Waters 2X5LF Customer Familarization Draft V2.0 BEW 8 14 - 2014

SFE 2X5LF System Customer Familiarization Draft Page # 1
SFE 2X5LF System Draft Customer Familiarization Guide

REVISION NUMBER Draft 2.0
DATE 14 August 2014
Waters Educational Services Draft Rev 2.0 BEW 8/14/2014

Table of Contents
Page # Section # Title of Section
3-4 A General information about the SFE 2X5LF Extraction System
5 B Tools Required for Installation and Operation
6-7 C SFE 2X5LF System Components
8 D The 2x5LF Recycler Overview
9-10 E General System Workflow for SFE Sample Extraction
11-14 F Pre-Setup for SFE Extraction of Sample Extraction
15-23 G Software and System Operation for SFE Extraction – No Recycler
24-26 H Software and System Operation for SFE Extraction – Recycler
27-29 I Collecting Extracts from Collectors
30-32 J Switching and Cleaning the Extraction Chambers
33-35 K System Troubleshooting
36-37 L Pm and Maintenance
38-40 M ABPR Alignment and Details
41 N Appendix: Schematics and Diagrams
42-47 O SFE Method Development Guidelines with the 2x5LF System
Vicam Botanical Instruction Guide

48-57

A: General Information about the SFE 2X5LF System
1. The SFE 2X5LF System is the following system configuration:
System Extraction Extraction Collection Collection Collection CO2 Co-Solvent Flow CO2
Configuration Vessel 1 Vessel 2 Vessel 1 Vessel 2 Vessel 3 Pump Pump Meter* Recycler*
SFE-2X5Liter 5000 mL 5000 mL 1000 mL 1000 mL 1000 mL P200 No Installed Installed
*The Flow Meter and CO2 Recycler are options
2. Supercritical CO2 Extraction = pressures above 1078 PSI and 31C.
Figure 1 General SFE Concepts - Part 1
3. Supercritical CO2 has the polarity equivalent of n-Heptane. Increasing CO2 pressure during
extraction and collection increases the ability to extract increasing stronger polar compounds.
Adding an optional co-solvent pump to the system will let you extract out the most polar
compounds.
Figure 2 General SFE Concepts Part 2

4. The system for extraction usually uses CO2 only with no co-solvent as most plant extracts are
non-polar. There is an option for a co-solvent pump.
a. Supercritical CO2 for extracts is a clean and safe extraction process that does not leave
any residual materials in the collected extract.
b. Most botanical products of interest are non-polar in nature, so CO2 is also the method of
choice for selectivity of a majority of different botanicals, and does not require the use of a
co-solvent pump.
c. The system does have an optional Co-solvent pump, though for botanical products the co-
solvent pump is not used.
2. Supercritical CO2 is added to botanical materials, which during the extraction process each
manual back pressure regulator will yield different ratios of components based upon polarity,
density and molecular weight.
3. The “SFE 2X5LF System” will use 1 CO2 tank approximately 90 minutes of operation (one
extraction) without a recycler. Each tank has about 2/3 of 50 pounds of useable CO2.
4. The “SFE 2X5LF System” will use 1 CO2 tank approximately 600 minutes of operation (about
seven extractions) with a recycler.
5. Safety Information and Details:
a. Lab safety glasses are mandatory and should be used at all times.
b. Use disposable gloves, booties, and lab coats, and optionally a face mask.

B: Tools Required for Installation and Operation:
Customer Required
~ 3 meters -12 feet of space for system

5 Power Connectors
Channel Locks (C02 Tank) 12/16”
Heat Gun
Funnel for Recycler Chiller
linelines
Shop Vacuum with long extension
Funnel with 3.0” Opening
Packer (to pack samples down)
Paint Brush
Grinder to ground samples up
Ethanol for cleaning Extractors -Collectors
Flasks
Chemwipes
50:50 Ethylene Glycol– 4 Liters (Antifreeze)
Marbles for extraction vessels - 6 packs
3 ¼ Brush for cleaning inside of vessels
CGA 320 fitting to CO2 tank
7 CO2 Tanks with dip-tube
Waste line to vent Exhaust outside
6 Plastic Coated Erlenmeyer Flasks 500ml
Containment Vessel for Collection Flask
Computer Cart - Stand

C: SFE 2X5LF System Components
1. The system configuration consists of the following components:

2. The basic flow path for installation and operation is as displayed below.

D: The CO2 Recycler for the 2X5 LF System
1. The CO2 Recycler will reclaim a large amount of the CO2 exhaust from the SFE 2X5LF System
exhaust point. CO2 not reclaimed is the residual CO2 in the extraction vessels and the CO2 inside the
Collection Vessels. The major components of the CO2 Recycler are displayed below:
1 From CO2 Tank 6 Pressure Relief Valve
2 From SFE 2X5LF System Exhaust 7 Level Sensor
3 Manual CO2 Supply Valve 8 Drain Valve
4 To SFE 2X5LF System CO2 Inlet 9 Pressure Gauge
5 Heat Exchanger 10 Level Sensor Display Box (not displayed)
2. Locate the Level Sensor System Box. Attach the Level Sensor cable from the recycler to either of the
In/Out connectors. This is located in the back of the level sensor system.
3. Attach the power supply to the Level Sensor System box.
Note: The Level Sensor System box will not show any power indicator until the CO2 recycler has been
filled with enough Co2 to activate the “Low Low” Level sensor. If the power cord is connected, then
assume the power is on.

