Training Manual

INDEX

Sr. No. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Description Chromatography Instrumentation A Review Basic Theory Practical hints GC General Information on GC Column Choosing a Suitable column Choice of liquid phase Selectivity of CG Phases according to McREYNOLDS Constants Diatomaceous Earth supports in Gas Chromatography List of Stationary Phases Suitable for selected Compounded Classes Porous Polymers in Gas Chromatography Where to use Derivatisation Ghosting effect in Gas Chromatography General Troubleshooting Practical Capillary Column Gas Chromatography High Pressure Liquid Chromatograph

Page No. 3 8 19 35 43 48 57 64 69 86 89 110 126 144

CHROMATOGRAPHY INSTRUMENTATION - A REVIEV Chromatography is the most accepted separation tool in modern analytical laboratories. The acceptance of Chromatography as an analytical tool for separation, identification and quantitation of mixtures is due to the simplicity of the equipment required and the ease of interpretation of. the data produced. This has been due to rapid technological advancement over the past 25 years. Some of these developments which has resulted in the simplification of very complex chromatographic separations are outlined below. The evaluation of Gas Chromatograph and Liquid Chromatograph and other chromatography techniques have been dealt with separately. Chromatographic separation occurs when sample component zones or bands travel through the column at different velocities. The parameters for the separation are quite a few and include velocity, temperatures, polarities, etc. Therefore, to achieve a meaningful measure of degree of separation, termed resolution, we shall discuss those parameters, which are instrumental in improving the same.

GAS CHROMATOGRAPH: Gas Chromatograph in recent years is one of the most efficient and convenient tools for separation, detection, and quantitative estimation of components present in a complex mixture. This is an analytical technique of separation based on the solubility or adsorption of components between a liquid stationery phase and mobile gas phase. The basic parts of the GC include :

A. B. C. D. E.

THE PNEUMATIC SYSTEM THE INJECTION SYSTEM OR THE SAMPLING SYSTEM THE COLUMNS THE DETECTOR THE RECORDER/DATA PROCESSOR SYSTEM

A.

Pneumatics

The carrier gas serves as the mobile phase in GC. Over the last two decades sophisticated pneumatics have been instrumental in achieving high degree of stability, and reproducibility. Since this requires constancy of gas flow, the following components have been largely responsible for achieving the same i) ii) iii) iv) Differential flow controllers Pressure regulators Flow restrictors Diffusion proof pressure gauges

The combined advantages of the above four in the pneumatics have resulted in reproducible retention time, stable base line in the temperature programming mode and constancy of flow at different temperatures. This coupled with high purity gas which has been made available of late has resulted in simplicity of trace analysis of large number of compounds.

B.

The Injection System / The Sampling System :

The conventional injection system incorporated a heated zone into which the sample was injected, transporting the same into the column with the help of the carrier gas. Subsequent developments in the injection system resulted in
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reduced dead volume injection system to reduce phenomena such as band broadening owing to dead volume. Further developments resulted in the oncolumn injection technique whereby the sample is introduced into the column head as a "Plug" thereby eliminating band broadening and also certain other disadvantages of the sample coming into contact with hot metal surface resulting in thermal decomposition. Recent developments in injection techniques involve septum purge facility to reduce the phenomena of ghost peaks. Advancements in column technology has resulted in the development of capillary columns and consequent modification in the injection system for capillary chromatography. Capillary gas chromatograph involves the split and the split less techniques and the more recently introduced Programme Temperature Vaporizer (PTV) technique. The necessity to introduce gas samples reproducibly into a continuously flowing carrier gas stream led to the development of the gas-sampling valve. However, these valves can also be used for other functions like back flushing, column or sample selection, concentrating vapors etc. With a certain degree of automation these valves have resulted in uninterrupted automated analysis. C. Columns : The column of a GC acts as the heart of the equipment. The separation mechanism is totally dependent on the contents of the same. Earlier models of Gas Chromatographs utilised 'U' shaped columns. This led to a limitation on the length and to the introduction of coiled packed columns. The resolution of Chromatography peaks is related to two factors : i) ii) Column efficiency and Solvent efficiency
5

The former results from the column design and the operating conditions and the latter results from the solute solvent interaction and determines the relative position of solute bands on a chromatogram. The necessity to measure the efficiency of a column resulted in quantification of the same by number of plates. Hence to compare column efficiency one must specify the solvent, solute, temperature, flow rate and sample size. The plates concept is a carry over from distillation processes where the first efficient columns were in fact composed of discrete plates. In the laboratory however, the plate value is a theoretical concept introduced so that column performance can be evaluated. Figure one is an illustration of column and solvent efficiency. The development of stationary phases covering a wide range of polarity along with solid support has resulted in improved column efficiency. Attached are two charts, 'one relates to some of the most commonly used stationery phases for the separation of various compounds and the other a table of selected McReynolds constants.

Application of McReynolds constants : The McReynolds constants are present in the form of tabulations of- I for l0 test probes. The phases are arranged according to increased polarity. A valuable use of the table is the choice of substitute stationery phases of similar selectivity. While McReynolds tables do not answer every question concerning to a GC separation, however, they provide the best information available for the comparison of the selectivity of the GC phase. They can also be used to suggest other phases which might be used to improve a separation.
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D.

Detector :

The Thermal Conductivity Detector began as being the most popular and widely used detector system when GCs were introduced. This was followed by the Flame Ionization Detector. Subsequent developments in detector technology resulted in the introduction of speciality detectors like the Electron Capture Detector, the Nitrogen Phosphorous Detector, Flame Photometric Detector. These selective detectors have been particularly useful in the trace analysis of compounds and also in pollution studies. Other recent developments include interfacing the GC with Mass spectrometers.

E.

Recorder

The Recorder serves the function of identification and quantification of various components of the mixture to be analysed. The development of digital integrators to complement recorders, was responsible for accurate quantification of the concentrations of the different components. These along with development of specific algorithms for integration of different types of peaks like leading peaks, skewed peaks, tailing peaks help not only in quantifying but also in minimising the time of analysis. Current developments in data processor technology includes computation in whatever format that may be desired by the use of specific programmes designed to meet the chromatographers requirement e.g. area percentage, computation using international standard methods, external standard methods, area normalization method etc.

BASIC THEORY AND PRACTICAL HINTS GC Nth The number of theoretical plates is a measure for the quality of a column. It is calculated from the chromatogram. N tr to t HETP The length of the column divided by the number of theoretical plates gives the height equivalent to a theoretical plate. HETP = L/N A more efficient column has a small value of HETP. Van Deemter equation The value of HETP of an actual column depends on the carrier gas velocity and the type of carrier gas. Van Deemter formed an equation which describes relation between HETP and the linear velocity G. HETP = A + B + C. A= Eddy diffusion, depends on the particle size and the vegularity of the A = O for open tubular columns. = = = = number of theoretical plates = 5.54 [tr/W ] 2 bruto retention time peak width at half of the peak height gas hold up time tr to

W =

packing.

B= C=

diffusion of the carrier gas. resistance to mass transfer.

In practice the chosen velocity is slightly higher than the optimum velocity. Using a dense gas like nitrogen, smaller values for HETP are obtained at the optimum, however using helium or nitrogen as a carrier gas the analysis can be carried out quicker and the optimum is a longer range. K The capacity ratio K indicates how many times longer the nett retention time of a compound is compared to the gas hold up time of the column. For packed columns K should be at least 5 for optimum resolution R. Increasing of K can be attained by decreasing the oven temperature or increasing the amount of liquid phase. The relative retention or selectivity is the ratio of nett retention time of two components. It is characteristic for a given stationary phase. R The measure of separation of two components is the resolution R. R= tr2, - tr1 ______________ W1, + W2, = tr2 / trt = k2 / k1

Two peaks are separated for 98% if R = 1. if R = 1.5 the peaks are separated for 99.7%. This is only valid for symmetrical peaks. R = K1 x (-1) Nth _________ ______ K1+1 4

Calculation of column efficiency Coating efficiency is defined as : HETP min theor x 100% HETP min actual The theoretical minimum plate height can be calculated from : HETP min theor =

1+ 6k + 11k2 d 3(1 + K)2

An ideal coated column will give in the very optimum of the H-G curve a coating efficiency of 100%. The phase ratio and film thickness values as recorded for the open tubular columns are no efficiency parameters, but they determine the column capacity, Phase ratio is defined as : B = Volume of mobile phase / volume of stationary phase in the column. The relation between film thickness and phase ratio is given in the next expression : = 250 x d df d = column internal diameter df = film thickness (micrometer)
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Temperature programmed analysis A parameter that can be used as a measure for column efficiency in a temperature program is the separation number. This parameter gives the maximum number of peaks that can be separated between two sequential homologs (peak 1 and peak 2). SN1.2 = tr2 tr1 - 1 (W ) + (W )

Type of column WCOT Narrow bore

ID (mm) 0.10 0.22 0.25

Nth/m 9500-12000 3300-5500 3000-5000 2500-4000 1500-2500

SN/25m 65 43 40 35 25

WCOT Medium bore WCOT Wide bore

0.32 0.53

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The Kovats Index From the beginning of gas chromatography, people tried to define the polarity of a liquid phase. Mostly this was carried out by comparing the retention data of two components. If the elution of the components was in order of their boiling points the liquid phase was called non polar; if a polar component was more retarded than a non polar one with the same boiling point the liquid phase was called polar. This of course was not a very precise method. An improvement of characterizing a liquid phase was made by using the Kovats indices. If log retention times or log retention volumes of n-hydrocarbons are plotted against the number of carbon atoms times 100, an almost straight line is obtained. To find the kovats index of a certain compound on the liquid phase in question log tr is plotted and the abscissa is interpolated. In equation : I = log tr - log t r(n) Log tr (n+1) - log t r(n) tr is the nett retention time of the compound eluting between the normal hydrocarbons CnH2n+2 and Cn+1H2n+4. - 100+100n

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Test Chromatogram The test report for a pretested column can be used in evaluating the column, it is never a waste of time to duplicate this standard test before the column is used for its proper application. Any failure of the system will cause a drop in performance of the column with respect to efficiency, peak shape and base line drift by regular repeating this standard test, the quality of the column can be followed and overall quality can be watched. (For your convenience a test sample as used for the standard test is supplied with the columns). Malfunctioning will often be caused by dead volumes. Optimization Optimization can be done by making a H- curve. The plate height is plotted against the carrier gas velocities and an optimum velocity is chosen from this plot. If the separation of some key components is insufficient lowering the column temperature can improve the separation. Especially with capillary columns, this will often produce a good result since capacity ratios are generally quite low. To Improve a seperation the next steps can be made Increasing K, to K = 1 effected by increasing the amount of liquid K + 1 phase or lowering the temperature. effected by choosing a more selective liquid phase. Increasing L Decreasing HETP effected by lengthening the column. effected by taking into account the Van

Increasing

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Deemeter equation. Optimum Operation Condition for Nitrogen Internal Diameter 0.10 mm 0.22 mm 0.25 mm 0.32 mm 0.53 mm Average carrier gas Velocity 15-20 cm/sec 12-17 cm/sec 10-15 cm/sec 7-11 cm/sec 4-8 cm/sec Inlet Pressure 1.4 bar/10 m 0.6 bar/25 m 0.5 bar/25 m 0.3 bar/25 m 0.15 bar/25 m

Optimum Operation Condition for helium Internal Diameter 0.10 mm 0.22 mm 0.25 mm 0.32 mm 0.53 mm Average carrier gas Velocity 27-32 cm/sec 25-30 cm/sec 22-27 cm/sec 16-21 cm/sec 11-16 cm/sec Inlet Pressure 2.8 bar/10 m 1.2 bar/25 m 1.0 bar/25 m 0.7 bar/25 m 0.4 bar/25 m

Optimum Operation Condition for Hydrogen Internal Diameter 0.10 mm 0.22 mm 0.25 mm 0.32 mm 0.53 mm Average carrier gas Velocity 45-55 cm/sec 45-50 cm/sec 40-45 cm/sec 29-34 cm/sec 20-25 cm/sec Inlet Pressure 2.8 bar/10 m 1.2 bar/25 m 1.0 bar/25 m 0.7 bar/25 m 0.4 bar/25 m

14

Sample Capacity The amount, which can be injected on to a capillary column, is a function of the film thickness rather than the column radius Internal Diameter 0.10 mm 0.22 mm 0.32 mm 0.50 mm Film Thickness 0.12 m 0.12 m 0.4 m 0.2 m Max. amount for one component 1-10 ng 20-50 ng 5-100 ng 30-300 ng

15

THERMAL CONDUCTIVITY OF GASES AND ORGANIC VAPOURS Type of Compound GASES Air Hydrogen Helium Nitrogen Oxygen Argon Carbon monoxide Carbon di Oxide Nitric Oxide Sulphur dioxide Hydrogen Sulphide Carbon di Sulphide Ammonia HYDROCARBONS Methane Ethane Propane n-Butane Isobutane n-pentane Isopentane n-Hexane 7.2 4.3 3.6 3.2 3.3 3.1 3.0 3.0 10.9 7.3 6.3 5.6 5.8 5.3 5.0 5.8 41.6 34.8 5.8 5.9 4.0 5.6 3.5 5.7 2.0 3.1 3.7 5.2 7.5 53.4 41.6 7.5 7.6 5.2 7.2 5.3 7.8 0 degree C 100 degree C

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n-Heptane Cyclohexane n-Hexane Ethylene Acytelene Benzene ALCHOLS Methanol Ethanol KETONES Acetone HALOGEN COMPOUNDS Carbon tetrachloride Chloroform Dich loromethane Methyl Chloride Methyl Bromide Methyl Iodide Difluoromethane (Freon 12) Ethyl Chloride Ethyl Bromide Ethyl Iodide ETHERS Methyl ethyl Methyl Butyl

2.5 4.2 4.5 2.2 3.4 2.4 1.6 1.6 2.2 1.5 1.1 2.0 2.3 1.7 1.4 -

4.4 4.3 4.7 7.4 6.8 4.4 5.5 5.3 4.2 2.2 2.5 2.7 4.0 2.6 1.9 4.1 5.8 5.0

17

Ethyl popyl Ethyl butyl Propyl Isopropyl Propyl butyl Butyl ESTERS Methyl acetate Ethyl acetate AMINES Methyl Di methyl Ethyl Propyl Trimethyl Diethyl Isobutyl n-Amyl di-n-popyl Triethyl

1.6 3.8 3.6 3.4 3.0 3.3 3.0 3.0 2.8 2.6 2.7

5.4 4.7 4.6 4.8 4.3 4.0 4.1 -

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GENERAL INFORMIATION ON GC COLUMNS

A.

Sources Of Gas Chromatographic Retention Data

There is always the problem of selecting the proper gas chromatographic (GC) stationary phase to solve the analytical problem. The best way is to simply use a procedure that has already been reported. The following summaries of GC separation date can be used as an aid in selecting suitable phases for a particular analysis. 1. "Gas Chromatography Literature, Abstracts and Index", Preston Technical Abstracts Company, P.O. Box 312, Niles, Illinois 60648. Appears monthly and subscription rate is $324.00 per year. The abstracts are current and a yearly index is provided. Information on the column and conditions is usually given in the abstract. Microfilm copies and a computerized index are available from the publisher. 2. "Gas and Liquid Chromatography Abstracts", contact the Executive Secretary, Chromatography Discussion Group, Trent Polytechnic, Burton Street, Nottingham NGI 4BU, United Kingdom appears quarterly and the cost is 10 pounds Sterling by regular mail, or 14 pounds Sterling by air. mail. The yearly index is first-rate, but the original literature has to be referred to for details. 3. "Gas Chromatography Data Compilation", AMD 25A; "Gas Chromatography Data compilation , First supplement, "AMD 25A Sl. Published by the Amer Society for Testing and Materials , 1916 Race St ., Philadelphia , Pa, 19103.
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Cost is $40.00 per volume. These references cover the older literature. AMD. 25A covers the literature through 1965, while AMD 25A Sl covers the years 1966 to Jan,1969. The usefulness of these references lies in the listing of retention data and retention indices for many compounds on many GC Phases . 4. "Gas Chromatographic Retention Data", by W.O. McReynolds, Preston. Technical Abstracts Co., Niles, Ill., 1966 Provides retention data for a number of phases, but with no index. 5. Technical journals devoted- to chromatography are "Journal of Chromatographic Science", "Journal of Chromatography", "Chromatographia", "Journal of High Resolution Chromatography , and Chromatography Communications . " Other journals of interest are "Analytical Chemistry", "Analyst", and "Analytical Letters", and "Journal of the Association of Official Analytical Chemists". 6. Review articles appear in "Chromatography Reviews", now published pert of "Journal of Chromatography". Annual review articles appear in the April issue of "Analytical Chemistry". In the latter issue, developments in Chromatography are viewed in even years, and applications are reviewed in' odd years. 7. Many books are being published which discuss the use of chromatography in solving analytical problems. The Annual Buyer's Guide Issue of "Analytical Chemistry" (published in August) provides book lists and references to book reviews in ACS Publications. .
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8.

"GC Applications Library", published by Varian Instruments, Palo Alto, CA, 1977, cost $350.00. Contains more than 50,000 references.

9.

"CRC Handbook of Chromatography", edited by G. Zweig and J. Sherma, Volumes I and II, CRC Press Inc., Cleveland Ohio, Volume 1, $45.95; Volume II, $39.95.

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B.

Practical Aspects Of Gm Chromatographic Column Operation

1.

