Gas Chromatography (GC) (IUPAC Compendium of Chemical Terminology):

Lecture 3. Gas chromathography. 

Gas Chromatography (GC) 

(IUPAC Compendium of Chemical Terminology): 

A separation technique in which the mobile phase is a gas. Gas chromatography 

is always carried out in a column. 

Gas-liquid chromatography, GLC. 

Comprises all gas-chromatographic methods in which the stationary phase is a 

liquid dispersed on a solid support. Separation is achieved by partition of the 

components of a sample between the phases. Mostly used nowadays. 

Gas-solid chromatography, GSC. 

Comprises all gas chromatographic methods in which the stationary phase is an 

active solid (e.g. charcoal, molecular sieves). Separation is achieved by adsorption 

of the components of a sample. In gas chromatography the distinction between gasliquid 

and gas-solid may be obscure because liquids are used to modify solid 

stationary phases, and because the solid supports for liquid stationary phases 

affect the chromatographic process. For classification by the phases used, the term 

relating to the predominant effect should be chosen. 

The very first advertisement of a commercial gas chromatograph (PerkinElmer’s 

model 154) used a chromatogram of the C1–C5 hydrocarbons, including all the C4 

saturated and unsaturated isomers. Instead of a large gas sample, this analysis 

needed only 1–5 ml gas samples. And instead of many hours, the analysis was 

finished in 23 min. 

1


Application area and Instrumentation. 

Mobile phase: carrier gas. 

He, N 2 , H 2, CO 2 , Ar. The carrier gas must be chemically inert. The choice of carrier 

gas is often dependent upon the type of detector which is used. The carrier gas 

system also contains a molecular sieve to remove water and other impurities. 

Stationary phase: nonvolatile liquid, sometimes solid. 

Two kinds of column are used: packed and open tubular (capillary). 

Packed columns contain a finely divided, inert, solid support material (commonly 

based on diatomaceous earth) coated with liquid stationary phase. Most packed 

columns are 1.5 - 10m in length and have an internal diameter of 2 – 4mm. 

Capillary columns have an internal diameter of a few tenths of a millimeter. The 

inner walls are coated with thin layer of stationary phase. 

Analyte: gas or volatile liquid. 

Hydrocarbons, fatty acids, flavor compounds, essential oils, environmental 

pollutants (pesticides), especially modified substances. It is estimated that 10-20% 

of the known compounds can be analyzed by GC. To be suitable for GC analysis, a 

compound must have sufficient volatility and thermal stability. If all or some of a 

compound or molecules are in the gas or vapor phase at 400-450°C or below, and 

they do not decompose at these temperatures, the compound can probably be 

analyzed by GC. 

Flow controller 

Injection port 

Recorder 

Carrier 

gas 

Column oven 

Detector 

2


Advantages of GC 

● 

● 

● 

● 

● 

non-destructive method of analysis; 

analysis is fast; 

analysis is sensitive; 

high resolution; 

method is compatible with many types of detectors, including MS. 

Drawbacks of GC 

● 

● 

suitable mostly for analytical purposes; 

restricted choice of eluent “polarity”; 

Variable parameters in GC: 

● 

● 

● 

● 

column; 

carrier gas; 

gas flow rate; 

temperature. 

3


Columns. 

Open tubular (capillary) columns. 

Typical length is 15 to 100 m. Inner diameter is 0.10 to 0.53 mm. Narrow columns 

provide higher resolution but require higher operation pressure and have less 

sample capacity. 

WCOT (wall-coated open tubular) columns. 

Column wall 

Stationary 

liquid phase 

This type of columns features a 0.1 to 5 μm thick film of stationary liquid phase on 

inner wall of the column. Decreasing the thickness of the stationary phase increases 

resolution, decrease retention time, and decrease sample capacity. Most capillary 

columns are made of fused-silica with a polyimide outer coating. These columns are 

flexible, so a very long column can be wound into a small coil. 

SCOT (support-coated open tubular) columns. 

