HPLC COLUMNS - Homepage | United Chemical · 2020-02-10 · HPLC COLUMNS INNOVATION THROUGH...
Transcript of HPLC COLUMNS - Homepage | United Chemical · 2020-02-10 · HPLC COLUMNS INNOVATION THROUGH...
SELECTRA® PFPPPENTAFLUOROPHENYLPROPYL STATIONARY PHASE
HPLC COLUMNS
I N N O VAT I O N T H R O U G H C H E M I S T R Y
U C T
Product Benefits• Can be used for Reverse Phase, Normal
Phase, or HILIC separations
• Excellent first-choice column for method development
• Excellent for LC/MS/MS• Strongly retentive for basic compounds• Special selectivity compared to C18 phases• May exhibit pi-pi overlap• Fully endcapped
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S E L E C T R A ® P F P P H P L C C O L U M N S
Selectra® PFPP HPLC ColumnsDimensions Particle Size Part Numbers
50 x 2.1 mm 1.8 µm SLPFPP50ID21-18UM
100 x 2.1 mm 1.8 µm SLPFPP100ID21-18UM
50 x 4.6 mm 1.8 µm SLPFPP50ID46-18UM
100 x 4.6 mm 1.8 µm SLPFPP100ID46-18UM
10 x 2.1 mm guard (sold as a 2 pack) 1.8 µm SLPFPPGDC20-18UM
10 x 4.6 mm guard (sold as a 2 pack) 1.8 µm SLPFPPGDC46-18UM
50 x 2.1 mm 3 µm SLPFPP50ID21-3UM
100 x 2.1 mm 3 µm SLPFPP100ID21-3UM
50 x 4.6 mm 3 µm SLPFPP50ID46-3UM
100 x 4.6 mm 3 µm SLPFPP100ID46-3UM
150 x 4.6 mm 3 µm SLPFPP150ID46-3UM
10 x 2.1 mm guard (sold as a 2 pack) 3 µm SLPFPPGDC20-3UM
10 x 4.6 mm guard (sold as a 2 pack) 3 µm SLPFPPGDC46-3UM
50 x 2.1 mm 5 µm SLPFPP50ID21-5UM
100 x 2.1 mm 5 µm SLPFPP100ID21-5UM
50 x 4.6 mm 5 µm SLPFPP50ID46-5UM
100 x 4.6 mm 5 µm SLPFPP100ID46-5UM
150 x 4.6 mm 5 µm SLPFPP150ID46-5UM
250 x 4.6 mm 5 µm SLPFPP250ID46-5UM
10 x 2.1 mm guard (sold as a 2 pack) 5 µm SLPFPPGDC20-5UM
10 x 4.6 mm guard (sold as a 2 pack) 5 µm SLPFPPGDC46-5UM
HPLC Guard HolderHPLC Guard Cartridge Holder SLGRDHLDR
UHPLC Guard Cartridge Holder SLGRDHLDR-HP
Replacement Peek Tip for Holder SLGRDHLDR-TIP (2/pk)
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The Selectra® PFPP (pentafluorophenylpropyl) phase interacts strongly with basic, halogenated and nitrogen-containing analytes. The pentafluorophenyl ring exhibits higher selectivity for electron rich aromatic containing molecules as compared to other sorbents. This phase also shows affinity for molecules with fluorine and other halogen atoms. The PFPP phase uses multiple selectivity mechanisms including hydrogen bonding, dipole-dipole, pi-pi overlap, hydrophilic (HILIC), and hydrophobic interactions.
UCT’s Selectra® PFPP HPLC column is an excellent choice for the retention and selectivity of amine-containing compounds, and charged bases. Typical compound classes include nucleosides, nucleotides, purines, pyrimidines, [beta]-blockers, tricyclic antidepressants, fluorinated corticosteroids, and taxanes. Selectra® PFPP columns are ideal for pharmaceutical and natural products research.
UCT’s Selectra® PFPP is well suited to LC-MS instrumentation due to its reliability and efficiency with acidic mobile phases. The Selectra® PFPP shows excellent peak shape in neutral mobile phases as well. The Selectra® PFPP columns are available packed with 3 or 5 µm particles and are offered in various column dimensions.
Retention Profile of Imipramine on a
RP Region HILIC Region
C18 does not have HILIC character, whereas, PFPP does (one of PFPP’s major advantages).