The Carrier Gas

The carrier gas must be pure and contain no more than 5 ppm oxygen and1.2 ppm water. Starting from the gas supply, place molecular sieves drier, then an oxygen adsorption trap in the carrier gas lines to remove these impurities. All organic phases used in gas chromatographic columns will degrade in the presence of oxygen at high temperatures. Trace water will cause depolymerization of condensation polymers such as polyethers, polyesters, and silicones at high temperature. Oxygen is an electron-capturing species and will affect the electron-capture detector. Moreover, oxygen will burn out filaments on a thermal conductivity detector. Each cylinder of carrier gas has its own impurity level. Every now and then, a tank with higher amounts of impurities will appear which will rapidly overcome an oxygen adsorption system and destroy a column. A new tank and oxygen absorption unit will improve this situation which will arise in an unpredictable way. A two-stage regulator is needed to reduce the tank pressure to the operating pressure, and it should have a stainless steel diaphragm. Rubber or plastic diaphragms permit oxygen, water, or organic components from the diaphragm, to diffuse into the carrier gas. Set the second stage of the regulator to 40-60 psig in temperature programmed operation. When using this technique, the resistance to flow in the GC column increases with increasing temperature. Pressures of 40-60
22

psig on the second stage are necessary to maintain constant gas flow through the column. Higher pressures might be required to maintain flow when using longer columns (l0 ft.or so), or fine mesh packing (finer than 120 mesh). Always change the tank when the pressure is less than 200 psig. As the total pressure in the cylinder decreases, there is an increase in the partial pressure of the water and other impurities adsorbed on the inner walls of the gas cylinder. As a result, the last amounts of gas delivered from the gas cylinder contain higher levels of impurities. Periodically, check the fittings on the carrier gas lines to be sure they are tight. Air can diffuse through loose fittings and into the carrier gas, even though the gas is under a pressure of 40-60 psig. When using the flame ionization detector, leaks in the fittings or septum can be detected by the following method. Set the detector for high sensitivity and blow butane gas (from a lighter) or propane at the fitting. Butane (or propane) will diffuse rapidly through the leak into the carrier gas and be carried to the detector, where its presence will produce a "blip" on the chromatogram trace. This is proof of the fact that ambient gases, as air, will diffuse into a gas chromatographic system through leaky fittings even though the system is under a pressure of several atmospheres.

2.

The Injection Port

The purpose of the injection port is to introduce the sample into the gas Chromatographic column by instantaneous volatilization following injection from a syringe. If the injection port is too hot, which is a common tendency,
23

thermal degradation and rearrangement of the sample can occur. Reduce the injection port temperature. If the peaks become too asymmetrical or broad, then the temperature has been set too low, and must be increased slightly. Another problem can arise if the injection port or detector is hotter than the recommended temperature for the liquid phase in the column. In many instruments, the part of the column nearest the injection port or detector will become too hot, leading to column bleed. The column performance will degrade and peak tailing will appear in the chromatogram if this situation proceeds too far. A glass liner placed inside the injection port will eliminate sample contact with hot metal walls, which can catalyse thermal degradations. Any debris left in the liner, especially from biological samples, can be sources of excessive sample adsorption. If a liner is used, the debris can be easily removed by replacing the liner. Problems With Septums

3.

The rubber septum is one of the weakest parts of the gas chromatographic system. When installing the septum do not over tighten the retaining nut. Change the septum every day. If the septum is punctured too many times, it can leak. The result is a change in flow rate (a change in retention time) and loss of sample (irreproducible peak heights). Air can diffuse into the column through a heavily punctured septum, even though the system is under a pressure of 40-60 psig. By using a needle guide, the number of puncture holes in the septum is decreased, and the septum life is increased.

24

Ghost peaks can be observed in temperature programmed runs. These false peaks are artifacts produced by septum bleed, and appear in the chromatogram of programmed runs, even when no sample is injected. This can be shown by the disappearance of ghost peaks after replacing the septum with a lead disc (injection temperature below 300C). Septum bleed can be decreased by using either air-or water-cooled septum retaining nuts, or by using a septum flush head. Adsorption of sample on the face of the septum can be minimized by using septums with Teflon or polymide inner faces.

4.

Syringe Injection Technique

In the usual microsyringe, there is dead volume in the needle. In the 10 micvoliter syringe, the dead volume in the needle is 0.8 ul. This has to be accounted for when injecting the sample. One injection procedure is to first pump the sample back and forth vigorously in the syringe, in order to wet the Plunger and to remove air bubbles. Then withdraw the sample back into the syringe so that the entire volume can be read on the volume marks of the syringe barrel. When charging the syringe, never leave sample solution in the needle. Otherwise, the sample will boil out of the needle as it is inserted into the hot injection port. (This is somewhat of a problem with sub micro liter syringes, in which the entire sample volume is held within the needle. The sample is very small, and it boils out of the needle at once. A precise injection technique is necessary ) .

25

In a better procedure, the syringe is loaded in the following order: solvent, air, sample, air, and finally with only air remaining in the needle. Now, when the sample is injected, solvent is the last to leave the syringe and rinses out sample residue in the needle. . Wipe off the syringe needle before injecting. After injecting the sample, leave the needle in the injection port about two seconds, and withdraw at once. The reproducibility of peak heights upon repeat injection should be within 5%, and within 1% for skilled operators. Clean the syringe by injecting solvent into the Hamilton heated syringe cleaner. In this device, the needle is heated and sample residues are baked out, thus insuring complete removal of traces adsorbed impurities. Often the tip of the needle becomes bent, forming a fish hook. This can be detected by running a finger past the end of the needle. A few strokes on a sharpening stone will- remove the fish hook. Read the manufacturer's instructions on the use of the syringe.

5.

On-Column Injection

Higher efficiencies are always observed if the column is packed for on column injection. In this technique, the column is packed so that there is empty space at the injection port end. The column is pushed up into the injection port so that the end is 1/8 in from the septum. The void space at the column. end is of a length such that the injection needle just penetrates into the glass wool plug. Upon injection, the sample enters directly into the
26

column. When using this technique never set the injector temperature higher than the maximum operating temperature of the liquid phase. When conditioning the column, set the injection port at the conditioning temperature.

6.

Minimum And Maximum Recommended Operating Temperatures

In table "A" list of stationary phases, minimum and maximum operating Temperatures are recommended. Below the minimum temperature, the phase will behave as either a very viscous liquid of solid, and less efficient separation will be observed. The chromatographic result will be broader peaks in the gas chromatogram . Maximum operating temperatures are suggested to serve as a general indication of the temperature limits. The limits were mostly determined with a TC detector. As a result, this limit does not necessarily apply when using a very sensitive detector at the suggested maximum temperature. The observed maximum temperatures. will depend on many experimental variables, such as column conditioning, phase loading level, analysis temperature, sensitivity setting of the detector, purity of the carrier gas, etc. In temperature programmed runs, the column can sometimes be operated for short periods about 25C above the maximum temperatures. This will result in shorter column life, but it can help in finishing a short-term analytical project.

27

Above the maximum temperature limit, the phase will begin to bleed off the column and the observed result will be a drifting baseline or excessive spiking in the -baseline. Under these conditions, liquid phase is being blown off from the column, and this will eventually lead to changed retention times. Other signs will be poorer resolution of very close peaks and peak tailing, as uncoated surface is exposed by removal of liquid phase. Thus, the column life is shortened and it will have to be replaced. In extreme cases, the bleeding will result in fouling the detector and connecting lines.

7.

General Procedure For Conditioning New Columns

In the conditioning process, the new column is heated to remove any trace solvent or other volatile components in the gas chromatographic phase. In this process, the phase distributes itself evenly over the surface like butter on hot toast. Certain precautions must be taken during this process. Never connect the column to the detector in the conditioning stage. Any volatiles blown off the column might form deposits in the detector and its line connecting to the column. Heat the column to 100C, and flush air out by passing carrier gas at normal analytical flows through the column for 30 min. (Remember that any organic liquid phase will decompose at high temperatures in the presence of oxygen). Next, heat the column to the final conditioning temperature at a rate of 4C per min., and maintain this temperature overnight with normal carrier gas flow. The conditioning temperature should be at least 20-30C above the analytical temperature, but not higher than 10-15C below the maximum recommended temperature (Part 6, above).

28

Special no-flow conditioning procedures are sometimes recommended for silicone polymers as SE-30, OV-17, OV-I, SE-52, and UC-W98. This procedure is not generally recompensed for condensation polymers as polyethylene oxides, and polyesters. The procedure starts off the same way as conventional conditioning procedures. Pass carrier gas through the column at 100C for 30 min. Next, turn off the carrier gas and heat the column at 310C for 1.5 hr., then cool the column to 100C. Turn the carrier gas on, and program the column temperature to the conditioning temperature at 4C/min. It is claimed that this procedure provides improved columns for the analysis of drugs and steroids. The column is now ready for use. If baseline drift -is observed at the highest sensitivity settings, a longer period of high temperature conditioning should improve this situation. Other sources for poor baseline response are a dirty detector, air leaks in the gas line fittings, a much punctured septum, or chemical decomposition of the phase due to presence of trace acid or base on the support in the phase or on the inner column walls. Remember, the lower the column temperature, the longer column life. Many of the liquid phases are commercial grade materials and conditioning might require several days before the noise level is low enough to provide acceptable baselines at high sensitivity. The gas chromatographic grade silicones are recommended since they have been carefully cleaned up and long periods of conditioning are not necessary. These materials are the OV, and Silar-series of silicones, Chromatographic Grade SE-30, and AN-600.

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8.

Precautions For Columns After Use

Never heat gas chromatographic columns with air in them. When a column, which has been used before, is first placed in a gas chromatograph, flush out any air, which may be in the column, with carrier gas for a period of 15-30 min. Then heat the column to the desired operating temperature. Always cool the column to room temperature before removing from the gas chromatograph. If you intend to use the column again, cap the ends during storage to prevent diffusion of air into the column. Plastic caps, can be used for this purpose.

9.

Column First Aid

Upon continued use, the column performance can worsen, as shown by peak broadening or tailing, or gradual merging of adjacent peaks. The problem usually arises because something has happened to the front end of the column. The injection port temperature might have been too high and overheated the injector end of the column, thus destroying liquid phase and exposing bare uncoated support surface. Residues or decomposition products, might have built up on the glass wool plug, which should be replaced. (Do not handle the glass wool plug with greasy fingers). The front end of the Column packing might have degraded due to oxidation or buildup of residue. When using glass columns, remove and replace the first few inches of packing. The performance of metal columns can frequently be improved by carefully cutting off several inches of the injection side of the column, and repacking with fresh packing and a new silanized glass wool plug.

30

The silanized glass wool plug itself can cause problems. In a few cases, it is the source of compound decomposition. The adsorption of acidic compounds as barbiturates, free organic acids, and phenols, can be decreased by coating the glass wool with trace phosphoric acid. Glass wool can be the source of extraneous peaks in trace analysis.

B. A) 1. 2.

How To Improve Column Efficiency Choice Of Support And Percent Loading Use small particle size support with a narrow particle size range. Use the liquid phase of similar McReynolds Constants, which has the lower viscosity, i,e. OV-101 instead of OV-I. However, the maximum Operating temperature might mean that only one phase will be suitable.

3.

Prepare a stationary phase of uniform coating at a 3-10% loading. A Power loading provides a more efficient column and a lower elution temperature will be observed for the compound being separated. There is an optimum percent loading of the stationary phase on the support, which is best defined by experimentation using the mixture to be analyzed.

B) 1.

Column Diameter And Preparation Use a narrow-bore column. A 1/8-in OD column is more efficient than a 1/4-in OD column. Improved resolution can be obtained with the Analabs Hi-Plate Columns , 1 /8- in OD, of 20- and 30- ft, lengths.

31

2.

In preparing the packing and loading the column, no fragmentation by abrasion of the particles against each other should occur. The diatomaceous white supports as Anakrom Q, Anakrom A, Anakrom ABS and the Chromosorb W's, are very friable and must be handled very carefully to prevent fragmentation. Smaller size particles or "fines" result in a less efficient column. There should be no void space within the packed column. Use the electric vibrator very sparingly, and at very low amplitudes of vibration.

3.

Install the column so that there is a minimum of free space between the septum and the column packing and between the column packing and the detector. Older gas chromatographs are poorly designed in this respect.

C) 1.

Choice Of Analysis Conditions With the flame ionization detector, nitrogen as the carrier gas is capable of providing lO-2O % more efficiency than helium. (In the case of the thermal conductivity detector, helium is recommended). However, the analysis time will be twice as long as using nitrogen, so helium is the preferred carrier gas if fast analysis time is necessary.

2.

Plot the number of plates, "N", vs. the carrier gas flow rate. The best Practical flow rate is actually somewhat higher than the optimum flow rate indicated on the plot. Use the flow rate where the "N" value is about 95$ of its maximum value. A good test compound is a methyl ester, or better, a compound from the mixture being analyzed. Generally, for a 1/8-in O.D, column, the optimum flow is
32

approximately 30ml/min for helium and l5 ml/min for nitrogen. For a 1/4-in O.D. column, the approximate optimum flow rates are about 24 times these values. 3. Every GC analysis will have an optimum temperature in which the peaks of interest will separate in the shortest possible time. This temperature must be found by trial and error. However, observe the lowest temperature limit recommended for that phase. The retention times for the peaks of interest will be about half for a 20-30C increase in temperature. One disadvantage is that the adsorptive effects of the support surface may become more important at lower temperatures, which will result in peak broadening or tailing. The lower the temperature, the longer the life of the column. 4. The sharpest peaks will be observed with the smallest sample size. However, there are practical limits to the sample size with each column and detector. Adsorptive effects from the support surface will predominate at very low sample sizes and the result will be peak tailing. However, without this effect, more efficient separation will be obtained with small sample sizes . 5. Peaks of analytical interest should emerge at time corresponding to 46 times that required for solvent. Under these conditions, 80-85% of the maximum efficiency will be obtained in the shortest analysis time. 6. Adjust the column length to give the fastest analysis time possible. To

33

double the resolution, a 6-f't, column must be increased by four times to 24 ft. (22 X 6). Sometimes a shorter column will suffice. To halve the resolution, shorten a 6-ft, column to 1.5 ft., (1/2)2 X 6. If a partial separation is indicated on a certain column length and phase, a much longer column will improve the separation. In the latter case, the analysis time is certainly longer, but this approach might lead to a faster solution of the problem than spending more time searching for a more selective stationary phase.

34

CHOOSING A SUITABLE COLUMN The successful separation of a given mixture in a gas chromatograph primarily depends on the choice of the column. Both capillary and packed column are used. Capillary columns are open tubes of small diameter with a thin liquid film on the wall. Packed columns consist of solid particles alone or coated with a thin film of the nonvolatile liquid phase. The column tube may be glass, copper, stainless steel 316 or plastic of internal diameter in the range of 2 to 6 mm and length from 1 metre to several metres. The solid support, type end amount of liquid phase, method of packing, length, and temperature of the column are important factors which determine the desired resolution. Copper tubing sometimes reacts with the constituents of the sample or exerts a catalytic effect on decomposition. In such cases stainless steel 316 tube is preferred. Glass as column tubing has the advantages of being chemically inert and of allowing inspection of the state of the packing both during the preparation of the column and subsequent use. The function of the solid support is to give as large an interface as possible between the gas and the liquid phase so as to facilitate partition between them. Therefore it should have a high specific surface without enhancing adsorptive effects which would interfere with the portion and lead to peaks of unsatisfactory shape. The most widely,. used supports are diatomaceous earths, such as celite, chromosorb W, Embacel, Anakrom, etc, or crushed firebrick such as sterchamol and chromosorb P. The latter has a higher mechanical Strength but is not completely inert to polar samples resulting in
35

tailing. The removal of adsorption sites of the column support can be achieved by chemical treatment with a silylating agent such as hexamethuldisilazane or by acid or alkali washing. In choosing the stationery phase the principal objective is to have the components of the sample well separated. As a general rule, the retention of a polar solute on a polar stationery phase is greater than that of a non polar solute. The order of elution of a homologous series from a stationery phase is a function of their respective vapor pressures at the temperature of the column. Materials to be analysed can be classified according to their polarities given below :-

MOST POLAR : Water, glycol, glycerol, amino alcohols, hydroxy acids, polyphenols, dibasic acids,

POLAR : Alcohols, fatty acids, phenols, primary and secondary amines, oximes, nitrocompounds with a - H atoms, nitrites with a - H atoms, NH3, HF, N2H4 HCN.

INTERMEDIATE POLARITY : Ethers, ketones, aldehydes, esters, tertiary amines, nitro-compounds, nitriles

36

LOW POLARITY : CHCl3, CH2Cl2 aromatic hydrocarbons, olefinic hydrocarbons. NON POLAR : Saturated hydrocarbons Cs2, mercaptans, sulphides, CC14. Non-polar stationery phases separate components according to boiling point. However, components with the same or very close boiling points will not separate on a non polar column. A liquid phase loading of l0 to 20% on a chromosorbw type support is recommended for volatile, low molecular weight compounds that would have too short a retention time, on a low percent loaded column. For high molecular weight compounds and thermally labile substances, a liquid phase loading of about 3 to 5% is recommended. Many biomedical compounds, drugs and pesticides fell into this category. All columns should be preconditioned before usage for at least 6 to 8 hrs. at about 25 below the maximum temperature limit for the liquid phase, by passing the carrier gas at a low flow rate after disconnecting the column exit from the detector. Porous polymer beads with large surface area, uniform pore structure and varying polarity have found extensive use as stationery phases or as solid supports. Porapak series and Chromosorb century series of different mesh size and polarity are good examples of this class. A majority of compounds can be analysed on one of the six polymers (coated on suitable solid support) mentioned below. All these have a wide temperature range, reproducibility and have been well tried out.
37

1. Dimethyl-silicone (e.g. OV-101, SP-2100, SE-30, SF-96) 2. 50% phenylmethyl silicone (e.g. OV-17, SP-2250) 3. Polyethylene glycol (e.g. Carbowax) 4. Diethylene glycol succinate (DEGS) 5. 3-cyanopropyl silicone (e.g. Silar-10C, Apolar-10C, SP2340) 6. Trifluoro propyl methyl silicone (e.g. OV-210, SP-2401). A representative list of stationery phases is given below for various classes of compounds.: ACIDS : C1-C9 (free) Chromosorb 101, Porapak Q, SE-30 C1-C18 (free) : FFAP, Porepak QS Bile and urinary : OV-1, OV-17 Fatty acids methyl esters : DEGS, FFAP, Apiezon L, EGSS-X, Carbowax 20M. ALCOHOLS: C1-C5 : Porapak Q, Chromosorb 101, Carbowax 1500, Silicone oil, Carbowax 400, UCON 500-X, Glycerol C5-C18: Glycols: FFAP, Carbowax 20M, Fluoro Silicone oil Porapak Q, Chromosorb 101

Poly alcohols : OV-210 ALDEHYDES : C1-C5 : Silicone oil 550, Porapak N, Chromosorb l05, Digiyceroi, UCON 500-X, - ' oxydipropionitriie, Dialkyl phthalates C5-C18: Carbowax 20M, Fluorosilicone oil, Porapak Q.
38

ALKALOIDS : OV-17, OV-101, OV-210, Cyanosilicones, QF-1, SE-30

AMINO ACIDS : Free acid : DEGS, EGSS-X Esters : EGA, OV.17, tricresyl phosphate, carbowax 20M, LAC-2-R-446. .