E: System Workflow Summary for Plant Extractions
Note: The 2x5LF System workflow summary to complete an extraction is described below. The
general steps are detailed in this section, while the specific instructions for each are in
Sections J-N of this manual.
1 Fill Extraction Vessel with Sample

2 Turn on Process Suite
3 Turn on Heaters
4 Turn APR
5 Turn on Pump
6 Let System Equilibrate to Pressure
7 Set Manual BPR pressures for each Collection Vessel
8 Wait 75-90 minutes for extraction to complete
9 Depressurize System
10 Collect Fractions- Lowest Pressure First
11 Fill 2nd Extraction Chamber and Connect to System
12 Switch Extractions Chambers, Empty and Clean
13 Repeat Process with Recycler
1. Power on the Chiller and set the temp 5 C. Let the system re-circulate and stabilize before operation.
If necessary add more Ethylene Glycol/Water (50/50 mix) to the chiller.
2. Also make sure all modules on the system are connected to power and powered on in the system
(ABPR, P200 Pump).
3. Turn on the CO2 Heater temperatures for the extraction chamber you are using, HE 2 Heat
Exchanger, and collection chambers as listed below.
4. Open the CO2 Tank, and then check for leaks at the incoming connector to the SFE 2X5LF System
base unit.
5. For optimal extraction, expose as much surface area to the botanical product by grinding the
botanical product using a Coffee or other style grinder.
6. Pack the sample into the extraction chamber using a wooden or plastic rod. Be careful to not scratch
the sides of the extraction vessel.
7. Spray the Extraction end cap with Teflon spray before each use.
8. Turn on CO2 flow and use Process Suite to setup the generic method parameters as listed below:
2x5000 LF System without Recycler – Total Extraction Time ~90 minutes
ABPR HE2 Extraction Collection Collection Flow Manual BPR Manual BPR Manual BPR
(bar) Temp Temp Temp Temp (mls) (PSI) (PSI) (PSI)
(1-2) (3) Collector 1 Collector 2 Collector 3
200-350 50-60 40-50 50 30 150-180 2500-3500 1200-2000 30-100

2x5000 LF System with Recycler – Total Extraction Time ~90 minutes
ABPR HE2 Extraction Collection Collection Flow Manual BPR Manual BPR Manual BPR
(bar) Temp Temp Temp Temp (mls) (PSI) (PSI) (PSI)
(1-2) (3) Collector 1 Collector 2 Collector 3
200-350 50-60 40-50 50 30 150-180 2500-3500 1200-2000 ~800
*Collector 3 pressure will be dependent upon recycler pressure.
9. Allow the system to come up to pressure by comparing the ABPR pressure setting to the Extraction
chamber pressure gauge.
10. Once the Extraction pressure gauge and the ABPR have reached the same pressures, manually adjust
the collection manual back pressure regulators in order of MBPR 1, 2, and then 3 (using the pressures
settings as listed above). It is important to wait for the system to come up to pressure before
adjusting the back pressure regulators.
Note: If you overpressure the Manual back Pressure Regulators, you risk the chance of blowing the
fraction collectors burst disk.
11. Run the system for around 90 minutes.
12. Open up the Collection Valve to collect your sample using the following process:
a. Turn off the CO2 pump flow using the Stop System button.
b. Isolate the collection cyclone from CO2 to by closing the MV2 valve or closing the ABPR.
c. Open up Collection Valve 3 (Back collector) by turning the handle slowly and collecting the extracts.
Do not open the collection valve all the way.
d. Use a heat gun on the valves to get out the remaining extracts.
e. Close the Extraction Vessel 3 Valve, then repeat the process with Collector 2 and Collector 1.
13. To switch vessels, close off MV1 – V1 and MV1-2 then connect Extraction vessel 2 to MV2 valve.
14. Open both MV2 –V1 and MV2-V2 to the open position. This will bleed off the CO2 to other chamber.
15. Close V1 Valves and wait unit the pressure gauge on the cyclone, CS1-PT1, reads 0 Bar and CO2 is no
longer exiting the bleed valve.
16. When using the recycler, open the valve to the CO2 tank when the Level Sensor is Low-Low and refill
only to the Low position. Do not add CO2 above the Low position.

F: Pre-Setup for SFE Sample Extraction
1. Power on the System and set the System Chiller to 3 C. Turn on the Recycler chiller and set the
temperature to 11 C. Let the system re-circulate and stabilize before operation. If necessary add
more Ethylene Glycol/Water to the chiller.
2. Turn on the CO2 Tank fill and open the fill valve on the CO2 recycler until the “Low” light is displayed.
3. Check to make sure that the incoming pressure is ~50 psi higher than the recycler pressure.

4. Turn off the CO2 tank Manual Fill Valve when the “Low” light is displayed.
5. For optimal extraction, expose as much surface area to the botanical material by grinding the
botanical product using a Coffee or other style grinder.

6. Unscrew the Extraction vessel cap by turning counter-clockwise. Load the material using a funnel.
Pack as much plant extract into the extraction chamber using a wooden packer up the fill line. If you
don’t have enough plant extract to fill the chamber, use inert marbles to fill the remaining chamber
space. Use a paint brush to wipe the plant extract off the threads.
Fill Line
5. Spray the extraction cap fittings with PTFE spray lubricant each time during normal operation.

6. Hand tighten the end cap (do not use tools), then use secure the tubing from MV-2 – V1 to the
Extraction Chamber end cap. Be careful not to cross thread the connector.
7. Fully open MV1 – V1 and close MV1 – V2 valves. Fully open MV2 – V1 (extractor 1) and close MV1 –
V2 (extractor 2) valve. Note: Extraction can only use one extractor vessel at a time.

G: Software and System for Plant Extraction – No Recycler
1. Power on computer and login as: User: Administrator Password: waters (case sensitive)
2. Click on Process Suite and use Login: Admin Password: Admin
3. Familiarize yourself with Process Suite using the 6 main areas of navigation for the software.
1. Automatic Back Pressure Regulator Manual Control

2. CO2 Pump Manual Control
3. Heat Exchanger Manual Control
4. Method Settings: Pump Control, Back Pressure Control
5. Start Method
6. Stop all Flow and ABPR

4. Click on the Heat Exchanger icon to manually set the temperature control for the Heat
Exchanger and Extraction Chamber heaters (the Heater Control for the Collection
Chambers is setup in a separate temperature control).
5. Click on the Heater Control “Device Settings” button to set the extraction chambers zones
temperatures for the vessel you are using (do not set the other extractor temperature).
6. Click on the Heater Control 1 Device Settings button to set each zones temperatures. The following
zones are for heat control or the extraction vessels and heat exchanger.
Zone Description Temperature Settings

Zone 1 Vessel 1 Heater 40-55
Zone 2 Vessel 1 Internal Temperature - Read back View Only – No Need to Set
Zone 3 Vessel 2 Heater 40-55
Zone 4 Vessel 2 Internal temperature -- Read back View Only – No Need to Set
Zone 5 Heat Exchanger 2 – before the Extraction Chamber 45-65
Zone 6 Not Used

7. Adjust all temperature set points, and then click OK.
Note: Apply will keep the Heater Control setting window open and OK will close the settings dialog box.
8. Turn on 5L Vessel 1 Heater, turn on Vessel Internal temperatures, and Electric Heat Exchanger by
clicking on the toggle button.
9. Switch to the Process Suite Main window and from the menu bar, select “View- Heater Controller 2”.
This will be used to setup the Collector chambers temperatures.

