How to improve the GC&GC-MS

data quality by the automation of

sample workflows

Dr. Massimo Santoro,

Florianopolis (SC), Brazil - October 1st, 2012

2

Agenda

• Modern laboratory challenges

• Benefits of automated standard and sample preparation

• TriPlus RSH features and capabilities

• Using TriPlus RSH to automate GC, GC/MS workflows

• USP 467

• Food analysis

• Biodiesel analysis according to EN 14105 and ASTM D6584

• Summary

3

Tough challenges faced in the laboratory

• High sample loads/short

deadlines

• Keeping sample analysis

costs down with more and

more challenging LODs and

matrices

• Integrating and maintaining

new methods and

technologies into production

workflows to remain

competitive

4

Multiple sample prep steps and injection modes…

• Multiple sample injection

techniques can be used in GC to

fully characterize a sample; for

example, you’d run Headspace

analysis to determine the volatile

content, then liquid injection for

the Semi Volatiles and SPME to

further increase the sensitivity.

• Many analytical procedures

aimed at the quality assessment

or at the identification of

contaminants in a matrix require

the implementation of numerous

sample preparation steps prior to

the GC or GC/MS injection

5

Where automation can help?

• The possibility of performing the

sample preparation automatically,

by means of the same robotic

sampler used for sample injection

into the gas chromatograph,

allows running a quantitative

workflow for GC or GC/MS in

unattended way.

• An automated approach could

also allow scaling down the

volumes of reagents and solvents

used, thus also reducing the

waste generated, furthering

increasing the economical

benefits of such methodologies.

6

What‘s required...

• Adoption and

implementation to

automated sample and

standard injection and

preparation procedures

• Realization of the

productivity advantages

of high performance GC

and or GC-MS(/MS)

7

Benefits of automated standard and sample preparation

• The use of a modern robotic platform can:

• automate the preparation and analysis of standards and samples

• use a less error-prone approach to sample and standard preparation

• free the operator’s time

• guarantee increased throughput

• scale down sample and standard preparation volumes, for example by a factor of

10, lowering reagent costs and reducing waste chemicals.

8

Unattended, injection modes changes during a sequence: liquid

– HS - SPME

• ATC (Automatic Tool Change) Station for automatic recognition and

syringe exchanges during operation

Advanced sample handling and sample prep

Dilution, standard addition, internal standard additions,

derivatizations, etc.

Unattended, weekend long operations

• Up to 360 20-mL or 972 2-mL vials

Reliable, reproducible injections with microvolume samples

• <2%RSD and up to 3 injections possible with 5 ml sample volume in vial.

Start with the Autosampler : TriPlus™ RSH Autosampler

TriPlus RSH is an integrated Sampling System for:

9

TriPlus RSH : The highest precision

• More robotic X-Y-Z precise movement controls

Solvent: Ethyl Acetate

10 ul syringe

Liquid taken from washing station and

used to fill empty vials

Transfer per vial: 8 ul, 15 times

Typical repeatibility 0.066%

Solvent: water

100 ul syringe

Liquid taken from washing station and

used to fill empty vials

Transfer per vial: 100 ul, 1 time

Typical repeatibility 0.021%

Indipendently from GC performance

10

TriPlus RSH : The highest precision

• More robotic X-Y-Z precise movement controls

Solvent: 2-propanol

100 ul syringe

Liquid taken from washing station and used to fill empty vials

Volume Linearity > 0.99999

Indipendently from GC performance

11

TriPlus RSH : Ultimate productivity for reliable 24/7 operations

• The largest sample capacity, ensuring

true, weekend-long operations

• Multiple tray holders:

• Up to 972 2-mL vials

• Up to 360 10 or 20 mL vials

• The largest solvent washing capability

• Four different 10-mL solvent vials to

maximize cleaning efficiency

• Multiple 100-mL solvent bottles

stations for high sample throughput,

with waste collected externally

12

Excellent liquid injection repeatibility

10 injections overlay.