AMINES : General : Porapak R, Chromosorb 103 , Cyano silicones Diamines : Polyethylene glycol on methanolic KOH treated support Secondary and tertiary amines : Apiezon L on KOH treated support Pyridines, quinolines, anilines : Versamid 900 on KOH treated support, UCON HB 2000 on KOH treated support, Glycerol on KOH treated support. AMIDES : Versamid 900 AMMONIA : Chromosorb 103, Porapak P-S, Porapak Q with polyethylene amine

CARBOHYDRATES : TMS derivatives : OV-17, OV-210, OV-225

ESTERS : Mixed : Dinonyl phthalate, Porapak Q, LAC, Carbowax 400, Carbowax 20M, Triglycerides : OV-1 , Dexil 300 GC, OV-101
39

ESSENTIAL OILS : Carbowax 20M, LAC, Quadrol, DEGS. ETHERS : Carbowax 400, Tricresyl phosphate, LAC, Apiezon L, - ' oxydipropionitrile. HALOGEN COMPOUNDS : General Freons Aromatic : : : Dinonyl phthalete, Kel-F grease, silicone oil 550, OV-210, FFAP, Carbowax 1500 UCON polar, dibutyl tetrachlorophthalate, dinonyl phthalate Igepal 800, silicone oil 703, Apiezon L.

HYDROCARBONS : Aliphatic (C1-C5) : Molecular sieve, Silica Gel, Squalane, Diisodecyl phthalate, Porapak N, Poropak Q. Aliphatic (C5-C10) Aliphatic (C10-C18) Aromatics Olefins(C1-C8) Oiefins(C6 and up) Cis and trams alkenes : : : : : : Squalane, SE-30, OV-101 Apiezon L , Silicone Oil Tricresyi phosphate, Carbowax 20M, Apiezon L, Silicone Oil 550 AgNO3 Benzylcyanide, dimethyl sulpholane, - ' oxydipropionitrile Siiicdne oil 550, Silicone oil 703, Carbowax 20M AgNO3 Benzylcyanide.

40

Naphthalenes and biphenyls : Apiezon L, Diisodecyl phthalate, Bentone 34 Polynuclear aromatics : SE-30, OV-I, OV-101, FFAP, Polyphenyl ethers, Apiezon L KETONES : FFAP, Silicone Oil 550, Porapak Q, Chromosorb 101, Diglycerol, UCON 500X, Carbowax 20M. NITRILES : General : FFAP, OV-225, Porapak PS, EGS Paraffix wax on Haloport F Aliphatic nitrites : PESTICIDES : SE-30, Silicone Oil 200, OV-17, OV-101, OV-210, OV-225, QF-1, XE-60. PHENOLS : Dinonyl phthalate Carbowax 20M, OV-17, Apiezon M STEROIDS : OV-1, OV-17, .OV-210, OV-225, XE-60 SULPHUR COMPOUNDS : Tricresyl phosphate, Squalane, Silicone oil 550, Dinonyl phthalate, Carbowax 20M, FFAP, Porapak Q WATER : Any Porapak, Chromosorb 102

41

GASES : He-Ne Ar-O2 H2, O2, He, N2, CO, CH4 Air-CH4 CO2 H2-AIR-CO, CH4-CO2 CO2-H2S-CS2-COS-SO2 N2O-CO2-NO Halogens and interhalogen Compounds MISCELLANEOUS TYPE : Alkyl phenanthrenes : Silicone oil and neopentyl glycol succinate Chloronitrobenzenes : Carbowax 1000 Mono, di and tri carboxylic acids as methyl esters chromosorb Phenyl phenols and chlorophenyl phenols Isothiocyanates XE-60 Apiezon L, Squalane, Dinonyl phthalate. Butanediol succinate on silanised Molecular sieve 5A at dry ice temperature Molecular sieve 13X Silica Gel Porapak Q Porapak Q, Silica Gel Porapak Q, Chromosorb 102 Kel F oil on PTFE support

42

CHOICE OF LIQUID PHASE The liquid phase chosen depends on the composition of the sample. Hopefully, the type components likely to be present in the sample will be known before the analysis is started. The more one knows about a sample (suspected components, boiling range, structures) the easier it is to select the proper column and operating conditions. For an efficient, normal separation, the liquid phase should be similar in chemical structure to the components of the mixture. Example : Hydrocarbon compounds are best separated with a hydrocarbon solvent: Paraffins on squalane (a long chain hydrocarbon); polar compounds with a polar solvent: alcohols on Hallcomid (an amide). If the components of the mixture are of different chemical classes, but close in boiling point, liquid phases of different polarity must be used. By varying the polarity of the solvent, interaction forces may be brought into play to effect a separation. These forces are described in Chapter III and should be reviewed if they are unfamiliar to the student. Table IV-1 shows the various functional groups in the five classifications suggested by Ewell et al.

43

TABLE IV-I-SOULUTE CLASSIFICATION CLASS I (Most Polar) Water Glycol, Glycerol, etc. Amino alcohols Hydroxy acids Polyphenols Dibasic acid Class II (Polar) Alcohols Fatty acids Phenols Primary & Secondary amines Oximes Nitro Compounds with C-H atoms Nitriles with -H atoms NH3, HF, N2H4, HCN

Class III (Intermediate) Ethers Ketones Aldehydes Esters Tertiary amines Nitro compounds with No C-H atoms Nitriles with no -H atoms

Class IV (Low Polarity) CHCl3 CH2Cl2 CH3CHCl2 CH2ClCh2Cl CH2ClCHCl2 etc. Aromatic Hydrocarbons Olefinic hydrocarbons

Class V (Non Polar) Saturated hydrocarbons, CS2, Mercaptans, Sulfides, Halocarbons not in class IV such as CCl4

44

Class I consists of compounds capable of forming networks of hydrogen bonds. Class II is composed of compounds containing both a donor atom (O,N,F) and an active hydrogen atom. Molecules containing donor but no active hydrogen atom are in class III. Class IV is made up of molecules containing an active hydrogen but no donor atoms. Compounds exhibiting no hydrogen bonding capacity are placed in class V. Table IV-2 shows some commonly used liquid phases arranged in the classification given in Table IV-1. Table IV 2 LIQUID PHASE CLASSIFICATION

Class A (I) FFAP 20M-TPA Carbowaxes Ucons Versamid 900 Hallcomid Quadrol Theed Mannitol Diglycerol Castorwax

Class B (II) Tetracyanoethyl pentaerythirtol Zonyl E-7 Ethofat - Oxydipropionitrlle XE-60 XF-1150 Amine 220 Eppon 1001 Cyanoethyl sucrose

Class C (III) All Polyesters Dibutyl tetrachloro-phthalate

Class D (IV & V) SE-30 SF-96

45

SAIB Tricresyl phosphate STAP Benzyl cyanide Lexan Propylene carbonate QF-1 Polyphenylether Dimethylsulfolane OV-17

DOW 11 Squalane Hexadecane Apiezons OV-1

A solute will be retained more strongly by that liquid phase, which is closest according to its classification in Table IV-2, This means higher solubility and usually better separation.

46

TABLE IV 3 EFFECT OF CLASS ON RETENTION Solute-Liquid III C IV D V C or D IV C I II III II D IV B V ID IV A V Solute not retained by liquid phase, frequently very limited solubility. Solute not well retained by liquid phase. A or B Solute well retained by liquid phase. Solute generally retained by liquid phase. Quasi-ideal systems, solutes separate according to boiling points.

47

CHAPTER II

SELECTIVITY OF GC PHASE ACCORDING TO McREYNOLDS CONSTANTS McReynolds Constants have been reported for most of the liquid phases used in gas chromatography and can be used to arrange and compare the separating ability of these phases.

TABLE I Test Probes used in Determining RohrSchneider and McReynolds Constants Probes for Rohr Schneider System (determined at 100 degree C) Benzene Ethanol 2-Butanene Benzene 1-Butanol 2-Pentanone Probes for McReynolda Organic compounds System (determined at 120 degree C) expected to have similar behavior on two GC phases Aromatics, Olefins Alcohols, weak acids and phenols Keto Compounds, as Aldehydes, Ketones, esters Nitromethane Pyridine ---1-Nitropropane Pyridine 2-Methyl -2-Pentanol Nitro and Nitrile compounds Bases, partaromatic Nheterocycles Branchedchain

48

compounds, part alcohols ------------1-Iodobutane 2-Octyne 1,4-Dioxane Cis-Hydrindane Halogen compounds Acetylenes (Olefins) Ethers, bases Nonpolar Steriids, terpenes and nephthenic structures. McReynolds constants are reported as " I units, and a tabulation of selected phase is given in Table II. The larger the McReynolds constants, the greater the 'retention time for that particular compound. McReynolds based his procedure on an earlier suggestion by Rohr Schneider, but used a wider variety of compounds. The latter compounds gave a better fit to a larger number of observed compound class separations. Additional information is given by the McReynolds constants "b" and "r". The constant "b" is the slope of the curve obtained from the plot of retention time of decane and dodecane vs. the corresponding "I" valves, 1000 and 1200. The constant "r" is the ratio of the net retention times of adjacent nAlkanes. The "r" value is calculated from the square root of the ratio of retention times of dodecane to that of decane. A comparison of "b" and "r" values indicates the preferred phase for separation of the members in a homologous series of compounds.

49

APPLICATION McREYNOLDS CONSTANTS: The McReynolds constants are present in the form of tabulations of I values for the 10 test probes in Table I on a large number of stationary phases. The phases are arranged according to increased "polarity", defined as the average of the first 5 values of the McReynolds constants in Table II. Since a wide variety of compound Probes is used, an indication of the selectivity for a wide variety of compound types is available as indicated in Table I.

A.

Relative retention for particular compound types:

If one wants a column that will retain ketones more strongly than alcohols, a stationary phase is required which has a relatively high value for I 2Pentanone compared to I 1-Butanol. A scan of Table II shows that OV-210 meets this requirement. Similarly, high alcohol selectivity is shown by Quadrol, Theed, diglycerol, and Hyprose SP-80 on the basis of high relative values of I 1-Butanol as compared to I 1 2-Pentanone. The high I benzene values for N, N-bis (cyanoethoxy) formamide and1,2,3-tris (eynoethoxy) propane show that these phases will retain aromatic compounds of similar molecular weight. Table I gives a list of organic compounds expected to have similar behavior on GC phases as the McReynolds test compounds. Reasoning similar to that applied to alcohol phases above can be used to indicate preferred phases for these separations.

50

When using packed columns, one would not expect to see an improved separation using phases which show differences in McReynolds constants of less than 20. Differences of about 200 would begin to show significant separating ability. B) Selection of the phase which gives the best separation of a homologous Series: Apiezon L and SE-30 have almost identical McReynolds constants. Yet Apiezon L has higher "b" and "r" values. Therefore, Apiezon L should provide better separations of a homologous series of aliphatic compounds than SE-30. We would expect similar improved separations for any series of homologous compounds using the phase with the higher "r" value.

C)

Choice of a substituted phase:

A valuable use of the McReynolds constants is the choice of substitute stationary phase of similar selectivity. Similar McReynolds constants are shown by SE-30, OV-I, OV-101, DC-200 and any of these phases can be used to replace each other. GC separations reported using Emulphor ON-670 should be duplicated on Trition X-100 or Ucon 50-HB-2000 columns.

2.

LIMITATIONS:

'The McReynolds constants do not answer every question concerning a gas chromatographic separation. They give no information concerning relative efficiencies and peak shape (tailing). Even though two phases have the same McReynolds constants, the most efficient separation (sharpest peak) will be given by the phase with lowest viscosity at the analysis temperature. McReynolds constants are determined at 20 percent loadings to minimize
51

the surface effects of the stationary phase. However, most GC work is done at much lower loadings and the active (nonsilanized) surface of any support will have some effect on the selectivity. Selectively on capillary columns is reported to be somewhat different than indicated by the McReynolds constants. No information is given about the tailing of the more polar compounds, which can be caused by adsorption on the support. The polarity of these phases is different at temperatures higher than 120C, the temperature at which the McReynolds constants are determined. Recent work has shown that the type of support, phase loading, and sample size can influence the McReynolds constant.

3.

CONCLUSIONS

McReynolds constants cannot accurately predict results in GC separations. However, they provide the best information available for the comparison of the selectivity of GC phases. They can be used to suggest other phases which might be used to improve a separation.

52

SELECTED McREYNOLDS CONSTANTS TABLE II

I 2-Methyl 2-Pentanol

Liquid Phase Squalane Apiezon SF-96 SE-30 OV-1 W-982 SE-33 M&B Silicone Oil DC-200 OV-101 DC Silastic 401 Versilube F-50 SE-52 SE-54 OV-3 Fluorolube HG1200 Kelf Wax Halocarbon Wax OV-7 Di(2-ethylhexy) Sebacate Di (2-ethylexyl) Tetrachlorophthalate

I cis - Hydrindane

I 1,4 - Dioxane

I Nitropropane

I 1-Iodobutane

I 2-Pentanone

I 2-Octyne

I Benzene

I Pyridine

I Butanol

b 0.2891 0.2821 0.2525 0.2495 0.2407 0.2471 0.2478 0.2507 0.2509 0.2484 0.2483 0.2579 0.2548 0.2524 0.2547 0.2550 0.2594 0.2575 0.2570 0.2829 0.2874

r 1.945 1.914 1.788 1.776 1.766 1.766 1.769 1.781 1.782 1.771 1.771 1.811 1.798 1.788 1.797 1.799 1.817 1.809 1.807 1.918 1.938

0 21 12 15 16 16 17 14 16 17 17 19 32 33 44 51 55 55 69 72 109

0 22 53 53 55 55 54 57 57 57 58 57 72 72 86 68 67 71 113 168 132

0 15 42 44 44 45 45 46 45 45 47 48 65 66 81 114 114 116 111 108 113

0 32 61 64 65 66 67 67 66 67 66 69 98 99 124 144 143 143 171 180 171

0 42 37 41 42 42 42 43 43 43 46 47 67 67 88 118 116 123 128 125 168

0 13 31 31 32 33 33 33 33 33 34 36 44 46 55 68 73 70 77 135 104

0 35 0 3 4 5 4 2 3 4 4 7 23 24 39 12 16 16 68 68 75

0 11 21 22 23 23 23 22 23 23 23 23 36 36 46 53 57 57 66 49 45

0 31 41 44 45 46 46 46 46 46 48 50 67 68 84 104 109 110 120 107 137

0 33 -6 -2 -1 -1 -1 -4 -3 -2 -1 -1 9 10 17 3 4 4 35 11 34

53

TMP Tripelargonate Diisodecyl Phthalate OV 11 DC 710 Di (2-ethylhexyl) Phthalate Hallcomid M-18 Diisooctyl Phthalate OV 17 Versamid 940 Ucon LB-550-X Span 80 Castorwax OV 22 Trimer Acid Acetyitributyl Citrate Di (2-ethylextyl) Phthalate Didecyl Phthalate OV-24 OS 124 Tributyl Citrate OS 138 NPG Sebacate Squalene Tricresyl Phosphate SAIB GF 1 OV 210 Ethofat 60/25 Igepal CO 630 Ucon 50-HB-2000 Emulphor On-870 Triton X-100 Ucon 50-HB-5100 Siponate DS 10

84 84 102 107 92 79 94 119 109 118 97 108 160 94 135 135 136 178 176 135 182 172 152 176 172 144 146 191 192 202 202 203 214 99

182 173 142 149 186 268 193 158 314 271 266 265 188 271 268 254 255 204 227 286 233 327 341 321 330 233 238 382 381 394 395 399 418 569

122 137 145 153 150 130 154 162 145 158 170 175 191 163 202 213 213 208 224 213 228 225 238 250 251 355 358 244 253 253 251 268 278 320

197 218 219 220 236 222 243 243 212 243 216 229 283 182 314 320 320 305 306 324 313 344 329 374 378 463 468 380 382 392 395 402 421 344

143 155 178 190 167 146 174 202 209 206 268 246 253 378 233 235 235 280 283 262 293 326 344 299 295 305 310 333 344 341 344 362 375 388

143 133 100 107 143 202 149 112 225 177 207 202 133 234 214 200 201 144 177 226 181 257 248 242 264 203 206 277 277 277 282 290 301 466

77 83 103 108 92 82 92 119 112 96 94 105 152 94 112 126 126 169 169 119 176 156 140 169 147 136 139 168 172 173 179 181 185 114

55 59 92 98 66 48 69 105 57 91 66 73 132 57 102 101 101 147 135 102 136 109 101 131 128 53 56 131 136 147 140 145 155 61

127 130 164 174 140 106 147 184 150 177 191 196 228 216 207 202 202 251 266 229 273 257 265 254 276 280 283 279 288 289 289 304 316 437

18 24 59 60 26 16 24 69 78 40 41 49 99 60 26 38 38 113 103 29 112 73 64 76 65 59 60 73 78 80 80 83 86 63

0.2804 0.2812 0.2562 0.2595 0.2789 0.2860 0.2799 0.2551 0.2592 0.2644 0.2719 0.2684 0.2464 0.2684 0.2653 0.2715 0.2714 0.2428 0.2660 0.2666 0.2623 0.2550 0.2630 0.2630 0.2489 0.2094 0.2086 0.2551 0.2552 0.2483 0.2523 0.2521 0.2442 0.2527

1.907 1.910 1.803 1.817 1.9000 1.932 1.905 1.799 1.816 1.838 1.870 1.855 1.763 1.855 1.842 1.868 1.868 1.749 1.845 1.847 1.826 1.799 1.836 1.832 1.774 1.619 1.616 1.799 1.800 1.771 1.787 1.787 1.754 1.789

54

XE 60 OV 225 NPGA Ucon 75-H-90000 Igepal CO 880 Triton X-305 Cyclohexanedi Methanol -Succinate Quadrol NPGS Igepal CO 990 Carbowax 20M Carbowax 20M TPA Epon 1001 Carbowax 6000 Ethylene Glycol Isophthalate FFAP STAP Carbowax 1000 PEG 600 Butanediol Succinate EGA PDEAS Reoplex 400 LACIR 296 DEGAdipate LAC-2-R-446 Ethylene Glycol Pathalate DEGS LAC 3 R 728 THEED EGS Tetracyanoethyl

204 228 232 255 259 262 269 214 272 298 322 321 284 322 326 340 345 347 350 370 372 386 364 377 378 387 453 499 502 463 537 526

381 369 421 452 461 467 446 571 467 508 536 537 489 540 508 580 586 607 631 571 576 555 619 601 603 616 697 751 755 942 787 782

340 338 311 299 311 314 328 357 365 345 368 367 406 369 425 397 400 418 428 448 453 472 449 458 460 471 602 593 597 626 643 677

493 492 461 470 482 488 493 472 593 540 572 573 539 577 607 602 610 626 632 657 655 674 647 663 665 679 816 840 849 801 903 920

367 386 424 406 426 430 481 489 472 475 510 520 601 512 561 627 627 589 605 611 617 654 671 655 658 667 872 860 852 893 889 837

289 282 335 321 334 336 351 431 371 366 387 387 378 390 400 423 428 449 472 457 462 437 482 477 479 489 560 595 599 746 633 621

203 226 208 220 227 229 248 208 243 261 282 281 291 282 299 298 301 306 308 324 325 362 317 328 329 339 419 422 427 427 452 444

120 150 156 180 180 183 176 142 184 205 221 220 207 222 213 228 235 240 240 242 250 242 245 253 254 257 306 323 329 269 348 333

327 342 357 348 362 366 394 379 419 406 434 435 502 437 498 473 484 493 503 533 546 562 540 551 554 567 699 725 726 721 795 766

94 117 103 110 112 113 124 111 124 133 148 148 187 147 168 161 163 161 162 178 177 213 171 177 176 186 260 240 243 254 259 237

0.2237 0.2275 0.2362 0.2369 0.2414 0.2404 0.2351 0.2353 0.2267 0.2303 0.2235 0.2237 0.2261 0.2239 0.2159 0.2204 0.2178 0.2174 0.2180 0.2106 0.2091 0.2100 0.2131 0.2105 0.2105 0.2074 0.1929 0.1900 0.1891 0.1906 0.1807 0.1887

1.674 1.688 1.722 1.725 1.743 1.739 1.718 1.719 1.685 1.699 1.673 1.674 1.683 1.674 1.644 1.661 1.651 1.649 1.652 1.624 1.618 1.622 1.633 1.623 1.623 1.612 1.564 1.548 1.545 1.550 1.516 1.544

55

Pentaerythritol Cyanoethyl Sucrose BCEF 647 690 919 991 797 853 1043 1110 976 1000 713 773 544 557 388 371 917 964 299 279 0.1653 0.1951 1.463 1.593

The absolute values of the retention indices observed for squalane are Benzene 653, 1 Butanol 590, 2-Pentanone 627, 1 Nitropropane 652, Pyridine 699, 2-Methyl-2-Pentanol 690, 1 Iodobutane 818, 2 Octyne 841, 1,4 Dioxane 654, cis Hydrindane 1006.

