KROMATOGRAFI GAS

PRINSIP GC INSTRUMENTASI GC DETEKTOR GC

Gas Chromatography
an analytical separations technique useful for separating volatile organic compounds consists of :
Flowing mobile phase (inert gas - Ar, Ne, N) Injection port ( rubber septum - syringe injects sample)
kept at a higher temperature than the boiling point

Principles
Separation due to differences in partitioning behavior selective retardation

Key Information
organic compounds separated due to differences in their participating behavior between the mobile gas phase and the stationary phase in the column in contrast to other types of chromatography, the mobile phase does not interact with molecules of the analyte; its only function is to transport the analyte through the column

Gas Chromatography
Separation column containing stationary phase
since partitioning behavior independent of temperature - kept in thermostat - controlled oven

Detector

Schematic of a gas Chromatograph

The Beginning
concept of GC announced in 1941 by Martin and Synge (also did liquid partition chromatography) 10+ years later GC used experimentally 1955, first commercial apparatus for GC appeared on the market

Today
estimate : 200, 000 gas chromatographs are currently used through out the world. 30+ instrument manufactures 130 different models cost 1,500 to 40,000 dollars improvements: computers- automatic control open tubular columns-separate a multitude of analytes in relatively short times

Uses of Gas Chromatography

Determination of volatile compounds (gases & liquids) Determination of partition coefficients and absorption isotherms Isolating pure components from complex mixtures

Instrumentation

Instrumentation
flowing mobile phase injection port separation column detector

Gas Chromatography Overview

Sample is vaporised and injected onto head of a chromatography column.
Elution is effected by the flow of an inert gaseous mobile phase.

Separation is based upon the partition of the analyte between a gaseous mobile phase and a liquid phase immobilised on the surface of an inert solid (GLC) at a temperature above boiling point of analyte (multi-analyte: temperature programming).
Mobile phase does not interact with molecules of the analyte. Eluted analyte detected by a detector and recorded by PC Chemstation. GC columns are either packed (with silica particles coated in stationary) or capillary in nature.

Sample Injection
GC column efficiency requires that the sample be of suitable size (to prevent column over loading) and be introduced as a plug of vapour. Two common approaches include for introduction of 0.01 50 ml include: Microsyringe and valve loop. The syringe technique is most common and can be used with both gas and low viscosity liquid samples by inserting the needle through a rubber septum to the column inlet port.

The region into which the needle projects must be heated in order to flash vaporise the sample.
However, overheating of the rubber septum must be avoided to prevent out gassing. The most popular inlet for capillary GC is the split/splitless injector.

If this injector is operated in split mode, the amount of sample reaching the column is reduced (to prevent column overloading) and very narrow initial peak widths can be obtained.
For maximum sensitivity, the injector can be used in so-called splitless mode, then all of the injected sample will reach the column.

Injection may be manual or automated.

Split Splitless Injection

Septum purge outlet prevents components of previous injections from entering the column and minimizes the effect of septum bleed (low flow rate ~3 ml/min).
The sample is injected into the liner region where it is completely vaporised. Mostly glass liners zero dead volume The sample volume is then split between the column and the split outlet. Split injection is employed to dilute the sample and prevent column overloading. Typically 1:100 split ratios are employed with 99% of sample being vented to atmosphere. Method development: Some parameters of split/splitless injection that require optimisation, apart from instrumental design, are injector temperature, split ratio, split delay, injection volume, sample solvent and initial temperature of the column.

Sample Valve Injection

Sample valves are convenient for on-line gas stream analysis.

In position (a) the stream to be sampled flows through a loop of calibrated volume while the carrier gas alone passes through the column.

(a) Sample mode

In position (b) the loop is placed in the carrier gas stream and the entrapped sample is swept along to the column.
Sample valves are becoming more prevalent for quantitative work employing both liquids and gases to introduce a reproducible volume of sample onto a column. They are typically employed for smaller volumes, e.g., to prevent over loading of a column > 0.01 ml of a liquid sample is preferred volume - a precision syringe for this volume is both expensive and fragile. Valves may also be used in split splitless mode.