Splitless injections “Florida Mix”

C24H50

13

TG-ALC1

TG-ALC2

Columns:

ALC1 10m, 0.18 mmID, 1um

ALC2 10m, 0.18mmID, 0.63 um

Liner: 0.8mm for SSL

Precolumn

0.32 mmlD,

30 cm

Blood alcohol analysis by Headspace

14

Blood alcohol reproducibility and linearity

Replicate Area

1 7144775

2 7141844

3 7134720

4 7112455

average 7133448

std dev 14618

RSD% 0.20

TRACE 1310 GC

Oven temp: 40° isothermal (3.5min)

SSL injector: 220°C, split flow 80 ml/min

Carrier: costant pressure, 80 kPa

FID: 230°C

Triplus RSH

Agitator temperature: 80°C

Syringe temperature : 85°C

Incubation time : 10 min; Injection speed: 60 ml/min

Ethanol in water: 0.05 g/dL R² = 0.9911

R² = 0.9996

R² = 0.9948

R² = 0.9981

R² = 0,9999

R² = 0,9993

R² = 0,9997

R² = 0,9997

0

5000000

10000000

15000000

20000000

25000000

30000000

35000000

40000000

0,000 0,010 0,020 0,030 0,040 0,050 0,060

are

a

g/dL

methanol

acetaldehyde

ethanol

2-propanol

acetone

ethyl acetate

acetonitrile

MEK

Concentration range from 0.01 to 0.05 g/dL

15

ethanol_BAC1Y = 71740.4+1.75678e+008*X R^2 = 0.9997 W: Equal

0.00 0.02 0.04 0.06 0.08 0.10

g/dL

0

5000000

10000000

15000000

Are

a

Ethanol linearity

(0.0025 g/dL to 0.10 g/dL)

R² = 0.9911

R² = 0.9996

R² = 0.9948

R² = 0.9981

R² = 0,9999

R² = 0,9993

R² = 0,9997

R² = 0,9997

0

5000000

10000000

15000000

20000000

25000000

30000000

35000000

40000000

0,000 0,010 0,020 0,030 0,040 0,050 0,060

are

a

g/dL

methanol

acetaldehyde

ethanol

2-propanol

acetone

ethyl acetate

acetonitrile

MEK

Calibration results

Blood alcohol linearity test

16

SPME GC-MS/MS Quantitative Analysis of Food and Beverages for the Presence of Restricted Biologically Active Flavorings

17

TriPlus RSH: Higher quality results and performance

• Accurate injection from microvolume samples – ideal for trace POPs

analysis, biological fluids, explosive residues or when an expensive external

standard is used

• New bottom vial sensing capability delivers accurate and reproducible

injections from a sample as low as 5 ml

Powerful versatility in sample handling and injection

18

NEW bottom vial sensing capability: down to with 5 mL sample

5 mL sample in vial: C20-C40 Florida mix

5 replicates, 1 mL each

using the bottom sensing option

3 replicates possible with 5 mL sample: average RSD 1%

19

TriPlus RSH Autosampler: substantially reducing $/sample costs

• Unique ability to automatically handle multiple syringes

• Unattended syringe exchange during a sequence

• Zero carryover and no compromise in sample preparation

New Automatic Tool Changing (ATC) Station

ATC Station

20

Auto Syringe Tool Exchange video

• Insert video on ATC

21

ISTD

TriPlus RSH Sample/standard prep capabilities

• Sequential dilution: dilute a standard to

meet the analytical requirement

• Calibration dilution: prepare calibration

points adding internal standards

• (Internal) Standard addition: add precise

amount of liquid standards to any vials

(including HS and SPME), prior to the

analysis.

• Derivatization: add reagents and agitate

automating specific workflows

Automated sample preparation cycles

22

TriPlus RSH Sequence using custom cycles

1- Automation of USP 467 standard

preparation.

Unattended “Calibration Dilution”

24

1 - Unattended “Calibration dilution” capabilities

• The Calibration Dilution PrepCycle can be used

to prepare a serial dilution unattended, with or

without the addition of Internal Standard,

starting from a concentrated stock solution.

• This cycle guarantees optimal linearity and

reproducibility when performing unattended

sample preparation.

• This custom cycle has also been used, in the

automated preparation of the standards used for

the determination of Organic Volatile Impurities

in pharmaceutical products, following the USP

467 method.