56

DIATOMACEOUS EARTH SUPPORTS IN GAS CHROMATOGRPHY The most popular gas chromatographic supports are those prepared from .diatomaceous earth, which is also called diatomaceous silica or keselguhr. Diatomaceous earth consists of about 90 percent silica and is composed of the skeletons of fossilized diatoms, which are microscopic algae composed of only one cell the skeletons are extremely small, and are very porous with a relatively high surface area. Two different treatments are used to prepare the diatomaceous earth for use as gas chromatographic supports, which result in two types of supports, white colored and pink colored . The white colored supports are prepared from diatomaceous earth which has been calcined at temperatures above 900C with sodium carbonate as flux. In this heating process, the diatomaceous earth fuses and is held together by sodium silicate glass and the silica is partially converted to crystalline cristobalite. The resultant product is white in color due to the conversion of iron oxide into a colorless complex of sodium iron silicate These white products are used to prepare the most inert supports as Anakrom A, Anakrom Q and Chromosorb W supports. These white flux-calcined supports are fairly fragile and excessive handling in the course of sieving, coating, and column packing will cause the particles to abrade. This abrasion will produce finer particles, or "fines,"and tend to decrease the column efficiency. The pink colored supports, Anakrom C-22 or Chromosorbp prepared from crushed firebrick in which the diatomaceous earth has been calcined with a clay binder at temperatures above 1000C, The metal impurities remaining form complex oxides which contribute to the pink
57

color of these supports. These pink, calcined supports are denser than the White supports because of the greater destruction of the diatom structure during the calcining treatment. These supports are harder and less friable than the white supports and are capable of holding larger amounts of liquid phase (up to 30 percent) without becoming too sticky to flow freely. The surface of the pink supports is generally more adsorptive, and this material is not recommended for use as supports in the gas chromatographic analysis of polar compounds. However, pink supports provide excellent efficiencies in the analysis of hydrocarbons and organic compounds of low polarity. Neither the pink nor the white supports give generally acceptable analyses without further treatment. With the more polar compounds severe peak tailing is often observed, especially with the dense pink supports. This tailing is due to the presence of adsorptive and catalytic centers on all supports. These adsorptive sites are attributed to metal Oxides (Fe, Al) on the surface, which catalyze degradation, and to surface silanol groups, SiOH, which are capable of forming hydrogen bonds with Polar compounds. Mineral impurities are removed by acid washing with hydrochloric acid, which leaches out iron and aluminium on the support surface and provides the acid washed supports : Type Anakrom A (QV) Chromosorb W AW. The surface acid washed supports are less adsorptive. However, even with acid washing the pink supports are still more adsorptive towards polar compounds than the white type supports. Acid washing is sometimes followed by base washing which seems to do little towards removing the metal impurities, but is a good pretreatment for supports. Which are to be used in the analysis of basic compounds.

58

Neither acid nor base washing is effective in reducing peek tailing due to hydrogen bonding with surface silanol groups, -SiOH. - SIOH + Cl2SI(CH3)2 -SiOH Si O Si O Si(CH3) + 2 HCL

Thes< groups are masked by reaction with chlorosilanes to provide a partially silanized surface, thus providing Anakrom AS, Anakrom Q, end Chromosorb W AW DMCS. (The actual reaction is more complicated end is presented here in a simplified form). In earlier work, silanol groups were masked by treatment with hexemethyldisilazane, (CH3)3 SiNHSi (CH3)3, or trimethlychlorosilene, (CH3)3 ,Sicl, which are both less effective in producing inert surfaces than dimethyldichlorosilane. Special acid washed silanized supports, Chromosorb G & Chromosorb 750 are available from Manville -Products Corp. They are the most inert supports available. CAUTION : The silanized supports have hydrophobic rather than hydrophilic surfaces, which are found on the nonsilanized diatomite supports. As a result, polar gas chromatographic phases as polyesters, DEGS, end FGS, and the silicones with a high content of cyano groups, such as OV- 2'/5, do not wet the surface of the silanized supports, but do wet, and spread out better on the surface on the nonsilanized support to provide a more evenly coated surface. Nonsilanized acid washed supports give satisfaction with the most polar phases. At high temperatures, water and
59

frees acids, when injected into silanized supports, will remove the surface silyl ether groups and regenerate an active surface. The silanized supports should not be used as a support in high temperature applications because the surface silyl ether groups will bleed off at a temperature of 325 to 350 . In summary, pink acid washed silanized packing, Anakrom C 22 AS, and Chromosorb P AW DMCS provide the most efficient packings for nonpolar compounds and those of low polarity. In the medium to high polarity range of compound polarity, the white type support, especially the acid Washed, silanized versions are recommended. For the highly polar compounds, especially in the analysis of trace amounts (less than 1 micro-gram per injection), the top-quality Anakrom Q; Chromosorb W HP, Chromosorb G HP, or Chromosorb 750 are recommended. Physical Properties of Diatomaceous Earth Supports Chromosorb Property Color Packed Density Surface Area Pink-Type Pink 0.47g/ml 4.0m2/g 1.9m2/ml Maximum Recommended Loading 30wt-% 25wt-% 5wt-% 7wt-% White-Type White 0.3g/ml 1.02/g 0.24m2/ml G Oyster-White 0.58g/ml 0.5m2/g 0.29m2/ml 750 Off-White 0.3g/ml 0.8m2/ml 0.24m2/ml

60

Comparison Table Of Mesh Size

U.S. Sieve No. Metric Inches Opening Tyler Sieve No. Canadian sieve No. Nominal Aperture Nominal Mesh French Opening No. German Opening No. Japanese Opening

10 12 14 16 18 20 25 30 35 40 45 50 60 70 80

2000 0.0787 10 1680 0.0661 12 1410 0.0555 14 1190 0.0469 16 1000 0.0394 18 841 707 595 500 420 354 297 250 210 177 160 0.0331 20 0.0278 25 0.0234 30 0.0197 35 0.0165 40 0.0139 45 0.0117 50 0.0098 60 0.0083 70 0.0070 80 0.0063 90

10 12 14 16 18 20 25 30 35 40 45 50 60 70 80

2000 8 1680 10 1400 12

2000 34 2000 9.2 12 1250 32 1250 14 1000 31 1000 16 20 800 30 800 24 630 29 630 28 500 400 28 500 27 400 42 315 26 315 48 250 200 25 250 24 200 80 160 23 160 55 65 32 36

2000 1410 1190 1000 840 710 590 500 420 350 297 250 210 177

1600 33 1600 10.5 1680

1200 14 1000 16 850 710 600 500 420 355 300 250 210 180 18 22 25 30 36 44 52 60 72 85

61

100 149 135 120 125 115 140 105 170 88 200 74 230 63 270 53 325 4 400 37 1. 2. 3. 4. 5. 6.

.0059

100 100

150 125 105 90 75 63 53 45

100 120 150 100 21 100 90 80 20 80 170 200 72 240 300 50 18 50 45 40 17 40 350 63 19 63 59 125 22 125

100 120 145 170 200 250 280 325

149 125 105 88 74 62 53 44

0.0053 110 0.0049 120 120 0.0045 130 0.0041 140 140 0.0035 170 170 0.0029 200 200 0.0025 230 230 0.0021 270 270 0.0017 325 325 0.0015 400 400 U.S. sieve series ASTM Specification E-11-61 Canadian Standard sieve series 8-GP-16 British standards institution, London BS-410-62 French standard Specification AFNOR X 11 501 German Standard Specification DIN 4188 Japanese Standard Specifications JIS Z 8801

62

Conservation Chart Of Diatomaceous Earth Supports Based on Treatment Treatment Acidwash Acidwash & Silanized Specially Acidwashed DMCS treated (for pesticide & steroid work) C22F irebrick (Not acid washed) C22F irebrick (acid washed) C22F irebrick (acid washed & silanized) Anakrom C22AS ChromosorbP AW DMCS Gas Chrom RZ Anakrom C22A ChromosorbP AW Gas Chrom RA Anakrom C22U ChromosorbP NAW Gas Chrom R Anakrom Q ChromosorbW HP Supelcoport Gas Chrom Q Analabs Anakrom A Anakrom AS Manville ChromosorbW AW ChromosorbW AW DMCS Supelco Altech/Applied Science Gas Chrom A Gas Chrom Z

63

LISTS OF STATIONARY PHASES SUITABLE FOR THE GC SEPARATION OF SELECTED COMPOUND CLASSES. The following survey lists some stationary phases reported for the GC separation of selected compound classes.

Acids, Free: C1-C9: Chromosorb 101, Porapak OS, Porapak Q, NPGSB + H3PO4. C1-C28 : FFAP, I0% polyster ( DEGS , EGA, e t c . ) + 2-3%, H3PO4, l2% Stabilised DEGS. Alcohols C1-C5: Porapak P (or PS), Porepak Q (or OS), Porapek S (for normal and Branched alcohols), Chromosorb lot, Chromosorb l02, THEED, Quadrol, Hyprose SP-80, diglycerol, Hallcomid M-18, Carbowax 400/Porasil C, Carbowax 1500. C1-C18: Carbowax 20M, FFAP, Super Pak 20M, Igepal CO-880. Aldehydes Carbowax 1540, Carbowax 20M, Igepal CO-880, SE-30, Porasil C, Porapak PS, Super Pak 20M. Alkaloids SE-30, OV-I, OV-17, OV-210, lO% Apiezon L-2% KOH.

64

Amino Acid Derivatives N-T FA butyl esters, EGA ( stabilised ) , OV-17, Dexsil 400 ( capillary column ) , OV-I, OV-11, Apiezon M, OV-210, EGA, XE-60.

Amines Chromosorb 103, 4% Carbowax 20M + O.8% KOH, lO% Apiezon L + lO% KOH, 3% Carbowax 20M on Chromosorb 103, Triton X-305 + Na3PO4. Amides Versamid 900, Apiezon L.

Carbohydrates (as methyl ether TMS-ethers or TMS derivatives) OV-101, SE-30, SE-52, XE-60, OV-225, Apiezon L, EGS, OV-17, OV-210.

Drugs OV-I, SE-30, OV-17, OV-210, OV-225, Dexsil 300 GC, PDEAS, OV-101 Apiezon L, EGS, OV-17 + H3PO4 (barbiturates and anticonvulsants). Esters General: FFAP, OV-101, Porapak R, Carbowax 20M, SE-30. Fatty acid methyl esters: DEGS (stabilised), EGA (stabilised), Aplezon L, OV-275, Silar I0C. C20-C30 Fatty acid methyl esters: Dexsil 300 GC. Bile acids, methyl esters: OV-I, AN-600, Dexsil 300 GC, SE-30, NPGS, OV-210. Triglycerides steryl ethers & steryl esters: Dexsil 300 GC, SE-30 (GC Grade), SE-52.

65

Ethers Carbowax 20M, Carbowax 1500, SE-30 (GC Grade). Glycols Porapak P, Chromosorb 101, Chromosorb 102. Halogenated Compounds Carbowax 20M, OV-210, SE-30. Hydrocarbons Aliphatic, low boiling: Squalane, Chromosorb 102, Chromosorb104, Porasil B, n-Octane/Porasil C, Phenyl isocyanate/Porasil C. Silica Gel, n-Octane/Porasil C, Porapak Q, Ucon LB - 550X. Aliphatic, high boiling: Apiezon L, Dexsil 300 GC, OV-101, or SE-30. Olefins: Propylene carbonate, benzyl cyanide-AgNO3, N, Nbis(Cyanoethyl) formamide, dimethyl sulfolane, phenyl isocyanate/Porasil C, Porasil B, Ucon LB-550X. Aromatic low boiling: N, N-bis(2-cyanoethyl) formamide, tricresyl phosphate, = ' oxydipropionitrile, DC-200, Bentone 34, DIDP-Bentone 34, Porasil C, Carbowax 400/Poras it F , Super Pak 20M. Aromatic, high boiling: Apiezon L, polyphenyl ethers, 'FFAP, Dexsil 300 GC, Reoplex 400, Super Pak 20M.

66

Ketones FFAP, DC-550, Porapak Q, Chromosorb 101, Chromosorb 102, OV-210, Carbowax 20M, Triton X-305, 1, 2, 3-tris(cyanoethoxy) propane, Super Pak 20M. Nitrites FFAP, OV-225, AN-600, tris(cyanoethoxy) propane. Pesticides DC-200, OV-101, OV-I, SE-30, OV-17, OV-210, OV-225, and mixed phases of the preceding, Versamid 900, Dexsil 300 GC, SE-30, Carbowax 20M, Reoplex 400. Phenols OV-17, Carbowax 20M, Polyesters + H3PO4, XE-60, OV-I, OV-25, SE-30, OV-210, AN -600, O V-7 Steroids OV-1, OV-101, SE-30, OV-225, AN-600, Dexsil 300 GC, OV-210, OV-17. Sugars (as TMS derivatives) Dexsil 300, OV-17, OV-210, OV-225, AN-600, SE-30. Sulfur Compounds Porapak Q, Chromosorb 104, FFAP. Terpenoids Apiezon L, SE-30, Reoplex 400, Carbowax 20M, EGA, DOP, TCP, EGA, polyphenyl ether, AN-600, BDS, OV-210, OV-225, DEGS.

67

Gases and misc low mol wt compounds H2, O2, CO, CH4: Molecular Sieves 5A or13X, Spherocarb CO2, H2S, CS2, COS: Porepak Q. Chromosorb 101, Chromosorb 102, Spherocerb. Air, CO2, CH4, light eliphatics: Silica Gel, Porepek N, Porapek Q, Spherocerb. Oxides of nitrogen - Porapak Q, Chromosorb 104, Oxides of sulfur - Chromosorb 104. CH2O, H2O, CH3OH - Porapak T, Chromosorb 105. Air, CO2, NH3, H2O - Porapak N. HF, Cl2, CIF3, F2 - Kel F Oil No.3 or No.10, Kel F wax all on Anaport Tee Six.

68

Porous Polymers In Gas Chromatography Porous polymers prepared by the Johns-Manville Corporation (Chromosorb Century Series Chromosorb 101 to 108), and Waters Associates (Poropak Series) are especially designed for use in gas chromatography and provide unique separations. The use of these materials has been summarized in several review articles. 1,2,3,4,5,6 The use of porous polymers in gas analysis has been reviewed by Thompson 7. Porous polymers can be used directly in gas chromatographic columns without further -coating with a liquid phase. Separations can be achieved for a wide variety of compounds, including alcohols, water and free acids, as well as gases end all organic solvents. However, separations of these compounds are usually achieved at temperatures at least 100C higher than those required using conventional liquid phases on diatomaceous earth supports. The range of compounds is usually limited to those with boiling points of less than about 250C, or less than 10-12 carbons for hydrocarbons, or molecular weights less than about 200. Higher molecular weight materials will adsorb on porous polymers packings and probably change the retention characteristics. Both the Chromosorb 100s and the Porapaks are similar in that they are cross-linked polymers. They are produced by co-polymerizing styrene and divinyl benzene. They differ in pore size and surface area by varying the amount of cross-linking agent, divinyl benzene. For example, the composition of Chromosorb 101 and Chromosorb.102 differ only by the amount of copolymerized divinyl benzene, yet they differ in pore size (ca.
69

0.35 microns vs. 0.008 5 microns, respectively), and surface area (less than 50 m2/g vs. 300-400 m2/g, respectively). Functional groups can be chemicaUyincorporated into porous polymers by addition of vinyl monomers such as acrylonitirle, vinyl pyridine, vinyl pyrollidone and ethylene glycol dimethacrylate to the polymerization mixture. In this way, a series of polar porous polymers are available which differ not only in pore size and surface area, but in chemcially bound polar groups. All these factors combine to provide gas chromatographic phases with unique selectivities by functional groups, as well as size or shape of the molecules being separated, see Tables I and II. Recent experience in the use of porous polymers in gas chromatographic analysis is sum marized below. 1. Porous polymers are recommended for analyzing aqueous solutions containing organic compounds capable of hydrogen bonding, as amines, alcohols, glycols and free acids. In contrast to the conventional diatomite supports, no hydroxyl groups are present on the surface to contribute to strong adsorption (peak taking) of these hydrogen, bonding compounds. (In these applications, adsorption effects can be minimized by using glass injection port liners or by oncolumn injection to glass columns.) 2. Analyses below the 100-ppm level are usually difficult to achieve with porous polymers because of peak broadening. However, the peaks can be made sharper and retention times decreased, by coating the porous polymers with GC phases. In this way, free acids can be detected at levels of 1-10 ppm in water by modifying Poropak T with % FFAP. (8)
70

3.