10. In the Heater Controller 2 area, select Device Settings to setup the Collection Chambers
temperatures.
11. Set Collection Vessel 1 and 2 Zone settings to a set point temperature of 45-65C. Set Collection
Vessel 3 to a set point of 30C. Adjust all temperature set points, and then click OK.
Note: Apply changes the set point but keeps the window open, OK changes the set point but closes the
window.

12. Toggle each Cyclone Heater to the on position to enable the collection heaters.
13. Click on the Automatic BPR icon to manually control the ABPR.
14. Click on “Device Settings” to manually setup the ABPR pressure.

15. Enter the “Pressure Set Point” to 375 bars, and select “OK”.
16. Click on to enable the BPR.

17. In the Process Suite main menu, double click on the CO2 Pump icon to manually
control the CO2 Pump.
18. In the CO2 Pump dialog box, click on “Device Settings”.
19. In the Set Point area, enter the flow rate to 175 g/min, and select Apply.

20. In the CO2 Pump dialog box, click and the pump will start flow and the ABPR will start to
build pressure.
21. Check the ABPR pressure and Pump Flow Trend plots. Make sure that the ABPR pressure and pump
flow rate stabilize.
22. Check the Flow Rate versus the RPM reading. If the RPMS are high, then check the flow meter for CO2
Density (the RPM should be within 20% of the flow settings). The pump RPM may read different from
the pump flow rate until the extraction chamber and ABPR are at pressure.
Note: See the system troubleshooting section for other pump related issues.
23. Wait a few minutes for the Extraction chamber 1 get up to pressure. Use the CO2 gauge on top of the
extraction chamber to check the pressure steadiness. The extraction pressure should be about the
same as the ABPR setting.

Important: Let the Extraction chamber get up to the full ABPR pressure before completing the next
steps. If it is not at full pressure, it is possible you might blow a burst disk on the collection chambers
when pressuring the manual Back pressure regulators.
24. For Collector 1’s Manual Back Pressure Regulator, adjust the pressure to 2500-3500 psi by manually
rotating the valve clockwise to increase pressure on Regulator to get the following backpressures.
Notes:
- Turn the valve and wait for the pressure to adjust before turning the valve again. Wait 15-20
seconds for the collection ABPR to get to system pressure.
- The Valve is completely closed or open with about 9 turns.
- It is important that you do not pressurize the MBPR for collector 1 above ~5000psi, as you could
blow the collectors burst disk which is rated at 6000psi.
- If in danger or over-pressurization, open up the collection valve to bleed some CO2 pressure from
the system.
25. Repeat for collectors 2 and 3 adjusting to the pressures below:
Collector 2: 1200-1800 psi Collector 3: 30-100 psi– No Recycler Collector 3: ~800 psi -Recycler
Note: Setup of the system with the recycler is documented in the next section (Section L).
26. Let the system Pump CO2 with the backpressures and temperatures for ~90 minutes.
27. Next, if you do not have a recycler, go to section M, “Collection of the Extracted materials”.

H: Software and System Operation for Extractions with the Recycler
1. Repeat section K steps 1- 25. The procedure to run an extraction for pump flow, temperature,
pressures is the same without a recycler and with a recycler.
2. For Collector 1’s Manual Back Pressure Regulator, adjust the pressure to 2500-3500 psi by manually
rotating the valve clockwise to increase pressure on Regulator to get the following backpressures.
Note:
- Turn the valve and wait for the pressure to adjust before turning the valve again. Wait some time
for the collection ABPR to get to system pressure.
- The Valve is completely closed or open with about 9 turns.
- It is important that you do not pressurize the MBPR for collector 1 above ~5000, as you could
blow the collectors burst disk which is rated at 6000psi.
- If in danger or over-pressurization, open up the collection valve to bleed some CO2 pressure from
the system.
3. Repeat for collectors 2 and 3 adjusting to the pressures below:
Collector 2: 1200-1800 psi Collector 3: ~800 psi – with Recycler
Note: It is important that the collector 3 pressure be higher than the pressure on the recycler. This is to
keep any of the plant extracts from getting into the recycler and pump. If the sample gets inside the
recycler chamber, the chamber may become blocked and have to be cleaned.
4. Let the system Pump CO2 with the backpressures and temperatures for ~90 minutes. Times for
extraction may be longer or shorter depending upon the product you are extracting.
5. Check the Level Sensor System monitor.
Note: The level Sensor System Monitor will not show any power light until enough CO2 fills the recycler
to get to the “Low – Low position.

6. The recycler will need to be refilled from the CO2 tank when the Level Sensor System monitor is
display with just the low-low light is on. Fill the recycler to only where the Low system light is lit.
Warning: Do not fill the tank to the High or High –High levels.
7. The recycler system is like a toilet valve. There are specific levels that are needed for the recycler to
work properly. The levels in the recycler tank are:
Low - Low Low CO2 Open Fill Valve from CO2 Tank
Low Proper CO2 Range Close Fill Valve from CO2 Tank
High Overfilled CO2 in Tank Drain Excess CO2 from Recycler
High - High Overfilled CO2 in Tank Drain Excess CO2 from Recycler

8. If you do over fill the recycler to the High or High – High position, use the bottom valve (or side valve
if installed) of the recycler to vent CO2 until the Level Sensor System displays the Low position.
9. Collect your extractions. See section I for detailed instructions.
10. If sample from the CS3 valve did get into the recycler, open the recycler drain valve located at the
bottom of the recycler storage container. This will drain CO2 and any sample from the bottom of the
storage chamber.

I: Collection of the Extracted Materials
1. Use a plastic coated Erlenmeyer flask within a waste containment vessel to get your sample. The
sample can be at high pressure so it is advised to have a second containment vessel.
2. It is recommended to use a lab coat and gloves, as well as use the heat gun during this process. The
heat gun will be used on the tubing located by the collection vessels to ensure the extracted oils stay
in solution and not plugged. Caution: Overheating the lines may burn or convert the extracted
material, so use a low heat setting.
3. To get your extracted sample follow the following procedure:
4. Identify each Collection Chamber as displayed below in the diagram:

5. Turn off the CO2 pump flow using the stop flow to the system.
6. Turn off CO2 to the cyclone by closing MV2-1 or adjusting the ABPR to fully close.
Note: This isolates the extraction chambers, ensuring the only CO2 pressure are from the collection
vessels. Make sure that the Pump flow is off.
7. Locate the furthest back collector (CS3), and locate the MV10 Valve located at the collector outlet.
 Slowly open the MV10 Valve to get your extract. You may need to toggle the valve to get out
your extract. Note: Be careful opening the valves.
 Close the MV10 valve when the collection pressure is zero pressure.
 This extract may be loose and slightly runny. This will be the most non-polar sample extract.