Stock etc... 1/2 1/5 1/10 1/20 1/50 1/100

25

USP <467> Pharmacopeia Method

• The United States Pharmacopeia (USP) General

Chapter <467> Residual Solvents is a widely used

compendial method for identifying and quantifying

residual solvents when no information is available

on what solvents are likely to be present

• Drug products should contain no higher levels of

residual solvents that can be supported by safety

data

• Testing of drug substances, excipients, and drug

products for residual solvents should be performed

when production or purification processes are

known to result in the presence of such residual

solvents

26

USP <467>: Residual Solvent Classification

Class 1 Solvents to be avoided

Known or strongly suspected

human carcinogens

Environmental hazards

Solvents to be limited

Non-genotoxic animal

carcinogens

Suspected of other

insignificant but reversible

toxicities

Solvents with low toxic

potential

Low toxic potential to

humans; no health-

based exposure limit is

needed

Class 2

Class 3

Procedure A: Screening and

identification (phase G43)

Procedure B: Confirmation

(column phase G16)

Procedure C: Quantification

(column phase G43)

Water-soluble and water-

insoluble articles

Loss on Drying procedure or

a specific determination of

solvents

27

USP <467>: Identification and Quantification

Procedure A

Identification

Procedure B

Confirmation

Procedure C

Quantification

Prepare standard and test solutions

Perform procedure

under USP<467>

method

Residual solvent

peaks with an area

greater than the

standard?

Test passed.

No other actions

Perform procedure

under USP<467>

method

Test passed.

No other actions

Calculate amount

of residual

solvents present Residual solvent

peaks with an area

greater than the

standard?

NO NO

YES

YES

28

USP <467>: Solution Preparation

• Class 1 Standard Stock Solution Preparation:

•in 100 mL flask: 1 mL USP Class1 Residual Solvent Mix. + 9 mL DMSO + 90 mL H2O

•in 100 mL flask: 1 mL of the previous + 99 mL H2O

•in 100 mL flask :10 mL of the previous + 90 mL H2O

Water soluble articles

Class 1 Standard Solution: 1 mL of Class 1 Std Stock Sol. + 5 mL H2O in HS vial

• Class 2 Standard Stock Solution A (B):

in 100 mL flask: 1 mL USP Class 2 mix A (B) Residual solvent + 99 mL H2O

Class 2 Mix A Standard Solution:

1 mL of Class 2 mix A Std Stock Sol. + 5 mL H2O in HS vial

Class 2 Mix B Standard Solution:

5 mL of Class 2 mix B Std Stock Sol. + 1 mL H2O in HS vial

_ USP<467> revised method August 2011_

29

HS Analysis Instrument Method

Column: Thermo Scientific TRACE TR-V1 30

m x 0.32 mm ID x 1.8 µm (P/N 260V339P) _

equivalent to G43

TriPlus RSH, HS acquisition:

Injection volume: 1 mL

Agitator: 80 °C for 20 minutes

Syringe temp: 120 °C

TRACE 1310 GC:

Oven: 40 °C (20’) 10 °C/min to 240 °C (20’)

Injector: SSL, 140 °C, split ratio 1:5

Detector: FID, 240 °C

Carrier: Helium, constant flow: 2.1 mL/min

1_ methanol 2_ acetonitrile 3_dichloromethane 4_trans-1,2-dichloroethane 5_cis-1,2-dichloroethane 6_tetrahydrofuran 7_cyclohexane 8- methylcyclohexane 9_ 1,4-dioxane 10_toluene 11_chlorobenzene 12_ethyl benzene 13_ m-xylene/p-xylene 14_ o-xylene

30

Class 2 Mixture A

Class 2 mix A Compounds

analyzed solution conc (ppm)*

Safety Conc Limit (ppm)

RT (min)

area RSD% RT SD

(5 replicates) (5 replicates)

1 methanol 150 3000 2.31 2.7 0.004

2 acetonitrile 20.5 410 4.32 2.53 0.008

3 dichloromethane 30 600 4.47 1.62

0.004

4

trans-1,2-dichloroethane

47

1870

4.87 1.62 0.005

5

cis-1,2-dichloroethane

47 7.22 1.74 0.005

6 tetrahydrofuran 34.5 720 7.89 0.7 0.003

7 cyclohexane 194 3880 8.48 2.32 0.006

8 methylcyclohexane 59 1180 13.45 2.04

0.007

9 1,4-dioxane 19 380 15.45 2.83 0.004

10 toluene 44.5 890 21.91 1.47 0.004

11 chlorobenzene 18 360 26.43 0.98 0.001

12 ethyl benzene 18.4

2170

26.72 1.38 0.001

13 m-xylene 65.1

27.02 1.24 0.002 p-xylene 15.2

14 o-xylene 9.8 27.83 1.32 0.001

Standard solution manually prepared

concentration

analyzed 20 times

lower than the

safety conc. limit

of USP 467

RSDs% reported

are those of the HS

analysis run at

these

concentrations

31

Class 2 Mixture A

Standard solution prepared by TriPlus RSH autosampler

Class 2 mix A Compounds

analyzed solution conc (ppm)*

Safety Conc Limit (ppm)