In general, coating porous polymer supports can improve the peak shape. Coating Chromosorb 101 with carbowax 20M improved the analysis of aliphatic amides obtained from tobacco smoke, (9) and provided more symmetrical and sharper peaks for acetic acid (10) Dihydric phenols can be conveniently distinguished frommonohydric phenols using 3% Carbowax 20M on Poropak Pll in another study, (12) 2% Carbowax 20M on Poropak Q provided the most efficient loading and shortest retention time.

4.

Chromosorb 103 is unique for the analysis of alkyl and aryl amines, although glycols are adsorbed. Amine analysis at low levels can be improved by coating with 3% Carbowax 20M, (13) Chromosorb 103 can be used for, the analysis of diethylenetriamine and triethylenetetramine, (14) Improved separation of ammonia and water can be achieved by coating Chromosorb 102 with 10% Theed, (15) and improved amine analysis by coating Poropak Q with polyethyleneamine, or triethylenepentamine, (16,17,18).

5.

Free acids, 2-7 carbons long, can be determined at very low levels by introducing formic acid vapors in the carrier gas, (19,20) Improved peak shapes for formic and acetic acids have been reported when Porapak Q is coated with 3% phosphoric acid (21)

6.

Porapak P and Porapak Q are available as the silanized products, Poropak PS and Poropak as. This treatment decreases peak tailing of glycols, and free acids, and make these packings more suitable for trace analysis.
71

7.

Porous polymers can be modified by adding functional groups to the aromatic rings in the polymer chain by organic synthesis (22,23). As a result, different selectivities are obtained.

8.

Pretreatment of porous polymers can sometimes improve the performance. Un reacted vinyl groups on the Chromosorb 102 surface can be removed by addition of HF to the double bond, (10) This pretreatment decreases the tailing observed for amine groups. If Porapak QS is first washed with acetone, improved analysis is observed for sulfur containing gases (24) In general washing porous polymer with acetone or other organic solvents will improve peak shape.

9.

The surface areas of porous polymers range from about 25 to 700 m2/g and they thus have a higher surface area and sample load capacity than the usual diatomite supports. Since large sample loads can be tolerated, these materials are excellent packings for preparative gas chromatography and for use in trace analysis.

10.

Porous polymers can be used to trap volatile organic components from air at room temperature, or at subambient temperatures (25) they have the advantage of not trapping water vapor under these conditions. The adsorbed material can be removed by heating the porous polymer, collecting the evolved organic materials on a gas chromatographic column at room temperature and separating the components in a temperature programmed run. If extended use of the porous polymer
72

columns is intended particularly extended operation at high temperature, trace oxygen should be removed from the carrier gas by use of Oxisorb. Any oxygen in the carrier gas can oxidize the surface of Chromosorb 102 or Poropak Q, they form carbonyl surface groups which changes the retention of certain compounds.26 General applications for the porous polymers are given in Tables I and II. Other retention data has been summarized by Dave.2 The polarity of the porous polymers is compared by the retention indices of the mixture benzene, t-butanol, 2-butanone and acetonitrile, as summarized in Table III and IV. Poropak T and Chromosorb 104 are the most polar in their respective series of porous polymers. In some cases, the porous polymer in each series might appear to be identical on the basis of retention data in Tables I and II. However, one should expect slight differences in selectivity, retention times and of efficiency which may not be indicated by the limited retention date presented here.

73

Packing -and Conditioning Porous Polymer Columns

Columns may be packed by normal techniques since the porous polymer particles are hard, and are not easily crushed. Add the particles gradually to fill the column m 2-3-inch increments. . Consolidate the pecking before the next edition by either tapping or vibrating the column (electric vibrator, low magnitude of vibration), while applying vacuum to one end. Conditioning is necessary to remove polymerization solvents, as well as. any compounds adsorbed during handling or storage. Before use, condition the column by heating at least two hours at about 25-50C below the maximum recommended operating temperature with carrier gas flowing through the column. When operating at highest sensitivity settings of the detector, the column should be conditioned overnight or longer until a satisfactory baseline is obtained. During this time, the column should not be attached to the detector. Porous polymers tend to shrink upon heating. Therefore, maximum efficiency can be attained by packing the column with fresh polymer at the end of the conditioning stage. This is best accomplished by removing the column, attaching one end to a vacuum one, vibrating the column, and adding fresh packing to fill the newly created void space. The column is then reconditioned at elevated temperatures for an additional one half to one hour before use. Most porous polymers discolor upon heating, however, this does not effect the separations.

74

TABLE I Application, Polymer Type and Reported Indices for Chromosorb Century Series 101 - 108 Max. Temp (a,b) Polymer Type Benzene Fast efficient separation of free alkenes, esters, ketones, aldehydes, hydrocarbons, and ethers. No tailing of water, alcohols and most other oxygenated compounds, useful for light and permanent gases and low molecular weight compounds. High surface area. Fast and efficient separation of alcohols, aldehydes, hydrazines, amides and ketones, Glycols are adsorbed Separation of nitriles, nitroparaffins, aqueous H2s, xylenols, NH3 and oxides of nitrogen, sulfur and carbon. Trace water in benzene, Different selectivities, than 101, 102, and 103. Separation of aqueous solutions containing

Chromosorb

Application

Retention Indices (C) TButanol 2-Butanone Acetonitrile

101

273 C

Stryene Divinylbenzene

745

565

645

580

102

250 C

Stryeme divinylbenzene

650

525

570

460

103

275 C

Cross-linked polystrene

720

575

640

565

104

250o C

Acrylonitrile divinylbenzene

845

735

860

885

105

250 C

Polyaromatic

635

545

580

480

75

106

107

108

CH2O, separation of acetylene from lower hydrocarbons, separation of most gases and organic compounds in boiling range up to 200 C. Less polar then 104, but with different selectivity, i.e. acid before butanol (the reverse order of elution is obs erved on 104) Retains, benezene and non-polar organic compounds, separation of C2 to C5 fatty acids from corresponding alcohols. Intermediate polarity, providing efficient separation of various classes of compounds in general and of CH2O in particular. Separation of gases and polar materials, such as water, alcohols, aldehydes, ketones, glycols, etc. retention characteristic differ from other Chromosorbs.

250 C

Cross-linked polystyrene

605

505

540

405

250oC

Cross-linked acrylic ester

660

620

650

550

250 C

Cross-linked acrylic ester.

710

645

675

605

(a) (b) (c)

Maximum temperature in programmed temperature operation might be about 25 C higher for short duration. Most porous polymers discolor upon heating. This does not effect column performance. Data reported by S.Dave, J. Chromatogr, Sci., 7,389 (1969)

76

TABLE II Application, Polymer Type and Reported Indices for Poropaks Poropak Application Max. Temp Polar Type Benzene Least polar, separates a wide variety of carbonyl compounds, glycols and alcohols. Surface silanized verson of p which minimizes tailing. Separation of aldehydes and glycols. Most widely used. Particularly effective for hydrocarbons. Organic compounds in water, and oxides of nitrogen. Surface silanized verson of Q which eliminates tailing separates organic acids and other polar compounds with minimum tailing. Moderate polarity, long retention and good resolution observed for ethers. Seperation of esters, seperation of H2O form Cl2 and HCl. Seperation of normal branched Retention Indices (C) T-Butanol 2Butanone Acetonitrile

250 C

**

765

560

650

590

P-S

250 C

Na

Na

Na

Na

250 C

***

630

538

580

450

Q-S

250 C

***

625

525

565

445

250 C

Vinyl pyrolidone

645

545

580

455

250 C

Vinyl pyrolidone

735

605

705

595

77

chain alcohols. Seperation of CO2, NH3, H2O and of acetylene from other C2 hydrocarbons. High water retention. Highest polarity and greatest water retention. Determination of formaldehyde in aqueous solutions.

190C

Vinyl Pyrolidone

735

605

705

595

190 C

Cross-linked acrylic ester.

710

645

675

605

I. II. III.

Data reported by S. Dave, J. Chromatographic Sci. 7389, (1969). Prepared by polymerizing styrene divinylbenzene mixtures. Prepared by polymerizing ethylvinylbenzene and divinylbenzene.

78

REFERENCES 1. 2. 3. 4. S. B, Dave, I & EC Prod. Res. Dev., 14,85 (1975). S. B. Dave, J. Chromotogr. Sci., 7,389 (1969) O.L. Hollis, J. Chromotogr. Sci, 11,335 (1973) Brochure, "Chromosorb Century Series, Porous Polymers Supports", available from Analba. 5. Brochure, "Poropok GC Column Pocking lleteriels", available from Analabs. 6. "poropok, o Selected Bibliography of Applications", available from Anolobs. 7. B. Thompso,i, "Fundamentals of Gas Analysis by Gas Chromatography", Varian Associates, 1977. 8. 9. Reported by Varian Applications Laboratory. W. Johnson, R.W. Hale, and J.W. Nedlock, Tobecco, 175110), 32 (1973). 10. W. Hertl and M.G. Neumen, J. Chromatogr. 60,319 (1971).

79

11. 12.

V Kusy J . Chromotogr. 57,132 (1971). B.O.Jansson, K.C. Holgren end G. Widmorck, J. Chrometogr. Sci. 8,398 (1970).

13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

Analabs Application Sheet. A.A. Cesselmon and R.A.B. Bennerd J. Chrometogr. 88,33 (1974). E. Moretti, G. Leofenti, D. Andreozze N. Giordeno, J. Chrometogr. Sci., O.L. Hollis, and W.V. Heyes, J. Ges Chrometogr., 4,235 (1966). J. R. Lindsey Smith end D.J. Woddington, Anel. Chem. 40,552 (1968). R.G. Ackmen, J . Chrometog r. Science, 10,560 11972). Anelebs Application Sheet. W.R. White end J.A. Leenheer, J. Chrometogr. Sci, 13,387 (1975). J.S. Fritz end R.C. Cheng. Anel. Chem. 46,938 (1974). J.R. Lindsey Smith, et, al., J. Chrometogr., 148,353 (1978); ibid., 151,21 (1978);ibid,lsl,2711978).

23.

T.L.C, desouza D. Lane and S. P. Bhetie, Anal, chem. 47543 (1975).

80

24.

Literature Survey, "Trapping volatile Organic Compounds from Air Using Poroua Polymers", available from Anolobs.

24.

M.G. Neumann end S. Morales, J. Chromatogr., 74,332 (1972).

81

Relative retention on Porapak at 100C using 6' X 3/16" I.D. column. Sample P Q R S T

Air Methane Carbon dioxide Ethylene Acetylene Ethane (Minutes) Water Propylene Propane Methyl chloride Vinyl chloride Methyl alcohol Ethyleneoxide Ethyl chlor1de Acetone Ethyl alcohol Pentane Cyclopentane Butadiene Isopropyl alcohol Acetonitr11e Acrylonitrile Diethyl Ether Methylene chlor1de n-propyl alcohol t-butyl alcohol

0.57 0.72

0.33 0.67

0.24 0.35 0.59

0.31 0.40 0.60 0.85 0.92 1.00 (1.3) 2.46 2.77 2.77 3.46 5.46 6.00 6 .00 10.38 27.69 15.00 27.&9 37.69 8.46 30.77 15.38 24.62 26.92 22.31 50.00 56.92

0.42 0.45 0.85 0.95 1.40 1.00 (1.0) 7.1 2.6 2.6 4.2 6.8 10.0 9 .5 13.0 41.0 26.0 22.0 28.0 9.1 54.0 41.0 58.0 28.0 34.0 85.0 98.0

1.00

1.00 1.00

'

0.79 1.00 1.00 (0.95) 3.68 2.42

1.00 (0.54) 1.85

1.00 (0.88) 1.36

1.85 2.22 2.96 2.59

3.30 3.64 6.36 3.30

2.74 2.95 5.02 5.89 4 .4 2

5.00 9.81 5.19 12.41 18.52 4.44 9.63 9.07 12.96 12.22 10.19 14.26 14.26

1!.02 19.32 9.32

8.95 15.79 14.74 21.05 26.32

8.98 20.45 13.63 23.86 34.09 21.59 32.95 44.32

7.89 28.42 13.68 22.11 23.16 20.00 49.47 57.89

82

Relat1ve retent1on on Porapak at 175C us1ng 1 meter X 2.3 mm I.D. co1umn

Sample

P.S

Q.S

Water Methanol Formaldehyde Acetaldehyde Ethanol Formic ac1d Acetonitri1e Propylene oxide Propionaldehyde Acetone Isopropanol 0.351 Methylene chloride Acrylonitri1e Acetic ac1d Methyl acetate Propanol Pentane Isob utyraldehyde Butyraldehyde 2-Butanone Chloroform Ethyl acetate Iso-butanol Propionic acid Hexane (Minutes) Butanol Benzene Carbon tetrachloride Isopropyl acetate Propyl acetate

0.408 0.475 0.475 0.475 0.592 0.717 0.792 0.666 0.750 0.758

0.467 0.542 0.517 0.542 0.666 0.717 0.934 0.784 0.808 0.850

0.056 0.127 0.'34 0.169 0.218 0.225 0.287 0.314 0.338 0.343

0.082 0.'34 0.127 0.170 0.230 0.189 0.286 0.327 0.343 0.349

0.131 0.180 0.190 0.190 0.307 0.368 0.358 0.336 0.376 0.390

0.109 0.168 0.172 0.187 0.291 0.386 0.348 0.329 0.383 0.391

0.135 1.193 0.195 0.222 0.367 0.819 0.497 0.406 0.476 0.544

0.188 0.244 0.172 0.259 0.462 0.187 0.670 0.444 0.543 0.666

0.960 0.892 0.926 0.800 0.883 0.666 0.934 1.10 1.18 1.28 1.13 1.26 1.53 1.00 (0.944) 1.48 1.69 1.53 1.47 1.85

0.950 1.00 1.03 0.883 0.984 0.684 1.05 1.22 1.26 1.35 1.22 1.36 1.67 1.00 (0.945) 1.58 1.86 1.53 1.46 1.83

0.373 0.388 0.419 0.419 0.479 0.501 0.598 0.711 0.734 0.753 0.812 0.902 0.909 1.00 (4.93) 1.07 1.16 1.16 1.33 1.72

0.403 0.404 0.379 0.434 0.478 0.536 0.623 0.710 0.730 0.718 0.852 0.900 0.843 1.00 (5.50) 1.07 1.16 1.14 1.43 1.83

0.407 0.474 1.31 0.445 0.660 0.481 0.670 0.776 0.820 0.854 0.864 1.24

0.438 0.475 1.91 0.438 0.641 0.469 0.676 0.802 0.846 0.791 0.862 1.21

0.510 0.660 1.34 0.598 0.862 0.490 0.905 1.09 1.20 0.966 1.20 1.76

0.545 0.853 1.90 0.735 1.06 0.467 1.04 1.28 1.41 1.24 1.44 2.07 4.27

1.00 (4.67) 1.47 1.24 1.07 3.48 4.20

1.00 (4.97) 1.46 1.25 1.07 1.44 1.83

1.00 (6.94) 2.08 1.42 1.17 2.04 2.64

1.00 (4.84) 2.50 1.67 1.34 2.38 3.19

83

Iso pentano1 Heptane Pentanol Toluene Temp. limit for porpak

2.29 1.64 2.61 2.92 250C

2.27 1.58 2.63 3.18 250C

2.10 2.28 2.46 2.71 250C

2.09 2.28 2 .46 2.69 250C

2.91 2.18 3.38 2.43 250C

2.85 2.05 3.35 2.85 250C

4.25 2.18 4.93 3.24 190C

5.10 2.20 5.86 3.65 190C

84

PORAPAK SERIES
Relative retention on Porapak at 30C using 1 meter X 2.3 mm I.D. Column Sample P P-S Q Q-S R S N T

Oxygen Nitrogen Argon Hydrogen Carbon monoxide Nitric oxide Methane Nitrogen dioxide Carbon dioxide Nitrous oxide Acetylene Ethylene Ammonia Ethane (minutes) Water Carbonyl Sulphide Difluoroethane Chloro-difluoromethaneSulphur dioxide Methyl chloride Propylene Propyne Dich1oro, difluoroethane Ethylene oxide Chloro-difluoroethane D;ch1oro-tetrafluoro ethane

0.426 " " 0.426 0.500 0.486 0.647 0.662 0.780 0.986 0.838 1.22 1.00 (0.535) 2.44 2.13 2.15 2.27 4.66 3.90 2.59 2.59 2.65

0.484 " " 0.484 0.500 0.500 0.532 0.694 0.806 1.02 0.887 1.36 1.00 (0.488) 2.85 2.21 2.47 2.50 4.52 4.57 2.06 2.06 3.10

0.153 " " 0.153 0.187 0.231 0.374 0.409 0.478 0.720 0.720 0.950 1.00 (1.60) 1.33 2.08 2.44 2.70 3.67 3.85 4.02 4.52 4.64

0.176 " " 0.050 0.176 0.213 0.273 0.432 0.420 0.534 0.705 0.739 0.636 1.00 (1.39) 1.17 2.31 2.77 2.40 3.65 3.99 3.84 4.51 5.03

0.204 " " 0.071 0.231 0.238 0.279 0.361 0.517 0.65 1.00 0.776 1.62 1.00 (1.16) 6.75 2.44 3.42 4.83 9.35 4.94 3.82 4.31 4.93

0.180 " " 0.041 0.180 0.192 0.279 0.192 0.483 0.593 0.884 0.744 1.30 1.00 (1.36) 5.44 2.55 3.40 4.12 17.5 5.14 3.96 4.39 5.24

0.162 " "

0.154 " " 0.053

0.62 0.189 0.236 0.189 0.602 0.623 1.33 0.812 1.26 1.00 (1.50) 9.14 2.70 5.99 7.31 11.0 6.51 4.76 4.76 6.52

0.154 0.180 0.256 0.190 0.820 0.790 1.92 0.857 1.76 1.00 (1.54) 15.6 3.06 7.75 9.00 14.6 7.74 4.72 4.72 6.67

8.24 3.78

9.67 5.12

7.94 9.65

8.46 8.08

10.8 10.1

10.9 10.1

16.5 -

22.1 -

19.5

16.1

16.8

16.5

17.1

85

WHERE TO USE DERIVATISATION

Many materials are not suitable for direct analysis on a gas chromatographic column due to their high polarity, low volatility or thermal instability. Derivatives of highly polar compounds are prepared in order to reduce the polarity and to increase the vapour pressure. Compounds having zwitterions or strong hydrogen bonded structures cannot be analysed without preparing their derivatives. Among the alternate derivatives that can be prepared, the choice will ultimately depend on the optimum separation achievable of the components in a given mixture. Alcohols , phenols, glycols , polyhydroxy compounds , steroids , carbohyd rates, amines, amino alcohols and amino acids are generally converted to their trimethyl silyl derivatives using a mixture of trimethyl chlorosilane (TMCS) and hexa methyl disilazane (HMDS) in a suitable solvent such as pyridine, dimethyl formamide, dimethyl sulphoxide and tetrahydrofuran. The time for complete conversion is usually less than 10 minutes. To avoid any possible reaction between the TMS derivatives and the stationary phase of the column, it is a common practice to inject repeatedly several aliquots, of TMCS and HMDS on to the column containing silanised support and thus passifying any active centres in the column, before injecting the derivatised sample. The following table gives a list of volatile derivatives for various classes of compounds which are in general use :

86

VOLATILE DERIVATIVES FOR G.C. ANALYSIS Class of compound Carboxylic acid CH2N2 ClCH2CH2OH : BF3 Alcohols and phenols HMDS + TMCS Trifluoroacetyl imidazole (CF3CF2CO)20 Acetic anhydride Carbohydrates Amines and amino hydroxy compounds HMDS + TMCS Acetic anhydride CF3COCH3 (CF3CO)20 HMDS Amino acids Trimethylsilylderiva tives Acetyl derivatives. Schiff's base Trifluoroacetyl derivatives Trimethylsilyl derivative (CF3CO)2O followed by Trifluoroacetyl CH2N2 TMCS on Na aminomethyl esters salt Amino alcohols HMDS+TMCS followed by (CF3CO)2O followed by CH3COCH3 Trimethylsilyl derivative Trifluoroacetyl amino trimethyl the imine silyl ether HMDS Trimethyl silyl ether of Reagent CH3OH : HCl Or BF3 Derivative Methyl ester Methyl ester - chloroethyl ester. Trimethyl silyl ether Trifluoroacetate Pentaf1uoropropionate Acetate

87

Aldehydes Ketones Lipids and glycerides Steroids

CH3OH-HCl/Ag2O N-amino Plperidine by esterification Acetic

Acetals/acid Schiffs base

Saponification followed Fatty acid esters anhydride Acetylderivative Trimethylsillyl deravitive.

