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Why Do We Do Sample Preparation?
Biotage® Analytical Sample Preparation
Products
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Sample Preparation
 Sample Filtration: ISOLUTE Filter+
 Protein Precipitation: ISOLUTE PPT+
 Phospholipid Removal: ISOLUTE PLD+
 Extraction: ISOLUTE SLE+, SPE
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• Cleanliness • Compatibility to analysis
• Reduce matrix interferences • Environmental
• Eg. Complex food matrices • Eg. Water samples by GC
• Concentrate sample • Ensure system robustness
• Longer column lifetimes
• Improves sensitivity
• Less maintenance on detector
• Relatively clean matrices
• Autosampler less likely to block
• Eg. Environmental contaminants
• Less contamination
Confidence in analytical results
ISOLUTE FILTER+
 Provide sample clean up by physical filtration
 Perfect for use prior to LC/MS analysis.
 Prevent sample particulates from reaching the
analytical LC column, protecting the analytical
system from particulate build up over time.
 Suitable for aqueous biological samples (urines, blood samples, etc)
 Compatible with HPLC Reversed Phase solvent (MeOH, ACN, acid / base
modified aqueous samples / solvents) 7
Before
After
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ISOLUTE PPT+ ISOLUTE PLD+ Remove proteins and phospholipids

 Remove proteins  Remove proteins and phospholipids

 Protein precipitation plates  Overcome the problems associated with matrix

interference by phospholipids
 Designed to overcome the time consuming sample
 Produce cleaner extracts; retain proteins and
handling steps associated with traditional PPT using phospholipids but allow small molecule analytes to
pass through unretained
organic solvents, strong acids (TFA), or salts such as zinc sulphate
 Traditional PPT would take 45 minutes for 96 samples. Load sample > Add crash  Increase analytes sensitivity (signal-to-noise (S/N))
solvent > Mix > Centrifuge > Further processing  “Solvent first “ methodology
 The functionalized bottom frit holds up organic solvents, allowing the  Available in 96 Fixed Well Plates and Columns
precipitating solvent to be dispensed into each well prior to sample addition 10

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ISOLUTE SLE+ Why use SLE?

 Supported Liquid Extraction

 Many Advantages over LLE:
 Alternative to traditional liquid-liquid extraction (LLE)
 Faster
 Extraction of analytes from aqueous samples.  No emulsions formed
 SLE utilized inert support material (modified diatomaceous  More efficient / higher recoveries / higher sensitivity
earth) packed in plates or column to mimic the liquid-liquid  Higher productivity / Automation (96 well plate format)
extraction process  Less sample needed
 Available in 96 Well Plates, Array Wells and Columns  Less solvent needed
 Need to purchase Array Base Plate (#120-1000-P01) when using
ISOLUTE SLE+ Array Wells
 Seal with base plate sealing strips (#120-1200) for empty well on
base plate

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Solid Phase Extraction (SPE) Solid Phase Extraction (SPE)

• What is it?
• Targeted extraction technique for isolation of a
“Separation or removal of an analyte or analytes from a mixture of compound(s) from complex matrices e.g. biological, food
compounds by selective partitioning of the compounds between a • Typical formats are SPE tubes or 96 well plates
solid phase (sorbent) and a liquid phase (solvent).”
• How does it work?
Condition Load Wash Elute
Products:
1. ISOLUTE SPE
2. EVOLUTE EXPRESS SPE

Analyte
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Biotage SPE Products Biotage SPE Products – pH Stability

 EVOLUTE EXRESS sorbents have a much wider working range than

ISOLUTE SPE EVOLUTE EXPRESS SPE
the silica based ISOLUTE sorbents and also when used in load, wash,
 Traditional SPE  Simple Load > Wash > Elute elute mode do not require conditioning
 No Condition and Equilibrate steps
(Optional)
EVOLUTE EXPRESS pH 0 to 14
ISOLUTE (Silica) pH 2 to 8

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EVOLUTE EXPRESS SPE EVOLUTE EXPRESS SPE

 Sample preparation for extracting a diverse range of neutral, acidic and
basic analytes from biological and aqueous based samples.
 EVOLUTE® EXPRESS sample processing is extremely simple using
just a Load > Wash > Elute procedure.
 EVOLUTE® EXPRESS products combine the powerful EVOLUTE® chemistry with
optimized component features which can further enhance productivity by eliminating the
need for some traditional steps in the SPE procedure.
EVOLUTE ABN for simultaneous extraction of acidic, basic and/or neutral analytes.