Column wall 

Solid support 

coated with 

stationary 

liquid phase 

This type of columns has solid particles coated with stationary liquid phase and 

attached to the inner wall. 

4

Film thickness, μm 


PLOT (porous-layer open tubular) columns. 

Column wall 

Stationary 

solid-phase 

particles 

In this type of columns the porous layer is the stationary phase. The surface area is 

higher, and larger samples can be handled. the performance of PLOT columns is 

between wall-coated and packed columns. 

Internal diameter, μm 

100 200 320 530 

1 

10 

Narrowbore 

FSWCOT 

Ultra-high resolution 

{ Small sample capacity 

0.3 

Capacity, ng 

100 

Conventional 

FSWCOT 

1.0 

1000 

Low resolution 

High sample capacity} 

Wide-bore 

FSWCOT 

2.5 

10000 5000 3000 1500 

Efficiency, N/m 

5


Stationary phases – capillar columns. 

Polarity and Selectivity. 

Polarity: 

physical characteristic of stationary phase. Polarity is determined by stationary 

phase structure, polarity of functional groups and amount of each group. 

Polarity 

Stability 

Temperature range 

Selectivity: 

solute interactions and separations. Determined by dispersion, dipole-dipole and 

hydrogen bonding interactions. 

Dispersion interaction is determined by differences in solute heat of vaporization 

ΔH vap . The value can be approximated from vapor pressure of the solute. 

Dipole-dipole interaction is determined by dipole moment of the molecule. Smaller 

differences require a stronger dipole phase. 

Cl 

Cl Cl Cl 

Hydrogen bonding interactions. Strong: alcohols, carboxylic acids, primary and 

secondary amines. Moderate: aldehyde, ketones, esters. Weak: hydrocarbons, 

halocarbons, ethers. 

6


POLYSILOXANES: 

R 

* Si O 

R 

n 

* 

Polysiloxanes are the most common stationary phases. They are available in the 

greatest variety and are the most stable, robust and versatile. 

The most basic polysiloxane is the 100% methyl substituted. When other groups are 

present, the amount is indicated as the percent of the total number of groups. 

Cyanopropylphenyl percent values can be misleading. A 14% cyanopropylphenyldimethyl 

polysiloxane contains 7% cyanopropyl and 7% phenyl (along with 86% 

methyl). The cyanopropyl and phenyl groups are on the same silicon atom, thus 

their amounts are summed. 

POLYETHYLENE GLYCOLS: 

* CH 2 

CH 2 

O * 

n 

Polyethylene glycols (PEG) are widely used as stationary phases. Stationary phases 

with "wax" or "FFAP" in their name are some type of polyethylene glycol. 

Polyethylene glycols stationary phases are not substituted, thus the polymer is 

100% of the stated material. They are less stable, less robust and have lower 

temperature limits than most polysiloxanes. 

With typical use, they exhibit shorter lifetimes and are more susceptible to damage 

upon over heating or exposure to oxygen. 

The unique separation properties of polyethylene glycol makes these liabilities 

tolerable. Polyethylene glycol stationary phases must be liquids under GC 

temperature conditions. 

7


Selectivity – Interaction strength 

Phase Dispersion Dipole H-Bonding 

Methyl -CH 3 Strong None None 

Phenyl -C 6 H 5 Strong None Weak 

Cyanopropyl -C 3 H 6 CN Strong Strong Moderate 

Trifluoropropyl -C 2 H 4 CF 3 Strong Moderate Weak 

PEG -OCH 2 CH 2 O- Strong Strong Moderate 

Compounds – Properties 

Compounds Polar Aromatic H-Bonding Dipole 

Toluene No Yes No Induced 

Hexanol Yes No Yes Yes 

OH 

Phenol Yes Yes Yes Yes 

OH 

Decane No No No No 

Naphtalene No Yes No Induced 

Dodecane No No No No 

8


Nonpolar to intermediate polarity stationary phases. 