(CF3CO)2O + HMDS

88

GHOSTING EFFECT IN GAS CHROMATOGRAPHY

ABSTRACT Ghosting effect has a big effect upon quantitativeness, resolution, and baseline stability in high sensitivity and/or programmed temperature gas chromatography (PTGC). The causes of ghosting effect are: 1) Non-volatile materials in injected samples remain in the injection ports and are carbonized into absorptive compounds. Newly injected sample is adsorbed by the adsorptive compounds and is eluted continuously. . 2) Some of the injected sample vapor is adsorbed on the surface of the rubber septum and is carried into the column continuously. 3) Some organic vapors are originated from the heated rubber septum and carried into the column continuously. 4) Impurities in carrier gas and contamination of gas piping.

The construction and handling of injection ports and method of sample injection, recommended for prevention of ghosting effect, are also discussed.

89

Introduction With the sensitivity enhancement of thermal conductivity detectors and flame ionization detectors, and the development of various other highly sensitive detectors, the technique of gas chromatographic analysis of tracequantity-level components has become very popular in research and routine. Also, various liquid phases have been developed that can be used at very high temperatures, such as OV-1, OV-17 and Dexsil 300GC. In high-sensitivity, high-temperature GC, a phenomenon called "ghosting effect or "repeating effect" has become an important problem. There exists the phenomenon that peaks of the components which do not exist in the sample are recorded in chromatograms. This naturally induces errors in quantitative and qualitative works. Some times it causes great baseline drift and makes high-sensitivity detection utterly impossible. The causes of "Ghosting effect" are many and varied. In some cases, the cause is unsuitable design of the instrument itself, but in many cases this can be prevented by suitable operation. This article describes our experiments to prevent the ill effects.

90

1.

How to Identify Ghost Peaks. The term "ghosting effect" means the phenomenon in general that

false peaks which do not correspond to the sample components appear. The phenomenon that the false peaks correspond to the components of the sample analyzed by the same apparatus previously is particularly called "repeating effect". In this report, we call all of these phenomena "ghosting effects" end the false peaks "ghost peaks". Fig, 1 shows a representative chromatogram of a ghost peak in isothermal GC. After ethanol containing butanol was analyzed, pure ethanol was analyzed in the next run. In Fig, 1(b), a butanol peak is traced through the sample does not contain butanol. Fig, 1(a) shows the normal chromatograms obtained under the same operational conditions. In isothermal GC, ghost peaks appear only when a new sample is introduced. Fig. 2 shows a representative chromatogram of ghost peaks in programmed temperature GC (PTGC). After a fatty acid methyl ester sample was analyzed, baseline was recorded at the same operational conditions, without introducing a sample. The ghost peaks appear at the positions corresponding to the components of the sample analyzed in the previous run. Thus, drawing a baseline in a blank test can recognize ghosting effect. If, another sample is analyzed in the next run; it is impossible to discriminate false peaks from the real ones. Fig. 3 is the chromatogram of amino acid TFA-Busters analyzed just after the analysis of fatty acid methyl esters. (The operational conditions are the same.) The ghost peaks of the methyl esters are recorded in the

91

chromatogram of amino acid esters (Fig. 3-(b)). The ghost peaks can be recognized only by comparison with a normal chromatogram (Fig. 3-(c)).

Fig.1 Ghosting Effect in Isothermal operation Comparison of clean and contaminated sample injection parts

Fig.2 Ghost peaks in Temperature operation.

programmed

Fig.2 Ghosting Effect in programmed Temperature

be recognized by drawing a baseline in a blank test. In, another sample is analyzed in the next run; it is impossible to discriminate false peaks from the real ones. Fig. 3 is the chromatogram of amino acid TFA-Busters analyzed just after the analysis of fatty acid methyl esters. (The operational conditions are the same.) The ghost peaks of the methyl esters are recorded in the chromatogram of amino acid esters (Fig. 3-(b)). The ghost peaks can be recognized only by comparison with a normal chromatogram (Fig. 3-(c)).

92

As it is clear from Figs, 1 and 3, ghost peaks may be overlooked if the operator is not careful enough. He may confuse ghost peaks with the real ones in data handling for identification or quantitation. The ghosting effects so far described are due to the components of the sample analyzed by the same apparatus in previous analyses (repeating effect). In PTGC, there are ghosting effects caused by other factors, Fig. 4 shows a ghosting effect accompanied by a great baseline drift, which is not due to the sample analyzed previously.

Characteristics of Ghosting Effect. To investigate the causes of the ghosting effect, it is necessary to study its characteristics by changing various operational conditions. Some of our experiments carried out for this purpose are described in this chapter. Methyl stearate was analyzed with an apparatus which 'gave a ghost peak in PTGC and then PTGC baseline was drawn repeatedly to study the relation of the height of the ghost peak and the time (Fig. 5). Fig. 5 shows that the height of the ghost peak decreases with time after the first sample introduction. The ghost peak in Fig, 1 has the similar characteristics. Next, the relation of the height of the ghost peak and the sample injection port temperature was studied. As shown in Fig. 6, the ghost peak is greater when the injection port temperature is higher. In this experiment, methyl stearate was analyzed at 250C injection port temperature, and then the

93

injection port temperature was rapidly cooled to the specified temperature, and the PTGC baseline was drawn.

Fig. 4 Ghosting Effect l d i j d Fig. 5 Relation of lapse of time and height of ghost peaks dieseline was

Fig 6 Effect of injection port Temperature on Height at Ghost Peak After analysis of methyl stearate at 250C injection port temperature, the injection port was rapidly cooled to the specified temperature and the PTGC baseline was d

Fig 7

Effect of holding time at

initial Temperature on height of ghost peak Methyl stearate was

It is noteworthy that the relation curve of the ghost peak height and the injection port temperature is very similar to the vapor pressure curve of methyl separate. The relation of the length of the holding time at the initial temperature to the height of ghost peak was studied with the injection port

94

temperature kept constant. The result in Fig. 7 shows that the ghost peak increases in height with the duration of the holding time at the Initial temperature non-linearly, however.

Fig 8. Effect of injection port Temperature on Ghost Peaks unrelated to injected sample

The ghost peaks, shown in Fig. 4, which occur in no relation to the samples analyzed before, are also influenced by the injection port temperature. They decrease when the injection port temperature is low , as shown in Fig. 8. The two types of ghosting effects are similar to the extent that they decrease with time and increase with the length of holding time at initial temperature. From these experiments, it is presumed that the cause of ghosting effect is in the upstream of the column, probably in the injection port.

95

Cause of Ghost Peaks. The characteristics of the ghosting effect so far described can well be interpreted on the assumption that comparatively. high boiling compounds (contaminants) are continuously carried into the column. When the column temperature is high, since the partition coefficient of the high boiling compounds (contaminants) contained in the carrier gas is very small, the contaminants are eluted continuously and recorded as a part of background signal not as ghost peaks, as shown in Fig. 9-(a). When the Column temperature is low, on the other hand, because of the large partition coefficient, the contaminants are trapped and accumulated at the inlet of the column as shown in Fig. 9-(b). In PTGC, such accumulation is accomplished in the isothermal step at the initial temperature. And, when the column temperature rises in the following programming step, the accumulated compounds begin to travel through the column end are eluted and recorded as peaks in the same way as samples of ordinary PTGC. In isothermal GC, the ghosting effect can be interpreted as follows. The contaminants are eluted continuously and are recorded as a part of background signal as shown in Fig. 9-(a). If the vapor of contaminants increases when sample is injected (the sample causes the increase of contaminant vapor), the increase will be recorded as a positive peak at the same retention time as the contaminants as in Fig, 10-(a). On the other hand, when a sample is injected into the carrier gas stream which contains contaminant vapor, a band is formed in which the concentration of the contaminant vapor is lower than in the other part of carrier gas stream in the column. This band moves through the column at the same speed as the
96

contaminant and is recorded -as a negative peak at the same retention time as the contaminant Thus, the assumption that the ghosting effects are caused by the compounds (contaminants) contained in carrier gas and carried continuously to the column can explain all of the ghosting effects. Next, we proceeded to investigate the source of the contaminants of the carrier gas end how to prevent ghosting effects.

Fig 9 Cause of Ghosting Effect (I)

Fig 10 Cause of Ghosting effect

4.

Discussion

Table I shows the possible causes of the continuous inflow of contaminants into columns. Table I .A part of by the same apparatus. injected sample is a) Cause related to samples analyzed adsorbed on the carbonized residue in the sample injection port end is sent to the column continuously et e

97

vapor pressure according to the adsorption coefficient. b) A part of injected sample is Then the adsorbed compounds are adsorbed on the cold spots or active carried to the column in the same sites in the injection port. way as (a). a) Vapors generated from the flow line kept at a high temperature are Causes not related to the previous samples. carried continuously into the column. b) Impurities of carrier gas itself or contaminants of its flow line are carried continuously to the column.

4.1

Ghosting Effect caused by Contaminated Sample Injection Port.

Most samples analyzed by gas chromatography contain nonvolatile materials. The nonvolatile compounds are accumulated in the sample injection port and their amount becomes great after a long use. Being kept at a high temperature and in inert gas atmosphere, these contaminants are gradually carbonized into very adsorptive compounds. This is proved by the fact that glass wool or glass insert tube kept in an injection port for some time is -found stained very dark. The carbonized contaminants adsorb some part of the injected sample and then generate its vapor according to the adsorption equilibrium. If another component that is equally adsorbed by the contaminants comes, it displaces the component that has been adsorbed and is recorded as a ghost peak like
98

the one in Fig, 1-(b). In case of Fig. 1, it is presumed that the newly introduced methanol has displaced the butanol that was adsorbed. This ghosting effect is not observed, therefore, in case of a clean injection port, as shown in F 1 (a). Though it is possible that sample vapor is adsorbed not only by the carbonized contaminants in the sample injection port but also by the inside wall of the column and the surface of the solid support, the following experiment shows that the contaminants in the sample injection port is the main adsorbent.

Fig 11. Relation of peak area and injection Number. Fig. 12 Relation of peak area and injection number for various injection ports

Fig, 11 shows the change of the sizes of the main and ghost peaks against the number of times of sample introduction, using propionic acid and valeric acid as sample and diethylene glycol succinate added with phosphoric acid as column material. Two columns of the same column packing were prepared (Column I and Column II). Propionic acid was injected to Column I repeatedly till the peak areas were stabilized. Then, valeric acid was repeatedly introduced instead of propionic acid. The propionic acid was
99

recorded as a ghost peak which gradually reduced in size with repeated number of the introduction of valeric acid. When the peak size of the valeric acid became constant, propionic acid was again introduced repeatedly till its peak height became equal. Then, Column I was replaced with Column II to which no sample had been introduced, (Column II was connected to the same injection port as Column I in the above step.) Valeric acid was introduced repeatedly to the new column (Column II). There were recorded ghost peaks of propionic acid, though NO propionic acid had been introduced to the column. These experiments show that the cause of the ghosting effect lies in the injection port, not in the column. Fig, 12 shows the relation of the conditions of injection port and ghosting effect. Fig, 12-1 shows a case where the injection port is contaminated. The peak area changes greatly, and it is necessary to repeat sample introduction many times to obtain equal peak areas. The ghost peaks are large and to eliminate them requires many repeated sample introductions. Fig, 12-2 shows the data after the injection port used in Fig, 12-1 is cleaned With solvent. The results are almost the, same. Fig, 12-3 shows the data after the injection port is cleaned with a wire brush and Fig, 12-4 the data with glass insert tube in the injection port. Some, though Incomplete, elimination of ghosting effect can be observed. Fig, 12-5 shows the data obtained by injecting the sample directly to the top of the column (on-column injection): the peak areas are constant from the first Injection and no ghosting effect can be observed.

100

As is clear from these data, the contaminants of the injection port cannot be removed by cleaning with solvent. A method to remove contaminants by oxidization in oxygen stream at a high temperature is reported. But this method has an adverse effect due to the formation of metal oxide on the inside surface of the injection port. Polishing may be the best method. The adsorption by contaminants in the injection port is a cause of ghosting effect in PTGC. Some of the compounds adsorbed by the contaminants are kept at a adsorption equilibrium in the gaseous phase at the injection port temperature, and are carried into the column continuously. These vapors are "trapped" continuously near the inlet of the column whose temperature is comparatively low. When the column temperature has risen to some degree, the concentrated components begin to travel through the column, separate, and are recorded as ghost peaks. The vapor of the adsorbed components emerging into the gaseous phase ought to decrease with time. This is proved by the decrease of ghost peak in size with time, shown in Fig. 5, and by its increase with the length of time at initial temperature. When the injection port temperature is high, the concentration in the gaseous phase becomes high. The relation of the injection port temperature and the size of ghost peak shown in Fig. 6 prove this. It is impossible to prevent sample injection ports from contamination completely. The only countermeasures are to remove the remaining nonvolatile compounds before they are carbonized. A popular method is to put a glass insert tube into an injection port (Fig, 13) and to replace it from
101

time to time. Another is to use a double-tube sample injection port as shown in Fig, 14. Since the inner tube in which samples are vaporized can be removed easily, it can be cleaned at any time.

Fig 13 injection port with glass insert tube

Fig 14. Injection port with Removable inside tube

The on-column sample injection systems, which is often used in combination with a glass column to prevent isomerizatlon and/or decomposition of chemically unstable samples, is not superior to ordinary sample Injection system from the viewpoint that nonvolatile components contaminate the part where samples are vaporized. Though the top of a glass column is less contaminated than ordinary injection port, which is commonly used for all samples, it is advisable to renew the glass wool at the inlet -of glass columns from time to time.

102

In preparative GC, it is not advisable to use the same injection port as that used in analytical operation, because a single sample size corresponds to the sample volume injected in a whole year in analytical works resulting in much contamination .

4.2 Ghosting Effects due to Adsorption Inside the Injection Port and its Vicinity. Besides the ghosting effects caused by the contaminants in the sample injection port described in the previous chapter, there are ghosting effects caused by clean injection port. The causes are the adsorptive surface inside the injection port and its vicinity, such as : 1) 2) 3) Injection port rubber septum and its vicinity Glass wool at the inlet of column Oxidized surface in the injection port in case of old apparatus.

Since part of the rubber septum and its vicinty are exposed to the outer atmosphere, its temperature is generally lower than the injection port temperature. If some sample vapor is flushed back to the vicinity of the rubber septum when the sample is vaporized under a high temperature, it may be adsorbed on the surface of the rubber septum which has similar properties as a liquid phase. This may become a cause of ghosting effect. Fig, 15 shows the data obtained for inspecting the relation of the rubber septum and ghosting effect, using rubber septa which have an especially large surface area for this experiment. The injection port was kept at 250C and the rubber septum temperature was found 146C. A mixture of fatty acid methyl esters was analyzed and then the baseline was recorded in PTGC to
103

observe the ghost peaks ( Fig, 15-2 ) Then the same experiment was made with a rubber septum whose surface was covered with fluoro resin film almost completely. The ghost peaks were then much smaller. It may seem that this phenomenon can be prevented by raising the injection port temperature. But this countermeasure is not recommended because the upper temperature limit of the septum is rather low and it may evolve gases as described in the next chapter. Rubber septa coated with fluoro resin may be very useful but are too expensive to be a consumption item. The only reliable method is to design the injection port so that the surface area of the rubber septum exposed to carrier gas is minimized and that the rubber septum is located at a place where the vaporized gas does not reach it when flushing back during evaporation.

