freestyle thermelute
TRANSCRIPT
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Visit us at www.LCTech.de!
Aflatoxin & Ochratoxin Analysis
A Completely Automated Solution with the
Freestyle, ThermELUTE, and SMARTImmunoaffinity Cleanup Columns
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Background
• Pickering Labs primarily makes HPLC Post-column
instruments and reagents for a variety of post-columnapplications.
• LC Tech has been the European distributor for Pickering Labs
for about 20 years.
• Recently Pickering Labs has been distributing LC Tech
products in North America along with help from
Chromatographic Specialties in Canada.
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What LCTech does
supplies innovative products
and methods for thepreparation and analysis of
food, feed and environmental
samples.
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Introduction
The goal: totally automated system
for Aflatoxins (B1, B2, G1, G2) and/or Ochratoxin A
HPLC
From raw extract to chromatogram
=> in 15 minutes
=> without manual steps
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Introduction
The Components
UVETM
UV derivatiser (only for Aflatoxins)
ChromatogramFREESTYLETM with ThermELUTE
and SPE modules
Raw extract
quaternary or
isocratic pump
sampler
column
oven
detector
HPLC
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Instrumentation
Sample preparation Analysisand
FREESTYLE Basic
SPE module
ThermELUTE module
Racks and vials
AflaCLEAN and/or OtaCLEAN
SMART Immunoaffinity Cleanup
Columns
Any HPLC
Pump(s)
Autosampler
Column oven
Derivatizer (e. g. UVETM)
Fluorescence detector
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Sample preparation with large volume injection
FREESTYLE Basic Automated and unattended handling
of the immunoaffinity column:
- loading of a precise volume
- washing
- drying in case of AflaCLEAN SMART
- column transfer into the ThermELUTE module
ThermELUTE module heating = breaking the antigen-antibody bond
- elution of the analytes with water and
- large volume injection into HPLC sample loop
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Analysis Instrumentation
Any HPLC possible
tested with DIONEX Ultimate 3000
Pump Isocratic mode
Autosampler Only needed for standard injection
Column switching
valve
If Aflatoxins and Ochratoxin A should be analyzed
a switching valve is recommended
Column Oven For two columns (Afla & Ochra)Column(s) Standard guard and analytical columns
Derivatizer UV derivatizer, photochemical derivatization
Fluorescence detector Standard
Software Standard
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Dionex Ultimate 3000 details
Pump LPG 3400a
Sampler WPS 3000 TSL
Mobile Phase Methanol/Water/Acetonitrile, 30/60/15
Flow rate 1.2 mL/min
Column Oven 36 °C
Column LCTech HPLC column (LCTech P/N 10522)
Flourescence detector RF 2000, FLD
Fluorescence wavelength 365 nm excitation
460 nm emission
Derivatizer LCTech UVE
ThermELUTE – chromatograms
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The goal:
Completely automated system
for Aflatoxin and/or Ochratoxin A analysis
Step 1:
Replace 1 mL or 3 mL immunoaffinity columnswith SMART Immunoaffinity columns
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What is so smart about SMART columnns?
SMART columns - advantages
Original size:25 mm They are completely comparableto 1 mL or 3 mL immunoaffinity
columns
But,
-less t ime is needed-less sample is needed
-less solvent is needed
-Less waste
And-toxin concentration is equivalent
to 1 mL or 3 mL format
S M A R
T C o l u m n s
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What is so smart about SMART columnns?
They meet AOAC requirements
SMART columns - advantages
Loading capacity
100 ng B1
Loading capacity
100 ng Ochratoxin A
Recovery > 80 % Recovery > 80 %
S M A R
T C o l u m n s
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What is so smart about SMART columnns?
Reduced Cleanup time
SMART columns - advantages
Cleanup process down
to less than 9 minutes
Cleanup process down
to less than 13 minutes
Incredible time reduction
- less sample volume = less time for sample loading
- high flow rate
S M A R
T C o l u m n s
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What is so smart about SMART columnns?
They are applicable for all regulated matrices
SMART columns - advantages
All kinds of nuts:
Hazelnut, walnut, brazil nut,
peanut, etc.
pistachios, almonds, raisins, figs
All kinds of cereals, corn, rice
All common spices
etc.
All kinds of coffee
Red wine
Beer
Raisins
Liquoriceetc.