8. Select the middle collector (CS2), slowly open the MV9 Valve to get your extract.
 This extract will be tooth paste in consistency. Toggle the valve to get out your extract.
 This will be medium non-polar sample extract. Close the valve after collecting the extract.
9. Select the front collector (CS1), slowly open the MV8 Valve to get your extract.
Note: This valve will have the highest collection pressure, so use caution when turning the valve.
 This collection extract will be the lowest non polar sample and may be thick and gooey in
consistency. Once you have collected the extract close the valve.
 Use a heat gun on the valve tubing to aide in getting all the collected extract.
10. Once all collection vessels have been depressurizes, continue onto to step J, Switching and Cleaning
the Extraction Modules.

J: Switching and Cleaning the Extraction Chambers
1. Click on the “Stop All” to turn off the CO2 pump, ABPR.
2. Select the Heater Zone 1 and Heater Zone 2 and toggle off the heaters for the Extractor and the
collection chambers. The heaters do not turn off with the “Stop All” command”
3. Fill the 2nd 5 Liter Extraction Vessel (V2) with the plant extract and attach the 2nd 5 Liter Extraction
Vessel (V2) to the MV2 valve.

4. Slowly open MV1- V2 and keep open MV2 –V. Also open MV2 –V2 and keep open MV2-1. This will
send residual CO2 from Extraction Vessel 1 to Extraction Vessel 1.
5. Wait until pressures on both gauges equalize. This will re-use some of the CO2 from extraction
chamber 1 to extraction chamber 2.
6. Keep MV1-V2 valve open and close MV1-V1.
7. Close MV2-V1 valve for extraction vessel 1 and keep MV2-V1 open for extraction vessel 2.
8. This will close the CO2 to Extraction Vessel 1 so it can be cleaned and refilled.

9. Drain the residual CO2 by opening the MV6 vent valve located on the left backside of the extraction
vessel. Open the valve gradually, as there will be high pressure in the vessel.
Note: This valve must be exhausted to an outside vent.
Vent Line
10. Use the following steps as stated below:
a: Open the vessel by turning counterclockwise.

b: Remove the residual plant material waste from the extraction vessel.
c: Inspect the end frit and o-ring for any issues or damage.
d: Reload the next batch of material as stated in section E: Preset up of Plant Extraction.
e: Operate the system as stated in the procedures

K: System Troubleshooting
1. Check the Mass Flow Meter use the selection button to display Density on the flow meter.
The CO2 Density should be >0.87 g/cm3. If the density is lower, the CO2 tank or recycler may need
to be refilled to the “Low” position. The Tank may also be empty, or you have a system leak.
2. Compare the Pump CO2 Flow rate to the RPM value. The RPM of the pump should be within ~10% of
the flow rate. If the values are less, check the items listed on the next page.

3. When the RPM are higher or lower than the flow rate selected, the following could be the issues:
a. The recycler may need to be refilled with CO2 from the supply CO2 tank.
b. Look below the Extraction chamber and make sure there is not ice buildup. There may be blockage
inside the changer or the heat exchanger is not functioning.
c. Make sure the tubing connectors do not have leaks or bad fittings.
4. The incoming CO2 line from the tank/recycler into the SFE 2X5LF System base unit has a filter that
can be blocked and cause high pressure. If this is the case, remove the top nut and replace the filter
as needed. The part number is 700007354 Filter Kit, 5 micron for part# 06013 (skid inlet).

5. Troubleshooting the CO2 Recycler Chiller – Local Lockout Issue - To disable local lockout
- Press and Hold the Select Know Key until “Can” appears momentarily as local lockout changes
from enabled (LLo) to disabled (about 5 seconds).

L: Preventative Maintenance Information
Note: PM, Maintenance and Repair should be completed by a trained Waters Field Service
Engineer.
1. Recommended PM is 4x a year with 24/7 operation or 1x a year with 8/5 operation.
2. There is not a SFE 2X5LF System pm kit, though there are individual pm kits, as listed below:
SFE PM Kit Summary SFE 2X5LF System System (Number indicates kits required)
Module p/n SFE SFE 2X5LF System Unit
P-200 700007688 1
ABPR-200 700007693 1
High Pressure Vessel 5L 700006619 2
High Pressure Cyclone 1000 ml (per cyclone) 700006566 3
3. The recommended spare parts kits are PN 205001332 - Kit, SFE Bio-Botanical Spare Parts. Below is a list of
the parts:

4. The Collection modules have specific gauges, rupture disks and manual BPR’s. Below is a listing of the
description and part numbers for these components (these are already installed).
Vessel # Vessel Label Pressure Gauge Rupture Disk Manual BPR

Collection 1 CS1 6Kpsi - 70010093 6KPSi - 700007356 6Kpsi Max 70010094
5. The rupture disk breaks if you overpressure the Manual Back Pressure Regulator. Below is the location
of the rupture disk on the collection chamber.
6. The Extraction Vessels may need the filters replaced. Below is a list of replacement and consumable
parts for the extraction chambers.

M: ABPR Alignment Procedure and Details
1. The Automatic Back Pressure Regulator (ABPR) is an electronically motor driven controlled needle
valve that keeps the CO2 in supercritical state (pressures above 73.7 bar and temperatures above
33C for the extraction vessel).
2. The ABPR operates by closing a needle into a seat to build pressure.
3. The ABPR needle position controls the flow through the valve by adjusting the gap between the valve
needle and the seat which corresponds to a needle count of between 0 and 6000.
a. “0” position is fully closed (no flow). This builds the back pressure to the extraction vessel.
b. “6000” position is fully open. This means the system will be unpressurized full flow.
4. Once the valve reaches the desired pressure for the extraction vessel the valve slowly opens in a
controlled state so as to keep the material in the extraction vessel at a supercritical state, but allow
CO2 flow out to the 3 collection vessels.
5. The needle position can be viewed in the “Pressure Regulator” section of Process Suite.