Column 1 Selectra® PFPP, 5 cm x 2.1 mm, 3 µm
Column 2 Selectra® C18, 5 cm x 2.1 mm, 3 µm
Mobile Phase 10mM NH3HCOOH: ACN
(adjusted to pH = 4 with Formic Acid)
Detector UV@254nm
Flow Rate 0.2 mL/min
Sample Imipramine
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S E L E C T R A ® P F P P H P L C C O L U M N S
Comparison of Benzodiazepenes on a C18 vs. PFPP
C18
PFPP
M...1CozB41021040 fo )pm 002 elps 81CozB41021040( 3 elpmaS morf 1 mapezanolC onimA-7 :DI aD 3.222/1.682 :)sriap 03( MRM+ fo CIX ax. 2.5e4 cps.
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5Time, min
0.00
5000.00
1.00e4
1.50e4
2.00e4
2.50e4
3.00e4
3.50e4
4.00e4
4.50e4
5.00e4
5.50e4
6.00e4
6.50e4
7.00e4
7.50e4
8.00e4
8.50e4
9.00e4
9.50e4
1.00e51.03e5
Inten
sity,
cps
aM...ozB41021040 fo )pm 002 elps PPFPozB41021040( 4 elpmaS morf 1 mapezanolC onimA-7 :DI aD 3.222/1.682 :)sriap 03( MRM+ fo CIX x. 7090.0 cps.
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5Time, min
0.00
5000.00
1.00e4
1.50e4
2.00e4
2.50e4
3.00e4
3.50e4
4.00e4
4.50e4
5.00e4
5.50e4
6.00e4
6.50e4
7.00e4
7.50e4
8.00e4
8.50e4
9.00e4
9.50e4
1.00e5
1.05e5
1.10e5
1.15e51.18e5
Inten
sity,
cps
Analyte C18 RT (min) PFPP RT (min)1. 7 Aminoclonazepam 1.11 1.26
2. Clonazepam 2.02 2.86
3. Lorazepam 2.16 2.16
4. Oxazepam 2.20 2.36
5. Alpha Hydroxy Alprazolam 2.22 2.45
6. Nordiazepam 2.70 3.18
7. Temazepam 2.75 3.12
8. Alprozolam 2.79 3.41
9. Diazepam 3.74 4.50
Column C18 100 x 2.1 mm, 5 µm
Mobile Phase A = 40% H2O w/ 0.1% Formic Acid B = 60% MeOH w/ 0.1% Formic Acid
Flow Rate 0.5 mL/min
Column Temp 50 °C
Injection Volume
5 µL
Column Selectra® PFPP LC 100 x 2.1 mm, 5 µm
Part Number SLPFPP100ID21-5UM
Mobile Phase A = 40% H2O w/ 0.1% Formic Acid B = 60% MeOH w/ 0.1% Formic Acid
Flow Rate 0.5 mL/min
Column Temp 50 °C
Injection Volume
5 µL
1
2
3
4, 5
6, 7
8
9
1
2
3
4
5
67
8
9
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Gabapentin/Pregabalin/Baclofen
XIC of +MRM (6 pairs): 160.2/97.1 Da ID: pregabalin from Sample 1 (10232013GabaPregaBaclo std1) of 10232013GabaPregaBaclo3.wi... Max. 1.5e6 cps.
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0Time, min
0.0
2.0e5
4.0e5
6.0e5
8.0e5
1.0e6
1.2e6
1.4e6
1.6e6
1.8e6
2.0e6
2.2e6
2.4e6
2.6e6
2.8e6
3.0e6
3.2e6
3.4e6
Inten
sity,
cps
Analyte Precursor ion
Product ion 1
Product ion 2
Gabapentin 172.1 137.1 67.1
Baclofen 214.01 150.8 65.0
Pregabalin 160.2 124.1 97.1
Instrument: Agilent 1200 Binary Pump SL
Detector: ABSciex 4000 Qtrap MS/MS
Mode: ESI +
Column: Selectra® PFPP, 100 x 2.1 mm, 5 µm (UCT p/n SLPFPPID21-5UM)
Flow Rate: 0.5 mL/min
Column Temp: 50 °C
Injection Volume: 10 µL
Mobile Phase A: 0.1% Formic Acid in H2O
Mobile Phase B: 0.1% Formic Acid in MeOH
Isocratic: A:B (85:15)
Gabapentin
Pregabalin Baclofen
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Cocaine / Benzoylecgonine / Cocaethylene
XIC of +MRM (21 pairs): 290.1/105.1 Da ID: BE from Sample 3 (10182013PFPPCoc std2) of 10182013PFPPCOCISOcratic2.wiff (Turbo ... Max. 1.4e6 cps.
