Application Book Volume 4 Biodiesel Quality Control...14110. Reproducibility of the measurement...

Quality Control According to DIN EN 14103, 14105, 14110

Application Book Volume 4Biodiesel Quality Control

SEG-A-081

III.

II.

I. Determination of methanol in biodiesel 5

Fatty acid methyl esters (FAME) Determination of total ester content and linolenic acid methyl ester contents 9

Determination of glycerol and glycerides 14Part 1: Introduction and sample preparation 15Part 2: Instrument configuration 17Part 3: Optimization of the gas chromatographic method 20Part 4: Signal identification 22Part 5: Evaluation and quantification 25


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Contents

Biodiesel quality control according to DIN EN 14110I. METHANOL

6

Biodiesel production hasincreased considerably inEurope in recent years.

In 1996, production was nearly0.5 million tonnes. Today, theproduction capacity is muchhigher. This development has alsobeen supported by the automo-bile industry. In fact, several mil-lion passenger cars and commer-cial vehicles have been approvedby their manufacturers to run onbiodiesel fuel or higher biodieselblends with mineral diesel. Com-pliance with the European Bio-diesel standard DIN EN 14214ensures a continuously high qual-ity level.

The production of biodiesel isbased on transesterification ofplant oils or animal fats to fattyacid methyl esters (FAME, Fig-ure 1). At a temperature ofapproximately 60 °C, the plantoil, consisting mainly of glycerinesters, is esterified usingmethanol and a catalyst (alkalihydroxide or alkali alcoholate).In order to quantitatively shiftthe equilibrium reaction towardsthe biodiesel or FAME side,methanol is added in excess.

After the reaction is finished, the excess methanol togetherwith several by-products must be removed.

Determination of methanol in biodiesel

The DIN EN 14214 standardspecifies all legal limits for possi-ble by-products. For safety-relat-ed reasons and in order to com-ply with requirements of theautomobile industry, the legallimit for methanol residue hasbeen defined as 0.2 mass percent-

ages (% m/m). DIN EN 14110describes the required method formethanol determination.

GC with headspace injection

Biodiesel can consist of up to 100different fatty acid methyl esters,depending on the plant oils usedduring production. For the deter-mination of methanol in such acomplex matrix, gas chromatog-raphy in combination withheadspace injection is the recom-mended method. This enablesseparation of low-boiling pointmethanol from the high-boilingpoint fatty acid methyl estersalready during the sample prepa-ration step.

The biodiesel samples are heatedin a gas-tight 20 mL vial. Metha-nol is a low-boiling point com-pound and is enriched in the gas-phase while the high-boilingpoint compounds remain in theliquid phase (Figure 2). After aspecific time, equilibrium is

H2C – O – CO – R1

HC – O – CO – R2

H2C – O – CO – R3

Triglyceride

CH3OH

H2C – O – H

HC – O – H

H2C – O – H

Glycerine

H3CO – CO – R1

H3CO – CO – R2

H3CO – CO – R3

FAME (Biodiesel)

Figure 1: Equilibrium reaction of the transesterification of plant oils (triglycerides) to fatty acid methyl esters (biodiesel)

Figure 4: GC-2014 and HT200H

Figure 2: Equilibrium between the liquid- and gas-phase during headspace sample preparation. Although component A is present at a lower concentrationin the liquid phase, the concentration ratio in the gas-phase may shift, depend-ing on the vapor pressure of the individual components. Correspondingly, thechromatograms resulting from headspace injection usually produce differentsignal ratios when compared with those resulting from liquid sample injection.

P

A B

A PB

A

B

� System with headspace autosampler for high precisionresults

� Requirements of DIN EN 14110exceeded

0.5000.00485

0.97

reached and a constant volumecan be sampled from the gas-phase (headspace) of the vial andinjected into the GC system.

Calibration with minimum complexity

Since the biodiesel matrix caninfluence the equilibriumbetween the methanol concentra-tions in the liquid- and gas-phase,calibration standards must beprepared in methanol-freebiodiesel. The DIN EN 14110biodiesel standard recommendspreparation of a calibration curvecontaining 0.01, 0.1 and 0.5 %(m/m) methanol in FAME. Aninternal standard should alwaysbe used when applying manualheadspace injection. When usinga headspace autosampler, aninternal standard will not be necessary but is, however, recom-mended to increase the reliabilityof the results. The additionalwork is kept within reasonablelimits: to each standard and each biodiesel sample, 5 µL of 2-propanol is added (Figure 3).

All measurements are carried outon a Shimadzu GC-2014AFscsystem in combination with theHT200H headspace autosampler(Figure 4). Both instruments areeasy to operate and designed fordaily routine analysis.

For the calibration, 5 g of therespective standard solution aswell as 5 µL of 2-propanol areadded to a 20 mL headspacecrimp vial. Corresponding to 5 gof biodiesel sample, 5 µL of 2-propanol is added as internalstandard. The HT200H can han-dle up to 40 sample vials per tray.

Automated sample preparation

Sample preparation is fully auto-mated, whereby each sample isshaken and heated to 60 °C for

a given time. The autosamplerprepares several samples simulta-neously to ensure the highestpossible sample throughput andto prevent hold-up at the gaschromatograph. A gas volume of500 µL is sampled from theheadspace of each vial and inject-ed into the GC-2014AFsc systemwith a split ratio of 1:10.

Additional parameters are recom-mended according to DIN EN14110:Temperature split injector:150 °CColumn oven (isothermal):50 °CFlame ionization detector:150 °CCarrier gas (helium) linearvelocity: 35 cm/sSeparation column used for the measurements: Restek, Stabilwax-DA 30 m, 0.32 mm, df 1.0 µm

At a methanol concentration of0.01 % (m/m) a signal-to-noiseratio of higher than 2000 :1 wasobtained. Thus, the techniqueoffers many options for improve-ments via application of largersplit ratios or faster chromato-graphic separation.

Results exceeding the standard requirements

The calibration curve in Figure 5demonstrates that the combina-tion of the HT200H autosamplerand the GC-2014 results in highlinearity over the concentrationrange recommended in DIN EN14110. Reproducibility of themeasurement values for biodieselsamples is higher than therequirements according to DINEN 14110 (Table 1). Biodieselsamples with very low methanol

content were spiked with variableamounts of methanol.

Samples with the lowest concen-trations showed the highest dif-ference of two out of six meas-urement values 0.00012 % (m/m).DIN EN 14110 allows for0.00151 % (m/m), a ten-foldhigher deviation.

The combination of the HT200Hand GC-2014AFsc offers an effi-cient as well as accurate automa-tion solution for the routine anal-ysis for methanol determinationin biodiesel. Smaller scale bio-diesel producers will profit fromthis robust analytical system withits excellent price/performanceratio.

7

Biodiesel quality control according to DIN EN 14110 I. METHANOL

Figure 3: Chromatogram of 0.01 %(m/m) methanol in biodiesel. 2-pro-panol was added as internal standard.

Figure 5: Calibration curve of methanol in biodiesel in accordance with DIN EN14110 using internal standard calibration

Table 1: Biodiesel sample spiked with three different methanol concentrations. Table 1 shows the reproducibility over 6 measurements of the same sampleusing the same method on the same instrument.

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

0.0 2.5 5.0 7.5

Met

hano

l

2-Pr

opan

oluV (x 100,000)

0.0

1.0

2.0

3.0

4.0

5.0

0.0 2.5 5.0 7.5 10.0

Area Ratio

Minutes

2

3

Conc

entr

atio

n Ra

tio

Y = aX + ba = 4,601507e-002b = 0.0R^2 = 0.9999938R = 0.9999969

3

Average concentration % (m/m)Reproducibility standard deviation % (m/m)Reproducibility % RSD

0.0090.00005

0.54

0.1000.00100

0.99

Sample 1 2

Biodiesel quality control according to DIN EN 14103

Fatty acid methyl esters (FAME)

II. FAME

10

In the last years, biofuels havebecome well established asan independent source of

energy in the industrial countries.The percentage of biofuels in theenergy mix is expected to in-crease considerably within thenext 10 years in order to reducedependency on fossil fuels and tomaintain energy prices at long-term affordable levels.