6. If the ABPR pressure is erratic and the P200 pump pressure is steady, the needle and seat may need
to be “re-aligned” to as to properly place the needle into the seat. This is the ABPR Alignment
procedure.
7. If the Alignment does not work the ABPR needle and seat may have to be rebuilt using the “ABPR 200
PM Kit” which can be ordered using Waters part number 700007693.
8. Below and the next several pages detail how to complete the ABPR Alignment procedure.
9. Setting up for the Alignment and materials needed:
Tubing, 1/8"OD X .035"WL. Straight 316SS 01829

Fitting, 1/8" L/P B Gland Nut 700006662
Fitting, 1/8" L/P B Ferrule 700006663
500 ml plastic beaker with Water n/a
wrench n/a
linelines
8. Disconnect the outlet of the ABPR tubing using a ½ wrench on the outside and a 9/16” wrench on the
backing connector. Replace with the tubing, nut and ferrule as listed above. In addition you will need
a ½ filled beaker of water.

ABPR Alignment
1. ABPR Alignment is used to adjust the zero position so there is no flow of CO2 from the system. This
means the needle should be completely in the seat. If CO2 is still flowing then make the following
adjustment.
2. Exit Process Suite.
3. Open the ABPR Alignment program on the PC.
4. In the ABPR main program area, click the beaker on the right hand side to make large needle moves
(Figure 36). Do this several times till the flow is lower (Small amount of bubbles).
5. Click the beaker the middle to make medium needle moves (Figure 36). Do this several times till the
flow is still lower (Smaller amount of bubbles).
6. Click the beaker on the left hand side to make small needle moves (Figure 36). Do this several times
till the flow is stopped (No amount of bubbles).
• For the ABPR 200 a few CO2 bubbles is permitted.
• For the ABPR 20 it is critical that all CO2 is stopped at the zero position.
Notes: The ABPR Alignment program automatically calculates an Overshoot which is used to
calculate the position for the needle to slow down and end up overstepping the motor position.
For example, if zero is at needle position 300, the motor will be stopped at 288 and the needle
can drift to 300 (closed position).
7. Exit the software by clicking on the [File/Exit] menu. The program will force the ABPR to reset.
8. Turn the ABPR off and then on, waiting 15 seconds before restarting Process Suite.

N: Appendix Section – System Schematics
E SFE 2X5LF System Electrical Connections:

O: SFE Method Development Guidelines with the 2x5LF System
The SFE extraction process is empirical by nature and successful compound extraction will require
changing a number variables. The chart below shows the different variables that can affect an SFE
extraction. These method development parameters will be discussed in this section.
1. Pre-test your botanical sample for mycotoxins and mold. Use the AflaCheck Test Kit – Available from
Vicam Corporation (see attached brochure and procedure).
 Test for Ochratoxin A - Toxic to the Kidney – 20 ppb limit

 Test for B1,B2,G1.G2 Aflatoxins - Cancerous mold – 20ppb limit

2. Knowledge of Your Sample: Key items to determine - Is your sample soluble in CO2?
 Sample solubility in Supercritical CO2 is about the same as Heptane.

 Trying adding ethanol or methanol to the sample? Does it go in solution?
 Listed below is the Polarity of some common co-solvents to aide in solubility:
3. Determine Sample Prep for the Sample.
Polarity Prep Procedure- Advantages

1 Non Polar or Polar Filter Plant Extract or Sample from Soil
2 Non Polar or Polar Grind Sample to expose as much surface area – aides in CO2 Diffusion
3 Non Polar or Polar Sizing Particles– aides in CO2 Diffusion
3 Non Polar or Polar Air Dry Sample
2 Non Polar or Polar Freeze Dry Sample
4 Non Polar or Polar Low Heat ~70-80C*
6 Polar Only Pre-Soak in Extraction Vessel with Ethanol or Methanol for 30 minutes **
* Volatile Components may evaporate
**Optional: Use Co-Solvent Pump if available
4. Determine the Extraction Technique. There are 3 styles of extraction for the SFE system:
Dynamic Static Extraction Dynamic – Static - Dynamic

Coffee Maker Style Tea Cup Style Combination
Continuous CO2 flow through CO2 is pumped into the extraction Continuous flow until the
Sample Extraction Chamber to chamber then samples is Extraction chamber is full, then
the Collection Chamber “Soaked” with CO2. No Flow after Static for a time, then Dynamic.
Chamber filled until collection.
Advantage: Fast Great for Non- Advantage: May help less non- Advantage: Ability to get
Polar Compounds polar compounds – aides in separate fractions and aides in
diffusion of CO2 diffusion, iterative
Disadvantage: Contamination Disadvantage: Longer Time Disadvantage: Longest Time
may build up at the Collection
Vessel
Disadvantage: Volatiles may be Disadvantage 2 : May not Extract Must Experiment with both
blown from the Collection all components Static and Dynamic Times
Vessel

5. Determine the Extraction and Collection Temperatures:
a. The general temperatures settings for extraction are between 40-70C.

b. The Pre-extraction Heater (HE2) should be set ~5 degrees higher than the extraction
Chamber temperatures.
c. Collection temperatures are usually 5-10 degrees higher than the extraction temperature.
6. Determine the Extraction Pressures (ABPR and Extraction Chamber Pressures):
a. The general ABPR settings for SFE extraction is between 100-375 bar.
b. When adjusting ABPR pressures, wait for the extraction chamber to equilibrate to the same
temperatures as the ABPR before changing other pressures.