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5Time, min
0.0
1.0e5
2.0e5
3.0e5
4.0e5
5.0e5
6.0e5
7.0e5
8.0e5
9.0e5
1.0e6
1.1e6
1.2e6
1.3e6
1.4e6
1.5e6
1.6e6
1.7e6
1.8e61.9e6
Inte
nsity
, cps
AnalytePrecursor
ionProduct
ion 1Product
ion 2
Benzoylecgnonine 290.1 168.1 105.1
Cocaine 304.1 182.1 105.1
Cocaethylene 318.2 196.4 82.7
Instrument: Agilent 1200 Binary Pump SL
Detector: ABSciex 4000 Qtrap MS/MS
Mode: ESI +
Column: Selectra® PFPP, 100 x 2.1 mm, 5 µm (UCT p/n SLPFPP100ID21-5UM)
Flow Rate: 0.5 mL/min
Column Temp: 50 °C
Injection Volume: 10 µL
Mobile Phase A: 0.1% Formic Acid in H2O
Mobile Phase B: 0.1% Formic Acid in MeOH
Isocratic: A:B (30:70)
S E L E C T R A ® P F P P H P L C C O L U M N S
Cocaine
Benzoylecgonine
Cocaethylene
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Bath Salts
Analyte
MRM Transitions
Relative Retention Time
(minutes)Q1 Q3
1. Flephedrone 182.1 164.2 4.78
2. Methylone 208.1 160.1 5.26
3. Ethylone 222.0 131.0 5.84
4. Mephedrone 178.1 145.0 6.10
5. Butylone 222.0 131.0 6.36
6. Methcathinone 192.2 144.0 6.93
7. MDPV 276.2 126.1 8.67
8. Pyravalerone 246.2 105.2 8.83
Time %A %B
0.00 98 2
1.00 65 35
5.00 65 35
7.00 0 100
10.00 0 100
10.20 98 2
15.00 STOP
Instrument: Thermo Scientific Dionex Ultimate 3000 LC
Detector: Thermo Scientific TSQ Vantage tandem mass spectrometer
Mode: ESI +
Column: Selectra® PFPP, 100 x 2.1 mm, 5 µm (UCT p/n SLPFPP100ID21-5UM)
Flow Rate: 0.3 mL/min
Injection Volume: 10 µL
Mobile Phase A: 0.1% Formic Acid in H2O
Mobile Phase B: 0.1% Formic Acid in MeOH
Gradient:
1
2
34
5
6
7
8
Ephedra Alkaloids
RT: 0.00 - 24.99 SM: 7G
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24Time (min)
0
20
40
60
80
1000
20
40
60
80
1000
20
40
60
80
100
Rel
ativ
e Ab
unda
nce
0
20
40
60
80
1004.00
5.38 6.26 7.07 7.782.96
10.15
11.41
12.888.71 13.87
15.1117.03
19.7213.79
20.54
23.7018.66
NL: 1.82E4TIC F: + c ESI SRM ms2 168.104 [90.969-90.971, 106.969-106.971] MS 100ppb_std
NL: 1.27E4TIC F: + c ESI SRM ms2 152.141 [91.019-91.021, 115.019-115.021] MS 100ppb_std
NL: 2.83E4TIC F: + c ESI SRM ms2 166.173 [91.009-91.011, 115.019-115.021] MS 100ppb_std
NL: 1.51E4TIC F: + c ESI SRM ms2 180.175 [90.989-90.991, 147.039-147.041] MS 100ppb_std
S E L E C T R A ® P F P P H P L C C O L U M N S
AnalyteRT
(min)Precursor
ionProduct
ion 1Product
ion 2
Synephrine 4.00 168.1 91.0 107.0
Norephedrine 10.15 152.1 91.0 115.0
Norpseudoephedrine 11.41 152.1 91.0 115.0
Ephedrine 15.11 166.2 91.0 115.0
Pseudophedrine 17.03 166.2 91.0 115.0
Methylephedrine 20.54 180.2 91.0 147.0
Instrument: Thermo Scientific Dionex Ultimate 3000 LC
Detector: Thermo Scientific TSQ Vantage tandem mass spectrometer
Mode: ESI +
Column: Selectra® PFPP, 100 x 2.1 mm, 3 µm (UCT p/n SLPFPP100ID21-3UM)
Guard Column: Selectra PFPP, 10 x 2.0 mm, 3 µm (UCT p/n SLPFPPGDC20-3UM)
Flow Rate: 0.3 mL/min
Column Temp.: 40 °C
Injection Volume: 8 µL
Mobile Phase A: 10mM Ammonium Acetate
Mobile Phase B: MeOH
Isocratic: A:B (80:20)
Synephrine
Norephedrine
Norpseudoephedrine
Ephedrine Pseudophedrine
Methylephedrine
Diquat/Paraquat
Norm.