Based on the amount of freighttransportation, the need for dieselfuels and consequently biodiesel,is significant compared to otherbiofuels in the European Union.Should the European Parliamentagree with the proposal of theEuropean Commission, EUmember states will add at least 10 % biofuel to fossil fuels beforethe year 2020. In Europe, thedemand for biodiesel will furtherincrease, leading to a continuoussearch for suitable plant oils andanimal fats that can be used asraw materials. Experience showsthat the quality of the raw mate-rial has an enormous impact onthe quality of biodiesel. Continu-ous quality control thereforeremains necessary in order tomeet the demands of moderndiesel generators.

According to DIN EN 14103biodiesel must consist of 90 %(m/m) or more of fatty acidmethyl esters within the range ofC14:0 up to C24:1. The designa-tion Cx:y refers to the carbonnumber or C-number (x) of thecorresponding fatty acid as wellas the number of double bonds(y). When y is larger than 0, it isan unsaturated fatty acid.

Accordingly, the polyunsaturatedlinolenic acid (Figure 1) is desig-nated as C18:3. The content of

linolenic acid methyl ester is alsodetermined. While this compoundis a welcome essential acid in sal-ad oils, it is an interfering reactivecompound in biodiesel that mightinfluence long-term stability ofbiodiesel.

Instrumentation and measurement

A Shimadzu GC-2010AF withAOC-20i autosampler (Figure 2)was used for FAME analysis. TheGC is equipped with a Split/Splitless injector (SPL) as well asa Flame Ionisation Detector(FID). Control of the GC-2010and AOC-20i autosampler as wellas data acquisition was performedby GCsolution software. Alterna-tively the AOC-5000 sampler canbe used if the system will also beused for quality control accordingto DIN EN 14410 (headspaceinjection technique).

Using the special Restek FAME-WAX column it is possible tocompletely separate almost allfatty acid methyl esters fromC6:0 to C24:1. For identificationof the individual fatty acidmethyl esters the Supelco stan-dard “37 Component FAMEMix” (Cat. No. 47885-U) wasused. The standard contains 37

Figure 1: Structural formula for linolenic acid methyl ester (methyl cis,cis,cis-9,12,15-octadecatrienoate)

Figure 2: GC-2010AF with AOC-20i+s

fatty acid methyl esters in therange from C4:0 up to C24:1.

The chromatogram in Figure 3shows the complete separationexcept for C4:0 and the oleic acidmethyl ester C18:1 cis/trans iso-mers. The chromatogram timecan be reduced drastically since a complete separation of all fattyacids methyl esters is not re-quired for the evaluation accord-ing to DIN EN 14103. Good sep-aration is required for linolenicacid methyl ester and the internalstandard C17:0 which is added toeach biodiesel sample. All otherfatty acid methyl esters fromC14:0 to C24:1 are evaluated assum area. Therefore, separationof single peaks is not needed.

Problems could occur if animalfats are used as feedstock forbiodiesel production. In contrastto plant oils which consist of fat-ty acids with even chain lengthnumbers, animal fats include alsouneven numbers including C17:0.Thus co-elution with the addedinternal standard amount must becorrected for if necessary.

Another problem might occurwith plant oils containing fattyacids with chain lengths higherthan C24. The DIN EN 14103-

Determination of total ester content and linolenic

� Determination of iodine value

� Requirements of DIN EN 14103exceeded

� GC system prepared for future challenges

C = x 100 %( ∑ A)-AISTDx

CISTD x VISTD

AISTD m

11

Biodiesel quality control according to DIN EN 14103 II. FAME

Equilibrium time: 1.0 min

2003 determines that all peakswithin the chromatogram rangefrom C14:0 to C 2 4 :1 are evalu-ated for fatty acid methyl estercontent C but peaks outside thisrange must not be included in theevaluation (for calculation of Csee equation on the right). Thusbiodiesel produced from plantoils containing a considerableamount of fatty acids C25:0 orhigher might fail in quality con-trol according to DIN EN 14103.

Gas chromatographic method

GC: GC-2010AF Autosampler: AOC-20iInjection vol.: 1 µLColumn: Restek

FAMEWAX30 m, ID 0.25 mm, df 0.1 µm (Cat. No.12497)

Carrier gas: HeliumControl mode: Constant

linear velocityInjection mode: Split 1:50Linear velocity: 35 cm/s (constant)

Septum purge: 3 mL/minInjector temp.: 250 °C

Oven temperature program FID settings:

Figure 4 on Page 12 shows themeasurement of a biodiesel sam-ple using the method described.For evaluation, the sum of all sig-nal areas between C14:0 andC24:1 is calculated, taking alsointo account the areas of uniden-tified signals. Using the followingequation, the fatty acid methylester content C in % (m/m) canbe calculated according to DINEN 14103 as:

acid methyl ester contents

Hold time(min)1.006.00

Temp. (°C)

150.0240.0

Rate(°C / min)

5.00

Temperature: 250 °CH2 flow: 40 mL/minAir flow: 400 mL/minMakeup flow: 30 mL/min

(He)Sampling rate: 40 msFilter time const.: 200 ms

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

µV (x10,000)

2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5

Minutes

Figure 3: Supelco standard “37 Component FAME Mix” separated on a RestekFAMEWAX column. Not separated are the cis/trans isomers C18:1 (16). C4:0 notseparated from the solvent and the signals (1) - (6) representing the fatty acidmethyl esters C6:0 up to C13:0 are not required for the quality control accordingto DIN EN 14103. It was measured with the described gas chromatographicmethod except for the split ratio was 1:20. The naming of the individual compo-nents is given in Table 3 on Page 13.

∑A total signal area of allmethyl esters from C14:0 up to C24:1(15734000 µV*s)

AISTD signal area of the internalstandard heptadecanoicacid methyl ester C17:0(2644200 µV*s)

CISTD concentration of the heptadecanoic acid methylester in the internal standard solution used (10 mg/mL)

VISTD volume of the internalstandard solution added(5 mL)

m mass of the weighedbiodiesel sample (250 mg)

For the biodiesel sample analyzed(Figure 4), a fatty acid methylester content of 99.0 % wasdetermined. A fatty acid methylester content of 90 % or higher isrequired. The linolenic acidmethyl ester content L is calcu-lated using the following equa-tion as:

L = x 100 %AL

( ∑ A)-AISTD

AL signal area of linoleic acidmethyl ester C18:3(991200 µV*s)

∑A total signal area of allmethyl esters from C14:0to C24:1 (15734000 µV*s)

AISTD signal area of the internalstandard heptadecanoic acid methyl ester C17:0(2644200 µV*s)

The result is 7.6 % linolenic acidwhich is well within the accept-able range of 1 % up to 15 %(m/m). �

g iod / 100 g0.2

55.132.419.8

1.00.3

Biodiesel quality control according to DIN EN 14103II. FAME

12

iodine/100 g was determined (Table 1, the limiting value is 120 g iodine/100 g).

When the same biodiesel sampleis measured repeatedly with thesame instrument, DIN EN 14103allows a maximum difference inmethyl ester content of 1.6 %(m/m) for two arbitrary results.Using the GC-2010 in combina-tion with the AOC-20i autosam-pler, 24 measurements resulted ina range over all results of 0.2 %(m/m) – the difference betweenthe largest and the smallest meas-ured value was therefore 8 timesless than the required value in thenorm.

For the determination oflinolenic acid, a similar result isobtained. Over 24 measurementsthe range over all results wasonly 0.01 % (m/m).

The norm allows for a value tentimes higher i.e. 0.1 % (m/m). All statistical results of the per-formed measurement sequenceare summarized in Table 2.