RT (min)

area RSD% RT SD

(5 replicates) (5 replicates)

1 methanol 150 3000 2.31 5.43 0.005

2 acetonitrile 20.5 410 4.32 3.51 0.008

3 dichloromethane 30 600 4.47 1.12

0.004

4

trans-1,2-dichloroethane

47

1870

4.87 2.04 0.004

5

cis-1,2-dichloroethane

47 7.22 1.32 0.005

6 tetrahydrofuran 34.5 720 7.89 0.82 0.003

7 cyclohexane 194 3880 8.48 3.3 0.005

8 methylcyclohexane 59 1180 13.45 4.53

0.007

9 1,4-dioxane 19 380 15.45 5.02 0.004

10 toluene 44.5 890 21.91 3.95 0.003

11 chlorobenzene 18 360 26.43 4.03 0.001

12 ethyl benzene 18.4

2170

26.72 6.35 0.001

13 m-xylene 65.1

27.02 6.23 0.002 p-xylene 15.2

14 o-xylene 9.8 27.83 5.22 0.001

concentration

analyzed 20 times

lower than the

safety conc. limit

of USP 467

RSDs% reported

are those of the

automated

standard

preparation + HS

analysis run at

these

concentrations

32

1. TriPlus RSH prepares each standard

dilution independently and directly into

the pre-crimped HS vial

2. Replicates are run by TriPlus RSH

• Result: Instrument precision controls both

sample preparation and sample analysis.

• Advantages:

Lower chance for errors

Unattended operations: Triplus RSH

prepares and injects samples

Chromeleon audit trail registers how

the dilution is performed (CFR21)

Manual vs automatic sample prep: is there any difference?

1. An operator prepares the dilution

once, manually

2. 5 HS replicate vials are manually

prepared from this single diluted

solution

3. Replicates are run by TriPlus RSH

• Result: if an error is made during the

manual standard dilution, it is then

reflected in each of the replicates

• Disadvantages:

Possibly inaccurate results

Several manual steps

Time spent by the operators

Error prone

Standard solution manually prepared TriPlus RSH prepares standard solutions

33

Linearity Check

Class 2 Mixture A Calibration Levels Prepared by the TriPlus RSH Autosampler

with the Prep Cycle Calibration Dilution:

Class 2 mix A Concentration (ppm)

Compound name level 1 level 2 level 3 level 4 level 5 R2

1_ methanol 150 300 750 1500 3000 0.9978

2_ acetonitrile 20.5 41 102.5 205 410 0.9996

3_dichloromethane 30 60 150 300 600 0.9994

4_trans-1,2-dichloroethane 47 94 235 470 940 0.9987

5_cis-1,2-dichloroethane 47 94 235 470 940 0.9987

6_tetrahydrofuran 34.5 69 172.5 345 690 0.9999

7_cyclohexane 194 388 970 1940 3880 0.9943

8_ methylcyclohexane 59 118 295 590 1180 0.9938

9_ 1,4-dioxane 19 38 95 190 380 0.9998

10_toluene 44.5 89 222.5 445 890 0.9974

11_chlorobenzene 18 36 90 180 360 0.9947

12_ethyl benzene 18.4 36.8 92 184 368 0.9917

13_ m-xylene/p-xylene 65.1 130.2 325.5 651 1302

0.9910 15.2 30.4 76 152 304

14_ o-xylene 9.8 19.6 49 98 196 0.9983

R² = 0,9994

0

5000000

10000000

15000000

20000000

25000000

0 200 400 600 800

pe

ak a

rea

conc (ppm)

dichloromethane

R² = 0,9999

0

5000000

10000000

15000000

20000000

25000000

0 200 400 600 800

pe

ak a

rea

conc (ppm)

tetrahydrofuran

34

Summary

• Full compliance with USP <467> has been demonstrated

• Unprecedented level of sequence automation to run HS and liquid samples

in a single sequence

• TriPlus RSH autosampler automates sample preparation steps

• Reduces solvent and standard consumption

• Less time spent by operators

• Lower chance of errors

• Unattended operations for both preparation and injections

• Full traceability in a 21 CFR part 11 compliant environment: from

standard preparation steps to reporting

• TRACE 1300 Series GC: innovating routine analysis, redefining GC

usability

2- Trace analysis of PCB in food.