BONDED PHASE Temp °C GENERAL USE OF PHASE 

O 

CH 3 

Si * 

* 

X 

CH 3 

Methyl polysiloxane 

Methyl 5% Phenyl Polysiloxane 

CH 3 

* O Si O Si * 

X 

CH 3 

Y 

50-325 

50-325 

Nonpolar. Optima 1. 

Most frequently used phase in GC. Low 

selectivity, separates compounds according to 

boiling points. Excellent thermal stability. 

Nonpolar. Optima 5. 

Similar to methyl polysiloxane but slightly 

more selective due to phenyl content. Excellent 

thermal stability. 

Methyl 50% Phenyl Polysiloxane 

CH 3 

* O Si O Si * 

6% Cyanopropylphenyl 94% 

Methylpolysiloxane 

X 

CN 

CH 3 

CH 3 

* O Si O Si * 

X 

Y 

CH 3 

Y 

40-325 

30-320 

Intermediate polarity. Optima 17. 

Added selectivity due to higher phenyl content. 

Usually retains similar compounds longer than 

methyl silicone. Provides efficient separations 

of PAHS and biomedical samples such as 

drugs, sugars and steroids. Good thermal 

stability. 

Intermediate polarity. Optima 1701. 

An additional choice for a general purpose 

phase with nominal selectivity for polarizable 

and polar compounds. Good thermal stability. 

9


Intermediate polarity to strongly polar stationary phases. 

BONDED PHASE Temp °C GENERAL USE OF PHASE 

Methyl 7% Cyanopropyl 7% 

Phenyl Polysiloxane 

CN 

CH 3 

* O Si O Si * 

X 

CH 3 

Y 

280 

Intermediate polarity. 

Unique selectivity of cyanopropyl and phenyl 

groups provide efficient separations of 

derivitized sugars and many environmental 

samples. Not truly a polar phase. Good thermal 

stability 

Methyl 25% Cyanopropyl 25% 

Phenyl Polysiloxane 

CN 

CH 3 

* O Si O Si * 

X 

CH 3 

Y 

40-240 

Polar. Optima 225 

Provides efficient separations of polar 

molecules such as fatty acids and alditol 

acetate derivatives of sugars. Fair thermal 

stability. 

50% Trifluoropropyl 50% Methyl 

polysiloxane 

* 

CF 3 

CH 3 

Si O Si 

Y 

X 

* 

CH 3 

CF 3 

Polyethylene Glycol 

* CH 2 

CH 2 

O * 

n 

40-300 

20-260 

Polar. Optima 210 

Selectivity for compounds with lone pair 

electrons or carbonyl groups. Retains 

oxygenated compounds in the order ether, 

hydroxy, ester and keto Widely used as a 

confirmatory phase for chlorinated pesticides. 

Also suitable for PCB’s, phenols and 

nitroaromatics. Good thermal stability. 

Carbowax 20M is a polyethylene glycol phase 

which demonstrates unique selectivity 

hydrogen bonding-type molecules. Particularly 

useful for the analysis of complex oxygenated 

samples but is susceptible to oxygen 

degradation. Not recommended for the 

analysis of mixtures containing silylating 

reagents 

10


11


Column dimensions. Diameter, length, film thickness. 

Resolution= N 

−1 

k ' av 

4 1k ' 

e.g. resolution is proportional to square root of plates 

av 

number 

Diameter. 

I. D. mm Common Name 

0.53 Megabore 

0.45 High speed Megabore 

0.32 Wide 

0.20-0.25 Narrow 

0.18 Minibore 

for high flow situations 

for low flow situations, e.g. 

GC-MS 

12


Column length. 

Most common 15 – 60 m. Available 5 – 150 m. 

Column cost is rising with column length. 

Film thickness. 

Most common 0.1 – 3 μm. Available 0.1 – 10 μm. 

The effect of film thickness is described by Van Deemter equation: 

H mass transfer =Cu x =C s C m u x 

where C s describe the rate of mass transfer trough stationary phase 

C s = 

where d is the thickness of stationary phase. 

2 k ' 

3k '1 2 d 2 

D s 

13


Examples of film thickness effects. 