Summary

The price of biodiesel dependsvery much on the availability ofcheap feedstock for the produc-tion process. This forces the pro-ducers to also use used oils oranimal fats instead of fresh plantoil. Keeping up with this processwill be the future challenge ofquality control for biodiesel.Especially the importance of thequality control according to DINEN 14103 will grow with thisdevelopment.

The performance of GC-2010 in combination with AOC-20i –or alternatively with AOC-5000– not only meets today’s require-ments but is also prepared forfuture challenges. The system isable to separate almost the wholecomposition of fatty acids,including those with odd carbonnumbers from animal fat, in areasonable chromatogram time.This is the requirement for thecorrect determination of theiodine value.

The precision of the system wasshown to exceed the require-ments in the DIN EN 14103 by a factor of ten.

2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5

Minutes

µV (x10,000)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

Figure 4: Biodiesel sample measured with the gas chromatographic methoddescribed. For detailed information on the compounds see Table 3.

Table 1: Calculation of the iodine value for the sample analyzed (Figure 4). The factors for the respective unsaturated fatty acid methyl esters are takenfrom DIN EN 14214 Annex B. The mass percentages are calculated from thechromatogram in Figure 4.

Table 2: Reproducibility over 24 measurements of one biodiesel sample. The table shows the statistical data including the relative standard deviationand the range of all results (max.-min. value).

According to DIN EN 14214Annex B the iodine value can alsobe calculated from the methylester pattern obtained. However,in case of a doubt DIN EN 14111is the preferred method. For cal-culation according to DIN EN14214 Annex B a good separationof the compounds in Table 3marked with asterisk is required.For these compounds a factor tocalculate the iodine value is given.

The percentage by mass obtainedfrom the chromatogram is thenused to calculate the iodine valueof the sample, being the sum ofthe individual contributions ofeach methyl ester, obtained bymultiplying the methyl ester per-centage by its respective factor.

For the biodiesel sample in Fig-ure 4 an iodine value of 109 g

Factor iodine value0.950.861.7322.6160.7850.723

CompoundC16:1C18:1C18:2C18:3C20:1C22:1

Calculated iodine value: 108.8

% (mm)0.20

64.118.7

7.551.320.40

Methyl ester

content

98.9

0.06

99.0

98.8

0.2

Measuring value

Mean value % (m/m)

Standard deviation % (m/m)

Maximum value % (m/m)

Minimum value % (m/m)

Difference maximum/minimum

value % (m/m)

Linoleic acid content

7.55

0.06

7.56

7.55

0.01

CAS No.

106-70-7

111-11-5

110-42-9

1731-86-8

111-82-0

1731-88-0

124-10-7

56219-06-8

7162-64-1

90176-52-6

112-39-0

1120-25-8

1731-92-6

75190-82-8

112-61-8

112-62-9

2462-84-2

112-63-0

2566-97-4

16326-32-2

301-00-8

1120-28-1

2390-09-2

2463-07-7

55682-88-7

21061-10-9

2566-89-4

2734-47-6

6064-90-0

929-77-1

1120-34-9

61012-47-3

2433-97-8

2442-49-1

301-01-9

2733-88-2

13

Biodiesel quality control according to DIN EN 14103 II. FAME

Table 3: Components in the chromatograms in Figure 3 and 4. For the substances marked with an asterisk, the iodine value can be calculated with the given factor.

Peak No.

01

02

03

04

05

06

07

08

09

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

Alternative naming

Methyl caproate, Hexanoic acid methyl ester,

Methyl capronate

Octanoic acid methyl ester, Caprylic acid methyl ester

Decanoic acid methyl ester, Capric acid methyl ester,

Caprate

Undecanoic acid methyl ester

Dodecanoic acid methyl ester, Lauric acid methyl ester,

Methyl laurate

Tridecanoic acid methyl ester

Methyl myristate, Tetradecanoic acid methyl ester,

Myristic acid methyl ester

9-tetradecenoic acid, methyl ester, Methyl cis-9-

tetradecenoate, Methyl myristoleate

Pentadecanoic acid methyl ester

cis-10-Pentadecenoic acid methyl ester

Hexadecanoic acid, Methyl ester

Methyl cis-9-Hexadecenoate, Palmitoleic acid

methyl ester

Heptadecanoic acid methyl ester

Methyl cis-10-heptadecenoate

Methyl octadecanoate

Methyl cis-9-oleic methyl ester, Methyl cis-9-octa-

decanoate (Z)-9-Octadecanoic acid methyl ester

(E)-Methyl 9-octadecenoate

Linolenic acid methyl ester; Methyl linoleate,

cis,cis-Octadeca-9,12-dienoic acid methyl ester

trans, trans-octadeca-9,12-dienoic acid methyl ester

Methyl (Z,Z,Z)-6,9,12-octadecatrienoate,

6,9,12-Octadecatrienoic acid methyl ester

Linolenic acid methyl ester,

Methyl cis,cis,cis-9,12,15-octadecatrienoate

Eicosanoic acid methyl ester

Methyl cis-11-eicosenoate, cis-11-Eicosenoic acid

methyl ester

Dihomo- -linolenic acid methyl ester, Methyl-DGLA

Arachidonic acid methyl ester,

Methyl cis-5,8,11,14-eicosatetraenoate

cis-5,8,11,14,17-Eicosapentaenoic acid methyl ester

Heneicosanoic acid methyl ester

Docosanoic acid methyl ester

cis-13-Docosen, Methyl cis-13-docosenoate;

Erucic acid methyl ester

Tricosanoic acid methyl ester

Methyl tetracosanoate, Lignoceric acid methyl ester

Methyl cis-4,7,10,13,16,19-docosahexenoate,

DHA methyl ester

Nervonic acid methyl ester; Selacholeic acid

methyl ester, Methyl cis-15-tetracosenoate

Molecular formula

C7H14O2

C9H18O2

C11H22O2

C12H24O2

C13H26O2

C14H28O2

C15H30O2

C15H28O2

C16H32O2

C16H30O2

C17H34O2

C17H34O2

C18H36O2

C18H34O2

C19H38O2

C19H36O2

C19H36O2

C19H34O2

C19H34O2

C19H32O2

C19H32O2

C21H42O2

C21H40O2

C21H38O2

C21H36O2

C21H36O2

C21H34O2

C21H32O2

C22H44O2

C23H46O2

C23H44O2

C23H42O2

C24H48O2

C25H50O2

C23H34O2

C25H48O2

Saturation

C6:0

C8:0

C10:0

C11:0

C12:0

C13:0

C14:0

C14:1

C15:0

C15:1

C16:0

C16:1

C17:0

C17:1

C18:0

C18:1 cis

C18:1 trans

C18:2

C18:2

C18:3

C18:3

C20:0

C20:1

C20:2

C20:3

C20:3

C20:4

C20:5

C21:0

C22:0

C22:1

C22:2

C23:0

C24:0

C22:6

C24:1

Compound

Methyl hexanoate

Methyl octanoate

Methyl decanoate

Methyl undecanoate

Methyl dodecanoate

Metyl tridecanoate

Methyl tetradecanoate

Myristoleic acid methyl ester

Methyl pentadecanoate

Methyl cis-10-pentadecenoate

Methyl palmitate

Methyl palmitoleate*

methyl heptadecanoate

cis-10-heptadecenoic acid methyl ester

Methyl stearate

Methyl oleate*

trans-9-elaidic methyl ester*

Methyl linoleate*

Linolelaidic acid methyl ester*

-linolenic acid methyl ester*

Methyl linolenate*

Methyl arachidate

Methyl eicosenoate*

cis-11,14-Eicosadienoic acid methyl ester

cis-11,14,17-Ecosatrienoic acid methyl ester

cis-8,11,14-Eicosatrienoic acid methyl ester

Methyl arachidonate

Methyl 5,8,11,14,17-eicosapentaenoate

Methyl heneicosanoate

Methyl behenate

Methyl erucate*

cis-13,16-Docosadienoic acid methyl ester

Methyl tricosanoate

Methyl lignocerate

cis-4,7,10,13,16,19-Docosahexaenoic

acid methyl

Methyl nervonate

�

�

15

Biodiesel quality control according to DIN EN 14105 III. PART 1

Beef2819455

Comments

Derivatization of OH groups

Buffer

Solvent

Internal Standard 1

Internal Standard 2

Keep refrigerated at -20 °C

Keep refrigerated at -20 °C

Storage below 8 °C

Alternative naming

N-Methyl-N-(trimethylsilyl)-trifluoracetamide

Azine

Tricaprin

1-Mono[cis-9-octadecenoyl]-rac-glycerol

(monoolein)

1,3-Di[cis-9-octadecenoyl]glycerol

(diolein)

1,2,3-Tri[cis-9-octadecenoyl]glycerol

(triolein)

Biodiesel is an interestingalternative to the decreas-ing resources of mineral

fuels. It can be manufacturedfrom all kinds of plant oils oreven fats from fryers.