Automated Standard Addition

36

2- Automated Standard Addition preparation cycles

Automated basic sample handling and sample

prep

• Standard addition: automatically add precise amount of

standards to any vials.

• Ideal for HS & SPME quantitation with solid matrix

• Normally, different amount of standards are added to the

vials

• Internal Standard addition: add precise amount of

internal standards to any vials, prior the analysis

• Usually, the same amount of standard is added to all the

vials

Labor costs strongly reduced while improving accuracy

X µl

Standard or Internal Standard

37

Replicate injections of biological egg extracts

C13pcb81 C13pcb77 C13pcb169

827298 1065671 1604601

856177 1071542 1608853

843191 1057469 1621732

884641 1093632 1666368

873057 1091545 1711894

856872.8 1075972 1642689.6 avg

22887.47 15988.2 45784.259 stdev

2.67% 1.49% 2.79% RSD

RT: 21.71 - 22.98

21.8 21.9 22.0 22.1 22.2 22.3 22.4 22.5 22.6 22.7 22.8 22.9

Time (min)

0

50

100

0

50

100

0

50

100

Rela

tive A

bundance

0

50

100

0

50

100

RT: 22.30

AA: 1588745

RT: 22.29

AA: 1594165

RT: 22.29

AA: 1604418

RT: 22.29

AA: 1644553

RT: 22.29

AA: 1694740

NL: 3.65E5

TIC F: + c EI SRM

ms2 371.880

[301.935-301.945]

MS ICIS

pcbq_150811-03

NL: 3.62E5

TIC F: + c EI SRM

ms2 371.880

[301.935-301.945]

MS ICIS

pcbq_150811-04

NL: 3.70E5

TIC F: + c EI SRM

ms2 371.880

[301.935-301.945]

MS ICIS

pcbq_150811-05

NL: 3.73E5

TIC F: + c EI SRM

ms2 371.880

[301.935-301.945]

MS ICIS

pcbq_150811-06

NL: 3.81E5

TIC F: + c EI SRM

ms2 371.880

[301.935-301.945]

MS ICIS

pcbq_150811-07

analysis of non

ortho PCB,

showing 13C

labeled Internal

standards

data courtesy of Dr. Alexander Kotz, CVUA, Freiburg

38

Replicate injections of animal fat extracts

RT: 13.99 - 14.86

14.0 14.1 14.2 14.3 14.4 14.5 14.6 14.7 14.8

Time (min)

0

50

100

0

50

100

0

50

100

Rel

ativ

e Abu

ndan

ce

0

50

100

0

50

100

RT: 14.21

AA: 1989967RT: 14.67

AA: 2044297

RT: 14.22

AA: 1918038RT: 14.67

AA: 1989858

RT: 14.22

AA: 1838759RT: 14.67

AA: 1900249

RT: 14.67

AA: 1831155RT: 14.22

AA: 1767543

RT: 14.22

AA: 1744033RT: 14.68

AA: 1799326

NL: 6.55E5

TIC F: + c EI SRM

ms2 301.960

[232.015-232.025]

MS ICIS

pcbq_150811-10

NL: 6.32E5

TIC F: + c EI SRM

ms2 301.960

[232.015-232.025]

MS ICIS

pcbq_150811-11

NL: 6.11E5

TIC F: + c EI SRM

ms2 301.960

[232.015-232.025]

MS ICIS

pcbq_150811-12

NL: 5.90E5

TIC F: + c EI SRM

ms2 301.960

[232.015-232.025]

MS ICIS

pcbq_150811-13

NL: 5.73E5

TIC F: + c EI SRM

ms2 301.960

[232.015-232.025]