(b) Inject mode

Column Configuration

Packed Columns 2 to 4 mm I.D. and 1 to 4 meters long.

Capillary Columns 100 mm to 500 mm I.D. and 10 m to 100 m long Stationary phase is coated on the internal wall of the column as a film 0.2 mm to 1 mm thick Sharper peaks no eddy diffusion.

Packed with a suitable adsorbent.

Mostly used for gas analysis. Peak broadening due to zone (eddy) diffusion resulting from multitude of pathways a molecule can pass through column.

Up to 500,000 theoretical plates excellent separations.

Most popular type of column in use.

Advantages and Disadvantages of GC

Fast analysis
Typically minutes (even sec.)

Limited to volatile samples

6

High Resolution
Record N~1.3 x 10

T limited to ~ 380 C Need Pvap ~ 60 Torr at that temperature

Sensitive detectors (easy ppm, often ppb) Highly accurate quantification (1-5 % RSD) Automated systems Non-destructive
Allows online coupling to Mass Spec.

Small sample (mL) Reliable and relatively simple Low cost (~20,000)

Not suitable for thermally labile samples Some samples may require extensive preparation Requires spectroscopy (usually MS) to confirm peak identify

Classification of Chromatography Methods

Introduction to Chromatography
Separation of a mixture of components A and B by column elution chromatography. The detector signal at each stage is shown. The effectiveness of a column in Column separating two analytes depend in part on the relative rates at which the two components are eluted. These rates are determined by the magnitude of the equilibrium constants for the components partioned between the stationary phase and mobile phase. Sample M obile phase

Detector

A mobile A stationary

Time

Mechanisms of Partition to Stationary Phase

Equipment Overview
Sample injection Detector Mobile phase Packed column GC & HPLC Chem station

Capillary column GC only

Partition in Chromatography
Stationary phase, mobile phase, & analyte form a ternary system. Each analyte is distributed between the two phases (in equilibrium): Partition Coefficient

CS: concentration of analyte on the stationary phase CM: concentration of analyte on the mobile phase

Using the Partition Coefficient: Plate Theory

Column

Column divided into theoretical pieces (plates). Analytes are partitioned between SP and MP in each plate. Separation occurs as analytes move down the column.

Definitions: Prototype Chromatogram

Factors Influencing Retention

Are those that influence distribution Stationary phase: type & properties Mobile phase: composition & properties Intermolecular forces between Analyte & mobile phase Analyte & stationary phase

Temperature

Choice of GC Columns

Packed GC Columns

Open (capillary) GC Columns

Column Type Vs Separation

Comparison of Columns

Effect of Column Diameter & Film Thickness

Optimum Mobile Phase Velocity

Effect of Column, Film and Carrier Gas

Effect of Temperature on Retention Time

Two Decisions

How to Choose a Stationary Phase

Most Important: Stationary Phase Polarity

Effects of Stationary Phase Polarity

=>

Can change the order of elution, so need to verify elution times using standards

Separating Efficiency Peak Asymmetry

Resolution
Objective: accurate measurement of individual peak areas

Can One Have Too Much Resolution

RS = 2.5

Resolution and Selectivity

Quantification in GC

Quantification: Normalizing Peak Areas

Requires identical samples not very robust

Quantification: Internal Standard

Popular method: may be used as internal QC

Why?

Quantification: External Standard

Normally performed in conjunction with internal standard

Quantification: Standard Addition

Different concentrations of standard are added to ` sample aliquots. Samples are analysed Can be used to verify linearity

Q: why are separate sample aliquots prepared?

