• A gaseous mobile phase flows under pressure through a heated tube either coated with a liquid stationary phase or packed with liquid stationary phase coated onto a solid support.

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Chapter 11 Gas Chromatography

Shin-Hun P. Juang Ph.D.

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Principles of GC

• A gaseous mobile phase flows under pressure through a heated tube either coated with a liquid stationary phase or packed with liquid stationary phase coated onto a solid support.

• The analyte is loaded onto the head of the column via a heated injection port, where it evaporates. It then

condenses at the head of the column, which is at a lower temperature. The oven temperature is then either held constant or programmed to rise gradually.

• Once on the column, separation of a mixture occurs

according to the relative lengths of time spent by its

components in the stationary phase. Monitoring of the

column effluent can be carried out with a variety of

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Strengths Limitations Capable of the same quantitative

accuracy and precision as high- pressure liquid chromatography (HPLC), particularly when used in conjunction with an internal standard.

Only thermally stable and volatile compounds can be analyzed.

Has much greater separating power than HPLC when used with capillary columns.

The sample may require derivatisation to convert it to a volatile form, thus introducing an extra step in analysis and, potentially, interferants.

Readily automated Quantitative sample introduction is more difficult because of the small volumes of sample injected.

Can be used to determine compounds which lack chromophores.

Aqueous solutions and salts cannot be injected into the instrument

The mobile phase does not vary and MW < 400

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Types of GC Instrument

Gas-solid chromatography (GSC)

Gas-liquid chromatography (GLC, GC)

Stationary Phase Solid Immobilized Liquid

GSC based on physical adsorption of solute molecules onto a solid

usually lots of tailing due to non-linear process so has not found wide application

except for the separation of certain low-molecular-weight gaseous species

GLC, GC based on partition of analyte between a gaseous mobile phase and a liquid phase

immobilized on the surface of an inert solid. GLC is found in all fields of science and name is usually

shortened to just GC

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GC Instrument

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Gas Chromatography Instrument

1. Syringes

2. Injection systems

1. Packed column injection 2. Split/splitless injection 3. Cool on-column injection

4. Programmable temperature vapouriser (PTV)

3. GC oven

4. Type column

1. Packed column

2. Capillary column

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Syringes

• The volumes injected in GC are routinely in the range of 0.5-2 μl; the usual syringe volumes are 5 and 10 μl.

• The syringe needle can then be introduced into the injector and left for a couple of seconds to warm up

0.5 μl of solvent

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Injection Systems (I) Packed column injections

• Injection generally occurs through a resealable rubber septum. The injector port is held at 150-250℃ depending on the volatility of the sample and direct injection of 0.1-10 μl of sample is made onto the head of the column. The

amount of sample injected onto a packed column is ca 1-2 μg per component.

• All of the sample is introduced into the packed column. Thus, although packed

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Injection System (II) Split / splitless injection

• This type of injector is used in conjunction with capillary column GC. Injection takes place into a heated glass or quartz liner rather than directly onto the column.

– split mode: sample is split into two unequal portions, the smaller of which goes onto the column. Split ratios range

between 10.1 and 100: 1, with the larger portion being vented in the higher flow out of the split vent. This technique is used with concentrated samples.

– splitless mode: all of the sample is introduced onto the column and the injector purge valve remains closed for 0.5-1 min after injection.

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Splitless Model

Split Model: 20:1

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Cautions

1. Lack of precision: High temperature resulting from

decomposition of some of the components in a mixture before they reach the column. Ensure that the sample has minimal contact with metal surfaces during the injection process since these can catalyse decomposition.

2. No discrimination between more and less volatile components in a mixture in the split.

3. Sample backlashes in splitless injection: Inject volumes have to be kept below ca 2 μl in case the through rapid expansion of the solvent in which it is dissolved, into either the gas supply lines. Each 1 μl of solvent expands greatly upon vapourization, e.g. methanol ca 0.66 ml/μl or ethyl acetate ca 0.23 ml/μl at atmospheric pressure.