Because of the high viscositydirect use of those regenerativeenergy resources in diesel carsrequires expensive modificationsto engine and tank. Therefore, thetransesterification (Figure 1) ofthe glycerol esters to fatty acidmethyl esters (FAME) is pre-ferred. FAMEs are less viscousand remain fluid even at low tem-peratures. They can be used inmany diesel cars instead of min-eral diesel without changes to thecar. Presently the biggest marketfor biodiesel is the blending withmineral fuels (e.g. B5; up to 5 %biodiesel in mineral diesel).

Introduction and sample preparation

Biodiesel mainly consists ofmethyl esters of the followingfatty acids:

Palmitic acid C16 (saturated)Stearic acid C18 (saturated)Oleic acid C18 (unsaturated)Linoleic acid C18 (poly unsatu-

rated)

The distribution changes depend-ing on the plant oil used (Table1). In principle, all fatty acidswith chain lengths from C14 toC24 may be found (in case ofplant oils only the even num-bers).

FAMEs are the products of thereactions in Figure 1.

Dependent on process parametersthe transesterification is more orless quantitative. Generally, a cer-

tain amount of mono-, di-, andtriglycerides remains in thebiodiesel. Additionally, the sur-plus of methanol and the by-product glycerine must beremoved during the biodieselproduction. In quality controlaccording to DIN EN 14105 orASTM D 6584-00 the contents ofthe glycerine, mono-, di-, andtriglycerides are determined. The methanol content is meas-ured with headspace techniquedescribed in DIN EN 14110.

Sample preparation

Gas chromatography is a relativetechnique. Every componentmust be calibrated before it canbe quantified in unknown sam-ples. In the case of biodiesel onemono-, di-, and triglyceride iscalibrated, representative for �

Table 1: Approximate content of fatty acids in different oils and fats

Table 2: List of components required for biodiesel applications. Possible supplier: Sigma-Aldrich (Fluka). Due to derivatization with MSTFA all components must be water-free.

Figure 1: Transterification of triglycerols to FAME

Property fatty acid in %PalmiticStearicOleicLinoleic / Linolenic

Rape seed51

6030

Sun flower64

2861

Palm tallow425

4110

Compound

MSTFA

Pyridine

Heptane

1,2,4-Butanetriol

1,2,3-Tridecanolyglycerol

Glycerin

1-Monooleoglycerine

(Monoolein)

1,3-Dioleoylglycerine

(Diglyceride)

1,2,3-Trioleoylglycerine

(Triglyceride)

No.

1

2

3

4

5

6

7

8

9

CAS No.

24589-78-4

110-86-1

142-82-5

3068-00-6

621-71-6

56-81-5

111-03-5

2465-32-9

122-32-7

� Control of transesterification process

� Simplified standard preparation

� Improved reproducibility

H2C – O – CO – R1

HC – O – CO – R2

H2C – O – CO – R3

Triglyceride

CH3OH

H2C – O – H

HC – O – H

H2C – O – H

Glycerine

H3CO – CO – R1

H3CO – CO – R2

H3CO – CO – R3

FAME (Biodiesel)

Triolein

0.050.20.350.5

4100 µL

100 µL

Calibration levelVolume of stock solution A given into a 10 mL flaskAdd the given volume of stock solution C into the same flask

Biodiesel quality control according to DIN EN 14105III. PART 1

16

Table 3: Preparation of the four standard solutions

Table 4: Compound concentrations in the four calibration levels (given in mass %)

the other components of eachgroup. Table 2 gives an overviewof chemicals needed for prepara-tion of standard and unknownsamples. Required is a four levelcalibration of the components 6to 9. Glycerin (6) is calibratedrelative to the internal standard 1(ISTD1). The glycerides (7-9) arecalibrated relative to internalstandard 2 (ISTD2).

Preparation of standard solutions

In deviation from DIN EN 14105,we recommend the followingstandard preparation. Three stocksolutions A, B and C are pre-pared. Later, only the stock solu-tions A and C are needed forpreparation of standard levels andunknown samples.

Stock solution A: Internal standard50 mg 1,2,4-butanetriol and 400 mg tricaprin are given into a50 mL flask. Pyridine is added upto the 50 mL marking.

Stock solution B: Glycerin dilution50 mg glycerin are given into a 10 mL flask. Pyridine is added upto the 10 mL marking.

Stock solution C: Olein standard solution with glycerin200 mg monoolein, 50 mg dioleinand 50 mg triolein are given intoa 10 mL flask. 1 mL of the stocksolution B is added. Pyridine isadded up to the 10 mL marking.

The three stock solutions can bekept in a refrigerator for weeks.For preparation of the standard

levels add the given volumes ofstock solution A and C (Table 3)into 10 mL flasks.

Derivatization of the calibrationmixtures

Derivatization of the sampleswith MSTFA is crucial forimprovement of peak shape andchromatographic separation.

100 µL MSTFA are added to eachof the four flasks preparedaccording to Table 3. Close theflasks, tighten and shake themwell. At room temperature thederivatization requires a mini-mum of 15 minutes. After thattime add 8 mL of n-heptane toeach flask and shake again. Thesample is now ready for measure-ment.

Table 4 shows the concentrationsin mass-% for the given compo-nents in the respective calibrationlevels.

Preparation and derivatization ofunknown biodiesel samples

100 mg biodiesel is given into a10 mL flask. 100 µL of stocksolution A (internal standard) areadded to the flask. Then add 100µL of MSTFA, close the flask,tighten, and shake it well. Thederivatization takes at least 15minutes at room temperature.After that time add 8 mL of n-heptane to each flask and shakeagain.

Important

Derivatized standards or samplesmust be measured within 24hours.

1100 µL

10 µL

2100 µL

40 µL

3100 µL

70 µL

Monoolein

0.20.81.42.0

Calibration levelconc. in mass %

1234

Glycerin

0.0050.020.0350.05

Diolein

0.050.20.350.5

17


With this application theinternal standardmethod is used for

quantification so an auto sampleris not required, but recommend-ed for better reproducibility ofretention times.

The injection technique is “coolon-column” injection (OCI) toavoid discrimination effects. WithOCI injection a thin needle of 26gauge – outer diameter (OD) 0.47mm – is inserted into the columnand the sample is injected com-pletely on the column. Foraccommodation of the syringeneedle, the capillary column musthave a minimum internal diame-ter (ID) of 0.53 mm. In principle,a 0.53 mm column can be used asmain column for this application,but for better separation andfaster chromatogram times asmaller ID is preferred.

Therefore, a retention gap – usu-ally a 2 m uncoated fused silicacapillary with an ID of 0.53 mm– is used as pre-column. Connec-tion of the retention gap with themain column can be done with ametal connector (Figure 1). Thepreferred glass connectors cannotbe used with this application dueto the high oven temperatures –most glass connectors have amaximum temperature of 350 °C,

Instrument configurationan exception being the RestekVu2 connector with a maximumoperation temperature of 400 °C.