7. Determine the Extraction Times.
a. The time of extraction is dependent upon several variables including the style of
extraction, sample polarity, matrix, solubility and diffusion.
b. A suggested model for testing is to extract for 60 minutes and collect a fraction. Then,
extract the same sample for 30 minutes more and collect a fraction. Repeat additional 30
minutes segments until no additional materials are collected.
c. Another technique is to add a static extraction segment for 4 hours and collect.
One Sample Extraction Example* *
** May not be typical of your extraction

8. Determine the Collection Chambers Pressures.
a. Collection pressures have to be lower than Extraction pressures.

b. When setting collection pressures, it is important to make sure that the extraction
pressures have been equilibrated before adjusting the manual back pressure regulators.
c. For the 3 fraction collector system adjust the first (front) collector, then middle, then back
collectors.
d. If you have a CO2 recycler, you must have the back pressure regulator higher than the
incoming recycler tank pressure.
e. Sample collection will be a mix of your components though more of the nonpolar condense
at the lower pressures.
f. General starting pressures should be from 3000 psi for the first collector to 800 psi for the
last collector.
* Samples collected will be a mixture of different polarities with a tendency of your more nonpolar samples being
in the lower pressure collector.
g. General starting pressures should be from 1000 psi to 3000 psi for collection pressures.
h. If you stop the pump and have to depressurize the system before collection, drop the ABPR
pressures in 25 bar increments a minute (do not decrease pressure all at once). This is to
eliminate the sample from being exhausted out instead of collected.
9. Collection of the Sample Extract.
a. Stop the pump

b. Close the MV2 valve located between the Extraction vessel and the ABPR.
c. Start collection with the furthest back collector (CS3).
d. Open the collector valve slowly. Mark where the valve is open with a marker to determine
optimal collection.
e. Depressurize the chamber completely before starting with the next collector.
f. You may have to toggle the valve to get viscous samples collected.
g. Open the collector valve slowly. Mark where the valve is open with a marker to determine
optimal collection.

10. Post Extraction - Once the sample has been collected, there are numerous analytical techniques and
processes to identify or purify the components collected. These include:
a. Determine analytically the component or components that have been extracted. This can
be accomplished by using a Waters UPC2 or UPLC Chromatographic system. The addition of
a QDa mass detector can aid in compound identification by determining the Analytes mass.
b. Sub-fractionate the components using a SFC Chromatography System like the SFC Prep 80
system.

Mycotoxin Detection
for Botanicals
34 MAPLE STREET, MILFORD, MA 01757 USA

TEL: 800.338.4381, 508.482.4935
FAX: 508.482.4972 EMAIL: VICAM@VICAM.COM
TABLE OF CONTENTS
1.0 INTRODUCTION .........................................................................................................................2
1.1 Safeguarding the Efficacy and Quality of Botanicals................................................................................2
1.2 Mycotoxins and Human Disease..................................................................................................................2
1.3 Regulations...................................................................................................................................................2
2.0 METHODS FOR THE ANALYSIS OF AFLATOXIN AND OCHRATOXIN .................................................3
2.1 AflaCheck® 20 ppb Cutoff Procedure for Botanicals................................................................................3
2.2 AflaTest®.......................................................................................................................................................5
2.3 AflaOchra™ procedure for HPLC (AOAC method 2008.02 for Ginseng and Ginger)
proposed for Various Botanicals..................................................................................................................6
3.0 LIABILITY ..................................................................................................................................7

1.0 INTRODUCTION
1.1 Safeguarding the Efficacy and Quality of Botanicals
Ensure that Botanicals are free from unwanted contamination by aflatoxin, ochratoxin and other naturally occurring mold toxins. Botanicals
may become susceptible to mycotoxin contamination during cultivation, storage or processing. The natural byproducts of several mold
species, aflatoxin and ochratoxin present serious health risks to humans and animals – including liver toxicity, immune suppression, and
aflatoxin is a known carcinogen according the International Agency for Research on Cancer (IARC).
VICAM’s family of mycotoxin testing solutions offers your business a full range of versatile, practical solutions to aflatoxin and ochratoxin
monitoring. From simple, on-site screening strip tests to quantitative fluorometer data to highly sensitive HPLC or UPLC detection in the
laboratory – VICAM delivers proven aflatoxin and ochratoxin detection and monitoring to support your Botanicals business and the
analytical laboratories that you trust for quality and safety confirmation.
VICAM solutions empower you to:
§ Manage botanical quality with confidence
§ Ensure consistent product purity
§ Meet internal and industry regulatory requirements with ease
1.2 Mycotoxins and Human Disease

Mycotoxins are metabolic products of plant spoilage fungi that induce toxic responses when consumed by animals or people. Hundreds of
mycotoxins have been identified. They fall into many different chemical classes and induce a wide variety of toxic responses.
Aflatoxin, a chemical toxin naturally produced by Aspergillus flavus and Aspergillus parasiticus, is a Group 1 carcinogen proven to cause
cancer in humans. Major aflatoxin types are B1, B2, G1 and G2. The aflatoxins fluoresce strongly in ultraviolet light; B1 and B2 produce a blue
fluorescence whereas G1 and G2 produce green fluorescence.
Ochratoxin A is a chemical toxin naturally produced by Aspergillus ochraceus, Penicillium verrucosum and Penicillium carbonarius.
Ochratoxin is thought to be a kidney toxin and has been linked to Balkan endemic nephropathy. The International Agency for Research on
Cancer ranks ochratoxin as a group 2b, possible carcinogen.
1.3 Regulations
Most recently, the State of Colorado and the State of Connecticut have set regulations for total Aflatoxins B1, B2, G1 and G2 in medicinal
and recreational botanicals at less than 20 ppb. Comparatively, The United States federal government action level for aflatoxin in food for
human consumption or in dairy cow feed is 20 ppb. For milk for human consumption, the highest permissible level is 0.5 ppb aflatoxin M1.
The State of Connecticut has set a regulation of less than 20 ppb Ochratoxin A in medicinal botanicals.
Mycotoxin Detection for Botanicals [ 2 ]

