIQAH BINTI RUSMAN
4. ANWAR

GROUP AS2452S2
LECTURER’S NORAINI
NAME
DATE OF
EXPERIMENT
DATE OF
SUBMISSION
ABSTRACT
To achieve the efficient separation of compounds in gas chromatography is dependent on the
retention time, tR of the compounds traveling through the column and peak shape. Column
temperature and carrier gas flow rate are the factors that affect the efficient separation. So, in this
experiment the optimal flow rate and column temperature was determine by analysis at different
condition of flow rates of carrier gas and column temperature. Different flow rates was analyse
on isothermal temperature at 210oC at carries gas flow rate 30 cm sec-1, 50 cm sec-1 and last at 70
cm sec-1. From the analysis flow rates at 70 cm sec-1 to be the optimal flow rates for analysis.
After that, to find the optimal column temperature, the column temperature was run at isothermal
temperature of 170 oC, 190 oC and 210 oC throughout the analysis at optimal flow rates 70 cm
sec-1. The optimum column temperature is dependant upon the boiling point of the sample. As a
rule of thumb, a temperature slightly above the average boiling point of the sample. Minimal
temperature give good Rs, but increase elution times. From the result obtained, the optimal
temperature for this analysis is at 210 oC. Lastly, the individual compounds of methyl ester was
analyse at optimal flow rates and column temperature to get efficient separation.

OBJECTIVE
To determine the optimize flow rate and column temperature in Gas Chromatography (GC) and
to analyse individual standard methyl esters compounds ; methyl laurate, methyl myristate,
methyl palmitate, methyl stearate and methyl linoleate.

INTRODUCTION
Gas chromatography is a separation method based on the conversion of the sample to the vapour
phase, followed by the introduction of the sample vapour onto a column containing a stationary
phase material. The column is most often an open tubular capillary with the stationary phase
coated onto the capillary walls themselves (wall-coated open tubular (WCOT) column) or onto
support material that has been attached to the capillary walls (support-coated open tubular
(SCOT) column). The mobile phase is typically an inert gas such as helium. As in other column
chromatographic methods, separation occurs on the basic of interactions between the sample
constituent and the stationary phase. These interaction most often take the form of partitioning
between the mobile phase and the stationary phases, although some stationary phase operate by
adsorption interactions. Because the sample must be maintained in the vapour phase, the column
is contained within an oven.

Separation efficiency in gas chromatography is the ability to separate two mixture components
by GC depends on the choice of stationary phase, the flow rate of the mobile phase, and the
column temperature. Optimizing a separation depends on the optimization of column separation
efficiency and on altering the relative migration rates of the components.

The technique propose in this experiment has been employed in isothermal gas chromatography
with split injection since, the concentration of the analyte is high. Isothermal gas
chromatography is an analysis where the column temperature is remained constant throughout
the analysis. Resolution (Rs) is used to describe how well the species in the mixture are
separated. Ideal resolution is 1.5 that indicates adequate separation between species. Lower than
1.5 resolutions is when the two species is not completely separated while too high resolution will
need longer analysis time.

The resolution, Rs of column provides a quantitative measure of the ability to resolve two
components. Resolution is defined as :

2Δ𝑍 2[(𝑡𝑅 )𝐴 − (𝑡𝑅 )𝐵 ]

𝑅𝑠 = =
𝑤𝐴 + 𝑤𝐵 𝑤𝐴 + 𝑤𝐵

REAGENTS AND SOLUTIONS

1. Individual methyl ester compounds: methyl laurate, methyl myristate, methyl palmitate,
methyl stearate and methyl linoleate.
2. Standard mixture of methyl laurate (0.20 mg/ml), methyl myristate(0.20 mg/ml), methyl
palmitate(1.0 mg/mL), methyl stearate(0.70 mg/mL) and metyl linoleate(0.35 mg/mL).