MS ICIS

pcbq_150811-14

C13pcb81 C13pcb77 C13pcb169

1989967 2044297 1427341

1918038 1989858 1374218

1838759 1900249 1338351

1757543 1831155 1292773

1744033 1799326 1284212

1849668 1912977 1343379 avg

105027.4 103596.6 59345.69 stdev

5.68% 5.42% 4.42% RSD

analysis of non ortho PCB,

showing 13C labeled Internal

standards

data courtesy of Dr. Alexander Kotz, CVUA, Freiburg

3 – method-specific preparation cycles:

Automated In-line Sample Preparation

and GC Analysis of Biodiesel

According to

EN14105 and ASTM D6584

40

What is Biodiesel?

• An “alternative” fuel comprised of methyl or ethyl esters from

long chain fatty acids derived from vegetable oils or used

animal fats

• Pure biodiesel is designated B100 and must meet the

requirements of ASTM D 6751 or EN 14124.

41

How is Biodiesel Made? Transesterification mechanism

R1COCH2

O =

R2COCH

O =

R3COCH2

O =

+ 3 CH3OH 3 - - - - COCH3

O =

+ HOCH

CH2OH

CH2OH catalyst

tri-glycerides

methanol FAME

glycerol

Transesterification: exchanging the alkoxy group of the ester by another alcohol.

Reactions often catalyzed by the addition of an acid or base

Reaction of a vegetable oil or animal fat with an alcohol to yield

mono-alkyl esters and glycerin (then removed)

42

Method Description

EN 14103 Determination of ester and linolenic acid methyl ester contents

EN 14110 Determination of methanol content

EN 14105 Determination of free and total glycerol and mono-, di-, triglyceride

content

ASTM D6584

Determination of Total Monoglycerides, Total Diglycerides,

Total Triglycerides, and Free and Total Glycerin in B-100

Biodiesel Methyl Esters by Gas Chromatography

EN 14106 Determination of free glycerol content

GC Based biodiesel standard methods

43

• ASTM D6584 and EN14105 dictate all the steps for an accurate standard

curve preparation

• It takes 1-1.5 hours to manually prepare a single set of calibrants

• Multiple dedicated microliter syringes, various solvents and reagents must be

used.

Some considerations on standard preparation…

ASTM D6584:

EN14105:

44

Add 100µL of MSTFA (derivatizing agent)

ASTM D6584 – 10a: sample preparation procedure

Weigh 100 mg of sample directly into a 10-mL vial

Add 100 µL of 1,2,4-Butanetriol (Internal Standard 1)

Add 100 µL of Glycerol Tridecanoate (tricaprin, ISTD2)

Shake the vial, wait 15-20 minutes at room temperature

Add 8 mL n-Heptane and shake

Inject 1 uL into the cool on-column injector of a GC

45

Add 200µL of MSTFA (derivatizing agent)

EN 14105:2011: sample preparation procedure

Weigh 100 mg of sample directly into a 10-mL vial

Add 80 µL of 1,2,4 Butanetriol (Internal Standard)

Add 200 µL of standard glycerides solution

Add 200 µL of pyridine

Shake the vial, wait 15 minutes at room temperature

Add 8 mL n-Heptane

Inject 1 uL into the cool on-column injector of a GC

46

Practical issues?

• At least 1.5 hours of manual operations to prepare

the calibration points.

• 3 to 7 pipetting steps are required for each sample

to be prepared.

• Derivatization time sensibly limits the number of

samples prepared in parallel by an operator.

• Manual operations are performed “off-line” in a

preparation lab before moving to the instrument

lab.

47

Completely automated biodiesel workflows…

• Prepare standards, solvents, reagents and syringes

• Load biodiesel samples in pre-determined positions

Build SW sequence

• Load biodiesel cycle

• Prepare vial injection sequence

GC analysis

• Run samples via GC

• Obtain reports and evaluate data

…for ASTM D6584 and EN14105

TriPlus RSH

Set-up

48

TriPlus RSH set-up for biodiesel workflow…

Mount the syringes

on ATC station:

• 100 mL, for

reagents

• 10 mL, for GC

liquid injection

• 10 mL, for

heptane

49

solvent for syringes washes

to chemicals

waste collection

heptane, for derivatization

quenching

• Load empty vials for

calibration, then

biodiesel samples.