2.0 METHODS FOR THE ANALYSIS OF AFLATOXIN
2.1 AflaCheck 20 ppb Cutoff Procedure for Botanicals (Procedure Time: Less Than 10 Minutes)
Sample Extraction * Strip Test Pipettor Use (Steps 2 and 3 above)
1. Weigh 1 g of sample and add to an Extraction Tube 1. Squeeze the top bulb of the pipettor.
2. Measure 4 mL of 70% Methanol with a 10 mL Graduated 2. Draw up enough liquid to fill the barrel of the pipettor
Cylinder and pour the solution into the Extraction Tube. and have one or two drops of liquid spill over into the
3. Cover the Extraction Tube and shake the mixture well for Lower bulb.
1 minute. 3. At this point the barrel of the pipettor should be completely
4. Filter through (VICAM part #600001106) filter into full of liquid.
clean vessel using pressure to extract as much solution 4. To ensure accurate measurement, be careful not to let
as possible. any liquid drip out of the pipettor before dispensing.
5. Squeeze the upper bulb to dispense the liquid.
AflaCheck Procedure
6. Squeeze all of the liquid out of the barrel when dispensing.
1. Place a Strip Test Dilution Tube in the Paper Rack. When
the kit first arrives, the Paper Rack will be nested
upside-down inside the box. Interpretation of Results For 20 ppb
2. Add 250 μL of Distilled Water to the Strip Test Dilution Cutoff Procedure
Tube with a 250 μL Strip Test Pipettor.* Negative Results:
3. Transfer 250 μL of sample extract to the Strip Test Sample contains < 20 ppb of aflatoxin.
Dilution Tube using a new Strip Test Pipettor.* (Any visible test line indicates a negative result.)
4. Mix solution by capping the Strip Test Dilution Tube and
shaking by hand.
5. Insert an AflaCheck Strip Test (arrows pointing down)
Positive Results: TEST LINE CONTROL LINE
into the Strip Test Dilution Tube and allow the test
Sample contains ≥ 20 ppb of aflatoxin.
to develop.
6. A negative result (less than 20 ppb) can be determined
once you can see both a test line and a control line. This
can occur in as little time as 3 minutes. Invalid Results: Test is invalid. CONTROL LINE
7. To check for a positive result (greater than or equal to Repeat with new Strip Test.
20 ppb), allow the AflaCheck Strip Test to develop in the

Strip Test Dilution Tube for at least 5 minutes. If after
5 minutes no test line appears then the results can be
interpreted as positive.
[ 3 ] Mycotoxin Detection for Botanicals

AflaCheck Botanicals Kit (176003475)
Contents for 25 Tests:
25 AflaCheck Strip Tests (100000175)

50 Strip Test Pipettors (600000812)
25 Strip Test Dilution Tubes (600000813)
Filter Paper (600001106)
1 Paper Strip Test Rack
1 AflaCheck Instruction Sheet
Packaged Separately:
(50) 40 mL Extraction Tubes (600000827)
Purchase Separately:
10 mL Graduated Cylinder (G4047)

Digital Timer (G4036)
Source Locally:
ACS Reagent-Grade Methanol

Distilled Water
Store at room temperature (15°–30 °C). Strip test

and sample extract should be at room temperature
before use. For use in accordance with vicam testing
procedure. Not to be ingested or applied to skin.

2.2 AflaTest Procedure for Botanicals (Small Sample Size – 1 gram sample) (0 – 500 ppb)
(Procedure Time: Less then 30 minutes)
1.0 Sample Type: Botanicals 6.0 Extract Dilution:
2.0 Detection Instrument: 6.1 Pipet or pour 5 mL filtered extract into a
Fluorometer model VICAM Series 4 or Series 4EX clean vessel.
3.0 Calibration Settings: 6.2 Dilute extract with 20 mL PBS 2% Tween Wash
Green Red Yellow Buffer solution.
-2.0 110 54±5 6.3 Mix well.
4.0 Set up: 6.4 Filter dilute extract through 1.5 μm glass
4.1 Calibrate fluorometer. Make sure the yellow microfibre filter (VICAM part #31955) into a
Calibration Standard reading is in the range clean vessel.
listed above. 7.0 Column Chromatography
4.2 Prepare methanol:water 60:40 (by volume) 7.1 Pass 10 mL of filtered extract through the
solution every week or as needed. AflaTest column at a rate of about 1 drop/second
4.3 Prepare a PBS 2% Tween Wash Buffer solution leaving column resin bed slightly wet.
(VICAM part #G1105) in purified water every 7.2 Repeat previous step once more
week or as needed by following instructions (20 mL = 0.20 g sample equivalent).
on bottle. 7.3 Pass 10 mL of PBS through the column at a rate
4.4 Prepare a PBS solution (VICAM part #G1113) in of about 1-2 drops/second leaving column resin
purified water every week or as needed by bed slightly wet.
following instructions on bottle. 7.4 Pass 10 mL of purified water through the
4.5 Prepare AflaTest Developer solution every column at a rate of about 1-2 drops/second until
8 hours by following instructions on bottle. air comes through the column.
4.6 Make sure that reagent blank (1 mL 7.5 Place glass cuvette (VICAM part #34000)
methanol + 1 mL Developer in a cuvette) reads under column and add 1.0 mL HPLC grade
0 ppb on a calibrated fluorometer. methanol into glass syringe barrel.
5.0 Sample Extraction: 7.6 Elute column at a rate of 1 drop/second or

slower by passing the methanol through the
5.1 Weigh 1 g ground sample and place in
column and collecting all of the sample eluate in
extraction tube.
a glass cuvette.
5.2 Add 20 mL methanol:water (60:40).
7.7 Add 1.0 mL of AflaTest Developer to eluate in
5.3 Shake for 3 minutes.
the cuvette. Mix well and place cuvette in a
5.4 Filter through VICAM 31240 Filter calibrated fluorometer. Read aflatoxin
(Cut to 2 Inches) into a clean vessel. concentration after 60 seconds.

2.3 AflaOchra Procedure for HPLC (AOAC Method 2008.02 for Ginseng and Ginger)
Proposed for Botanicals
1.0 Sample Extraction:

1.1 Weigh 5 g ground sample and 1 g salt and place in a 50 mL centrifuge tube.
1.2 Add 25 mL methanol:0.5% sodium bicarbonate (NaHCO3) (70:30) solution.
1.3 Shake at 400 rpm for 10 minutes.
1.4 Centrifuge for 10 minutes at 7000rpm (5323g).
2.0 Extract Dilution:
2.1 Pipet 7 mL of the supernate into a 50 mL centrifuge tube.
2.2 Dilute extract with 28 mL 0.1 M sodium phosphate containing 1% Tween 20 buffer solution.
2.3 Mix well.
2.4 Filter dilute extract through 1.5 μm glass microfibre filter (VICAM part #31955) into a clean vessel.
3.0 Column Chromatography
3.1 Pass 25 mL of filtered extract through the AflaOchra™ column by gravity until column is dry
(1 g sample equivalent).
3.2 Pass 5 mL of 10 mM PBS through the column.
3.3 Pass 5 mL of purified water through the column, then force 3 mL air through column to remove all liquid.
3.4 Place 3 mL volumetric flask under column to collect eluate.
3.5 Elute column by adding 1.0 mL HPLC grade methanol into column and letting drip into volumetric flask.
Let column run dry. Let stand for 1 minute, then elute with an additional 1mL methanol and collect in the
same 3 mL volumetric flask. Force 10 mL air through column to remove all liquid.
3.6 Dilute eluate to 3 mL volume with water and inject onto HPLC or UPLC.
4.0 HPLC Conditions
4.1 HPLC set up for Ochratoxin:
4.2 System: Waters Alliance ® HPLC System
4.3 Column: reverse phase C18 (Waters NovaPak C18, 3.9 mm X 300 mm, 4 μm)
4.4 Mobile phase: acetonitrile:water:acetic acid (49.5:49.5:1, v/v/v), degassed
4.5 Flow rate: 1.0 mL/min.
4.6 Fluorescence detector: Waters 2475 Fluorescence detector
4.7 Detection wavelength: 333 nm excitation and 460 nm emission
4.8 HPLC set up for Aflatoxin:
4.9 System: Waters Alliance ® HPLC System
4.10 Column: reverse phase C18 (Waters NovaPak C18, 3.9 mm X 150 mm, 4 μm)
4.11 Mobile phase: water:methanol (55:45, v/v) isocratic degassed.
4.12 Flow rate: 0.8 mL/min.
4.13 Fluorescence detector: Waters 2475 Fluorescence detector, excitation 362 nm, emission 440 nm
4.14 Post column derivatization (PCD): PhCR Photochemical reactor VICAM product #600001222