120
100
80
60
40
20
0
2 4 6 8 10
DAD1 A, Sig=258,4 Ref=550, 100 (010914\PQDQ2.D)DAD1 B, Sig=308,4 Ref=550, 100 (010914\PQDQ2.D)
Paraquat Diquat
1.67
5
3.02
1
1.67
5
ParaquatDiquat
Instrument: HP 1050
Detector: HP Photo Diode Array
Column: Selectra® PFPP, 100 x 2.1 mm, 3 µm (UCT p/n SLPFPP100ID21-3UM)
Flow Rate: 0.2 mL/min
Injection Volume: 1 µL
Mobile Phase A: 10mM Ammonium Acetate (pH=4)
Mobile Phase B: Acetonitrile
Isocratic: A:B (99:1)
Monitoring Wavelength: Diquat: 308 nm
Paraquat: 258 nm
S E L E C T R A ® P F P P H P L C C O L U M N S
Sympathomimetic Amines
Analyte
MRM Transitions Relative Retention Time
(minutes)Q1 Q3
1. Amphetamine 136.1 91.2 5.41
2. MDA 180.2 163.1 6.24
3. Methamphetamine 150.1 91.2 6.35
4. MDMA 194.2 163.1 7.29
Instrument: Agilent 1200 Binary Pump SL
Detector: ABSciex API 4000 Qtrap MS/MS
LC Column: Selectra® PFPP HPLC Column 50 x 2.1 mm, 5 µm (UCT P/N SLPFPP50ID21-5UM)
Mode: ESI +
Flow Rate: 0.3 mL/min
Injection Volume: 10 µL
Mobile Phase A: 0.1% Formic Acid in D.I. H2O
Mobile Phase B: 0.1% Formic Acid in Methanol
Isocratic: A:B (30:70)
SELECTRA® PFPP HPLC Column
1
2
3
4
Column Care & Usage
Each UCT, LLC high performance liquid chromatography (HPLC) column is individually packed and tested to ensure superior performance. A HPLC CoA is included with each column. It contains a chromatogram, the column serial number, and the lot number of the packing material. Retain this information for as long as you have the column; it may be useful if troubleshooting is ever required.
Guard Columns & Filters
In-line filters and/or guard cartridges can extend the life of an analytical column. They are connected in-line prior to the analytical column. l Pressure Recommendation UCT, LLC HPLC columns are silica based. To ensure optimal column life, operating pressures of 3000psi or lower are recommended. Column pressures may increase as the column ages as particulates from the system accumulate on the column. Sudden increases in pressure are usually a result of a blocked frit. Pressure will vary with different mobile phases. For example Water/Methanol mixtures will generally give higher back pressure than Water/Acetonitrile mobile phases. (see Table I)
l Guard Cartridges Guard cartridges are used to capture impurities that may otherwise lodge on the HPLC column. Guard cartridges are especially useful with samples from biological sources as these may contain lipids and proteins that pass through frits can quickly block columns. Guard cartridges should have the same phase as the column they are protecting. Guard cartridges should be replaced when the chromatography begins to deteriorate or when the guard cartridge contributes excessive back pressure to the HPLC system.
Mobile Phase
When shipped, the column contains the storage solvent listed on its HPLC CoA. Before use the first time, ensure that your initial planned mobile phase is compatible with this solvent (see Table II). If it is not, you must flush the column with an intermediate solvent that is compatible with both the storage solvent and your planned mobile phase. Be especially cognizant if you are using buffers; the storage solvent for most columns contains greater than 50% organic solvent, and contact with a buffer could cause a salting out effect. The resulting precipitate can plug the column. Flow Direction & Flow Rate
The arrows on the column label indicate the recommended flow direction.
