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GAS CHROMATOGRAPHY

I R W I N HORHSTEIW

UN I T E D S T A T E S D E P A R T M E N T O F AGR I C U L T U R E

A major contributor t o food flavor is odor. Odors are produced by volat i le materials which impinge on the olfactory centers, of this sense is fantastic; man can detect concentrations as low as frac- t i o n a l p a r t s per b i l l ion of some substances and man has rather a poor sense of ~ m e l l as compared t o other members of the animal kingdom, flavors of foods are usually a complex mixture of organic compounds which vary in amount, s t ab i l i t y and Volatility. The classical approach by flavor chemists t o isolate and identify these materials has been t o use micro dis- t l l l a t i o n techniques in conjunction with methods of chemical analyses. Separations by d i s t i l l a t i on even with the wst elaborate setups and maximum precautions give incomplete resolution of complex mixtures and higher boil- ing materials are often badly decomposed. The introduction of chromato- graphic methods has been as great a boon t o the flavor chemist as t o the biochemist, and with the development of gas chromatography progress in the flavor f i e l d should move on apace.

The acuteness

The volat i le

Chromatography is a method of separating a mixture into i t s compo- nents by repeating msny, many times the unit step of parti t ioning a compound between two phases. form, composed of granules of very large surface area;the other i s a f lu id tha t flows throu@ this stationary phase.

One of these phases is stationary ana, if in column

In gas chromatography, the moving fluid, as the name iqplies, is a gas; the stationary phase may be ei ther a l iquid or a solid, chromatography i s the more useful form for most purposes and will be dis- cussed here.

Gas-liquid

I n the first slide, a schematic diagram of equipment normally used is sham. sion gas regulating valve and a pressure gauge. most commonly used method of sample introduction is t o inser t the needle of a micro syringe through a rubber septum cap and quickly indect the sample into the gas stream. For gaseous saxples, a known volume is introduced from a calibrated chamber by the carrier gas, The tube i t se l f may be glass or m e t a l and may vary in shape, length, an8 diameter. be msde of copper, 6' i n length and 1/4" 0. D. The inert granules should be of uniform size; the i r function is t o act as the support f o r the stationary l iquid phase with which it is coated, The column operating temperature is a function of the boiling points of the materials t o be separated. The vapor pressure of the stationary phase must be negligible a t t h i s colurrm tempera- ture. The gas i s the eluent, and depending on the detector is usually helium, nitrogen, or argon. the column depends on i ts normal vapor pressure as w e l l as the extent of i t s interaction w i t h the l iquid phase.

We have a tank of gas under high pressure, followed by a preci- For l iquid samples, the

Atypica l column might

The ra te of flow of a given component through

The components emerge in t h e form of

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bands separated by zones of car r ie r gas. gas stream i s constantly monitored by a suitable detector and the inform- t i on continuously recorded. slide. The peak areas represent a momentary change in a physical property of the gas stream when it contains a foreign material.

The composition of t he emerging

A typical chromatogram is shown i n the next

The ever-increasing use of gas chromatography is a resu l t of i t s inherent advantages over d i s t i l l a t i on and other forms of chromatography. The separations a re similar t o those obtained by d is t i l l a t ion , but wi th tremendously increased resolution, and they are carried out on micro amounts of material. viscosity of the gas permits the use of longer columns with a correspond- ing gain i n efficiency. It is easier t o detect changes i n composition of a gas than i n a l iqu id stream, and separations a re effected much more rapidly. The major areas of research i n the developrnent of gas chromatog- raphy have been finding the appropriate conditions and l iquid phases f o r carrying out a given separation and i n &eveloping more sensit ive detection devices.

Compared with other forms of chromatography, the low

Just as the heart of the gas chromatograph i s the column, the Since changes in the effluent gas stream may be brain i s the detector.

minute and rapid, the detector must have a fast response, high sens i t iv i ty and good s tab i l i ty . chromatography; those most commonly used a re flame, ionization, Beta-ray ionization, and thermal conductivity. Thermal conductivity, i n par t be- cause h is tor ica l ly it was the f irst r e a l l y pract ical detector, is the most widely used although the ionization type detectors a re more sensitive. In the hot-wire thermal conductivity detector, a filament heated by an accur- a t e ly controlled e l ec t r i c current is held i n the center of a small tube through which the car r ie r gas passes. A l l e lse constant, the thermal con- ductivity of the gas determines the temperature of the wire and consequent- l y i t s resistance; t h i s i s the property measured, When a foreign substance enters with the gas stream there is a change in the thermal conductivity of the gas and consequently a change i n the resistance of the wire. t i c e 2 gas channels and 2 wires that are as nearly --e as pssible are built in to a ce l l , Pure carr ier gas flows through one channel and an equal quan- t i t y of gas, which has passed through the column, through the second. Any differences i n resistance of the two wires due t o the effect of vo la t i le components i n the effluent are recorded. means of a Wheatstone Bridge circui t . For most organic compounds the change i n thermal conductivity f o r a given weight of compound i s a maximum when hydrogen is the car r ie r gas. Catalytic reaction at the hot wire may take place, and there i s some hazard i n i t s use. and none of i t s drawbacks and is the preferred choice f o r the car r ie r gas.