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4. Randomly discriminated exist: Because the volatility or

decompose properties between the internal standard and samples.

5. The sample boiling point > ca 50°C higher than the column starting temperature in thesplitless mode: The sample must be efficiently trapped at the head of the column. For this to occur, it must be sufficiently involatile, i.e. have a. If the sample is relatively

volatile, it has to be injected into the GC in a low volatility solvent, which will condense at the head of the column, trapping the

sample in the process.

6. Slow sample transfer: may be and it is important to take this into account when setting purge valve times.

7. Injection precision: Auto sampler is better to carry out injection.

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Injection System (III) Cool on-column injection

 Advantages:

1. Reduced discrimination between components in mixtures 2. No sample degradation in a hot injector

3. No backflash, hence quantitative sample transfer

 Disadvantages:

1. Samples have to be clean otherwise residues will be deposited on the column

2. The injector is mechanically more complex and requires more maintenance than a septum injection system

3. The syringe needle may damage the head of the column

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Injection System (IV)

Programmable Temperature Vapouriser (PTV)

 Designed to enable the injection of large volumes of sample (5 and 50 μl ) onto capillary GC columns. The injector being held at low temperature, e.g. 30°C. The solvent is then vented through a purge valve at high flow rate (e.g. 100 ml/min for 1 min).

 The less volatile sample components are retained in the injection port; the purge valve is then closed and the injector temperature is ramped up rapidly (e.g. to 300°C at 700°C/min). This is possible because is made of Silicosteel, which provides an inert surface but conducts heat much more rapidly than glass.

 The boiling point of the lowest boiling component in the sample should be at least 100°C greater than that of the solvent .

 Can be used in conjunction with fast GC. Fast GC utilizes the high efficiency of capillary GC by using very short columns so that

separations of complex mixtures may be achieved in less than a minute.

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Programmable Temperature Vapouriser

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GC Oven

 Purpose of the Fan: Provide the uniform heat distribution

 Purpose of Programmer: provide Isothermal conditions or Gradual increase in temperature

Advantages

1. Materials of widely differing volatilities can be separated in a reasonable time

2. Injection of the sample can be carried out at low

temperature, where it will be trapped at the head of the

column and then the temperature can be raised until it elutes.

GC ovens incorporate a fan

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Isothermal vs Temperature Gradient

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GC Column

Two major types of columns

1. Packed: internal diameters of 2-5 mm

2. Capillary: internal diameters of 0.15 - 0.5 mm

lengths between 12 and 50 m

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The columns are packed with particles of a solid support (diatomaceous earth) which are coated with the liquid stationary phase. Packed column GC affords a relatively low degree of resolution compared to capillary GC.

internal diameters of 2-5 mm

mobile phase: nitrogen with a flow rate of ca 20 ml/min

temperature limit : 280°C

Types of column (I)

Packed Column

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Types of column (II) Capillary Column

 internal diameters of 0.15 - 0.5 mm lengths between 12 and 50 m

 Capillary columns are made from fused silica, usually coated on the outside with polyamide to give the column flexibility. Coating on the outside with aluminum has also been used for high temperature (> 400°C) work.

 mobile phase: helium with a flow rate between 0.5 and 2ml / min

 wall-coated open tubular (WCOT) columns

 support-coated open tubular (SCOT) columns

 Fused-silica open tubular (FSOT) columns: most common type

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Typical form of a coiled column

FSOT

organo silicone polymers

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Selectivity of liquid stationary phases

Kovats indices (I-values) are based on the retention time of an analyte compared to retention times of the series of

n-alkanes.

The n-Alkanes have most affinity for non-polar phases and tend to elute more quickly from polar phases.

In contrast, a polar analyte will elute more slowly from a

polar phase and thus, relative to the n-alkanes, its retention

time, and thus its I-value, will increase as the polarity of the

GC phase increases.

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McReynold's constant is based on the retention times of

benzene, n-butanol, pentan-2-one, nitropropane and pyridine on a particular phase.

The higher the McReynold's constant, the more polar the phase.