Fig. 15 Effect of Septum Surface Area on Ghosting Effects

104

Fig 16 Glass type cartridge Type sample injection ports

Fig 17 Effect of Syringe Handling on peak Talling

Fig, 16 shows the glass cartridge type sample injection port which has both the advantage of the on column injection system to prevent decomposition and isomerization of samples and the advantage of the glass insert system to make it easy to remove nonvolatile compounds. The glass cartridge is pressed against the outlet of the injection port by the injection port rubber septum: thus a double tube system is established, where carrier gas flows outside the glass cartridge towards the septum and then into the inside of the cartridge. A spacer is placed between the septum and the glass cartridge to prevent the contact of sample vapor and the surface of rubber septum. The glass cartridge can be easily replaced from outside after removing the rubber septum and the spacer. Handling of micro syringes is also an important factor of ghosting effect. If a micro syringe is inserted into a heated injection port with the sample filled down to the tip of the needle, the sample in the needle is being inserted through the septum and is vaporized before the needle is fully inserted. This phenomenon is more conspicuous with solution samples using a low boiling solvent. To prevent this trouble, it is advisable to take an exact sample of the
105

necessary volume and then to pull beck the plunger a little to fill the needle with air. Fig, 17 Shows the effect of this operation: the tailing of the solvent peak is considerably reduced in case the needle is filled with air. The glass wool at the inlet of the column is another cause of ghosting effect. Since glass wool is generally basic, in case of acidic samples like lower fatty acids, adsorption to the surface of the glass wool cannot be neglected. The adsorption can be prevented through silanization or treatment with phosphoric acid in some cases, but the use of silica wool is more preferable. The oxidized metal surface inside the injection ports, which may cause ghosting effect due to isomerization and/or decomposition of samples, can be removed in the same way as the carbonized contaminants described in 4,1

106

4.3 Ghosting effect due to Effluent from Rubber Septum It seems that injection port septa have no influence upon the chromatogram because the septa are kept at a considerably lower temperature than the injection port temperature and the upper temperature limit of the material of the septa is claimed to be 300 350C. But the upper temperature limit of the septa is deiced from the physical and mechanical viewpoint: there is no guarantee that they evolve no vapor even below the limit temperature. The vapor from the silicone rubber in the heated part of the flow line would cause serious trouble in high-temperature, high-sensitivity GC.

Fig, 18 is the data of our experiment on the commercially-available silicone rubber septa. A restrictor tube was connected instead of an analytical column and the septum was fit to the injection port. The organic vapors evolved from the septum with the rise of the injection port temperature were directly detected by an FID. In this experiment where the injection port temperature was set to 300C, the rubber Septum temperature was 148C.
107

Fig, 18-A is the datum of the normal state: the organic vapor increases rapidly with the rise of the temperature and then decreases gradually. Fig. 18-B is the datum in the case that the surface of the septum exposed to the carrier gas was touched with finger tips: the amount of the evolved organic vapor is increased very much. This shows that injection port septa must be handled carefully. Fig. 18-C shows a case where the surface area of the septum exposed to the carrier gas is reduced: it is clear that the exposed surface area has a considerable influence on the stability. In Fig. 18-D, the stability is further improved by attaching a cooling fin to the septum fitting metal to lower the septum temperature. The data in Fig, 18 were obtained by direct detection of effluent from rubber septa. In actual isothermal analysis, the effluent is sensed as an unstable base line in high-sensitivity operation; while, in PTGC, it is sensed as ghost peaks accompanied by baseline drift. The several peaks recorded on the drifting baseline in PTGC seem to correspond to straight-chain silicone polymers, but it is not known whether they are formed under heat or whether they have been contained as low boiling components. The ghost peaks of this kind are large just after the rubber septum is renewed and they are reduced with time, though the reducing rate differs with the material of the septa. The ghosting effect can be minimized by suitable design of the injection port, it is reported that the evolved organic vapor can be reduced to one tenth by heat-treating septa in vacuum.

108

4.4 Ghosting Effect due to Impurities In Carrier Gas Commercially available cylinders of gases used as carrier gas sometimes contain unexpectedly large amounts of impurities due to the past ill handling of the cylinder. Since the impurities in this case are gaseous, they do not cause ghost peaks but unstable baselines, except in gas characterized by reserved peaks as shown in Fig, 10 (b). The same trouble is caused more often by contaminants in carrier gas flow line than by impurities of the carrier gas itself. Contaminants of this type can be removed by attaching a purification tube packed with a molecular sieve at the inlet of the carrier gas. But the most reliable method is to clean or replace the contaminated tubings. solid chromatography. The ghosting effect due to continuous flow of impurities is

109

GENERAL TROUBLESHOOTING
Before any repairs are started, READ THE INSTRUMENT INSTRUCTION MANUAL, particularly check servicing or troubleshooting sections. Also check operating sections. Before ever attempting to operate e gas chromatograph, it should be thoroughly checked for gas leaks Carrier gas leaks give rise to many problems. The carrier gas exit lines should be capped. The carrier gas input pressure is then increased to 60 psig. When the pressure has stabilized, turn off the main cylinder valve. If no leaks are present, the pressure should remain constant for 15 minutes. If the pressure does not hold, repressurize and check for leaks with dilute soap solution. Detailed leak checking and procedures are usually given in the instruction manuals.

THE CHROMATOGRAPH RECORDER Whenever problems arise that could also be caused by the recorder, it should be checked first for proper operation. Read the recorder instruction manual for more details. MAKE SURE RECORDER INPUT LEADS ARE PROPERLY CONNECTED. Verification of proper recorder operation may be quickly made by disconnecting the gas chromatograph from the recorder input, and connecting a short piece of wire (a paper clip works fine) between the + and input connections. The recorder pen should go to electrical zero and

110

remain there without any oscillation. At this point, the indicating pointer on the. scale should indicate zero (mechanical zero) and the pen on the strip chart should indicate zero (chart zero), i.e. electrical, mechanical, and chart zero should all line up. The pen should not move with light finger pressure. make any recorder adjustments that might be necessary. Some common problems are :

a.

Sluggish pen movement 1) 2) 3) 4) GAIN adjustment too low, DAMPING adjustment over damped. Check reference battery ( where applicable ) . Check amplifier. Check for low level AC voltage signal to input. Correct by installing 0.25 (approximately) capacitor from either + or connection to ground. 5) 6) Ensure the CHASSIS GROUND is a true EARTH GROUND. If possible, connect to water pipe. Check input leads for improper connection.

b.

Jerky pen movement 1) Dirty slide wire. Clean the slide wire and contacts. Ethyl ether is a good solvent solvent. Do not oil after cleaning. 2) 3) Mechanical binding Check for any severe line power changes (line transients etc.) Isolate recorder and gas chromatograph, if use stabilized transformer if necessary.
111

c.

Pen oscillates 1) 2) 3) GAIN adjustment too high. Adjust DAMPING control also. Poor ground connection. Input leads not properly connected.

d.

Chart drive slips 1) Drive gear slipping on shaft. Tighten.

e.

Attenuation not linear 1) 2) 3) 4) 5) 6) 7) Poor ground connection. GAIN adjustment too low. Adjust DAMPING control also. Input leads not properly connected. Electrical and mechanical zero not at the same point and/or not at chart zero. Check amplifier. Check reference battery (where applicable). SPAN ADJUST out of calibration (where applicable).

Troubleshooting temperature control and programming system. The following problems are those most frequently encountered in heaters and temperature control system :

a.

No heat to detector (or column oven)

112

1) 2)

Check fuses - Main Power, oven and controller (or programmer) fuses. Replace defective fuses. Open heater element - check with ohmmeter by disconnecting one heater lead. Set ohmmeter to highest scale reading and read element resistance. Infinite resistance indicates and open heater element - replace. Zero ohms indicates heater element is O.K.

3) 4) 5) 6)

Check for loose wiring connections. Upper limit control switch set too low or defective. Defective temp. sensing probe. Check with ohmmeter. See instruction manual . Check tubes in detector oven controller (if applicable).
b. Full heat to detector (or column) oven regardless of position of control setting.

1) 2) 3)

Defective temp. sensing probe. Check with ohmmeter. Defective tubes in controller (when applicable) Defective silicone controlled rectifier (SCR) in controller ( When applicable ) .

c.

No heat to injectors 1) 2 3) Check fuses. Replace defective fuses. Open heater element. Check and, if necessary replace. Defective injector heater controller. See instruction manual.

113

d.

Temperature in ovens not stable. 1) 2) 3) Defective temperature sensing probe. Defective tubes in controller (when applicable) Gaps or holes in oven insulation. Crossover tubes should be capped when not in use.

5.

TROUBLESHOOTING THE THERMAL CONDUCTIVITY DETECTOR The following problems are those most frequently encountered in thermal conductivity system.

a.

Noisy baseline 1) 2) Carrier gas flow leaks. The most common places are column connections and septum. Contamination in columns or flow system, particularly injectors and any places in the flow system where cold spots might occur. 3) 4) 5) 6) 7) 8) Flow controller or cylinder regulator erratic. Loose -wires or connections. Dirty switch contacts. Clean with spray cleaner or eraser stick (pencil ) . Defective detector. Defective power supply. Defective recorder.

114

b. 1) 2) 3) 4)

Drifting baseline Carrier gas flow leaks. This is by far the most common cause of both drifting and noise. Contamination in column. Recondition. Flow controller or cylinder regulator defective. Detector oven temperature not at equilibrium temperature. This could be due to insufficient time being allowed for oven to reach a stable temperature. Also see comments in "4d".

5) 6)

Contaminated detector. Clean detector cell (see instruction manual). Poor carrier gas flow through either detector or reference side of detector.

c.

Recorder will not zero 1) 2) 3) 4) No carrier gas flow through reference side of detector Detector cell filaments out of balance or burned out. Recorder not connected and/or adjusted properly. Poor connection in the D.C. circuit of the instrument or interconnecting cable to recorder.

d.

No filament current 1) 2) Blown fuse in D.C. power supply. Check and replace if defective. Defective power supply or power supply switch causing no output from, supply .
115

3) 4) 5)

Burned out filaments. Can be isolated by checking schematic diagram in instruction manual. Test with ohmmeter. Defective current meter on instrument panel. Broken electrical connection in D.C, circuit of instrument.

e.

Attenuator not linear 1) 2) 3) Check resistors on attenuator for value and/or solder connections. Recorder electrical zero not at recorder scale zero. Poor connection between instrument and recorder, and/or recorder gain; damping, etc, out of adjustment. See section "3e."

Troubleshooting ionization detector systems The portions of the instrument ahead up stream of the detector are virtually identical to those used with thermal conductivity detectors, except that no reference gas is needed. Electrical systems used to control heaters, etc., are also the same, so are subject to the same troubleshooting methods outlined previously. An electrometer acts as an amplifier for the detector signal, and supplies an output compatible with the recorder. Therefore, the input must be matched to the detector and the output matched to the recorder.

116

If there is difficulty with a detector system using an electrometer, it is desirable to isolate the problem "to a particular component within the system (detector, electrometer, or recorder).

a.

Isolation of the component causing the problem 1) 2) Check recorder as outlined in section "3" of this section. If the recorder operates normally, reconnect it to the system. Check electrometer- by disconnecting signal input cable (leading from the detector) and shielding the input to the electrometer with a piece of aluminium foil. Be careful that the foil only shields the input and does not short -it to chassis ground. The foil serves to insure that no stray signals will be picked up, and amplified by the electrometer. If the operation agrees with specifications outlined in the instruction manuals, the electrometer is not at fault and the problem is being introduced into the system by the detector. If at this point the electrometer is found to be at fault, the problem is usually poor stability (-drifting or noisy baseline), or a problem with balancing the baseline et recorder zero. a) Drifting baseline Check the electrometer ICS and replace the. defective ones. Allow several hours for electrometer to restabilize before rechecking.
117

b)

Noisy baseline 1. 2. 3. Check for any defective ICS as above. Check for bad connection between electrometer and recorder. Check for any poor or dirty switch contacts in the electrometer.

c)

Cannot balance electrometer at recorder zero. 1. 2. Check 'electrometer the same as for noisy baseline. Check instruction manual and re-balance electrometer input circuit, if applicable.

If operation of the recorder end electrometer are found to be normal, check detector operation as outlined in the instruction manual. Some typical troubles with specific detectors are as follows :

b.

Flame ionization detector. 1) Flame will not stay lit. a) b) c) Flame burner tip restricted. Clean end check orifice size. Insufficient hydrogen. Check for any leaks and restriction in supply. Use more hydrogen. Insufficient air. Some as (b) above. Use more air.

118

d) e) 2)

Improper flow rates. Check the air, carrier gas, and hydrogen rates and adjust to specified optimum flows. Too large a sample injected into instrument.

Noisy baseline a) Inspect the detector for cleanliness. Dirt on the detector parts (especially electrical connectors) can cause the noise. Dirt can be caused by sample residue or liquid phase residue and is best removed by immersing the complete detector in an ultrasonic cleaner. b) Noise can originate from particles or residue in the flame base passageways. This is an often overlooked point. Clean the base just as you . would the detector. c) Excessive column bleed from poor columns or excessive temperature can often be identified by viewing the flame. A clean burning flame is not visible. A dirty flame burn orange or bluish. d) Dirty carrier gas, air or hydrogen can cause noise and should be investigated if normal checks do not eliminate noise. e) Improper balance of air, hydrogen, and carrier gas to the detector is a common cause of noise. Check instruction Manuel for optimum flow rates.

119

3)

Drifting baseline a) Drifting baseline is usually caused by column bleed or faulty flow system (leaks etc. ).

c.

Electron capture detector The following are some typical troubles encountered when using an electron capture detector. 1) Unstable baseline a) b) Carrier gas leaks. Check for leaks as with any other system. Column bleeding. Disconnect column from detector end condition column at approximately 50C higher than normal operating temperature. c) d) e) f) g) h) 2) Oven heat cycling. Check as outlined in section "4" of this chapter. Carrier gas filter is saturated with contaminants. Regenerate filter as outlined in instruction Manual. Air back diffusing into the detector. Faulty pressure regulator on carrier gas cylinder. Column oven cover is not properly closed. Poor earth grounding of complete instrument system.

Low standing current

120

a) b) c) d) e)

Improper connections to the detector. Low carrier gas flow. For optimum flow through detector check instruction manual. Tritium foil reversed in cell. Tritium foil contaminated by sample or column bleed. Clean as outlined in instruction manual. Carrier gas leaks. Check as with any other system.

D.

Trouble Shooting Distorted GC Peaks

In many cases, the appearance, or distortion, of the gas chromatographic peak indicates that instrument parameters, sample size, injection technique, etc, are not optimum. In this section, illustrations of problem peaks are shown, together with possible reasons and corrective action.

121

PEAKS

a) 0ne peak only, i.e. a) Reduce sample size. solvent overload. b) All peaks, dirty b) Clean detector. detector (Electron capture detector). c) TC detector c) Compound with different conductivity than carrier gas a) Flow controller not a) Increase inlet working. pressure and/or replace flow controller. b) Septum leaking b) Replace septum. during injection. c) 0verloaded gas c) Reduce sample size. sample d) TC detector d) Compound with different conductivity than carrier gas. a) FID-Gross overload. a) Dilute sample. Inject smaller sample. b) TCD-H2 in sample b) Use mixed carrier with He carrier. (8% H2 in He). c) Poor injection c) Inject sample method. quickly. a) Residual sample being eluted from previous sample injection. b) Syringe Contamination c) 0bserved when analyzing derivatives. Incomplete derivative formation, or partial hydrolysis of trimethy lsilyl ether derivatives. a) Allow sufficient time for previous sample to dilute. b) Clean syringe after injection c) Review derivatiza tion technique.

122

a) Sample overloading column.

a) Decrease sample size 10X. Increase detector sensitivity. b) Sample condensing b) Check injector, in chromatograph. column, and detector temperatures. Increase temperature if necessary. c) Poor sample c) Review syringe injection. techniques. d) Unresolved d) Lower column component. temperature 30C. Increase column length 3X. Use a more selective phase. Decrease sample size by 1/10 to improve efficiency. a) Thermal degradation of sample. a) Column and/or injector too hot, decrease temperature. Use glass liner in injection port. Bare, or active sites on column packing, replace packing. Use glass or nickel column, not stainless steel. a) Use column of different relative polarity. b) Clean injector. c) Increase temperature. d) Review syringe injection technique.

a) Wrong column: e.g. polar sample on non polar column. b) Dirty injector. c) Column/oven temperature too low. d) Poor sample injection technique.

123

e) Interaction of solid e) Use deactivated support with sample. support, e.g., silanized support with polar compounds. Phosphoric acid treated support with acid compounds; base (KOH, Na3P04) treated support for basic compounds.Deactivate support with 0.2% Carbowax 20H, 0.25% Epon 1001, or 0.2% polyster. f) Tailing due to f) Hake suitable hydrogen bonding of derivative. sample. g) Column too hot, g) Replace column. phase has been baked Check oven off revealing bare temperature . support surface. h) Unresolved h) Lower column component. temperature 20C and check peak shape. Lengthen column 3X. Use more selective phase. Decrease sample size 1/10 to see if separation improves. i) Too much dead i) Add glass liner to volume in injector. injection port or use on-column injection.

124

a) Narrow peak followed by wide peak (also asymmetrical peaks) possibility of 2 or more components in the wide peak. (note : Branched isomers usually give a wider peak when using porous polymer columns). a) Detector overload. Cigar and round peak shapes, ECD and FID. b) Flat top = saturation of electrometer a) Recorder gain too low. b) Physical damage to TCD filaments. c) Dirty slide wire on recorder. d) Line voltage change. e) AC interference.

a) Change column temperature by 20C to see if other peaks appear. Increase column length 3X. Use more efficient column. Use more selective phase.

a) Dilute sample or decrease sample size. b) Increase electrometer range by 10-10x. a) Readjust recorder gain. b) Replace filaments. c) Clean slidewire. d) Use voltage regulator. e) Check shielding and ground.

125

PRACTICAL CAPILLARY COLUWN GAS CHROMATOGRAPHY NOTES

1.

What is a chemically bonded fused silica capillary column? It is a capillary column prepared by chemical bonding between molecules of the liquid phase applied on the Inner wall of fused silica capillary tube, or between the liquid and Inner surface of fused silica capillary tube. The terms such as cross linked phase, immobilized phase, bonded phase and chemically bonded phase refer commonly to the liquid phase made by chemical bonding, and are substantially used synonymously. The chemically bonded fused silica capillary column presents the following features, as compared with an ordinary wall coated fused silica capillary column. 1) It resists higher temperature. For example : Max, operating temperature of the one equivalent to OV-1 :320C ( That of OV-1 : 280C ) Max, operating temperature of the one equivalent to PEG-20M : 250C ( That of P EG-20M : 180 C ) 2) Since bleeding of liquid phase vapor from the column is only slight, the base line is stable.

126

It is possible to perform the temperature. programming analysis with ECD or a single FID which was difficult with conventional column. 3) The column can be cleaned with solvent. Even the column contaminated with high boiling point molecules can be regenerated by cleaning.

2.

In what bores are fused silica capillary tubes available, end how are they used? Generally, the smaller the bore, the greater becomes the separating ability. But since the column load (the quantity of sample that can be injected) decreases, the columns of different bores must be used according to the applications as shown below.