To get the same retention, the temperature should be increased for thicker film. 

14


Effect of film thickness and capacity factor. 

For low k resolution is rising when film thickness increased: 

For high k resolution is decreasing when film thickness increased: 

15


BONDED AND CROSS-LINKED STATIONARY PHASES: 

Cross-linked stationary phases have the individual polymer chains linked via 

covalent bonds. 

Bonded stationary phases are covalently bonded to the surface of the tubing. 

Both techniques impart enhanced thermal and solvent stability to the stationary 

phase. Also, columns with bonded and cross-linked stationary phases can be 

solvent rinsed to remove contaminants. 

Most polysiloxanes and polyethylene glycol stationary phases are bonded and 

cross-linked. 

A few stationary phases are available in an nonbonded version; some stationary 

phases are not available in bonded and cross-linked versions. Use a bonded and 

cross-linked stationary phase if one is available. 

16


GAS – SOLID: PLOT Columns. 

Gas-solid stationary phases are comprised of a thin layer (usually


The Retention Index. 

Each chromatographic setup will vary to some degree. Retention times for a known set of 

species can be hard to reproduce even from instrument to another. 

Retention indexing helps to standardize the results. 

For alkanes C n H 2n+2 : n is proportional to log t' R 

By agreement, Kovats retention index for linear alkanes equals 100 times the number of 

carbon atoms. For the compound eluted between two linear alkanes with number of atoms n 

and N=n+1, is: 

I =100 

[ nN −n log t ' Runknown−log t ' R n 

log t ' R N −log t ' R n ] 

Example: 

t R (methane) = 0.5 min 

t R (octane) = 14.3 min 

t R (unknown) = 15.7 min 

t R (nonane) = 18.5 min 

Find the retention index for unknown. 

SOLUTION: 

t' R (octane) = 14.3 – 0.5 = 13.8 min 

t' R (unknown) = 15.7 – 0.5 = 15.2 min 

t' R (nonane) = 18.5 – 0.5 = 18.2 min 

[ 

log 15.2−log 13.8 

] 

I unknown =100 89−8 

log 18.0−log 13.8 =836 

Kovats retention indexes must be compared on the same or very similar phases. For phases 

with different polarity the order of elution and, therefore, the retentions indexes are very 

different! 

18


Temperature programming. 

By definition, programmed-temperature chromatography (temperature programming) 

A procedure in which the temperature of the column is changed systematically during a part or 

the whole of the separation. 

As stated before, the retention time of homologues increases exponentially with the number of 

carbon. With longer retention time, the peaks are broad and wide, making detection difficult or 

even impossible. 

C 10 

C 9 

C 12 

C 8 

C 11 

C 13 

C Isotermal 150 ºC 

14 

C 15 

0 10 20 30 40 50 60 70 80 90 100 

time, min 

Raising the column temperature: 

● decrease retention time; 

● sharpens peak. 

Factors to take into account for temperature programming: 

● Stability of stationary phase 

● Stability of solutes 

● Changes in flow rates 

● Changes in solute volatility 

● Changes in solute solubility 

Steps to create a temperature program: 

1. Determine initial temperature and time according to best possible separation of fast peaks. 

2. Determine final temperature according to best possible separation of last peaks. 

3. Find experimentally the optimal temperature gradient to account the middle peaks. 

C 10 

C 11 

C 14 

C 15 

C 12 

C 13 

C 8 

C 9 

Programmed temperature 

50 – 250 ºC at 8º/min 

C 16 

C 17C18 

C 19 C 20 C 21 

C 6 C 7 

0 4 8 12 20 36 

16 24 28 32 

time, min 

19


Example of temperature programming: 

20


Sample injection. 

For optimum column efficiency, the sample should not be too large, and should be introduced 

onto the column as a "plug" of vapour - slow injection of large samples causes band 

broadening and loss of resolution. The most common injection method is where a microsyringe 

is used to inject sample through a rubber septum into a flash vapouriser port at the head of the 

column. 

