CM2192 Analytical

Experiment 1:

Gas Chromatography (GC) for Qualitative and Quantitative Analysis

Prepared by Dr Emelyn Tan

Type Stationary Phase Mobile Phase Sample

GC Liquid on solid or solid beads in column

Inert gas e.g. H2 He or N2

Mixed in mobile phase (gas)

Compound starts in mobile phase

Column Chromatography: stationary phase is held in a narrow tube and the mobile phase is forced through the tube by gravity or under pressure. - Column, GC, HPLC and SCFC.

Separation based on rate of analytes movement through stationary phase.

2

Schematic Diagram of GC

Figure 31-1 Skoog

T is regulated

Requirements for the Analyte: - Volatile (low boiling point, high vapour pressure) - Thermal stability

Separation depends on: a) Volatility (MAIN FACTOR):

Higher volatility, shorter retention time (tR).

b) Differential interaction between analytes and stationary phase:

Weaker interaction (opposite polarity), shorter tR.

No interaction with the mobile phase i.e. inert carrier gas.

3

Instrument Parameters

1. Column type Packed with Chromosorb W

Chromosorb W is a white, polar, crumbled, naturally occurring, soft siliceous sedimentary rock and diatomaceous earth (contains a type of hard-shelled algae).

2. Coating 20% Free Fatty Acid Phase (FFAP): polyethylene glycol substituted with terephthalic acid

3. Diameter of packing particle (mm) 10 to 200 m

4. Inner diameter (mm) 2 or 3 mm

5. Length (cm) 100 or 200 cm

6. Temperature isothermal at 150 C

7. Carrier gas N2 8. Linear flow rate (cm/s) varied

4

Flame Ionisation Detector (FID)

Effluent from the column is directed to H2-air flame. Organic compounds form cations and electrons when pyrolyzed at the temperature of the flame. Pyrolysis is a decomposition of organic material at elevated temperatures without the participation of oxygen.

Collector electrode will capture the charge carriers (ions and electrons), the resulting current is measured by a picoammeter.

The response is proportional to the number of the carbon atoms in the sample. This is related to the effective carbon number (ECN).

Sample in

Figure 31-8 Skoog

5

Plate Theory

Plate theory supposes that the chromatographic column contains a large number of separate layers or plates, of a given plate height.

This theory, which is adopted from a distillation column, is an assumption as there is actually insufficient time for equilibration in an chromatographic column.

6

Figure 30F-2 Skoog

Column efficiency and separation improves:

as the plate count N increases

as the plate height H decreases

H

LN

N = plate count or number of theoretical plates

L = length of the column packing (cm) fixed

H = plate height or Height Equivalent of Theoretical Plate (HETP) (cm)

Column Efficiency, H

7

Because the chromatographic peaks are usually Gaussian, the column efficiency is reflected by the breath of the peaks i.e. the variance s2, per unit length of the column. The plate height i.e. column efficiency H:

L

H

2

Figure 30-11 Skoog

Plot showing distribution of molecules along the length of the column at the moment that the analyte peak reaches the end of the column/detector i.e. at the retention time, tR.

s2 = variance of Gaussian peak (cm2)

Gaussian Distribution

1s = 34%

t x uo = L

8

2

2

2

2

2

2

W

L16

)4(W

L

LN

No of theoretical plates:

Number of Theoretical Plates, N

H

LN

L

H

2

4W base, at width Peak

2

2

R

W

t16N tR

and W units are in minutes.

2

1/2

2

R

W

t5.54N

Peak width at height (PWHH), W1/2, is used when W is difficult to be accurately determined.

A larger N value represents better separation. Hence, a good separation is when narrow peaks are obtained.

W68% = 2s

linear flow rate (cm / min)

Confidence Level = 95.4%

Variables that affect Column Efficiency

A decrease of H relates to an increase in column efficiency and separation, less peak/band broadening.

Many variables affect H and various equations of H have been proposed that incorporate these complex physical interactions and effects.