Method Validation
Organisations regulating pharmaceutical industry: FDA (CFR 21) Irish Medical Board (IMB) Official Analytical Chemists (AOAC) US Environmental Protection Agency (USP) American Association of Official Analytical Chemists (AOAC) Resource Conservation and Recovery Act (RCRA ) European Pharmacopoeia Japanese Pharmacopoeia US Pharmacopoeia
The International Conference on Harmonization (ICH) of Technical Requirements for the Registration of Pharmaceuticals for Human Use has developed a consensus text on the validation of analytical procedures (1995)

ICH Guidelines
Validation requirements include: 1. 2. 3. 4. 5. 6. Specificity: Ability to measure desired analyte in complex mixture. Accuracy: agreement between measured and real value. Linearity: proportionality of measured value to the concentration. Precision: agreement between a series of measurements. Range: concentration interval where method is precise accurate and linear. Detection limit: lowest amount of analytes that can be detected (LOD). Quantitation limit: lowest amount of analyte that can be measured (LDQ). Robustness: reproducibility under normal but variable laboratory conditions.

7.
8.

What is the difference between repeatability and reproducibility?

Specificity

Selectivity in chromatography is obtained by choosing optimal columns and setting chromatographic conditions, such as mobile phase composition, column temperature and detector wavelength. UV-vis spectroscopy may be used to identify pure peaks

Accuracy and Recovery

Active Ingred. [ %] 100 >=10 >=1 Analyte ratio 1 10-1 10-2 Unit 100% 10% 1% Mean recovery [%] 98-102 98-102 97-103

>=0.1 0.01
0.001 0.0001 0.00001 0.000001

10-3 10-4
10-5 10-6 10-7 10-8

0.1 % 100 ppm

10 ppm 1 ppm 100 ppb 10 ppb

95-105 90-107
80-110 80-110 80-110 60-115

The accuracy of an analytical method is the extent to which test results generated by the method and the true value agree. Accuracy can be assessed by analyzing a sample with known concentrations, e.g., a certified reference material

Linearity

The linearity of an analytical method is its ability to elicit test results that are directly, or by means of well-defined mathematical transformations, proportional to the concentration of analytes in samples within a given range. Linearity is determined by a series of three to six injections of five or more standards whose concentrations span 80-120 percent of the expected concentration range.

Precision
Analyte % 100 10 1 0.1 0.01 0.001 0.0001 0.00001 0.000001 Analyte ratio 1 10-1 10-2 10-3 10-4 10-5 10-6 10-7 10-8 Unit 100% 10% 1% 0.1 % 100 ppm 10 ppm 1 ppm 100 ppb 10 ppb RSD (%) 1.3 2.8 2.7 3.7 5.3 7.3 11 15 21

0.0000001

10-9

1 ppb

30

The precision of a method is the extent to which the individual test results of multiple injections of a series of standards agree. The acceptance criteria for precision in pharmaceutical quality control of better than 1 % RSD is required.

Range, LDQ & LDQ

Range: The range of an analytical method is the interval between the upper and lower levels (including these levels) that have been demonstrated to be determined with precision, accuracy and linearity using the method as written. The range is normally expressed in the same units as the test results (e.g. percentage, parts per million) obtained by the analytical method. LDQ: Normally three times the baseline noise . LDO: The limit of quantitation is the minimum injected amount that gives precise measurements. This may be set as twenty times the baseline noise or determined using the Eurochem method viz A number of samples with decreasing amounts of the analyte are injected six times. The calculated RSD% of the precision is plotted against the analyte amount. The amount that corresponds to the previously defined required precision is equal to the limit of quantitation.

Head Space Analysis

Head space analysis is a technique where the vapours in the gas above, and in equilibrium with, a solid or liquid is sampled. The advantage of this approach is that GC can be used instead of HPLC, thus providing four to five orders of magnitude greater sensitivity. Procedure involves the extraction of a volume of the equilibrium gas over the sample (usually about 10 ml) by a syringe through either a vial containing a bed of an appropriate absorbent or a cryogenic trap. The vial/trap is the placed in line with a GC column, heated and the vaporised sample swept onto the column and the components separated. Used to identify spoiled food, fragrances from botanical material, the determination of plasticizers in plastics and for forensic samples involving arson.