S M A R
T C o l u m n s
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The goal:
Completely automated system
for Aflatoxin and/or Ochratoxin A analysis
Step 1:
Replace 1 mL or 3 mL immunoaffinity columns
by SMART Immunoaffinity columns
Step 2:
High end Completely automated solution
with FREESTYLE and ThermELUTE module
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Completely automated solution
ThermELUTE
module
Rack for SMART columns
Any HPLC
SMART
columns
SPE Gripper
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Completely automated solution
Width:110 mm
ThermELUTE , standard version (p/n 13572)
Heating element
Injection valve
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Rack (p/n 13497)
for 60 SMART columns
Rack (p/n 13497) in tray (p/n 11915)
for SMART columns
Completely automated solution
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ThermELUTE with Standards
Injection valve
Injection port
Extraction and direct
injection system
Cooled rack for standards
SMART Column
Gripper
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ThermELUTE module
Rack for up to 60 SMART columns
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SMART Column Gripper
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How the systemoperates:
SMART Column
gripper is taken upby SPE tool
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How the systemoperates:
gripper takes up the
SMART column
Used columns
Unused columns
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How the systemoperates:
The SMART column
is now tightlyconnected
to the system and
can be manipulated
by positive pressurecontrol
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How the systemoperates:
After handling steps
(loading, washing,drying…) column is
transfered into heating
element
Heating element (hidden)
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How the systemoperates:
Column is transfered
into heating element
Heating element (hidden)
The antigen-antibody
bond is broken by the
heat and the toxins are
eluted with water. No
organic solvent is
required.
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SMART columns - advantages
Original size:
25 mm
Normally you elute with organic
solvent; either methanol or acetonitrile.
This destroys the antigen-antibody
bond and elutes the toxins. The
disadvantage is that you cannot inject
high volumes of methanol onto the
Reverse Phase column. You usually
have to evaporate your cleaned sample
and re-suspend in a small volume prior
to injection onto the Reverse Phase
column.
When you are eluting with water you
are injecting all the toxin that is bound
to the SMART column (~700ul) directly
onto the Reverse Phase column.
S M A R
T C o l u m n s
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How the system
operates:
Column is now
in the heating element
The toxins are eluted into the
injection valve after thermalantigen-antibody bond
cleavage.
Sample was eluted as a
partial loop fil l with 400 µLinto 700 µL sample loop
Sample loop
Injection valve
Injection port
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Elution followed by whole sample large volume injection
Advantages
No loss of analytes. No storage between cleanup andanalysis
Large volume injection in water / all analytes are injected
straight from the column
- No evaporation
- No adsorption by the glass wall of storage vials
- No loss of recovery = highest sentivity
Less sample required to stay within FLD range.
- Less matrix on the immunoaffinity column
- Faster sample clean-up
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Elution followed by whole sample large
volume injection
Advantages
Suitable for very strict detection limits
for baby food
Maximum level according
to EU Regulation1881/2006 *
Detection limit with
FREESTYLE +ThermELUTE
Aflatoxin B1: 100 ppt Aflatoxin B1: 5 ppt
Ochratoxin A: 500 ppt Ochratoxin A: 30 ppt
* COMMISSION REGULATION (EC) No 1881/2006 of 19 December 2006
setting maximum levels for certain contaminants in foodstuf fs
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Elution followed by whole sample large volume injection
Advantages
Single-use AflaCLEAN and OtaCLEAN SMART Cleanupcolumns
- 100% initial performance of SMART columns for all samples
- No cross contamination
= Perfect analytical conditions
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Increased sample throughput by parallelization of
sample preparation and analysis
Completely automated solution
FREESTYLE sample preparation time: 15 min* / sample
1st sample
1st sample
Sample preparation
with FREESTYLETM
Analysis
with HPLC
2nd sample
2nd sample 3rd sample
HPLC analysis time: 15 min* / sample
4rth … etc
3rd … etc
* Ochratoxin A; Aflatoxin even faster
Automated
sample injection
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Throughput at i ts best
Completely automated solution - advantages
15 min / sample
4 samples per hour
96 samples per day
Monday Thuesday
Monday – Sunday
Day and night
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Loading capacity:
90 samples(1 SMART column /
sample
& samples presented
in 16 mL vials)
Quantity of samples
can be increased by
reduction of sample
volume and by choosing
smaller vials.