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Examples of the separation of mixtures by GC

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BPX-5 column selects mainly on the basis of molecular weight and

shape.

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Carbowax column is highly selective for

polar compounds.

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The monograph for almond oil states the composition of the fatty acids making up the triglyceride should be:

• palmitic acid (16:0) 4.0-9.0%

• palsnitoleic acid (16: 1) < 0.6%

• rnargaric acid (17:0) < 0.2%

• stearic acid (18:0) 0.9-2.0%

• oleic acid (18: 1 ) 62.0-86.0%

• linoleic acid (18:2) (7.0-30.0%)

• linolenic acid (18:3) < 0.2%

• arachidic acid (20:0) < 0.1 %

• behenic acid (22:0) < 0.1%.

Analysis of the fatty acid composition of

a fixed oil by GC

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Methanolysis of Triglycerides

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BPX-5

Stabiliwax

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Associate the following I-values obtained on an OV-1-type

column values: 1555, 2018, 2323 and 2457. Note: oxygen in an ether linkage is equivalent to ca 1 CH₂, unit. with structures of the local anaesthetics shown below. /-

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Chiral Selectivity

• Enantiomers are not separable due to the identical physical properties.

• Two approaches to separate and analyze – Chiral column

– Derivatisation

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Column with Chiral Center

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Using Derivatization for GC Masking the polar group

.

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*Silylating Reagents.

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• Carrier gas type / flow: Table 12

• Column temperature

• Column length

• Film thickness phase loading

• Internal diameter

Parameters governing capillary GC

performance

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A fixed temperature is used and the head pressure is

adjusted so that the linear velocity of a helium carrier gas through the following capillary columns is 20 cm/s: (i) 30 m x 0.25 mm i.d. x 0.25 pm OV-1 film; (ii) 15 m x 0.15 mm x 0.2 pm OV-1 film; (iii) 12 m x 0.5 mm i.d. x 1.0 pm OV-1 film.

a. List the columns in the order in which they would increasingly retain a n-hexadecane standard.

b. List the columns in order of increasing efficiency.

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GC detectors

most widely used

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Applications of GC in quantitative analysis

Analysis of methyltestosterone in tablets

Solution 1 : ca 0.04% w/v of methyltestosterone and ca 0.04% w/v testosterone in ethanol is prepared

Solution 2 : A weight of tablet powder containing ca 20 mg of methyltestosterone is extracted with 50 ml of ethanol

Solution 3 : dissolving tablet powder containing ca 20 mg of

methyltestosterone in exactly 50 ml of ethanol containing exactly the same concentration of testosterone as Solution 1.

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Data from Analysis of

Methyltestosterone Tablets

• Weight of 5 tablets = 0.7496 g

• Stated content of methyltestosterone per tablet = 25 mg

• Weight of tablet powder taken for assay = 0.1713 g

• Solution 1 contains: 0.04% wlv methyltestosterone and 0.043%

W/V testosterone

• Solution 3 contains: the methyltestosterone extracted from the powder taken for assay and 0.043% wlv testosterone

• Solution 1: Peak area testosterone = 216268; Peak area methyltestosterone = 212992

• Solution 3: Peak area testosterone = 191146; Peak area

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Analysis of Atropine in Eyedrops Analysis of Atropine in Eyedrops

BSA:

N,O-bistrimethylsilyl acetamide

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Quantification of Ethanol in a Formulation

Gas chromatography provides a useful method for

quantifying very volatile materials. Typically ethanol may be quantified against a related alcohol.

• Porapak Q: Porapak is an example of a porous polymeric

stationary phase which retains low molecular weight

compounds strongly. These types of phases are also

effective in separating gases such as CO

₂

, ammonia and acetylene.

• a thick film (e.g. 5 μm film) GC capillary column may be

used for this type of analysis

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Determination of Manufacturing and Degradation Residues by GC

Determination of pivalic acid in dipivefrin eye drops

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Determination of Dimethylaniline in

Bupivacaine Injection

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Determination of N, N-

Dimethylaniline in Penicillins

Used as a counter ion in the purification of penicllins by recrystallization.