Purge and Trap EPA method 5030C

5030C can be used for most volatile organic compounds that boiling points below 200 C and are insoluble or slightly soluble in water. Multiple sample aliquots are collected in sealed containers with minimum headspace and stored at 4 C or less in solvent free area. An inert gas is bubbled through aqueous sample and room or elevated temperature depending on the target analytes. The vapour is swept through a sorbent column where the analytes are captured. After purging, the sorbent column is heated (thermal extraction) and back-flushed with inert gas to desorb the components onto a GC column.

Solid Phase Microextraction

Solid phase microextraction (SPME) is suitable for sampling environmental contaminants with a wide range of physical properties in air, water and soil. A fused silica fibre with a polymer coating is exposed to the sample or the headspace above the sample. Organic analytes adsorb to the coating on the fibre. After adsorption equilibrium is attained, usually in 2 to 30 minutes, the fibre is withdrawn. The fibre is introduced into a GC injector, where the adsorbed analytes are thermally desorbed and delivered to the GC column. The amount of analyte adsorbed by the fibre depends on the thickness of the polymer coating and on the distribution constant for the analyte. Fibres with a range of different polarities are now commercially available.

Direct Thermal Extraction

Permits the direct thermal extraction of volatile and semi-volatile organics directly from small sample sizes (mg) without the need for solvent extraction or other sample preparation requirements.

The sample is maybe trapped on sorbent resisn or placed inside a preconditioned glasslined stainless steel desorption tube.
The desorption tube containing the sample is then connected to a short path thermal desorption system. The desorption tube is ballistically heated and carrier gas carries the analytes through the injection port and onto the GC column for analysis.

Comparison of Techniques

Gas Chromatography: Detectors

The Ideal Detector

Adequate sensitivity - range 10^-18 to 10^-15 g analyte/s good stability and reproducibility a linear response to analyte that extends over several orders of magnitude a temperature range from room temperature to at least 400 degrees C

Characteristics of Ideal GC Detector

Good stability and reproducibility. Linear response to analytes that extends over several orders of magnitude. Similarity in response toward all analytes. Temperature range from room temperature to 400 C. A short response time that is independent of flow rate.

Non-destructive.
High reliability and ease of use. No one detector exhibits all of these characteristics

The Ideal Detector

A short response time that is independent of flow rate high reliability and ease of use similarity in response toward all analytes of alternatively a higher predictable and selective response toward + classes of ananlytes nondestructive of sample

Types of Detectors
Thermal Conductivity Detector Thermionic Detectors Atomic Emission Detector Flame Ionization Detector Electron Capture Detector

Thermal Conductivity Detector (TCD)

Q. How does this work? Answer
Electronically measures the changes in the thermal conductivity of the gas. The thermal conductivity changes due to the presence of the analyte

The Modulate Single Filament (TCD)

A method to get rid of some noise ! How?
Both the analytical and reference gases are passed altrenately over a tiny filament This is done at a set frequency, that sets the frequency of the output of the electrical signal The amplitude fo the signal is proportional to the difference in the thermal conductivity of both gases

TCD
What is the best type of carrier gas for the TCD? He and H have high thermal conductivity therefore they show the presence of other components of the gas because of the large drop in thermal conductivity Other gases are not used in TCD

Advantages
simplicity large linear dynamic range ( about 10^5) general response to organic and inorganic samples non-destructive character

Limitations
Low sensitivity (10^-8 solute/mL carrier gas) Others exceed this by factor of 10^4 to 10^7

Thermionic Detectors (TID)

How does this work?
The effluent is mixed with H Passes through a flame tip assembly to ignite Hot gas flows around an electrically heated rubidium silicate bead Heated bead forms a plasma What occurs in the plasma to produce large amounts of ions for D and N containing molecules is not understood

TID
There is a large ion current that results

Advantages
Selective for organic compounds with P and N This is useful for detecting pesticides that contain P

Atomic Emission Detection (AED)