Another alternative is the “simpleon-column” injection techniqueoffered by Shimadzu. Instead ofretention gap a special glass lineris used. Design of the liner issimilar to a glass connector (seeFigure 2). The injection is doneinto a small liner compartmentdirectly above the column.

Column mounting with retention gap

If a retention gap is used the con-nection to the main column isoften done with a metal connec-

tor (e.g. SGE retention gap kitart. no. 052296).

For best performance both col-umns should meet in the middleof the connector. Since columnswith ID of 0.25 mm or smaller fitinside the ID 0.53 mm retentiongap, the optimum position is noteasy to find. A compromise couldbe pushing the main column upto 5 mm inside the retention gap(Figure 1). More than 5 mmwould lead to peak tailing andpoor results.

For accuracy and precision of the results it is essential that theconnection of the retention gapto the main column is gas tight. �

Figure 1: Mounting of retention gap kit with SGE metal connector

Column and retention gap connection with metal connector

� “Simple on-column” injectiontechnique

� Significantly simplified columnmounting

� More precise results throughglass liner

� Improved reliability

Figure 2: Mounting of column with simple on-column liner

Biodiesel quality control according to DIN EN 14105III. PART 2

18

Due to the high oven temperaturerange the metal connector mayeventually leak during operation.Therefore, it needs to be checkedregularly. Because of this uncer-tainty it is not recommended touse metal connectors in combina-tion with hydrogen as carrier gas.

Column mounting with liner

An alternative offers the “simpleon-column” injection techniqueoffered by Shimadzu. The task ofthe retention gap is taken by aglass liner inside the injector. Anycolumn ID from 0.1 to 0.53 mmcan be directly connected to theliner. The column sticks insidethe liner reduction and is addi-tionally tightened with a graphiteferrule on the injector capillaryadapter.

This simplifies the columnmounting significantly, and gastightness even with hydrogen ascarrier gas poses no problem any-more. Two different liners areavailable from Shimadzu depend-ing on the column inner diameter.

• 221-49381-02 for column ID ≥ 0.32 mm

• 980-00371 for column ID ≤ 0.25 mm

The injection is performed in asmall liner compartment abovethe column so a special on-col-umn syringe is not required. Anormal 23 gauge needle can beused.

Measuring the glycerol content inbiodiesel, more precise resultswere found using the liner com-pared to retention gap solution.

Disadvantage of the liner is thelower capacity for contaminationcoming from biodiesel samples.Retention gaps are good traps forhigh-molecular components in

biodiesel samples. If the first 20 -30 cm of the retention gap are cutfrom time to time the main col-umn is less affected by contami-nants.

With the liner the main column ismore affected by these contami-nants and it might be necessaryto cut 20 - 30 cm from the col-umn regularly. However, the linercan be recycled. Best is to clean itin an ultrasonic bath using firstheptane and then methanol assolvents. It can be dried using anitrogen gas stream or alterna-tively, by heating the liner in anoven. It is important that it doesnot come into contact with con-taminated surfaces. Deactivationof the glass liner is not necessary.

Capillary column

For good performance with thisapplication the GC oven is oper-ated at temperatures of 360 °C orhigher. Unfortunately, most metalcolumns or aluminium coatedcolumns show severe problemswith recovery of di- and triglyc-erides. Generally with fused silicahigh-temperature columns, therecoveries are better.

Often used is the HT5 capillarycolumn from SGE. It’s a fusedsilica column with temperaturelimits 380/400 °C – meaning thecolumn can be operated at 380 °Cfor longer times, at 400 °C onlyshort term. Also possible is theHT8 phase from SGE but due totemperature limit 360/370 °C thechromatogram time is extended.

An alternative to the SGE col-umns could be the new Restekhigh-temperature columns. Restek offers an RTX-BiodieselTG with a temperature limit of380 °C (Cat. No. 10294). Uponrequest they also offer Shimadzua Fast GC column of the same

Figure 3: Cross-section through a capillary column

19


type, which reduces the analysistime to below 17 min. Restek alsooffers metal columns for biodie-sel analysis e.g. MXT BiodieselTG (Cat. No. 70289). Due to aspecial deactivation process themetal columns show a goodrecovery for all biodiesel compo-nents including the triglycerides.The MTX column is a mega-borecolumn with an Integra-Gap,trapping contaminants beforethey enter the separation column.

Conditioning of the system

Before starting with measure-ments the conditioning of thesystem is essential. A new col-umn should be conditioned for12 hours using the temperatureprogram for injector and detec-tor. A metal connector should bechecked for leakage after the firsttwo conditioning runs.

Figure 4 shows a typical trend of a baseline over more than 20blank runs.

A temperature program was usedheating with 10 °C/min from 65 °C up to 380 °C. At the begin-

Figure 4: Column conditioning. Between red and green line the column wascycled about 20 times.

Figure 5: Baseline drift or “ghost peak” due to liner contamination

0.0

5.0

1.0

2.0

3.0

4.0

5.0uV (x 10,000)

35.030.025.020.015.010.0

Minutes

0.0

5.0

1.0

2.0

3.0

4.0

5.0

uV (x 10,000)

35.025.0 30.020.015.010.0

Minutes

Gly

ceri

n

But

anet

riol

ISTD

1

Mon

oole

in

Tric

apri

n IS

TD2

Dig

lyce

ride

Trig

lyce

ride

ning a baseline drift of about 30mV or more was observed. After12 hours it was reduced by 10 to20 mV. Figure 4 shows some con-ditioning steps with a 0.1 µm filmcolumn. Of course, a 0.25 µmfilm column might show a moresevere baseline increase. A lowerbaseline drift of about 5 - 15 mVwas observed with the RestekRtx-5 high-temperature column.

Ghost peaks

Even if the conditioning chro-matogram looks good it is recom-mended to inject pure heptaneonce before measuring biodieselsamples. Sometimes a baselineeffect might occur as shown inFigure 4.

Most probably the liner was con-taminated by contact with sur-faces covered by a fatty film (forexample the human skin). If theeffect occurs during operation ofthe system the reason may be thesamples themselves. For examplethe biodiesel additives, ifbiodiesel from a gasoline stationwas measured, overaged MSTFAwhich was used for derivatiza-

tion, or the chemicals and sol-vents used, which were notwater-free.

Normally, it is not possible to eliminate these “ghost peaks” byseveral injections of heptane. A new or cleaned liner has to beinstalled and 10 cm should be cutfrom the capillary column.

Hold time (min)1.003.00

III. PART 3 Biodiesel quality control according to DIN EN 14105

20

After leak check and con-ditioning of the instru-ment a first sample can

be injected. DIN EN 14105 recommends a non-polar columnwith length of 10 m, ID (innerdiameter) 0.32 mm, film thickness0.1 µm.

This is a good column to startwith, but most operators prefer a longer column (12 to 25 m).With smaller ID a better resolu-tion can be achieved even with ashorter column (e.g. 12 m HT5,ID 0.22 mm, 0.1 µm). In general,a smaller ID allows a faster chro-matography, but optimizing thegas chromatographic methodmight be more time-consuming.

Gas chromatographic method

GC: GC-2010AF with OCIAutosampler: AOC-20iColumn: HT5, 25 m, ID 0.32mm, df 0.1 µm with retention gapID 0.53 mmCarrier gas: Helium Injection mode: DirectLinear velocity: 50 cm/s (constant)

Septum purge: 3 mL/min

OCI temperature program

Optimization of the gas chromatographic method

FID settingsTemperature: 400 °CH2 flow: 47 mL/minAir flow: 400 mL/minMake-up gas flow: 30 mL/min(He)Sampling rate: 40 msFilter time constant: 200 ms

Oven temperature program

Autosampler

Heptane is used as solvent forbiodiesel samples. The lifetime ofmicro-syringes with biodiesel canbe extended if the syringe isrinsed regularly with a polar solvent (e.g. methanol) besidesheptane. With AOC-20i+s thiscan be automated by using thethree vial wash option. If noAOC-20s is used optional threewash stations can be inserted intothe AOC-20i long rack. Thewash cycle could be 1 x heptane, 1 x methanol and 1 x heptaneagain.