The limit set by B.P. : 20 ppm

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Determination of a Residual

Glutaraldehyde in a Polymeric Film

derivatizating reagent pentafluorobenzyloxime

This reaction stabilizes the analyte and increases its retention time into a region where it can be readily observed without interference from other components extracted from the sample matrix.

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Determination of Residual Solvents

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Determination of Residual Solvents and Volatile Impurities by Headspace Analysis

The sample, either in solution or slurried with a relatively involatile solvent with little potential for interference, e.g. water or

dimethylacetamide, is put into a sealed vial fitted with a rubber septum and heated and agitated until equilibrium is achieved. Then a fixed volume of headspace, e.g. 1 ml, is withdrawn. The sample is

then injected into a GC in the usual way.

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1) Partition equilibrium must be established by heating for an appropriate length of time and at an appropriate temperature 2) A clean room is required away from all other sources of

volatiles such as laboratory solvents

3) Potential interference from rubber septa must be checked 4) Reactive contaminants may react with the sample matrix at

high temperatures

5) If the sample is ground and mixed in preparation for the headspace analysis, care has to be taken to ensure that no volatiles are lost.

6) For best reproducibility the process should be automated and for quantitative accuracy it might be best to use the method of standard addition.

Several points are important to note:

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The Standard Phase used in Residual Solvent Analysis

dimethylsiloxane cynanopropyl phenyl

e.g. DB-1301 or Rtx-1301. The film thickness used is generally 3 μm.

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Solid-phase Microextraction (SPME)

It can be used to concentrate trace amounts of organic compounds either in the headspace of a sample or from an aqueous solution.

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Silicone OV‐1 為氣相層析管柱，其固定相為：

(A)Hydrocarbon (B)Polyethylene glycol

(C)Methylsilicone (D)50 % methyl, 50 % phenylsilicone

以氣相層析法分析時，下列何種管柱適用於旋光化合物之分離？

(A)Silicone OV‐17 column 　(B)Chirasil Val column (C)Silicone OV‐5 column 　 (D)ODS column

在執行GC對pseudoephedrine分析時使用trifluoroacetic anhydride

（TFAA）進行衍生化以改善波峰形狀，下列敘述何者錯誤？

(A)TFAA反應力強　 (B)TFAA沸點低

(C)會殘留TFAA在管柱　(D)過量的TFAA在GC分析前已揮發掉

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Kovats 指數（I 值）是下列何種層析法用於分析物滯留時 間與n‐alkane 系列之滯留時間的比較？

(A)氣相層析法 (B)薄層層析法 (C)高效液相層析法 (D) 毛細管電泳

下列薄荷油中之成分，經氣相層析法以carbowax 管柱分離

（I 為Kovats indices），則下列化合物何者滯留時間最短：

(A) menthone（I = 1480） (B) β ‐pinene（I = 1122）

(C) menthol（I = 1686） (D) limonene（I = 1206）

下列何種氣相層析管柱的McReynold's constant數值最大？

(A)Carbowax (B)Squalane (C)Silicone OV‐1 (D)Silicone OV‐17

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最廣泛使用的GC偵測器（detector）為：

(A)ECD（electron capture detector）　(B)FID（flame ionization detector） (C)FTIR（Fourier transform infrared detector）　

(D)TCD（thermal conductivity detector）

下列有關氣相層析儀之敘述，何者錯誤？

(A)毛細管柱比填充管柱的樣品承載量低 (B)氣相層析之衍生化需用到後置衍生裝置

(C)聚乙二醇（Carbowax 20M）管柱適合極性分析物的分析 (D)攜帶氣體需為化學惰性

下列有關氣相層析儀偵測器之敘述，何者錯誤？

(A)火焰離子化偵測器（FID）的離子室需要氫氣與空氣兩種氣體混合燃燒

(B)熱傳導偵測器（TCD）所用之熱絲元件常為鎢‐錸合金絲 (C)傅立葉紅外線轉換偵測器（FTIR）非為GC 之偵測器