Eluent introduced into a microwave energized helium plasma coupled to a diode-array optical spectrometer Plasma sufficiently energetic to atomize all of the elements in the sample and excite their characteristic atomic emission spectra

AED
Spectra observed using a spectrometer that employs a movable, flat diode array is capable of detecting omitted radiation between 170 to 1780 nm The positional diode array is capable of measuring 2 to 4 elements at any given setting Present software allows measurement of the concentration of 15 elements

Flame Ionization Detector

Consists of a stainless steel burner assembly installed in the detector compartment and a electrometer system in a separate unit adjacent to the gas chromatograph Often it is installed in the tandem with the thermal conductivity cell Effluent form the column enters burner base through millipore filters which remove contaminates

Flame Ionization Detector

hydrogen mixed with gas stream at bottom of jet and air or oxygen is supplied axially around the jet hydrogen flame burns at the tip, which also functions as the cathode and is insulated form the body by a ceramic seal Collector electrode is above the burner tip and is made of platinum

Flame Ionization Detector

In series with flame gases is a selection of resistors 10^7 to 10^10 ohms vibrating reed electrometer used to provide sensitivities up to 5 x 10^13 Amps Carbon counting device that produces a current proportional to number of ions or electrons formed in the flamed gases

Flame Ionization Detector

Responds to all organic compounds except for formic acid Response greatest with hydrocarbons and decreases with substitution Except for vapor of elements in Groups I and II, does not respond to inorganic compounds Sensitivity high due to low noise level

Flame Ionization Detector

Insensitivity to water, the permanent gases, and inorganic compounds simplifies the resolution of components in analysis of aqueous extracts and in air pollution studies

Electron Capture Detector

operates similar to proportional counter for measurement of X-radiation effluent form the column passes over a beta-emitter - I.e.) nickel-63 or tritium

ECD
electron from emitter causes ionization of carrier gas (N) produces a burst of electrons standing current between electrodes decreases in presence of organic species that capture the electrons

ECD
selective in its response and highly sensitive Hewlett Packard makes one with a detection limit of less than 8 fg/sec for lindane sensitive toward molecules with electronegative functional groups (halogens, peroxides, quinones, nitro groups) insensitive towards amines, alcohols and hydrocarbons

ECD
important application: detection and determination of chlorinated insecticides advantages
does not alter the sample significantly (in contrast to flame detection) quick and easy relatively cheap

ECD
disadvantage : linear response range is usually limited to around 2 orders of magnitude

Thermal Conductivity Detector

Exploits the changes in the thermal conductivity of a gas stream brought about by the presence of analyte molecules. The resistance of either a heated platinum wire or a heated semiconductor thermistor gives a measure of the thermal conductivity of the gas. Twin detector pairs are typically incorporated into two arms of a Wheatstone bridge. In the presence of a relatively small concentration of analyte a large decrease in thermal conductivity of carrier gas occurs resulting in a temperature rise in detector.

Thermal conductivities of He and H2 are ~ 6 10 times higher than most organic compounds. Necessitates the use of these gases as carrier gas.
Linear range of 10 and is suitable for organic and inorganic samples. Non-destructive and allows collection of sample -8 after detection but low sensitivity ~ 10 g/s analyte/gas
5

Flame Ionisation Detector

Most organic compound pyrolyse in H2-air flame and produce ions and electrons. A potential of a few hundred volts is applied across the burner tip and a collector electrode located above the flame. The resulting current is amplified and proportional to the number of carbon atoms in the flame. General detector for GC. However, carbonyl, alcohol, halogen and amine groups yield few electrons. Also insensitive to H20 CO2 SO2 NOX. Large linear response range (~ 10 ) and low noise (once detector has settled). Needs to be burning 24 hours before analysis. Exhibits very high sensitivity ~ 10 analyte/second
-13 7

Collector electrode

Insulator Connector nut Air H2-air flame Grounded jet H2 Inside oven wall

Exit end of column

g/s of