INSTRUMENT
Gas chromatography (Agilent Technologies 6890N) equipped with flame ionization detector
(FID) and 30 m x 250 µm x 0.25 µm HP5-MS capillary column.
ANALYTICAL PROCEDURE
a. The instrument is set up like below :

Injection port: split (40:1)

Injection port temperature: 250 oC
Column temperature: varies (170 oC, 190 oC, 210 oC)
Carrier gas flow rate: varies (30 m/s, 50 m/s, 70 m/s)
Detector temperature: 250 oC

b. Effect of carrier gas flow on isothermal GC separation of methyl esters.

The standard mixture was injected with 0.4μL at 210 oC isothermally and 30 m/s of gas
flow rate. For the second injection with 0.4μL volume of standard mixture, the gas flow
rate was increased to 50 m/s with same temperature of 210 oC. The same procedure was
repeated with 70 m/s gas flow rate. The resolution of 3 different flow rates at the same
temperature was calculated and compared to determine which gas flow rate give the
better separation of the compounds in the standard mixture.

c. Effect of column temperature on isothermal GC separation of methyl ester. 0.4μL

standard mixture was injected isothermally at 170 oC , followed by 190 oC at the optimal
carrier gas flow rate. The effect of column temperature on the separation, resolution and
analysis time was evaluated.

d. Identification of components in methyl esters mixture

Each methyl ester was injected individually to identify the various compounds in the
standard mixture using the optimized GC conditions.
RESULT AND CALCULATION
*Calculation of resolution is based on peak 2 and 3 as references
A. Effect of the variation of gas flow on the resolution.
Condition Injection Retention Peak width of Resolution Average
time of peak peak 2 and 3 ,Rs resolution
2 and 3 (min) (min)
30 cm/s, 1 4.594, 6.534 0.0503, 0.0780 30.2 30.1
210 °C 2 4.595, 6.536 0.0502, 0.0791 30.0
50 cm/s 1 2.758, 3.938 0.0602, 0.0765 17.3 20
210 °C 2 2.764, 3.943 0.0421, 0.0619 22.7
70 cm/s 1 1.980, 2.826 0.0358, 0.0514 19.4 18.65
210 °C 2 1.976, 2.822 0.0423, 0.0524 17.9
Table 1: The effect of the variation of gas flow on resolution.
From table (1) the optimized separation time of methyl ester is at 70 cm/s of gas flow rate.
B. Effects of the variation of column temperature at optimized column temperature on
resolution.
Condition Injection Retention Peak width of Resolution Average
time of peak 2 peak 2 and 3 ,Rs resolution
and 3 (min) (min)
70 cm/s 1 3.861, 7.683 0.0433, 0.0912 56.8 55.95
170°C 2 3.863, 7.686 0.0446, 0.0928 55.09
70 cm/s 1 2.583, 4.300 0.0324, 0.0516 40.9 40.55
190°C 2 2.593, 4.313 0.0321, 0.0534 40.2
70 cm/s 1 1.980, 2.826 0.0358, 0.0514 19.4 18.65
210°C 2 1.976, 2.822 0.0423, 0.0524 17.9
Table2 : The effect of the variation of column temperature at optimized column temperature on
resolution.
The optimized column temperature at 70 m/s of gas flow rate is 210°C column temperature
because it is produced the resolution nearest to the ideal resolution value that is 1.5 and also
shorter analysis time.
C. Retention time of standard compound of the methyl ester at optimum condition.

Standard Compound Retention time (min)

Methyl laurate 1.979

Methyl myristate 2.816

Methyl palmitate 4.527

Methyl stearate 8.010

Methyl linoleate 7.357

Table 3: The retention time of individual standard of methyl ester.