Completely automated solution - advantages
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Higher sensitivity compared to manual cleanup processing
Excellent chromatograms (less sample needed)
ThermELUTE – chromatograms
Ginger (10 ppb) withThermELUTE, 2.8 mL sample
Ginger (10 ppb), manualprocessing, 14 mL sample
Ginger
0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0
10,0
0,0
10,0
20,0
30,0
40,0
50,0
60,0
1 - 130206-FW- SOFTWARE 5.2 U SYSTEMWASC Emissi
ingwer halbe einwaag EmissimV
min
2
1
EM:460 nm
0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0
-50
100
200
300
400
ingwer blind 2,8ml halbe einwaage Emission
ingwer 10ppb 2,8ml halbe einwaage EmissionmV
min
21
EM:460 nm
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Higher sensitivity compared to manual cleanup processing
Excellent chromatograms (less sample needed)
ThermELUTE – chromatograms
Nutmeg (10 ppb) withThermELUTE, 2.8 mL sample
Nutmeg (10 ppb), manualprocessing, 14 mL sample
Spices: Nutmeg
0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0
-20
50
00
50
00
muskatnuss halbe einwa Emissi
muskatnuss halbe einwaa EmissimV
min
21
EM:460 nm
0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0
-50
0
50
100
150
200
250
300
muskatnuss blind 2,8ml halbe einwaageEmission
muskatnuss 10ppb 2,8ml halbe einwaageEmissionmV
min
21
EM:460 nm
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Higher sensitivity compared to manual cleanup processing
Excellent chromatograms (less sample needed)
ThermELUTE – chromatograms
Coriander (10 ppb) withThermELUTE, 2.8 mL sample
Coriander (10 ppb), manualprocessing, 14 mL sample
Spices: Coriander
0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0
-10
20
40
60
80
10
1 - 130206-FW- SOFTWARE 5.2 U SYSTEMWASC Emissi
koriander halbe einwaa EmissimV
min
21
EM:460 nm
0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0
-50
0
50
100
150
200
250
300
koriander blind 2,8ml halbe einwaageEmission
koriander 10ppb 2,8ml halbe einwaageEmissionmV
min
21
EM:460 nm
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Even low levels are detected. Not detected with manual
processing
ThermELUTE – chromatograms
Hazelnut, not spiked
Black:
ThermELUTE detects evenlowest level
Red:
Not detected with manual
processing
Nuts: Hazelnut
0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0
-50
0
50
100
150
200
250
300
Haselnuss 10ml blind Emission
2 - 130205-FW-THERMELUTE AFLACLEAN PISTAZIE_HASELNUSS_ROSINE_ERDNUSS #50mV
min
21
EM:460 nm
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Even low levels are detected. Not detected with manual
processing
ThermELUTE – chromatograms
Peanut, not spiked
Black:
ThermELUTE detects evenlowest level
Red:
Not detected with manual
processing
Nuts: Peanut
0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0
-50
0
50
100
150
200
250
300
Erdnuss 10ml blind Emission
Erdnuss blind manuell 3ml EmissionmV
min
21
EM:460 nm
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Less sample needed, thus faster processing
ThermELUTE – chromatograms
Corn, 10 ppb
Black:
ThermELUTE, 2.5 mLRed:
ThermELUTE, 1 mL
Orange:
Manual processing, 3 mL
Cereals: Corn
0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0
-100
125
250
375
500
700
Probe 4 10ppb 2,5ml Emission
Probe 5 10ppb 1ml Emission
10ppb Probe mais manuell 3ml Säule EmissionmV
min
321
EM:460 nm
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Less sample needed, thus faster processing
ThermELUTE – chromatograms
Hazelnut, 10 ppb
Black:
ThermELUTE, 2.5 mLRed:
ThermELUTE, 1 mL
Orange:
Manual processing, 3 mL
Nuts: Hazelnut
0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0
-100
125
250
375
500
700
Haselnuss 2,5ml 10ppb EmissionHaselnuss 1ml 10ppb Emission
Haselnuss 10ppb manuell 3ml EmissionmV
min
321
EM:460 nm
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Less sample needed, thus faster processing
ThermELUTE – chromatograms
Raisins 10 ppb
Black:
ThermELUTE, 2.5 mLRed:
ThermELUTE, 1 mL
Orange:
Manual processing, 3 mL
Raisins
0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0
-100
125
250
375
500
700
Rosinen 2,5ml 10ppb EmissionRosinen 1ml 10ppb Emission
Rosinen 10ppb manuell 3ml EmissionmV
min
321
EM:460 nm
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Less sample needed, thus faster processing
ThermELUTE – chromatograms
Pistachio, 10 ppb
Black:
ThermELUTE, 2.5 mLRed:
ThermELUTE, 1 mL
Orange:
Manual processing, 3 mL
Nuts: Pistachio
0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0
-100
125
250
375
500
700
Pistazien 2,5ml 10ppb EmissionPistazien 1ml 10ppb Emission
Pistazien 10ppb manuell 3ml EmissionmV
min
321
EM:460 nm
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Less sample needed, thus faster processing
ThermELUTE – chromatograms
Peanut, 10 ppb
Black:
ThermELUTE, 2.5 mLRed:
ThermELUTE, 1 mL
Orange:
Manual processing, 3 mL
Nuts: Peanut
0,0 2,0 4,0 6,0 8,0 10,0 12,0 15,0
-100
200
400
600
800
Erdnuss 2,5ml 10ppb EmissionERdnuss 1ml 10ppb Emission
Erdnuss 10ppb manuell 3ml EmissionmV
min
321
EM:460 nm
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Chromatogram of Peanuts Spiked with 10ppb total aflatoxin(G2/G1/B2/B1) (1ng G2; 4ng G1; 1ng
B2; 4 ng B1) extracted according to
LCTech extraction and clean-up
procedure, 10 mL processed using
AflaCLEAN SMART and
ThermELUTE.