TriPlus RSH set-up for biodiesel workflow…

50

TriPlus RSH set-up for ASTM D6584

Glycerin

stock

solution

Wash for

glycerin

stock

solution

Monoolein

stock

solution

Wash for

monoolein

stock

solution

Diolein

stock

solution

Wash for

diolein

stock

solution

Triolein

stock

solution

Wash for

triolein

stock

solution

Butanetriol

(ISTD 1)

solution

Tricaprin

(ISTD 2)

solution

Wash for

ISTD 1

solution

Wash for

ISTD 2

solution

Wash for

MSTFA MSTFA Heptane

•ASTM D6584

reagents and

standards are

placed in the first

set of 15 10-mL

vials.

•Additional 10-mL vials are placed in similar trays for calibration

and samples preparation

51

TriPlus RSH set-up for EN 14105: 2011

Glycerin

stock

solution

Wash for

glycerin

stock

solution

Pyridine

Wash for

pyridine

Butanetriol

(ISTD 1)

solution

Glycerides

stock

solution

Wash for

ISTD 1

solution

Wash for

std.

glycerides

solution

Wash for

MSTFA MSTFA Heptane

Empty

slot Empty

slot

Empty

slot Empty

slot

•EN14105:2011

reagents and

standards are

placed in the first

set of 15 10-mL

vials.

•Additional 10-mL vials are placed in similar trays for calibration

and samples preparation

52

Build the software sequence

53

Build the software sequence

54

TriPlus RSH cycle: standard preparation ASTM D6584

• TriPlus RSH prepares unattendedly the five standard solutions by transferring

the specified volumes, directly into 10-mL vials.

• TriPlus RSH adds to each vial 100 μL of MSTFA.

• TriPlus RSH shakes and allow vials to stand for 15-20 min at room

temperature.

• TriPlus RSH adds 8 mL n-Heptane to the vials and shakes them.

55

• 100 mg of sample are placed directly into a 10 mL vial.

• TriPlus RSH adds 100 μL each of the two internal standards.

• TriPlus RSH adds 100 μL of MSTFA.

• TriPlus RSH shakes the vials, and allow to set for 15-20 min at room

temperature.

• TriPlus RSH adds 8 mL of n-Heptane to the vials and shakes them.

• For both sample and standards, each step is

optimized so to guarantee the 15-20’ derivatization.

TriPlus RSH cycle: sample preparation ASTM D6584

56

TRACE 1310 GC conditions for ASTM D6584

• Oven Ramp: 50 ºC, for 1.0 minute

15 ºC/min to 180

7 ºC/min to 230

30 ºC/min to 380 ºC, for 10 minutes

• On-Column Inlet: 50 ºC, oven tracking to 380 ºC

• FID : 380 ºC

• Carrier Gas : He@ 3.0 ml/min

• Sample injected: 1.0 mL

57

Biodiesel samples according to ASTM D6584

58

Calibration curves prepared by TriPlus RSH

y = 1,3867x + 0,0967 R² = 0,995

0

0,5

1

1,5

2

0 0,5 1 1,5

Are

a r

ati

o

Amount ratio

monoglycerides

y = 1,3802x - 0,0204 R² = 0,9964

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0 0,1 0,2 0,3 0,4 0,5 0,6

Are

a r

ati

o

Amount ratio

glycerin

y = 1,0842x - 0,0558 R² = 0,9968

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0 0,2 0,4 0,6 0,8

Are

a r

ati

o

Amount ratio

diglycerides

y = 0,4824x - 0,0104 R² = 0,9871

0

0,05

0,1

0,15

0,2

0,25

0,3

0,35

0 0,2 0,4 0,6 0,8

Are

a r

ati

o

Amount ratio

triglycerides

Correlation coefficients > 0.9 as required by the ASTM method

59

Repeatability (r) and Reproducibility (R)

“The difference (Δ)between successive runs obtained by the

same operator with the same apparatus, under constant

operating conditions on identical test material would exceed the

calculated r value in one case in twenty”

“The difference (Δ)between two single and independent results

runs obtained by different operators, working in different labs on

identical material, in the normal operation of the test method

would exceed the calculated R value in one case in twenty”

Calculation example: • r =(2.339/100)*(% mass+0.0001)0.48888 For Free Glycerol

• r = (5.405/100)*(% mass+0.5164) For Total Glycerol

60

ASTM D6584 Repeatability

0

0,0005

0,001

0,0015

0,002

0,0025

0 2 4 6 8 10 12

%m

ass

Sample #

Free Glycerol

r

Δ [mass]

0,0000

0,0100

0,0200

0,0300

0 2 4 6 8 10 12

% m

ass

Sample #

Total Glycerol

r

Δ [mass]

All tested sample meet method’s precision criteria.