Worn Septum 

An injection port septum should last between 100 and 200 injections. Higher injection port 

temperatures shorten the septum's lifespan. A leaking septum adversely affects the GC 

instrument's sensitivity. 

If a portion of the specimen leaks back out of the septum, the amount of the specimen is not 

recorded. This event makes any eventual quantitative result erroneous. If air should leak into 

the injection port through a worn septum, the oxygen and water contained in air may skew the 

results. Any oxygen may react with the specimen components. If this happens, the GC 

instrument will provide results indicating the presence of this unintended reaction product, 

instead of the original compounds present in the specimen vial. Any water in the column 

adversely affects the GC instrument's ability to separate components. 

Injection Port Temperature. 

The temperature of the GC injection port must be high enough to vaporize a liquid specimen 

instantaneously. The temperature of the sample port is usually about 50°C higher than the 

boiling point of the least volatile component of the sample. If the temperature is too low, 

separation is poor and broad spectral peaks should result or no peak develops at all. If the 

injection temperature is too high, the specimen may decompose or change its structure. If this 

occurs, the GC results will indicate the presence of compounds that were not in the original 

specimen. 

For packed columns, sample size ranges from 0.1 to 20 μl. Capillary columns, on the other 

hand, need much less sample, typically around 10 -3 ml. 

Types of injection for capillary GC: 

● 

● 

● 

● 

split injection 

splitless injection 

on-column injection 

21


Split Injection. 

Used for samples with analyte concentration > 0.1 %. 

Only 0.2 – 2 % of the sample is delivered to column. 

Injector temperature is high, e.g. 350 ºC. 

102 ml/min 

1 ml/min 

100 ml/min 

1ml/min 

The sample is injected rapidly through the septum into evaporation zone. The injector 

temperature is kept high to promote fast evaporation. A brisk flow of the carrier gas sweeps 

the sample through the mixing chamber. At the split point, small fraction of vapors enters the 

column but most passes to waste vent. Split ratio (the proportion of the sample that does not 

reach the column) is typically 50:1 to 600:1. 

Septum purge gas flow: prevents the column during injection and chromatography from hot 

rubber septum gases and the excess of the sample vapors. 

● 

● 

● 

Advantages of split injection: 

narrow solute peaks; 

suitable for qualitative analysis; 

minimize the solvent effect. 

Drawbacks: 

● 

● 

● 

requires rather high concentration of analyte; 

split ratio makes the quantitative analysis more complex; 

not suitable for very expensive or toxic compounds. 

22


Splitless injection. 

Used for traces analysis with analyte concentration < 0.01%. 

Volume of solution injected is ~ 2 μl. 

Injection time is ~ 2 sec (SLOW INJECTION). 

Injector temperature ~220 ºC. 

~ 80% of the sample is applied to the column. 

2 ml/min 

0 ml/min 

1 ml/min 

1ml/min 

Solvent trapping. The initial column temperature is set 40 ºC below the boiling point of the 

solvent. Therefore the solvent condenses in the beginning of the column and traps the analyte 

to produce a narrow plug in the beginning of the column. For solvent trapping, the analyte 

concentration should be < 0.01%. 

Cold trapping. The initial column temperature is 150 ºC than the boiling points of analytes of 

interest. Solvent and low-boiling components are eluted rapidly, whereas high-boiling solutes 

remain as narrow band. The column is then rapidly warmed to initiate chromatography of highboiling 

solutes. Stationary-phase film thickness must be ≥2 μm. 

Cryogenic focusing is the variation of cold trapping for low-boiling solutes. The column is 

initially cooled with N 2 or CO 2 . 

● 

● 

● 

● 

● 

Advantages of splitless injection: 

suitable for quantitative and qualitative analysis; 

narrow peaks of analyte. 

Drawbacks: 

broad solvent peak; 

retention times depend on solvent evaporation speed; 

solvent affects the shape of the peaks. 

23


On-Column injection. 

Used for samples that decompose above their boiling point. 

Preferred for quantitative analysis. 