 EVOLUTE CX combines non-polar & strong* Cation eXchange functionality (-SO3 )- for extraction of
basic analytes.
 EVOLUTE WCX combines non-polar & Weak** Cation eXchange functionality (-COOH/-COO-)
(pKa~5), for extraction of strongly basic analytes.
 EVOLUTE AX Combines non-polar & strong* Anion eXchange functionality (-N(CH3)3 +for extraction
of acidic analytes.
 EVOLUTE WAX Combines non-polar and Weak** Anion eXchange functionality (-NH2/NH3 +)
(pKa~10), for extraction of strongly acidic analytes.

 * - Strong ion exchange sorbents carry a permanent charge, under all pH conditions.
 ** - Weak ion exchange sorbents carry a charge which can be eliminated by changing pH.
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EVOLUTE EXPRESS SPE

The Options – Workflow & Cost
Sorbent Selection Plate
Workflow Cost/sample
 96-Fixed Well plate with same new frit material as all other EVOLUTE Number of steps, time (USD)
EXPRESS products required & complexity Based on 96 WP

 Contains the four mixed mode ion-exchange sorbents, CX, WCX, AX and WAX Less Less
on one plate  Protein precipitation (PPT+)
Minimum 2 workflow steps.
 Designed to be used for screening with the Sorbent Selection Screening
method  Phospholipid depletion (PLD+)
 Once screened methods should be optimized Minimum 2 workflow steps.

with the usual EVOLUTE generic methods  Supported Liquid extraction (SLE+)
 Simple, quick and multipurpose method Minimum 3 workflow steps.
development for multiple analytes on one
plate  EVOLUTE EXPRESS
Minimum 3 workflow steps.

More  ISOLUTE More

Minimum 5 workflow steps.
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Sample Preparation Instruments and Processing Manifolds Manual Sample Processing: VacMaster™

Manual Sample Automated

AutomatedSample Evaporation
Evaporation  Vacuum manifolds for SPE, SLE and filtration
Processing Processing
Sample Processing applications
VacMaster TurboVap VacMaster-10 and 20
 For column format
Gravity Rack SPE Dry 96
 Standard Manifold:
Extrahera
 Stainless Steel Needle ④
 No Stopcock ①
 Recommend to buy:
PRESSURE+
 PTFE Stopcock/Needle Unit (#121-0001) ②
 Need to specify rack size:
RapidTrace+ V-10  VacMaster-10 : 10, 12, 16, 27 mm rack
 VacMaster-20 : 10, 12, 16 mm rack or a blank plate
for customisation
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Manual Sample Processing: VacMaster™ Manual Sample Processing: PRESSURE+

 Vacuum manifolds for SPE, SLE and  Positive Pressure to move both low and high viscosity liquids through
filtration applications
SPE plates and columns.
VacMaster-96
 Flow restrictors built into each port to standardize motive force through
 Plate format & Array well
each well or column
 1 mL (tabless) columns
[need Tabless 1 mL Column Holder  Gas input requirement:
(#121-9620)]
 Air compressor or N2 gas (Preferred)
 Standard Manifold:
 Recommended: 80 psi (5.5 bar)
 Without vacuum control
 Range: 60 -100 psi (4.1 - 6.9 bar)

19  Two Model: PRESSURE+ 48, PRESSURE+ 96 40

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Manual Sample Processing: PRESSURE+ Extrahera