3.0 LIABILITY
The analytical methods described above have been developed by VICAM to be used exclusively with the reagents in this test. The user
assumes all risk in using AflaTest®, AflaCheck®, and AflaOchra™ analytical procedures and products. VICAM makes no warranty of any
kind, express or implied, other than that AflaTest, AflaCheck, and AflaOchra products conform to VICAM’s printed specifications and
quality control standards. VICAM will at its option repair or replace any product or part thereof which proves to be defective in work-
manship or material. VICAM’s undertaking to repair or replace such products is exclusive and is in lieu of all warranties whether written,
oral expressed, or implied, including any implied warranty of merchantability or fitness for a particular purpose. VICAM shall have no
liability for anticipated or lost profits or any loss, inconvenience or damage whether direct, incidental, consequential or otherwise, to
person or property, or for strict liability or negligence arising from or in connection with the use of these assay procedures or AflaTest,
AflaCheck, and AflaOchra products.
The foregoing notwithstanding, protocols and other products developed by VICAM are periodically improved and revised in order to
maximize reliability and optimize customer use and satisfaction. When an improved, new or substitute version of a protocol and product
is available, VICAM shall not be held liable or responsible for any earlier protocol or product, even if use of earlier product or protocol
be within the expiration date. Please inform yourself about any new protocols by either e-mailing, faxing or phoning VICAM or your
local VICAM distributor. VICAM shall not be liable or responsible for any unsatisfactory or faulty results or performance involving the
use of VICAM protocols or products if the testing or sampling in question is not conducted properly. The customer is solely and fully
responsible for educating oneself about the proper testing and sampling procedures using VICAM protocols and products.
All VICAM products are protected by worldwide patents and trademarks.

KEY LOCATIONS
Headquarters:
34 Maple Street
Milford, MA 01757
USA
Tel.: +1 800 338 4381
+1 508 482 4935
Fax: +1 508 482 4972
Orders:
1848 N. Deffer Drive
Nixa, MO 65714
USA
Tel.: +1 877 228 4244
+1 417 725 6588
Fax: +1 417 725 6102
Technical Service and Support:

email: techservice@vicam.com
www.vicam.com
©2014 Waters Corporation. Waters, VICAM, AflaTest and AflaCheck

are registered trademarks of Waters Corporation. AflaOchra is a
trademark of Waters Corporation.
715004495EN Rev A March 2014 SC-PDF

Waters 2X5LF Customer Familarization Draft V2.0 BEW 8 14 - 2014

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SFE 2X5LF System Customer Familiarization Draft Page # 1

SFE 2X5LF System Draft Customer Familiarization Guide

DATE 14 August 2014

Waters Educational Services Draft Rev 2.0 BEW 8/14/2014

Page # Section # Title of Section

3-4 A General information about the SFE 2X5LF Extraction System

5 B Tools Required for Installation and Operation

6-7 C SFE 2X5LF System Components

8 D The 2x5LF Recycler Overview

9-10 E General System Workflow for SFE Sample Extraction

11-14 F Pre-Setup for SFE Extraction of Sample Extraction

15-23 G Software and System Operation for SFE Extraction – No Recycler

24-26 H Software and System Operation for SFE Extraction – Recycler

27-29 I Collecting Extracts from Collectors

30-32 J Switching and Cleaning the Extraction Chambers

33-35 K System Troubleshooting

36-37 L Pm and Maintenance

38-40 M ABPR Alignment and Details

41 N Appendix: Schematics and Diagrams

42-47 O SFE Method Development Guidelines with the 2x5LF System

Vicam Botanical Instruction Guide

Waters Educational Services Draft Rev 2.0 BEW 8/14/2014

A: General Information about the SFE 2X5LF System

1. The SFE 2X5LF System is the following system configuration:

2. Supercritical CO2 Extraction = pressures above 1078 PSI and 31C.

Figure 1 General SFE Concepts - Part 1

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5. Safety Information and Details:

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B: Tools Required for Installation and Operation:

~ 3 meters -12 feet of space for system

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C: SFE 2X5LF System Components

1. The system configuration consists of the following components:

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D: The CO2 Recycler for the 2X5 LF System

1 From CO2 Tank 6 Pressure Relief Valve

2 From SFE 2X5LF System Exhaust 7 Level Sensor

3 Manual CO2 Supply Valve 8 Drain Valve

4 To SFE 2X5LF System CO2 Inlet 9 Pressure Gauge

5 Heat Exchanger 10 Level Sensor Display Box (not displayed)

3. Attach the power supply to the Level Sensor System box.

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E: System Workflow Summary for Plant Extractions

1 Fill Extraction Vessel with Sample

2x5000 LF System without Recycler – Total Extraction Time ~90 minutes

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2x5000 LF System with Recycler – Total Extraction Time ~90 minutes

11. Run the system for around 90 minutes.

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F: Pre-Setup for SFE Sample Extraction

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G: Software and System for Plant Extraction – No Recycler

2. Click on Process Suite and use Login: Admin Password: Admin

1. Automatic Back Pressure Regulator Manual Control

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Zone Description Temperature Settings

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7. Adjust all temperature set points, and then click OK.