Syringe needle 

1 ml/min 

0 ml/min 

0 ml/min 

At initial oven temperature, e.g. 50ºC 

1ml/min 

Solution is injected directly into column, without going through a hot injector. 

Initial column temperature is low enough to condense solutes in narrow zone. Warming the 

column initiate chromatography. 

The special thin-needle syringe is required to use good resolution columns (column diameter 

0.2 - 0.32 mm). 

● 

● 

● 

● 

● 

● 

Advantages of on-column injection: 

narrow peaks of analyte; 

good accuracy and precision for quantitative analysis; 

no thermal destruction of the sample; 

little loss of high-boiling components. 

Drawbacks: 

non-volatile impurities harm the column; 

shape of the peaks depends on solvent. 

24


Comparison of different injection methods. 

Solvent 

A 

Solvent 

B 

3 

2 3 

2 

Split injection 

Split vent closed 

3 

Solvent 

C 

Solvent 

2 

D 

2 

3 

Split vent opened 

after 30 s 

Solvent trapping 

A: standard Split injection, peaks are sharp. 

B: split vent closed, injection liner was purged slowly, sample was applied over a long time, 

peaks are broad and tail badly. 

C: same as B, but split vent was opened after 30 s to rapidly purge all the vapors from the 

injector liner. 

D: same as C, but the column was initially cooled to r.t. to trap solvent and solutes; to be 

proper splitless injection, the sample should be much more diluted. 

25


Solid phase microextraction (SPME). 

Minimizes sample preparation and concentrates volatile analytes in a solvent-free manner. 

SPME was developed by Pawliszyn's research group at the University of Waterloo in the late 

1980s. SPME is a sensitive, reproducible, cost efficient, solventless technique that incorporates 

extraction, concentration, and sample introduction into a single step. 

A syringe-like device with an outer septum piercing needle and a plunger houses a fused silica 

fiber coated with a stationary phase. 

The fiber can be inserted into the sample matrix (aqueous samples) or the gaseous phase above 

the sample (headspace). 

Liquid sampling can be performed by inserting the fiber directly into the solution. Volatile 

analytes from solids can be sampled by inserting the fiber into the headspace region above the 

sample. 

Analytes are partitioned between the stationary phase coating and the gas phase when 

equilibrium is established. 

After concentration of analytes on the fiber, the syringe assembly is inserted into the injection 

port of a gas chromatograph where the analytes are thermally desorbed from the fiber and coldtrapped 

on the head of the capillary column. If an unknown sample has volatile components 

that can be detected by the human nose, SPME coupled to GC or GC/MS might be employed 

to identify and quantitate those compounds. Applications of SPME have included extraction 

of environmental contaminants from aqueous matrices, headspace extraction of flavor and 

fragrance compounds, and forensic investigations of drugs of abuse in biological fluids. 

26


Purge and trap. 

Purge-and-trap is the method of choice for extracting and concentrating volatile organic 

compounds (VOCs) from almost any matrix. 

This procedure is particularly useful for concentrating VOCs that are insoluble or poorly 

soluble in water and have boiling points below 200°C. 

The procedure can also be used with water soluble VOCs, but quantification limits are 

generally much higher for these analytes, because of their poor purging efficiency. 

Generally, longer purging times and heating the sample are required to increase the purging 

efficiency of water soluble, often polar compounds. 

The purge-and-trap procedure involves bubbling an inert gas, such as nitrogen or helium, 

through an aqueous sample (solids must be suspended in water) at ambient temperature. This 

liberates the VOCs, which are efficiently transferred from the aqueous phase to the vapor 

phase. During this purge step, the inert gas flow sweeps the vapor through a trap containing 

adsorbent materials which retain the VOCs. 

A few systems offer the ability to dry purge the trap after the purging step. The dry purge step 

continues to pass the purging gas through the trap, bypassing the purge vessel, for a set time to 

remove water that may have accompanied the VOCs into the trap during the purging process. 