PRESSURE+ 48
 Direct collection into tubes w/o
needles
 Column format: 1mL, 3mL, 6 mL
 Requires to order at least one
Column rack(s) and one Collection
rack(s) separately
PRESSURE+ 96
 Positive pressure (PRESSURE+ 48/96) + pipetting function
 Capable of handling
 24 Positions: Tabless column (400 µL, 1 mL, 3 mL, 6 mL)
 96 Positions: 96 well / 48 well plate
 User friendly, Less expensive
 12-inch touch screen
 PDF reporting function
 96 well plates
 Requires N2/Air (possible to have 2 gas inlet)
 1 mL (tabless) [ need Tabless 1 mL
Column Holder (#PPM-A96-CH)]  Small footprint
 86（61） x 57（51） x 73cm, 75kg
 Oasis μ-Elution plate holder is
（without touch screen）
available (#PPM-A96-SPCR) for
µElution Plates  Solvent bottles on right side of the system
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Extrahera Extrahera - Upper deck sample processing area

Sample Prep
Solvent tips Solvent Tips Waste
 Pipette Plate or
position Reservoir Position
Columns
 8 channel for 96 well plate
 4 channel for 24 syringe columns
 Can easily change 8 channel  4 channel
 Sample tip is one off, re-use for solvent tip

 Pipetting volume: 50-1000 µL, ≤1% CV, ≤± 2% accuracy

 Possible to set <50μL

Sample tips Sample Tube Additional 5 x

position or Plate Solvent or IS
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Extrahera - Lower deck sample processing area Extrahera - Collection plate lifter

Collection Flow Through

Carrousel
Plate Plate (Waste)

3CxoF
lleeracctitoionn Waste Tray lifts the
coPllelactteion collection/Waste Plate
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Extrahera Design Details TurboVap

 N2 blow evaporator - for tubes

TurboVap LV
TurboVap II
TurboVap EH – compatible w/ Extrahera

Positive Head unit seal

Pressure Head onto the plate

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TurboVap TurboVap

 Vortex shearing technology  Simple and easy to use intuitive software

Increases gas/solvent interface  Built in colour 7” touchscreen control

 Able to store methods for regular use

10x faster evaporation than conventional techniques
 Gas flow gradient - features both ramp and
Precise gas flow
step change
 Temperature control by regulator
 Smaller footprint versus old style system
 Suitable for installation on benchtop or fume-
Vortex shearing
hood

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TurboVap TurboVap LV

 Improved visibility of samples  Vial size: 1.5 mL to 60 mL

 Multi Racks:
 Visual and an audible alarm/waring for
evaporation completion  48 position for 10 – 16 mm mini vials (#415489)
 48 position for 10 – 20 mm vials (#414964)
 Sleep mode with automatic start-up
 24 position for 21 – 30 mm vials (#415129)
 Easy water bath temperature calibration
 Comparable or improved performance
compared to old style system 415000
(includes 415408
manifold, but no rack)

415489
414964 415129
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TurboVap II Vials for TurboVap II

 Vial size: 50 mL and 200 mL, 0.5 & 1 mL endpoint

 Multi Racks:
 6 position w/ endpoint sensors for 200 mL tubes (#415100)
 6 position w/ endpoint sensors for 50 mL tubes (#415535)
 6 position for 50 or 200 mL tubes (#415494)

415001
(includes 415222 manifold,
but no rack)

Customer deals with large sample volume

415100 415535 415494 - Environmental
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- Food

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Summary

A range of sample preparation techniques appropriate for LC-MS/MS are available:

 Protein precipitation  Phospholipid depletion

 Quick, simple, but dirty
 Only protein removal
 SPE
 Many mechanism options to
 Quick, simple but clean
 Protein AND phospholipid
removal
 Not suitable for all analytes
Brand Statement "Excellence in Science"

improve selectivity  LLE/SLE W e in the Shimadzu Group have delivered products and services to enable our customers around the world to develop a
diverse range of new products, to protect and improve the environment, and to improve the health and lives of mankind. This

 Removes proteins and  Fast and simple brand statement represents our sense of pride in this endeavor. It is our commitment to society and ourselves that Shimadzu
remains dedicated in our pursuit of technology and accumulation of knowledge, so that we can offer even more outstanding
phospholipids  Removes proteins and technologies, products, and services, so as to be recognized for excellence in the field of science.

 More complex procedures phospholipids

 Limited by analyte solubility
Choice of sample prep technique depends on assay requirements 35

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