M y detectors have been developed fo r use i n gas

In prac-

This i s conveniently done by

However, hydrogen has i t s drawbacks.

Helium has almost as high a thermal conductivity as hydrogen

The principal of the Beta-ionization detector can br ie f ly be sum- marized as follows, A source of Beta rays of re la t ive ly low energy i s present i n the detector; tritium dissolved i n titanium i s a convenient source. A portion of the carrier gas, which i n t h i s case i s argon, i s raised t o a high energy metastable s t a t e by t h i s radiation on entering the detector. most organic vapours.

This excitation leve l is higher than the ionization potent ia l of Organic vapours present i n the carr ier gas are ionized

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on collision with excited argon atoms as they enter the detector; t h i s causes a considerable increase in current through the cel l , and it is t h i s ionization current tha% is amplified and recorded. A t first glance helium would appear t o be a bet ter choice fo r the carr ier gas but i t s metastable state i s so energetic that impurities normally present i n the gas are ionized, rendering it unstable for use. The sensi t ivi ty here is about 300 times that of the thermal conductivity cell . However, not a l l materials w i l l ionize under these conditions, and it is therefore not as universal as the thermal conductivity detector.

The operation principles of the flame ionization detector are as follows. Sample components are swept by the stream of carr ier gas, usually nitrogen, into a burner assembly where it is mixed with hydrogen gas1 The mixture burns in an atmosphere of a& or oqgen. - The temperature of the hydrogen flame causes a camplex ionization to take place, There is a ring- type collector electrode above the flame Snd the ion current between the burner and the collector is amplified and measured. The magnitude of the output signal is proportional t a the nuniber of carbon atoms passing through the flame; in essence, t h i s I s a cerbon atom counter, The sensit ivity here may even exceed that of the Beta ionization detector. Again, not a l l materials w i l l be detected; f o r a given analysis t h i s may o r may not be advantageous.

A few words about the columns - Packed columns my vary in length from, say, 3 t o 60 feet; usually a 6 t o 8 foot column w i l l be satisfactory. Increasing the length of the column improves t h e separatory power, but not proportionately, aupport may be diatomaceous earth or ground fire-brick frequently, 30 - 68 mesh size, t h a t a similar i ty in the chemical nature of the l iquid phase and the materi- al t o be separated should exist@ Capillary colmns were introduced rather recently. Here the column length m y run t o 100 feet , The I. D. i s about .01 inch and the stationary l iqu ia phase is coated direct ly on the inner w a l l of the capillary. m e resolution on these columns is higher than that of packed columns. Their capacity is huwever low and they must be used i n combination with the more sensitive ionization type detectors. Probably the most eff ic ient and sensitive separation technique currently available i s this combination of capillary column and ionization detector.

The column width is usually about l/4 inch. The iner t

A pract ical rule for the choice of stationary l iquid phase i s

I wish t o ennphasize at th i s point that gas chromatography is essentially a separaeion technique, A completely unknown mixture placed on a GLPC column may be separated but no infommtion concerning the chemical nature of the compounds will be obtained. eluted components and t o identify them either by classical chemical methods o r by physical measurements such as inf'ra-red and ultra-violet spectroscopy o r mass spectrometry o r by a combination of methods depending on the ingen- u i t y of the chenist. materials, then gas chromatography can be used both t o separate and identify.

It is necessary t o t rap out the

However, should one have a mixture of homologous

I would now l ike t o describe Just such an application of gas chromatography in which t h i s technlque was used to separate, identify, and quantitati.vely determine the f ree f a t t y acids present in pork and beef fat. Our interest in th i s problem arose fromthe observation that the differences

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i n the flavor of these meats apparently resided i n the f a t , the lean of these meats contributing an identical basic "meaty" flavor and the fat the distinguishing flavor overtones. acids might i n pa r t contribute t o flavor d i rec t ly or a c t as precursors for the formation of flavor compounds such as carbonyls.

It was our thought that the free f a t t y

The problem resolved i t s e l f into finding a satisfactory method of quantitatively analyzing a mixture of the pure f ree fatty acids and in developing an adequate cleanup procedure for the separation of small amounts of the f a t t y acias from large amounts of fat and subsequently converting the acids t o the i r methyl esters. The methyl esters, because of t he i r lower boiling points, can be separated at lower column temperatures than the cor; responding acids. a re required t o effect separation, thus limiting the materials that can be successfully used as the "liquid" phase. Our choice of l iquid substrate was a commercially available polyvinyl acetate of 1500 molecular weight. The base l i n e d r i f t i n a J l experiments was negligible, The Gas chromatograph used was a Beclanan GC-2. connected t o a 1-mv. recorder. In the next s l ide tbe retention times and separation factors a re reported for commonly found C 1 2 through C i a f a t t y acid methyl esters. 83 ml,/min., temperature 205%. , column packing 15$ polyvinylacetate on Chromsorb "R", column 1/4" 0 , D. and 8' long. The retention time repre- sents the time required f o r a given component to be eluted from the column at the operating conditions described. Retention times are determined by the use of authentic s a q l e s . retention times f o r two components and measure the resolving power of the column. value from unity, a C1, reference acid.

hasn't been so. Chromasorb I'R''. The next several batches gave poorer results. Eventually, we used Chromosorb W. More recently we have used the 30-60 mesh fract ion obtained by sieving Celite 545. 12OoC., and then exposed t o vapor8 of dichlorodimethylsilane. The polar i ty of the Celite surface is mwkedly decreased by t h i s treatment and columns prepared with the support have shown improved separation factors, sharper peaks, and even lower retention t i m e s .