Next, the adsorbent trap is rapidly heated to the desorb temperature and the valve is switched 

to align the carrier gas flow in-line with the trap. The trap is then held at the desorb 

temperature for an optimal time to thermally desorb the analytes into the carrier gas. The 

vaporized contents are swept into the GC column in a tight band, ensuring superior 

chromatographic separation of analyte. 

27


Derivatization. 

● 

● 

● 

Aim of derivatization: 

improved volatility; 

better thermal stability; 

lower limit of detection due to improved peak symmetry. 

● 

● 

● 

● 

Derivatization demands: 

quantitative; 

rapid; 

reproducible; 

formation of only one derivative. 

● 

● 

● 

● 

● 

alcohols; 

phenols; 

amines; 

amides; 

carboxylic acids 

Derivatization subjects: 

● 

● 

● 

acylation; 

alkylation; 

silylation; 

Derivatization methods: 

28


Derivatization reagents. 

Function method derivative recommended reagents 

Alcohols, silylation R'O tms BSA, MSTFA, MSHFBA, 

TSIM 

Phenols, R'OH acylation O TFAA, HFBA, MBTFA, 

R'O C R 

MBHFBA 

alkylation 

TMSH 

sterically hindered silylation R'O tms TSIM, BSTFA 

Amines 

primary, secondary 

silylation 

R' 

R'O R 

N tms 

R'' 

BSA, MSTFA, MSHFBA 

R' 

N H R'' 

acylation 

R' 

O 

N C 

R'' 

R 

TFAA, HFBA, MBTFA, 

MBHFBA 

hydrochlorides silylation R' N tms MSTFA 

R'' 

Amides silylation NOT STABLE 

O acylation 

O O 

R' C NH 2 

R' C N C 

H 

R 


MBHFBA 

Amino acids silylation H O BSA, BSTFA, MSTFA, 

R' C C tms MSHFBA 

H O 

N 

R' C C OH 

H 

tms 

NH 2 

alkylation (a) 

+ acylation (b) 

R' 

H O 

C C 

N 

H R 

OR 

a) MeOH/TMCS, TMSH 

b) TFAA, HFBA, MBTFA, 

MBHFBA 

29


Function method derivative recommended reagents 

Carboxylic acids 

salts 

silylation 

alkylation 

silylation 

susceptible to hydrolysis 

susceptible to hydrolysis 

BSA, MSTFA, MSHFBA, 

TMCS, TSIM 

DMF-DMA, MeOH/TMCS, 

TMSH 

TMCS 

Carbohydrates silylation MSTFA, TSIM, HMDS 

acylation 

TFAA, MBTFA, 

Steroids silylation BSA, TSIM 

acylation 

R' 

R' 

R' 

O 

C O 

O 

O 

C O 

tms 

C O R 

tms 


MBHFBA 

F 3 

C 

F 3 

C 

F 7 

C 3 

F 7 

C 3 

F 3 

C 

F 3 

C 

F 7 

C 3 

F 7 

C 3 

O 

O 

O 

O 

O 

O 

O 

N 

O 

O 

N 

O 

Reagents for acylation. 

TFAA – trifluoroacetic anhydride. Used for alcohols, phenols, carboxylic 

acids, amines, amino acids and steroids, forming volatile, stable derivatives 

suited for FID and ECD detection. 

HFBA – heptafluorobutyric acid anhydride. Used for alcohols, phenols, 

carboxylic acids, amines, amino acids and steroids, forming volatile, stable 

derivatives suited for FID and ECD detection. 

MBTFA – N-methyl-bis(trifluoroacetamide). Recommended for alcohols, 

primary and secondary amines, as well for thiols under mild, neutral 

conditions. MBTFA forms very volatile derivatives with carbohydrates. 

MBHFBA – N-methyl-bis(heptafluorobutyramide). Recommended for 

alcohols, primary and secondary amines, as well for thiols under mild, 

neutral conditions. 

30


Reagents for alkylation. 