Chromatogram represents 0.28 gram
matrix equivalents.
Clean-up time consists of
10 mL sample loading (3mL/min)
2 mL washing (3mL/min)
6 min ThermELUTE treatmentReady to inject within 10 min
Methods - Aflatoxin
Nuts: Peanut
M th d Afl t i
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Chromatogram of Peanuts Spiked with 10ppb total aflatoxin(G2/G1/B2/B1) (1ng G2; 4ng G1;
1ng B2; 4 ng B1) extracted according
to LCTech extraction and clean-up
procedure, 2.5 mL processed using
AflaCLEAN SMART and
ThermELUTE.
Chromatogram represents 0.07 gram
matrix equivalents.
Clean-up time consists of
2.5 mL sample loading (3 mL/min)
2 mL washing (3mL/min)
6 min ThermELUTE treatmentReady to inject within 7 5 min
Methods - Aflatoxin
Nuts: Peanut
M th d Afl t i
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Spiked with 10ppb total aflatoxin
(G2/G1/B2/B1 (1ng G2; 4ng G1;
1ng B2; 4 ng B1) extracted according
to LCTech extraction and clean-up
procedure, 10 mL processed using
AflaCLEAN SMART and
ThermELUTE.
Chromatogram represents 0.28 gram
matrix equivalents.
Clean-up time consists of
10 mL sample loading (3mL/min)
2 mL washing (3mL/min)
6 min ThermELUTE treatmentReady to inject within 10 min
Methods - Aflatoxin
Chromatogram of Hazelnuts
Nuts: Hazelnut
M th d Afl t i
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Spiked with 10ppb total aflatoxin
(G2/G1/B2/B1) (1ng G2; 4ng G1;
1ng B2; 4 ng B1) extracted according
to LCTech extraction and clean-up
procedure, 2.5 mL processed using
AflaCLEAN SMART and
ThermELUTE.
Chromatogram represents 0.07 gram
matrix equivalents.
Clean-up time consists of
2.5 mL sample loading (3 mL/min)
2 mL washing (3mL/min)
6 min ThermELUTE treatmentReady to inject within 7 5 min
Methods - Aflatoxin
Chromatogram of Hazelnuts
Nuts: Hazelnut
Ochratoxin
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10 mL instant coffee sample after
extraction using methylene chlor idepartitioning, spiked at the regulated
level of 10 ppb.
The chromatogram represents
0.01 gram matrix equivalents.
By using PICKERING post-column
derivatization sensitivity could be
increased by a factor of approx. 4 and
chromatography (injection peak/matrix
interference could be dramatically
reduced).
Ochratoxin
Chromatogram of Instant Coffee
Coffee
Methods Ochratoxin
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10 mL roasted cof fee sample after
extraction using methylene chlor idepartitioning, spiked at the regulated
level of 5 ppb.
The chromatogram represents 0.05
gram matrix equivalents.
Methods - Ochratoxin
Chromatogram of Roasted Coffee
Coffee
Methods Ochratoxin
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10 mL White Wine sample extracted
according to LCTech OtaCLEANSMART extraction and clean-up
procedure loaded onto OtaCLEAN
SMART column, spiked at the
regulated level of 2 ppb.
The chromatogram represents 1 grammatrix equivalents.
Methods - Ochratoxin
Chromatogram of White Wine
Wine
Methods Ochratoxin
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10 mL Beer sample extracted
according to LCTech OtaCLEANSMART extraction and clean-up
procedure loaded onto OtaCLEAN
SMART column, spiked with 1 ppb
The chromatogram represents 1.42
gram matrix equivalents.
Methods - Ochratoxin
Chromatogram of Beer
Beer
Completely automated solution - further features
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SPE Module
EVAporationModule
GPC Module
ThermELUTE
Module
Completely automated solution - further features
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Visit us at www.LCTech.de!
www.freestyle-robotic.comwww.chromspec.com