Even so, column temperatures i n the neighborhood of 200 C.

The detector is of the thermal conductivity type

The operating conditions were helium flow ra t e a t ex i t

The separation factors are the ratios of the

The far ther apart the peaks, the greater the deviation of this I n t h i s instance, the separation factors are measured t o

The column support is theoretically iner t , but i n our studies t h i s The resul t8 Just shown were obtained with one batch of

'phis fraction was acid washed, dried a t

The analfiicaJ. procedure developed involves the adsorption on a strong anion exchange resin of the free f a t t y acids from a solution of fat i n petroleum ether t o which a known amount of the C17 s t ra ight chain satur- ated acid has been added, then, decanting the fat plus petroleum ether and preparing the methyl es te rs direct ly on the resin by treatment with anhydrous methanol - El. petroleum ether solution and chromatographed. d i rec t ly re la ted t o the quantity of es te r preseiit. Peak areas a re taken as one-half peak width times peak height. Calibration factors a re obtained by i n i t i a l l y chromatographing standard solutions of authentic esters. The C17 acid added t o an unknown i s used as an internal standard. recovery of the C

The es te rs we , a f t e r ap7ropriete cleanup, obtained i n a The areas under the peaks are

The per cent acid carried through the procedure gives a correction

factor fo r a l l ac ii 8 found. Absolute recoveries range from 60-95jb. In the

90 . next sl ide, recovery data fo r a known mixture of f a t t y acids covering a concentration range similar i n ratio t o that found i n la rd i s shown. t i t a t i v e resu l t s without the internal standard would be unobtainable. It i s interesting tha t with the thermal conductivity detector 2 7 can be readily detected and 257 determined quantitatively. 1/2 milligram is an adequate sample size,

Quan-

In general, 1/4 t o

Free f a t t y acids i n beef and pork fat were determined pr ior t o and after heating i n air at 100°C. the presence of air, the amounts were roughly doubled but no new acids were found. i s the greater amount of f ree unsaturated acid i n pork.

After heating fo r 4 hours a t 100°C. i n

O f in te res t The quantitative results a re shown in the next slide.

Beef f r ee f a t t y acids are s h m i n the next slide, the peaks labeled tetradecenoic, pentadecenoic, hexadecenoic, and heptadecenoic are assigned on the basis of t he i r retention volume rather than by comparison with authentic samples. plied by the flow rate.

The retention volume i s the retention time multi-

It has been shown that by plot t ing the log of retention volume

Thus, f a t t y acid e s t e r s with no double bond fall on one l ine, In the next slide, t h i s

Assigned structures The good agreement with The chromatogram fo r The major quali tative

against the number of C atoms f o r a homologous series, s t ra ight l i nes a re obtained. those w i t h one double bond on another, and so on. plot is shown f o r the peaks in the preceding slide. are black dots, c i rc les are fo r known compounds, prediction renders probable the assignments made. pork f ree f a t t y acids is shown in the next slide. difference is the absence of the C14 unsaturated acid and the presence of l inolenic acid.

Gas chromatography can also be useful f o r quali ty control. The extreme sens i t iv i ty of the capfllary column and ionization detector could be used t o sample the air space above the contents of a canned meat product. One characterist ic chromatogram f o r the trace amounts of vo la t i les might mean a sat isfactory product, another type a defective product. stance, neither quali tative or quantitative knowledge of the separated compounds is important, but the information gained is valuable.

In t h i s in+

Gas chromatography may ultimately unravel the tangle of odor com- pounds that contribute t o meat flavor, and an artist-chemist (he's a bet ter chemist than artist, I hope) at our Laboratory has suggested tha t the eventual chromatogram of beef flavor may resemble that shown i n the next and f ina l slide.

CHAIRMAN PEARSON: Thank you, Dr. Hornstein, particularly f o r the enlightening material on beef flavor.

Our next speaker t h i s afternoon is from the Pioneering Research Division, Quartermaster Research and Engineering Center i n MEbssachusetts.

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He has long been doing work on meat flavor and i s w e l l known, particu- l a r ly for his work on irradiated meat. hear from Dr. Charles Merritt, Jr., who w i l l speak t o us on the use of Mass Spectroscopy as a tool for separating and Identifying meat flavor components. Dr. Merritt.

A t t h i s time we are going t o

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