H C 

N CH 3 3 

H O O CH 3 

C 

3 

DMF-DMA – N,N-dimethylformamidedimethylacetal. Methylation 

with DMF-DMA can be applied for fatty acids, primary amines and 

(partially) amino acids forming N-dimethylaminomethylene amino acid 

methyl esters. 

H 3 

C 

S + CH 3 

H 3 

C 

OH 

TMSH – 0.2 M trimethylsulfoniumoxide in methanol. Methylation with 

TMSH is recommended for free acids e.g. fatty acids , 

chlorophenoxycarboxylic acids , their salts and derivatives as well as 

for phenols and chlorophenols, which can be detected in very small 

amounts. One great advantage is simplification of the sample 

preparation. Lipids or triglycerides can be converted to the 

corresponding fatty acid methyl esters (FAMEs) by a simple 

transesterification. 

31


O 

N 

Si(CH 3 

) 3 

Si(CH 3 

) 3 

Reagents for sylilation. 

BSA – N,O-bis(trimethylsilyl)acetamide. BSA is a strong silylation 

reagent, which can be used to form very stable TMS derivatives of a 

wide variety of compounds such as alcohols, amines, carboxylic acids, 

phenols, steroids, biogenic amines and alkaloids. 

F 3 

C 

(CH 3 

) 3 

Si 

O 

O 

N 

H 

N 

N 

Si(CH 3 

) 3 

Si(CH 3 

) 3 

Si(CH 3 

) 3 

F 3 

C Si(CH 3 

) 3 

X H 

O 

X Si(CH 3 

) 3 

O 

+ 

N 

F 3 

C Si(CH 3 

) 3 

N 

+ 

O 

F 3 

C 

N 

H 

F 7 

C 3 

Si(CH 3 

) 3 

Si Cl 

BSTFA – N,O-bis(trimethylsilyl)trifluoroacetamide. BSTFA is a 

powerful trimethylsilyl donor with approximately the same donor 

strength as the unfluorinated analog BSA. Reactions of BSTFA are 

similar to those of BSA. The major advantage of BSTFA over BSA is 

the greater volatility of its reaction products. 

HMDS – hexamethyldisilazane. HMDS is a weak TMS donor. Used 

alone its action is slow and not very effective. However, after addition 

of catalytic quantities of TMCS (e.g. 1%) it becomes a fast and 

quantitative reagent for trimethylsilylation of organic compounds. 

MSTFA – N-methyl-N-trimethylsilyl-trifluoroacetamide. MSTFA is the 

most volatile trimethylsilyl amide available. BSA and BSTFA, which 

have been used most frequently in GC silylation, can often be replaced 

by MSTFA. MSTFA offers the following advantages: 

1. For almost all compounds the reaction proceeds to the right side of 

the equation 

2. Even without a catalyst the reaction rate is several times higher than 

with other TMS donors such as hexamethyldisilazane (HMDS). 

3. As for BSTFA, the by-product of the silylation reaction (Nmethyltrifluoroacetamide) 

features the advantage of high volatility and 

short retention time. 

MSHFBA – N-methyl-N-trimethylsilyl-heptafluorobutyramide. 

MSHFBA is similar to MSTFA in reactivity and chromatography. It 

may be used for the general purpose trimethylsilylation of carboxylic 

acids, alcohols, phenols, primary and secondary amines and amino 

acids. 

TMCS – trimethylchlorosilane. TMCS is often used as a catalyst with 

other trimethylsilyl reagents. Without additives it can be used for 

preparing TMS derivatives of organic acids. 

Si N 

N 

TSIM – N-trimethylsilyl-imidazole. TSIM is considered to be the 

strongest hydroxyl silylator and is the reagent of choice for 

carbohydrates and most steroids (even highly hindered steroids react). 

The reagent is unique in that it reacts quickly and smooth with 

hydroxyl (even tert. OH) and carboxyl groups, but not with amines. 

TSIM is used in the trimethylsilylation of alcohols, phenols, organic 

acids, steroids, hormones, glycols, nucleotides and narcotics. 

32


33

Gas Chromatography (GC) (IUPAC Compendium of Chemical Terminology):

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