Bore (Inner diameter) (mm) 0.1

Length (m)

Film thickness (um)

Application

10 15

0.1

High load.

separation,

fast

analysis, very small column 0.2 0.3 25 50 25 50 0.25 0.5 0.5 1.0 High separation, generally used in split type analysis Used in split less oncolumn injection method. Column load is larger than in 0.2mm

127

0.5

10 12

1.0 5.0

Separation ability equal to that of packed column. Large column load.

The 0.5 mm column. (wide bore column) does not aim at high separation, but is intended to make use of the advantage of inertness of stationary phase, not available in packed column, while maintaining a property close to that of packed column, and to obtain the reproducibility of performance between columns.

3.

The relationship between bore of capillary column and number of theoretical plates.

The number of theoretical plates which express the operating ability of a column varier with the bore of the column, film thickness of liquid phase, and capacity ratio (k). Here, omitting theoretical discussions, is shown the relationship between the number of theoretical plates (Nexp) per I m and linear velocity (U) of carrier gas. (He) measured by using several actual columns (Fig.1)

128

NOTE : The linear velocity (U in the unit of cm/sec) of carrier gas and capacity ratio (k) are calculated as follows, from the result of analysis of methane on the column.

In the case of capillary column : tR = to (1+ 10/200) = to X (1+ 0.05) = 1.05X to It is thus known that, In the capillary column, the object component is hardly separated from the air or methane.

5.

Why Is He used as carrier gas in capillary column analysis? Concerning the separation performance of Column, He or H2 is superior to N2 as Carrier gas. The separation ability of a column is expressed by the number of theoretical plates or its inverse number, that is, the theoretical height plate (HETP), but in the case of capillary column, the HETP curve varies significantly with respect to the flow rate (or linear velocity) depending on the kind of carrier gas.
129

As shown in Fig. 2 in the case of nitrogen carrier gas, since the curve is bent quite sharply, the separation performance is extremely inferior, as compared with that of other two gases, unless the carrier flow rate

is set at the optimum value (the value to give the minimum value In Fig. 2l'which Is, however, actually very difficult. If successfully set at the optimum value, since the flow rate is small, a longer time is needed for analysis.

By contrast, in the case of helium carrier, the separation performance is not lowered so much if the flow rate is increased and, what is more, the optimum flow rate to give the highest separation ability is relatively large, Hence by using helium carrier, the time for analysis can be shortened, and the sample recovery may be improved in split less injection because the flow rate is relatively large.

130

On the other hand, hydrogen carrier is advantageous in that the time for analysis may be much more shortened that in the case of helium carrier, but It cannot be recommended generally in consideration of its danger. 6. The relation between carrier gas flow rate and column temperature The carrier gas may be regulated either by mass flow controller or by preassure controller, In the case of temperature programming analysis, generally, the mass flow control is preferable because the set flow rate may be maintained if the column temperature is changed. However, In the Grob injection method which is widely used in capillary column gas chromatography, since the flow rate may depend on the column temperature because of its regulation by the Pressure controller, it is necessary to understand its temperature dependency. Fig. 3 shows the flow rate changes due to variations of column temperature in the case of helium carrier gas. The flow rate should be set so that it may be optimum at an actual operating temperature, and in the temperature programming analysis, it should be set so as to be optimum at an intermediate temperature of temperature programming or around a temperature where the maximum separation is required. To do so, it is necessary to know the linear velocity of the carrier gas, rather than its flow rate. The liner velocity is, as seen from eq. ( 3-1 ) , obtained by dividing the column length by the retention time of ethane gas (in the case of FID). For easier measurement, instead of the retention time of methane, that of solvent peak may be used.
131

7.

What is the solvent effect? In the Grob's splitless injection method, the liquid of the solvent condensed at the top of capillary column acts like stationary phase, and traps the sample components to elute the peaks more sharply, which is called the solvent effect. To make use of this effect, it is essential to keep the initial temperature of the column about 20 to 40C lower than the boiling point of the solvent.

This example is shown in (A) and (B) of Fig. 4 (A) and (B) are chromatograms obtained in the identical conditions, except that
132

hexane is used as solvent in (A) and heptane in (B). In the case of IA), since n-hexane with boiling point at 69C cannot form a liquid film in a column of which initial temperature is 70C, the solvent effect does not occur. As a result, the peak of undecane (C11 ) spreads widely. On the other hand , in ( B ) , since the boiling point of n-heptane is 96C, the solvent effect takes place sufficiently at this column oven temperature, so that the peak of undecane peak is very sharp.

133

8.

What is the reverse solvent effect? Whether split or splitless method, a phenomenon of broadening of the peak of eluting immediately before the peek of principal component is called the reverse solvent effect. An extremely thick film of the overloading principal component which acts as a kind of stationary phase decrease the column efficiency in that portion and broadens the peaks. The phenomenon is likely to occur when a component having a considerably higher boiling temperature than the column temperature is injected in a large quantity.

134

Fig 5 is an example chromatogram of analysis of impurities in mxylene. As compared with the boiling point of 138.8C of m-xylene in the principal component, the column temperature is 80C, and it is known that the peaks of ethylbenzene and p-xylene eluting before mxylene are spread broadly.

9.

Peak distortion end peak Splitting in cold on-column Injection method In the cold on-column injection method, the sample is usually injected upto 2 ml, but when the amount of injection is further increased, the peak may be distorted or split.

The sample injected directly to the top of capillary column is carried into the column in the form of liquid, which at this time forms, not one homogeneous plug, but a band of sample solution composed of numerous heterogeneous liquid drops as shown in Fig. 6. When the sample amount reaches about 5ml, the band of sample solution grown lm or longer Even after the vaporization of the solvent in the solution, the sample components continue to be distributed long and. unevenly

135

while advancing through the column, thereby forming irregular peeks as mentioned above. A countermeasure is to remove stationary liquid phase from this unevenly distributed portion of the column (which is called flooded zone), or to add another capillary tube without any coating before, the separation column (this method is called retention gap) so that the peak profile may be improved.

10.

What is the Christmas tree effect? This phenomenon is observed when a component having a higher boiling point than the column temperature is injected far over the load capacity. The zigzag profile of the leading side of the peak is expressed by Schomberg as being "like a Christmas tree," and this effect is named accordingly.

The small wall thickness of fused silica capillary columns, since the heat capacity of the tube is small, allows their - inside be affected by the forced circulation of hot air in the column oven, and causes to
136

change the moving speed of the components advancing through the column, which is regarded as the cause of this phenomenon.

One of the simple remedies with a certain effect expected is to wrap the whole capillary column with aluminum foil (Fig. 7). However, excess use of aluminum foil may sacrifice the temperature follow-up characteristic of the column, and, as a result, worsen the repeatability of retention time.

137

In Fig. 8 (A) shows a chromatogram with Christmas -tree effect, and (B) is an improved chromatogram by a column wrapped with aluminum foil, in which the "zigzag" profile at the leading side is eliminated.

11.

Repeatability of retention time in capillary column gas chromatography An example of actual measurement is shown below. Normal paraffins (C14 to C32 ) were selected as the samples to be analyzed, and GC-9APF gas chromatograph, AOC-9 auto-sampler, and C-R2A data processor C hromatopack were used . Using fused silica column OV-1 01 ( 25m, O. 2mm) , the samples were analyzed ten times, and the repeatability of retention time was measured, and the results as shown in Table 2 were obtained.

TABLE 2 (Repeated analysis of Paraffins)

Retention Time (min.) Sample injection time

17 : 33 18 : 15 18 : 57 19 : 38 20 : 20 21 : 02

Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 (C14)

2.035 2.035 2.003 2.032 2.032 2.032

(C19)
6.745 6.744 6.743 6.745 6.742 6.743

(C21)
8.623 8.632 8.633 8.633 8.632 8.632

(C25)
12.007 12.01 12.007 12.005 12.008 12.007

(C28)
14.225 14.225 14.233 14.233 14.227 14.227

(C3 0)
15.572 15.573 15.575 15.573 15.575 15.577

(C 32)
16.835 16.838 16.835 16.833 16.833 16.837

138

21 : 43 22 : 25 23 : 06 23 : 48 Mean value (x) Standard deviation (6) x 10-3 Coefficient of variotation (CV % = 6/x x 10 2

2.032 2.033 2.033 2.032

6.74 6.745 6.743 6.742

8.632 8.633 8.633 8.63

12.01 12.008 12.012 12.008

14.225 14.228 14.227 14.123

15.577 15.575 15.578 15.575

16.837 16.842 16.837 16.837

2.033

6.743

8.632

12.008

14.226

15.575

16.836

1.197

1.595

0.919

2.000

2.211

1.944

2.646

0.0589

0.0237

0.0106

0.0167

0.0155

0.0125

0.0157

Thus, in the peaks 1 to 7 of which retention time is about 2 to 17 minutes, the repeatability of retention time was very stable, being within the coefficient of variation (CV) of 0.01 to 0.05%. Condition of analysis Column temperature : 100 to 280C, at 10C / min Column type : Fused silica capillary column OV 101 0.2mm N25m injection detector temperature : 300C Detector : FID

12.

Repeatability of peak area percentage in split method

139

The Grob test mixture was measured seven times by using GC-9APF gas chromatograph, CLH-702 split type capillary column holder, AOC-9 automatic liquid sample injector, and C-R2AX data processor, end the repeatability of area percentage of ten peaks was studied. The results are shown in Table 3. As clear from Table 3, the coefficient of variation (CV) was somewhere between O.1 end O.4, which suggested a favorable repeatability. Table 3
Peak No. / Run No. 1 1 2 3 4 5 6 7 Mean Value Standard deviation Coefficient of 2 3 1.544 4 10.261 5 9.996 6 10.543 7 1.816 8 9 10 8.532 10.499 11.002 7.123 10.955

8.368 10.530 10.567 10.259 10.012 10.461 10.805 10.987 7.112 10.901 8.322 10.496 10.547 10.239 10.003 10.449 10.780 10.999 7.149 10.016 8.346 10.494 10.535 10.260 10.023 10.499 10.805 10.968 7.155 10.695 8.346 10.506 10.557 10.278 10.011 10.433 10.800 10.979 7.153 10.911 8.369 10.504 10.549 10.272 10.001 10.440 10.805 10.978 7.151 10.931 8.336 10.474 10.530 10.279 10.002 10.436 10.789 10.981 7.201 10.972 8.351 10.501 10.547 10.264 10.007 10.447 10.800 10.985 7.149 0.017 0.20 0.016 0.15 0.011 0.11 0.013 0.13 0.001 0.10 0.007 0.7 0.012 0.11 .012 0.11 0.026 0.37 1.950 0.037 0.34

140

variation (%)

13.

Repeatability of peek area percentage in splitless method A hexane solution containing traces of components in the order of ppm was consecutively measured eight time by using SPL-G9 Grob type split/splitless sample in,jector, AOC-9 automatic liquid sample injector, and C-R2AX data processor, end the repeatability of area percentage of eight peaks (excluding solvent peaks) was studied. The results are shown in Table 4. From Table 4, the repeatability of trace components of ppm or smaller is also known to be excellent.
Table 4

Peak No. / Run No. 1 1 2 3 4 5 6 7 8 Mean value 1.057 0.1044 0.1036 0.1035 0.1063 0.1062 0.1060 0.1042 0.1050 2 0.0905 0.0899 0.0900 0.0905 .0898 0.0905 0.0902 0.0889 0.0900 3 0.0921 0.0931 0.0922 0.0926 0.0920 0.0937 0.0926 0.0924 0.0926 4 0.2315 0.2318 0.2316 0.2324 0.2321 0.2327 0.2319 0.2318 0.2320 5 97.4807 97.5619 97.5818 97.6118 97.6220 97.6169 97.5983 97.5940 97.5819 6 0.0757 0.0754 0.0755 0.0755 0.0754 0.0749 0.0752 0.0755 0.0754 7 0.0925 0.0919 0.0922 0.0916 .0915 0.0907 0.0927 0.0920 0.0919 8 0.9554 0.9602 0.9604 0.9597 0.9590 0.9582 0.9590 0.9597 0.9590

141

Standard deviation (X10-3) Coefficient of variation %

1.125

0.506

0.533

0.381

77.0

0.228

0.593

1.525

1.07

0.56

0.58

0.16

0.08

0.30

0.65

0.16

14.

What is a multidimensional capillary column gas chromatograph? It is a capillary gas chromatograph incorporating applied flow lines. That is, back flushing, fore flushing, precutting, and other operations are effected by changing over the flow of gas by means of four-way, six-way or eight-way column switching valve with' small capacity, or flow switching mechanism using stop valve.

142

An example configuration for analysis of, naphtha is shown in Fig. 9.

15.

Simple precutting capillary column gas chromatography In the GC-9AMPF gas chromatograph, the temperature can be set independently in the sample injection port and the detector. By inserting a short pecked column, instead of glass insert, into the sample injection port in the analysis.

143

Module of GC-9AM, the undesired components of high boiling point can be precut and discharged out of the system by means of this packed column pre cutting column) kept at an adequate temperature, so that the capillary column may be protected and that the time for analysis may be shortened, Fig 10 shows a flow configuration for such analysis. This is also e kind of multidimensional capillary column gas chromatography.

144

HIGH PRESSURE LIQUID CHROMAIOGRAPH

High pressure Liquid Chromatography which is an extension to the conventional column chromatography technique is considered as a complementary technique to Gas Chromatography rather then replacement of the same. The advantages of High Pressure- liquid chromatograph over Gas Chromatography coupled with some innovations in the last decade has made it one of the most exciting analytical technique in chemical and biochemical separations. The advantages may be listed as , a) b) c) Capability to analyse samples having high boiling point. Capability to analyse samples having high molecular weight. Capability to analyse thermally labile compounds.

This makes it the ideal choice for the analysis of macromolecules and ionic species. The basic parts of the HPLC are : a) b) c) d) e) Solvent Delivery System Sampling 'System Column Detectors Data handling system

145

A.

SOLVENT DELIVERY SYSTEM :

The first component of a modern liquid chromatograph is a reservoir of pure degassed solvent to be delivered to the pump. Basic requirements for an LC pumping system are pressure capability to at least several thousand psi and good solvent compatibility. The ideal pumping system for LC should also provide accurate, precise, pulse-free solvent delivery over a wide range of flow rates, be easy to change to a new solvent, be generally convenient to use and maintain, be able to drew from a large external reservoir, and be easily adapted to gradient elution. Solvent delivery system may be generally described as being one of two types, constant pressure (pneumatic) or constant volume (mechanical). Constant-volume pumps are sometimes referred to as metering pumps, because they deliver a calibrated flow of a liquid to the column. Both types of Pumps usually operate by displacing liquid from a chamber by a hydraulic piston. The evolution of the pump which may be considered to be the most important component of the HPLC is as outlined below : a. b. c. d. e. Gas Displacement Pump followed by Pneumatic amplifier pump followed by Screw-driven syringe pump followed by Single piston reciprocating pump followed by Dual piston reciprocating pump

146

The last mentioned has been found to be to date the most suitable pumping System having the advantages of compatibility with the microprocessor. Whereas the earlier systems improved two pumps or more for a gradient system. The advent of sophisticated microprocessor has resulted in better capability with reduced hardware. The modem pumping system utilizes a single pump to achieve the gradient of two or more solvents.

B.

SAMPLING SYSTEM :

The purpose of. injection system is to introduce the sample on to the pressurised column as sharp column plug with little loss in efficiency. The three common means of sample introduction are shown in table given below Sample Introduction Methods Syringe / 0n-Stream Variable volume Single, low cost Injection of small Minimal band spreading Syringe / less Advantages Not injecting Precise, injections reproducible 0perator convenience Easy to automate High pressure Can accommodate large sample against pressure Allow syringe injection at high pressure Minimal band spreading Longer Septa life Valves

147

amounts
Dis - Advantages

Column and syringe easily plugged Limited to low pressure(2,000 psi) Blow back of sample Limited to syringe precision Spurious Septa peaks Short Septa life Hard to automate

Limited tosyringe precision Inconvenient with some pump designs Hard to automate Still have Septa bleed, column Plugging

Sample waste when loading must be careful to avoid band spreading Inflexible sample size

It will be observed that the injection system utilizing valves has proved over the years the ideal choice of the LC manufacturers. Manufacturers are now designing injection system combining the flexibility of the valve with the advantages of the syringe system.

148

C.

COLUMN AND OVENS :

A good understanding of a chemical and physical interaction that takes place between sample, mobile phase and stationery phase is important if a chromatographer is to quickly make the correct mobile and stationery phase selection. The attached table "B" will serve as a column selection guide. Other parameters Which play a very important role in the separation mechanism will depend On the separation techniques involved namely : a. b. c. d. Adsorption Reverse phase Ion exchange Size exclusion

The parameters which play an appreciable role in the LC separation are a. b. c. d. e. Mobile Phase Composition Stationery phase Flow Rate Column material Temperature

Other parameters, which will have to be considered to achieve separation include :a. b. c. solubility functional group molecular weight

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D.

DETECTOR : The function of the Detector is to provide an electrical signal to the recorder and data system from which the qualitative and quantitative analysis is made. The most common L/C detector is RI and UV followed by the Infrared, conductivity, and the Electro chemical detector and fluorescence detector.

RI Detector The RI detector passes light through sample and reference cells in such a way as to observe the difference in refractive index of the contained liquids. RI detectors are much less sensitive than UV detectors for UV absorbing compounds but when the maximum molar absorptivity is less than 10, the RI detector may be preferred, although it is essentially useless for gradient elution. RI detectors are extremely sensitive to changes of temperature, flow and polarity. However, RI detectors are ideal for the analysis of compounds like sugar, certain grades of polymeric material and aliphatic compounds.

E. UV DETECTOR The UV detector is a photometer with a micro sample cell. Since this is very much similar to the conventional UV-vis. Spectrophotometer excepting for the cell design, the features available in modern
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spectrophotometers have been incorporated into the UV detectors used in LC like scanning, ratio recording etc.

F. RECORDERS / DATA PROCESSORS The Recorder serves the function of identification and -quantification of various components of the mixture to be analysed. The development of digital integrators to complement recorders, was responsible for accurate quantification of the concentrations of the different components. These along With development of specific algorithims for integration of different types of peaks like leading peaks, skewed peaks, tailing peaks help not only in quantifying but also in minimising the time of analysis. Current developments in data processor technology includes computation in whatever format that may be desired by the use of specific programmes designed to meet the Chromatograph requirement, e,g, area percentage, computation using international standard methods, external standard methods, area normalization method etc.

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