AOC-20i settingsInjection volume: 1 µL# Rinses solvent post-run: 2

or alternatively:

# Rinses solvent post-run: 3(if three vial wash option is used)

# Rinses with sample: 2Plunger speed (suction): LowPlunger speed (injection): HighSyringe insertion speed: HighInjection mode: NormalPumping times: 5

Rate (°C/min)

20.00

Temperature (°C)60.0

380.0

Table 1: OCI advanced settings fan: 100 °C

Table 2: Equilibrium time: 0.5 min

Table 3: Naming of biodiesel components

Hold time (min)1.000.000.009.00

Rate (°C/min)

15.008.00

15.00


170.0270.0390.0

The next step is the optimizationof the method according to thecolumn used. The first step isinjection of the standard solution4 (highest concentration).

The standard contains one representative component foreach group of glycerides.

Butanetriol and tricaprin areadded as internal standards. Evenwhen refrigerated, the stock solu-tions for the standards alter slow-ly. This is the reason for the addi-tional peaks in the chromatogram(Figure 1).

Since the glycerin concentrationcounts significantly in the finalevaluation, a good separationfrom the solvent peak is crucial(Figure 1: more than 1 minutebetween solvent peak and glyc-erin).

Typically the baseline driftbecomes steeper if the oven tem-perature rises above 340 °C (Fig-ure 1: chromatogram time 23 - 26

Used namingGlycerinButanetriol (ISTD1)MonooleinTricaprin (ISTD2)DiglycerideTriglyceride

Alternative naming

1-Mono[cis-9-octadecenoyl]-rac-glycerol (monoolein)1,2,3-Tridecanolyglycerol1,3-Di[cis-9-octadecenoyl]glycerol (diolein)1,2,3-Tri[cis-9-octadecenoyl]glycerol (triolein)

� Optimization of the methodaccording to columns used

� A good separation is crucial

III. PART 3

21


Figure 1: Chromatogram standard solution 4 Figure 2: Chromatogram standard solution 1

Table 4: Variation possibilities of the oven temperature program for optimization of retention times

-0.5 50.0

75.0

100.0

125.0

150.0

175.0

200.0

225.0

250.0

275.0

300.0

325.0

350.0

375.0

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.0 10.0 15.0 20.0 25.0 30.0 35.0

uV (x 10,000)

Column Temp. (Monitor)

Gly

ceri

n

But

anet

riol

ISTD

1

Mon

oole

in

Tric

apri

n IS

TD 2

Dig

lyce

ride

Trig

lyce

ride

Minutes

°C

-0.5 50.0

75.0

100.0

125.0

150.0

175.0

200.0

225.0

250.0

275.0

300.0

325.0

350.0

375.0

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.0 10.0 15.0 20.0 25.0 30.0 35.0

uV (x 10,000)

Column Temp. (Monitor)

Gly

ceri

n

But

anet

riol

ISTD

1

Mon

oole

in

Tric

apri

n IS

TD 2

Dig

lyce

ride

Trig

lyce

ride

Minutes

°C

min). Fortunately, the drift is nottoo large here. Therefore, it isacceptable that the diglycerideselute within this drift. A biggerdrift could affect the integrationaccuracy of the whole group ofdiglycerides eluting from 23 - 24minutes. In such cases an increaseof the linear velocity (up to 70cm/s) or changing of the oventemperature might help.

The oven temperature programcan be slowed down by changingthe end temperature (Table 4,blue marked temperature).

Alternatively, the temperaturerate in line 4 can be decreased

Hold time (min)1.000.000.00

X

Rate (°C/min)

15.008.00

10.00


170.0300.0390.0

(red marked value in Table 4). A combination of both is alsopossible. In any case the holdtime X in line 4 has to be adjust-ed so that the chromatogram timedoes not become too long but thetriglyceride signals are stillrecorded.

Extending the temperature pro-gram has a disadvantage: the tri-glycerides remain too long on thecolumn. The peak might disap-pear completely or become verysmall.

After optimization of the temper-ature program the standard solu-tion 1 (lowest concentration) is

injected to check the detectionlimits.

In the high-temperature range theFID noise is 40 µV. Peak heightof the triglyceride peak is 2002µV. Thus, the signal-to-noiseratio is 50. This is acceptable fora 0.32 mm column, but could beimproved by accelerating thechromatography for exampleusing a higher linear velocity.


22

Since the standards containonly one component ofeach group (mono-, di- and

triglycerides, Figure 1) the identi-fication of all components mustbe carried out in a real biodieselsample (Table 1).

Four groups are evaluated in thebiodiesel quality control – “free”glycerin, the monoglycerides,diglycerides and triglycerides.

Signal identification

Glycerin and monoglycerides areevaluated as single peaks andgrouped by the software. Di- andtriglycerides are integrated as onegroup respectively.

Identification

The biodiesel chromatogram(Figure 3) starts with glycerin andthe first internal standard butane-triol. After the FAME peaks

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.0 10.0 15.0 20.0 25.0 30.0

uV (x 10,000)

Gly

ceri

n But

anet

riol

ISTD

1

1-M

onoo

leoy

l-ra

c-gl

ycer

ol

Tric

apri

n IS

TD 2

1,3-

Di[

cis-

9-oc

tade

ceno

yl]g

lyce

rol

1,2,

3-Tr

i[ci

s-9-

octa

dece

noyl

]gly

cero

l

Minutes

Figure 1: Standard chromatogram measured on a 25 m HT5, ID 0.32 mm, df = 0.1 µm with retention gap

Table 1: Naming of biodiesel components

Figure 2: Glycerin and butanetriol peak zoomed from the biodiesel chromatogram in Figure 3

0.5

1.0

1.5

2.0

2.5

3.0

3.25 3.50 3.75 4.00 4.25 4.50

uV (x 10,000)

Gly

ceri

n

But

anet

riol

ISTD

1

Minutes

Used naming

Glycerin

Butanetriol (ISTD1)

Monopalmitin

Monolinolenin

Monoolein

Monostearin

Tricaprin (ISTD2)

Diglyceride

Diglyceride (rest)

Triglyceride

Triglyceride (rest)

Alternative naming

“Free” Glycerin

1-Monopalmitoyl-rac-glycerol

1-Monolinoleoyl-rac-glycerol

1-Monooleoyl-rac-glycerol

1-Monostearoyl-rac-glycerol

1,2,3-Tridecanolyglycerol

1,3-Di[cis-9-octadecenoyl]glycerol

All other diglycerides

1,2,3-Tri[cis-9-octadecenoyl]glycerol

All other triglycerides

� Four groups to be evaluated inbiodiesel quality control

� Correct identification of monoglycerides is challenging

III. PART 4

23


(from 10 to 14.5 minutes) themonoglycerides elute followed bythe second internal standard tri-caprin, and the di- and triglyc-erides.

From the standard chromatogram(Figure 1) the retention times ofglycerin, butanetriol (Figure 2) aswell as tricaprin are known.

Additionally one component ofeach group• 1-monooleoyl-rac-glycerol

(monoglycerides) • 1,3-di[cis-9-octadecenoyl]

glycerol (diglycerides) • 1,2,3-tri[cis-9-octadecenoyl]

glycerol (triglycerides) is identified in the standard chro-matogram.

In principle, four monoglyceridesshould be found in the chro-matogram. In case of the diglyc-erides up to 10 separate peaks arein the chromatogram but for thetriglycerides seldom more thantwo peaks are found. In thebiodiesel sample (Figure 3) onlyone major triglyceride peak wasfound.