Skoog

phase Mobile

phase Stationary

t

t k

Rate theory: The van Deemter equation

The column efficiency can be approximated by:

A: coefficient that describes multiple path effects (eddy diffusion)

B: longitudinal diffusion coefficient

CS: mass transfer coefficient for stationary phase

CM: mass transfer coefficient for mobile phase. At high flow rate, CM 0.

u: linear flow velocity (cm/s)

u )C (Cu

BAH Ms

10

In Plate theory, the number of theoretical plates, N, and plate height, H, is related closely to the efficiency of the chromatography column. Plate theory, however, does not provide any information on the effect of flow rate on H, whereas Rate theory does.

The Multiple Path Effects: Eddy Diffusion

A molecule can travel through a packed column using different paths, thus the residence times of each molecule is variable and band broadening occurs. This effect is called eddy diffusion.

The heterogeneity in axial velocities is related to:

- particle size and uniformity

- geometry of packing

Using small and uniform particles which give a

tighter and more consistent packing

will minimize this effect.

11

Figure 30-14 Skoog

Molecule 2 will arrive later at B.

uCu

BAH s

Eddy Diffusion:

pd 2A

l is packing factor, ranging from 0.8 1

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The Multiple Path Effects: Eddy Diffusion

l is a constant accounting for the consistency of the packing. A more consistent packing gives a smaller value for l which range from 0.8 to 1.

dp is the average diameter of the packing particles.

Longitudinal Diffusion

In chromatography, the diffusion results in movement of the solute from the concentrated center of a band to less concentrated regions on both sides.

This results in band broadening and is more evident as time increases.

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uCu

BAH s

MD 2B

g is a constant related to the column packing which range from 0.6 to 0.8. DM is the solutes diffusion coefficient in the mobile phase.

Mass Transfer

The Cu term comes from the finite time required for solute to equilibrate between the two phases.

Thicker film on particles, smaller diffusion coefficient i.e. solute travels slower, larger Cs.

uCu

BAH s

S

2f

2S D

d

1)3(k

2kC

k: retention factor, tS/tM df: thickness of film on stationary phase DS: diffusion coefficient of solute on stationary phase

pd 2A MD 2B S

2f

2S D

d

1)3(k

2kC

uCu

BAH s

14

van Deemter equation

NLH

tR and W units are in seconds. 2

1/2

2

R

W

t5.54N

Run GC at different carrier gas flow rates cm3/min 20 cm3/min

tR = 157.02 s

W1/2 = 11.99 s

N = 950.13

H = 200 / 950.13 = 0.210 cm

ruAuF 2oo uo = F / r

2

= (20/60) / 0.152

= 4.72 cm/s

Plot HETP (or H) vs. Carrier gas linear flow rate (or uo)

uCu

BAH s

Take 3 points, (uo, H), and solve simultaneously for A, B and C.

http://math.cowpi.com/systemsolver/3x3.html

Part 1: Determination of HETP Analyte: ethylbenzene

/min)(cm F 3

(cm/s) uo

)(cm A 2

4.72C4.72

BA0.21 s(4.72, 0.21)

(2.83, 0.19) (1.18, 0.19)

2.83C2.83

BA0.19 s

1.18C1.18

BA0.19 s

1 2 3

1 2 1.89C)2.83

B

4.72

B(0.02 s

1 3 3.54C)1.18

B

4.72

B(0.02 s

4 5

4 x (3.54/1.89) 4 x 1.873

3.54C)1.51

B

2.52

B(0.0375 s 6

6 5 0.048 B )1.18

B

4.72

B()

1.51

B

2.52

B(0.0175

0.014 C Sub B into 4, 5 or 6

Sub B and C into 1, 2 or 3 0.13 A 0.014u

u

0.0480.13H

(uo, H)

17

uCu

BAH s

H (

cm)

u (cm/s)

B/u

A

Csu Mass transfer

Eddy diffusion

Longitudinal diffusion

Optimum linear flow rate and maximum column efficiency, when H and band broadening is minimum.

18

Part 2: Qualitative and Quantitative Analysis

Sample A: equal volumes of toluene and ethylbenzene

Sample B: 3 mL cyclohexane, 4 mL of n-propanol and 3 mL o-xylene

Sample C: equal volumes of ethanol, n-propanol, n-butanol and n-pentanol

Sample D: ???