61

TriPlus RSH cycle: standard preparation EN 14105

• Standard Solutions— TriPlus RSH prepares the four standard solutions in

unattended mode by transferring the specified volumes by means of 100 μL

syringes, directly into 10-mL septa vials already capped.

• TriPlus RSH adds to each calibration point 150 μL of MSTFA.

• TriPlus RSH shakes and allow vials to stand for 15 min at room temperature.

• TriPlus RSH adds 8 mL n-Heptane to the vial and shakes them.

62

TriPlus RSH cycle: sample preparation EN 14105

• 100 mg of sample are placed into a 10 mL vial.

• TriPlus RSH adds 80 μL of Butanetriol (internal standard).

• TriPlus RSH adds 200 μL the standard mix of mono, di and triglycerides.

• TriPlus RSH adds 200 μL of pyridine

• TriPlus RSH adds 200 μL of MSTFA.

• TriPlus RSH shakes the vial, and allows to set for 15 min at room

temperature.

• TriPlus RSH adds 8 mL of n-Heptane to the vial and shakes it.

• For both sample and standards, each step is

optimized so to guarantee the 15’ derivatization.

63

TRACE 1310 GC conditions for EN 14105

• Oven Ramp: • Initial- 50 ºC, hold 1.0 minute

• 15 ºC/min to 180 ºC,

• 7 ºC/min to 230 ºC,

• 10 ºC/min to 370 ºC, hold 15.0 minutes

• On-Column Inlet: 50 ºC, oven tracking to 370 ºC

• FID detector: 380 ºC

• Carrier Gas Flow: 3.0 ml/min Helium

• Sample size: 1.0 mL

64

EN 14105: Biodiesel sample chromatogram

Glycerol

Monoglycerides Diglycerides

Triglycerides

65

Calibration curve prepared by TriPlus RSH

y = 0.0753x + 0.0052 R² = 1

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0 1 2 3 4 5 6 7 8 9

Are

a R

ati

o

Amount Ratio

Glycerol Calibration

Correlation coefficient > 0.9 as required by the EN14105

66

-0,000110

0,000090

0,000290

0,000490

0,000690

0 5 10 15 20

mass %

Sample #

Free Glycerol

r

Δ [mass]

EN 14105 Repeatability

-0,00001

0,00099

0,00199

0,00299

0,00399

0,00499

0,00599

0,00699

0 5 10 15 20

mass %

Sample #

Total Glycerol

r

Δ [mass]

All tested sample meet method’s precision criteria.

67

EN 14105 Reproducibility

-0,0005

0,0015

0,0035

0,0055

0,0075

0 5 10 15 20

mass %

Sample #

Free Glycerol

R

Δ [mass]

0

0,005

0,01

0,015

0,02

0,025

0,03

0 5 10 15 20

mass %

Sample #

Total Glycerol

R

Δ [mass]

All tested sample meet method’s precision criteria.

68

Considerations on the automated biodiesel workflows

• When running ASTM D6584 or EN14105 several manual steps

must be executed for the preparation of calibration standards

and samples.

• TriPlus RSH can automate these operations and perform the

GC injections in unattended fashion, saving precious time and

laboratory space.

• The use of a modern robotic platform guarantees the necessary

data quality requirements are consistently met.

69

Unattended, injection modes changes during a sequence: liquid

– HS - SPME

• ATC (Automatic Tool Change) Station for automatic recognition and

syringe exchanges during operation

Advanced sample handling and sample prep

Dilution, standard addition, internal standard additions,

derivatizations, etc.

Unattended, weekend long operations

• Up to 360 20-mL or 972 2-mL vials

Reliable, reproducible injections with microvolume samples

• <2%RSD and up to 3 injections possible with 5 ml sample volume in vial.

TriPlus™ RSH Autosampler

70

Thank you for your attention!

Questions?

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