Figure 4a shows the monoglyc-eride pattern stated in DIN EN14105. For comparison Figure 4bshows the corresponding partzoomed out of Figure 3. Accord-ing to DIN EN 14105 the peakmarked with a green arrow ismonolinolenin. In order to get apositive identification for allmonoglycerides a mixture fromSupelco was injected containingall four monoglycerides. Theresulting chromatogram showsthree peaks only. Using the purecomponents a co-elution ofmonolinolenin and monoolein onthe SGE HT5 column could beproven. �

Figure 3: Biodiesel chromatogram measured on a SGE HT5, 25 m, ID 0.32 mm, df 0.1 µm with a retention gap 2 m, ID 0.53 mm

Figure 4a: Monoglyceride patterntaken from DIN EN 14105

Figure 4b: Monoglycerides zoomed from the biodiesel chromatogram in Figure 3

0.00

0.25

0.50

0.75

1.00

1.25

1.50

2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0

uV (x 10,000)

Gly

ceri

n

But

anet

riol

ISTD

1

Mon

opal

mit

in

Mon

oste

arin Dig

lyce

ride

s

Trig

lyce

ride

s

Mon

oole

in +

Mon

olin

olei

n

Minutes

100

150

200

250

300

350Column Temp. (Monitor)

°C

Mon

opal

mit

in (1

6:0)

Mon

olin

olen

in (1

8:1,

2,3)

Mon

oole

in (1

8:1,

2,3)

0.00

0.25

0.50

0.75

1.00

1.25

1.50

1.75

2.00

14.0 14.5 15.0 15.5 16.0 16.5

uV (x 100,000)

Mon

opal

mit

in

Mon

oole

in +

Mon

olin

olei

n

Mon

oste

arin

Minutes


24

The same result was found onother non-polar columns (e.g.Zebron ZB 5HT). Therefore, a positive identification is recom-mended by using a monoglyc-eride mixture – for more infor-mation see Appendix A.

The diglycerides appear as onegroup of peaks in the chromato-gram (Figure 5a). In most casesthe biggest peak is the 1,3-di[cis-9-octadecenoyl]glycerol. Usingthis as marker the start and endof the “diglyceride peak group”can be identified according to thepattern shown in the DIN ENstandard (Figures 5a and 5b).

If identification of start and endof the group is uncertain it is better to implement one peak toomany than one too less. Mostlythe peaks are very small and donot contribute much to the finalconcentration.

It may happen that no triglyc-eride signals can be found in abiodiesel chromatogram. Rarely

four peaks can be found as shownin the pattern given in DIN EN14105 (Figure 6a).

Often only the signal of 1,2,3-tri[cis-9-octadecenoyl]glycerol isfound. This makes the definitionof the triglyceride group difficult.

According to the DIN EN stan-dard the middle of the group corresponds approx. with theretention time of the 1,2,3-tri-[cis-9-octadecenoyl] glycerol peakplus 0.4 minutes. We recommendevaluating all peaks within therange of the resulting retentiontime ± 1.5 minutes as triglycer-ides (Figure 6b). Dependent oncolumn and temperature programused also ± 1.0 minute could besufficient.

In Figure 6b the retention time of 1,2,3-tri[cis-9-octadecenoyl]glycerol is 30.27 minutes, plus 0.4 yields 30.67 minutes. Withinthe interval from 29.17 to 32.17minutes all peaks were evaluatedas triglycerides.

Appendix AThe mixture of monoglycerides from Sigma

Aldrich (Supelco) is custom designed.

It contains:

• 1-Monopalmitoyl-rac-glycerol 25 % w/w

• 1-Monolinoleoyl-rac-glycerol 25 % w/w

• 1-Monooleoyl-rac-glycerol 25 % w/w

• 1-Monostearoyl-rac-glycerol 25 % w/w

• Total of 100 mg per ampoule

C-34

:DG

C-36

:DG

C-38

:DG

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

22.25 22.50 22.75 23.00 23.25 23.50 23.75

uV (x 1,000)

1,3-

Di[

cis-

9-oc

tade

ceno

yl]g

lyce

rol

Minutes

C52:

TG

C54:

TG

C56:

TG

C58:

TG

7.5

8.0

8.5

9.0

9.5

10.0

10.5

28.5 29.0 29.5 30.0 30.5 31.0 31.5 32.0

uV (x 1,000)

1,2,

3-Tr

i[ci

s-9-

octa

dece

noyl

]gly

cero

l

Minutes

Figure 5a: Diglyceride pattern takenfrom DIN EN 14105

Figure 5b: Diglycerides zoomed from the biodiesel chromatogram in Figure 3

Figure 6b: Triglycerides zoomed from the biodiesel chromatogram in Figure 3

Figure 6a: Diglyceride pattern takenfrom DIN EN 14105

When ordering please specify custom-

designed according to Lot No. DE1675 made by

Sigma Aldrich Europe.

The components in the ampoules can be

dissolved in heptane. It may be treated as a

biodiesel sample. Just add internal standard

and MSTFA for derivatization.

25


Table 1: Biodiesel components used to determine response factors of the different groups

Figure 1: Diglycerides in a biodiesel chromatogram After identification of the

components in a biodieselchromatogram (Figure 3,

Part 4 on of this applica-tion manual) quantification willbe the next step. According toDIN EN 14105 a calibrationcurve is measured for one com-pound of each group. The calcu-lated response factors are adaptedfor all components of the respec-tive group.

Representative for all other com-ponents, the compounds in Table1 are calibrated by determinationof a four level calibration curve.

Quantitation parameters in GCsolution

The application uses an InternalStandard calibration with fourcalibration levels (concentra-tions). The curve fit type is set to “linear”. The calibration curveis not forced through zero. Aweighting method is not used andthe unit is mass-%.

For identification the “Band”method is used i.e. for everyidentified component an individ-

Evaluation and quantification

ual time interval, retention time ± ∆t, can be selected. Dependingon the column used a “DefaultBand Time” between 0.05 and 0.1min is sufficient. Relative reten-tion times should be selected as“Identification Method” – espe-cially if the samples are injectedmanually.

Under “Peak” select “All Peaks”and for grouping “ConcentrationSummation” is selected. In thenext step the compound table hasto be created – see Tab. 2 on Page26 as an example. The concentra-tions of the calibrated compo-nents are set according to Part 1(Page 15 ff) of this manual (sam-ple preparation). Uncalibratedcomponents are set to 1.

Under “Type” the Internal Stan-dard components are marked as“Internal Standard and Refer-ence” if relative retention timesare used. According to DIN EN14105 butanetriol is used as inter-nal standard for glycerin and tri-caprin for all other groups. Thiscan be set under “Internal Stan-dard Groups” in the compoundtable (ISTD groups, Table 2).

It is recommended to add thesame internal standard amount toall calibration standards as well asto all unknown biodiesel samples.This would allow setting theinternal standard concentrationsin the compound table equal tothe “Internal Standard Amount”of the unknown samples – theeasiest way is to set all to 1.