Sample Calculation :

Resolution for condition 70 cm/s at 210 oC

2(2.826 − 1.980)
𝑅𝑠(2,3) =
0.0358 + 0.0514

= 19.4 (𝑓𝑖𝑟𝑠𝑡 𝑖𝑛𝑗𝑒𝑐𝑡𝑖𝑜𝑛)

2(2.822 − 1.976)
𝑅𝑠(2,3) =
0.0423 + 0.0524

= 17.9 (𝑠𝑒𝑐𝑜𝑛𝑑 𝑖𝑛𝑗𝑒𝑐𝑡𝑖𝑜𝑛)

19.4 + 17.9
𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑟𝑒𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 =
2

= 18.65
DISCUSSION

In this experiment, the optimal GC conditions were determined for better separation and column
efficiency. This was to prevent band broadening of the peaks thus making the result
irreproducible. Other that, the method of split was also used due to the used of concentrated
sample where only small amount was needed.

Different flow rate of the mobile phase will affect the retention time of the compound for
example, the high flow rate of the gas will elute the analyte fast in shorter time but will cause
band broadening due to mass transfer (C-term) of Van Deemter plot, where the solute in the
column not completely interact and equilibrate. To reduce the band broadening and overlapping
peak of chromatogram, the optimum gas flow rate must be used for a better separation in short
retention time. First of all, the optimal flow rate was determined. The flow rate used was 30 cm/s
with 2100C of temperature. It gave resolution of 30.1 in averages. Next flow rate used was 50
cm/s with 2100C of temperature. The resolution given was 20 in averages. Lastly, for the flow
rate 70 cm/s with 2100C of temperature gave resolutions of 18.65 in averages. The optimal flow
rate was 70 cm/s which gave the fastest retention time compared to the other two and had the
lowest resolution time which yielded better separation.

In addition, the optimal temperature was also determined for better separation and faster
retention time but high temperature of column also will lead to column bleeding. So, the optimal
temperature need for column efficiency to get better separation of the mixture. The temperature
used were 1700C, 1900C and 2100C with the optimal flow rate of 70 cm3/s. For the temperature
1700C, the average resolutions was 55.95. While for the temperature 1900C, the average
resolutions was 40.55. Last at temperature 210oC, the average resolutions was 18.65. The
optimal temperature for this experiment was 2100C which gave the lowest resolution as well as
fastest retention time.

Besides that, the chromatogram result shows only three peaks detected. This is because the
sample only had three compounds. Five individual methyl esters compounds was analyze using
the isothermal column temperature method to separate each individual peaks. The order of
elution depended on the polarity of the analytes, stationary phase and mobile phase. The column
used was HP5-MS column which is less non-polar stationary phase. The elution order is depend
on the boiling point of the compound. The higher the molecular weight of the compound means
that the higher boiling point or simply we can say that boiling points increase as the number of
carbon is increased. From the result in the chromatogram methyl laurate is to be the first
compound that elute at retention time of 1.979 minutes because it has low boiling point follow
by methyl myristate with retention time 2.816 minutes then, methyl palmitate at retention time of
4.527 minutes. The two compound that not in the sample are methyl linoleate at 7.357 minutes
minutes and methyl stearate elute at 8.010.

CONCLUSION

From this experiment, we can conclude that the optimal GC conditions are the optimal flow rate
of 70 cm/s and 2100C for the optimal temperature.

REFERENCES

1. Nor’ashikin S., Ruziyati T., Mardiana S. (2012), Analytical Separation Methods

Laboratory Guide (2nd edition)
2. Prof. Rozita, Lecture notes, Gas Chromatography (GC)
3. Chemical Separations By Gas Chromatography And High Performance Liquid
Chromatography
http://www.phy.ohiou.edu/~small/c325/experiment3.pdf, retrieved on 17th May 2019.
4. Analysis of Fatty Acids ,
http://www.sigmaaldrich.com/analytical-chromatography/gas-chromatography/fatty-acid-
methyl-ester.html, Retrieved on 17th May 2019.

5. Skoog, D. A., Holler, F. J., & Crouch, S. R. (2007). Principle of Intrumental Analysis.
Canada: Cengage Learning.