Sample Compound Density / g mL-1

Mr BP / oC

Vol / mL

Weight / g

Weight %

n Mole

% Peak area

%

A

toluene 0.8669 92.15 110.6 5 x 10-4 4.3345 x 10-4

50.01 4.704 x 10-6 53.55 49.69

ethylbenzene 0.8665 106.17 136.2 5 x 10-4 4.3325 x 10-4

49.99 4.081 x 10-6 46.45 50.31

Inject 1 mL

Weight = Density x Vol

% 100 xWeight Total

Weight% Weight

Mr

mn

% 100 xn Total

n% Mole

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Sample Compound Density / g mL-1

Mr BP / oC

Vol / mL

Weight / g

Weight %

n Mole

% Peak area

%

A

toluene 0.8669 92.15 110.6 5 x 10-4 4.3345 x 10-4

50.01 4.704 x 10-6 53.55 49.69

ethylbenzene 0.8665 106.17 136.2 5 x 10-4 4.3325 x 10-4

49.99 4.081 x 10-6 46.45 50.31

Sample A: toluene and ethylbenzene have similar polarity. Hence elution order is dependent on boiling point.

% 100 xarea peak Total

area Peak% area Peak

toluene ethylbenzene

Retention time, tR

20

Sample C: equal volumes of ethanol, n-propanol, n-butanol and n-pentanol

Retention Volume (mL) = volumetric flow rate (mL/min) x retention time (min)

Sample Compound No. of C tR Retention volume/ mL Logarithm of retention volume

C

ethanol 2 1.4

n-propanol 3 1.6

n-butanol 4 1.7

n-pentanol 5 1.9

y = 0.16x + 1.09 R = 0.9846

1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2

0 1 2 3 4 5 6

Log

ret

enti

on

vo

lum

e (m

L)

Number of carbon atoms in the molecule

For n-hexanol : y = 0.16(6) + 1.09 = 2.05 Retention volume: 112 mL

Qualitative Analysis: identity of the analytes in Sample D can be determined by comparing the retention times, tR of the three unknown analytes in the Sample Ds chromatogram with the tR of nine known analytes in Sample A, B and C.

Sample A: equal volumes of toluene and ethylbenzene

Sample B: 3 mL cyclohexane, 4 mL of n-propanol and 3 mL o-xylene

Sample C: equal volumes of ethanol, n-propanol, n-butanol and n-pentanol

Sample D: ???

Quantitative Analysis:

C Sample in analyteof Volume xC Sample in analyteof area Peak

D Sample in analyteof area PeakD Sample in analyteof Volume

sample

standard standard sample

e.g. Analyte: n-propanol Volume of n-propanol in Sample D = L 0.5 L 0.25 x

10 x 2

10 x 45

5

% 100 xVolume Total

Volume% Vol

Volume area Peak

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Part 3: Effective Carbon Number

Flame Ionisation Detector (FID)

Current signal Number of ions produced

for organic compounds

Number of reduced carbon atoms in the

flame

Effective carbon number (ECN) = individual carbon atoms contributions + functional group contributions (Table 1-1 in lab manual).

a a

Atom Type ECN

C Aliphatic 1.0

C Aromatic 1.0

O primary alcohol 0.6

ECN: 6(1.0) + 1(1.0) = 7.0 e.g. toluene

ECN: 2(1.0) + 1(-0.6) = 1.4 e.g. ethanol

smallest ECN

Relative ECN ratio (expected): 7.0 / 1.4 = 5.00

Relative ECN ratio (expected): 1.4 / 1.4 = 1.00

Effective carbon number (ECN) (experimental) = n

area Peak

e.g. toluene

15

6-

10 x 1.56

)calculated y(previousl10 x 4.704

am)chromatogr (from .87317915310

tal)(experimen ECN

e.g. ethanol

14

6-

10 x 3.10

)calculated y(previousl10 x 4.274

am)chromatogr (from .41324364458

tal)(experimen ECN

smallest ECN (experimental)

Relative ECN ratio (experimental): 1.56 x 1015 / 3.10 x 1014 = 5.03

Relative ECN ratio (experimental): 3.10 x 1014 / 3.10 x 1014 = 1.00

Relative ECN ratio (expected): 7.0 / 1.4 = 5.00

Relative ECN ratio (expected): 1.4 / 1.4 = 1.00

% difference: 0.60 % % difference: 0.00 % 23