Alternative naming“Free” Glycerin1-Monooleoyl-rac-glycerol1,3-Di[cis-9-octadecenoyl]glycerol 1,2,3-Tri[cis-9-octadecenoyl]glycerol

Used namingGlycerinMonooleinDiglycerideTriglyceride

1.0

0.5

1.5

2.0

2.5

3.0

3.5

4.0

4.5

22.25 22.50 22.75 23.0 23.25 23.50 23.75

uV (x 10,000)

22.3 min 23.6 min

1,3-

Di[

cis-

9-oc

tade

ceno

yl]g

lyce

rol

Settings for di- and triglycerides

In Part 4 (Page 22 ff) of this man-ual the identification of the di-and triglycerides was discussed.For quantification of these “peakgroups” GCsolution softwareoffers a special tool using theidentification by band (Figure 1).�

� GCsolution “Band Method” for easy quantification of di- and triglycerides

� GCsolution “Grouping Function-ality” calculates the requiredtotal glyceride content

� Easy handling of multiple internal standards


26

According to the diglyceride pat-tern in DIN EN 14105 all peaksin the retention time range from22.3 to 23.6 minutes belong tothe diglyceride group. The 1,3-di[cis-9-octadecenoyl]glycerol ispositively identified by calibra-tion. The set retention time (RT)in the compound table is themiddle of this interval:

The band (∆t) is the differencedivided by two:

Since 1,3-di[cis-9-octadecenoyl]glycerol is the only positively

identified diglyceride it is recom-mended to quantify it separately.Therefore, the diglycerides havetwo entries in the compoundtable: diglyceride (1,3-di[cis-9-octadecenoyl]glycerol and di-glycerides (rest) representing allother components of this group.

It is important that diglycerideranges in the compound tablebefore diglycerides (rest). GCso-lution will then quantify diglyc-eride first and does not imple-ment this peak in the diglycerides(rest) group.

The calculation of the triglyc-erides is performed in a similarway. The determined retentiontime interval from 29.17 to 32.67minutes yields a retention timeRT = 30.67 minutes and band ∆t = 1.5 minutes (Table 2).

Conc. 2

0.05

1

1

1

2

1

1

0.5

1

0.5

1

Conc. 2

0.035

1

1

1

1.4

1

1

0.35

1

0.35

1

Name

Glycerin

Butanetriol ISTD1

Monopalmitin

Monolinolein

Monoolein

Monostearin

Tricaprin ISTD2

Diglyceride

Diglycerides (rest)

Triglyceride

Triglycerides (rest)

Type

Target

ISTD&Ref.

Target

Target

Target

Target

ISTD&Ref.

Target

Target

Target

Target

Ret. time

3.442

4.281

14.642

15.921

16.102

16.35

20.488

23.154

22.95

30.3

30.67

Band

Default

Default

Default

Default

Default

0.02

Default

Default

0.65

Default

1.5

ISTD group

1

1

2

2

2

2

2

2

2

2

2

Group ID No.

1

0

2

2

2

2

0

3

3

4

4

Conc. 1

0.005

1

1

1

0.2

1

1

0.05

1

0.05

1

Conc. 2

0.02

1

1

1

0.8

1

1

0.2

1

0.2

1

Table 2: Example of a compound table layout in GCsolution software. Clicking with the right mouse button on the compound table shows the function “Table Style.” This allows hiding of unnecessary columns and defini-tion of a convenient format.

Table 3: Response factor settings in the compound table after switching to Curve Fit Type: Linear RF (Linear)

Y = aX + b Internal standarda = 0.5415221 Calibration curve: Linearb = 4.398673e-003 Original: Not forcedR^2 = 0.9998509R = 0.9999255

Figure 2: Calibration curve for monoolein

0.000

0.003

0.005

0.007

0.010

0.013

0.015

0.018

0.020

0.0 1.0 2.0 3.0

Conc. ratio (x 100)

Area ratio

1

2

3

4

Y(0)00000000000

RF0.09161

00.541520.541520.541520.54152

00.627610.627610.81650.8165

NameGlycerinButanetriol ISTD1MonopalmitinMonolinoleinMonoolein MonostearinTricaprin ISTD2Diglyceride Diglyceride (rest)TriglycerideTriglyceride (rest)

RT = = 22.95 minRT1 + RT2 =

22.3 + 23.62 2

∆ t = = 0.65 min=RT2 - RT1

223.6 - 22.3

2

III. PART 5

27


Calibration

After measuring the four stan-dard samples with this methodGCsolution calculates a calibra-tion curve for each group (Figure2). It is important to check theoffset “b” of all calibrationcurves. If the deviation from zerois within acceptable tolerancesand the curve fit is also good, theresponse factors can be adaptedfor the other group constituents.

Quantitation method

In the GCsolution “CalibrationCurve Window” the calculatedresponse factors can be finallyapplied to the respective compo-nents. Under “Quantitative” the“Curve Fit Type” is changed to“Manual RF (Linear).” The cali-bration curves disappear and inthe compound table the “Conc.”rows are replaced by the rows“RF” and “Y(0).” RF is the res-ponse factor and equal to “a” inFigure 2. Y(0) is the offset andequal to “b.” For glycerin, mono-olein, di- and triglyceride the RFvalues are set equivalent to thevalues determined by the calibra-tion curves. These can be copiedto the other components respec-

Table 7: Limits for the content of the different glycerides and free glycerinaccording to DIN EN 14214

Table 4: Group table definition in GCsolution. Like all other tables in GCsolution the layout of the group table can be changed using the “TableStyle” function available by right mouseclick on the respective table.

Table 6: Group results table in GCsolution software

Table 5: Compound results table in GCsolution software

Group ID No.1234

NameGlycerinMonoglyceridesDiglyceridesTriglycerides

Unitmass %mass %mass %mass %

tively (Table 3). All offsets “Y(0)”are set to zero.

To calculate the total glyceridevalue according to DIN EN14105 the sum concentrations forall groups are needed. These areobtainable using another group-ing functionality of GCsolutionsoftware. In the compound table(Table 2) the groups are definedunder “Group ID No.” Thegroups must then be named inthe group table (Table 4). Themethod is now completed andcan be used for the evaluation ofbiodiesel samples.

Results

After evaluation of a biodieselsample GCsolution providesthree different result tables. Thepeak table lists all detected peaks– identified and not identified.The compound results table pro-vides concentrations for all com-ponents listed in the compoundtable.

Table 5 shows results from abiodiesel sample containingalmost no triglycerides. Only onepeak was detected for triglyceridebut no peak was detected in the

Peak No.137

1516172238

33, 34, 35, 36, 37, 39, 4045

NameGlycerinButanetriol ISTD1MonopalmitinMonolinoleinMonoolein MonostearinTricaprin ISTD2Diglyceride Diglyceride (rest)Triglyceride

Conc.0.0549

00.024

0.01220.30070.0229

00.03010.05580.0018

Unitsmass %mass %mass %mass %mass %mass %mass %mass %mass %mass %

triglyceride (rest) retention timeinterval.

A nice control option offers thePeak No. row in this table. Itshows that the peaks with thenumbers 33 to 40 are addedunder diglyceride (rest) with theexception of peak number 38which was already quantified asdiglyceride.

The group results table (Table 6)finally contains the informationrequired for total glycerol calcu-lation according to DIN EN14105.

The total glyceride value is calcu-lated according to the equation:

1.000* Free glycerol+ 0.255* Monoglycerides+ 0.146* Diglycerides+ 0.103* Triglycerides= Total glyceride

With the results from Table 6 atotal glyceride value of 0.159 iscalculated. Thus, the content offree glycerin in the biodiesel sam-ple measured is too high but allother requirements are metincluding the total glyceride limit(Table 7).

Summary

In routine analyses the “step bystep” procedure described is, ofcourse, automated in GCsolutionsoftware. The batch processingfunctionality is a powerful tool inthe development of calibrationcurves or quantification of asequence of unknown samples.Using export functionalities thecalculation of the total glyceridevalue can also be automated.

NameGlycerinMonoglyceridesDiglyceridesTriglycerides

Group No.1234

Conc.0.05490.35990.08590.0018

Unitsmass %mass %mass %mass %

Controlled contentsin biodiesel

MonoglyceridesDiglyceridesTriglyceridesFree glycerinTotal glyceride

Dimension

% (m/m)% (m/m)% (m/m % (m/m)% (m/m)

Max. value

0.80.20.20.02 0.25

Determinationaccording to

DIN EN 14105DIN EN 14105DIN EN 14105DIN EN 14105DIN EN 14105

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Tel.: +49 - (0)203 - 76 87-0Fax: +49 - (0)203 - 76 66 25Email: [email protected]

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