Begin by connecting the inlet end of the column to the injector or autosampler and allow mobile phase to flow from the outlet end of the column into a beaker for 10–15 minutes. Gradually increase the flow rate. For recommended flow rates refer to Table III. Then, stop the mobile phase flow and connect the column to your detector. Because every LC system is unique, especially when used in gradient mode, your results may slightly differ from those obtained in our laboratory. UCT, LLC Technical Service can assist you in optimizing your separations. Be sure to record the operating pressure before calling.
Increasing Column Lifetime
Silica based UCT, LLC HPLC column packing materials have a pH operating range of 2-8. Extended use of any column at extreme pH can shorten column lifetime.
The upper temperature limit for silica based HPLC columns is 80 °C. Elevated temperatures can improve efficiency by lowering solvent viscosity, but column lifetime may be compromised.
Use of HPLC-Grade solvents is strongly recommended. Residue and chemical contaminants in non-HPLC grade solvents can alter a column’s selectivity and, potentially plug the inlet frit leading to an increased system pressure. Mobile phase filtering and degassing (either off-line or in-line) is highly recommended. Column lifetime is also governed by stationary phase type. Hydrocarbon phases, such as C18, are relatively chemically inert. Polar phases, such as cyano or amino, require somewhat more care as they can be chemically active.
S E L E C T R A ® P F P P H P L C C O L U M N S
Column Maintenance
Columns should not be subjected to mechanical or pressure shock. This can cause irreversible damage to the column.
Columns should be run in the flow direction as marked on the column. The one exception is for column cleaning. The flow can be reversed to back-flush frits if blockages occur.
Do not store the column in an aqueous buffer, this will promote microbiological contamination. First flush the column with water and then with 50/50 organic solvent/water prior to storage.
Washing Procedure for Reverse-Phase Columns
Washing the column successively with non-polar eluents will usually remove accumulated impurities. Follow the washing sequence below, using 30mL of each solvent, to thoroughly clean the column.
1. Distilled water 90%, 10% Methanol
2. 0.5M H3P04 90%, 10% Methanol (Optional)*
3. Distilled water 90%, 10% Methanol (Optional)*
4. Methanol
5. Methanol/Chloroform (1:1) (Optional)
6. Methanol or Acetonitrile (Optional)
7. Distilled water 90%, 10% Methanol
8. Eluent to recondition column
*NOTE: Whenever step 2 is used, it must be followed by step 3.
It is recommended that columns be dedicated for the specific method when ion pairing reagents are used. This is because it is difficult to remove all of the ion pairing reagent.
l Protein Contamination
Proteins can adsorb onto columns causing loss of performance. In this situation rinse the column overnight with 20% 0.1M nitric acid/80% isopropanol at a flow rate of 1/5th the usual flow rate (i.e. at 0.2 mL/min for 4.6mm ID columns). Ensure that the rinse solution is directed directly to solvent waste and not through the detector.
l Lipid Contamination If lipids or other highly hydrophobic compounds have contaminated the column use the full washing procedure except replace step 5 with 100% chloroform or dichloromethane.
2
1.8
1
1.6
1.4
1.2
0.8
0.6
0.4
0.2
0
2
1.8
1
1.6
1.4
1.2
0.8
0.6
0.4
0.2
00% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
MeOH/Water
MeCN/Water
MeCN/MeOH
III: Mobile Phase Viscosity ComparisonVi
scos
ity, c
PTable I: Mobile Phase Viscosity Comparison
S E L E C T R A ® P F P P H P L C C O L U M N S
Table I: Solvent Miscibility Chart
Ace�c AcidAcetone
AcetonitrileBenzene
n-ButanolButyl Acetate
Carbon Tetrachloride Immiscible SolventsChloroform
Cyclohexane1,2-Dichloroethane
DichloromethaneDimethylformamide
Dimethyl SulfoxideDioxaneEthanol
Ethyl AcetateDi-Ethyl Ether
HeptaneHexane
MethanolMethyl-t-Butyl EtherMethyl Ethyl Ketone
Pentanen-Propanol
Iso-PropanolDi-Iso-Propyl Ether
TetrahydrofuranToluene
TrichloroethyleneWater
Xylene
Acet
ic Ac
idAc
eton
eAc
eton
itrile
Benz
ene
n-Bu
tano
lBu
tyl A
ceta
te
Carb
on Te
trach
lorid
eCh
loro
form
Cyclo
hexa
ne
1,2-
Dich
loro
etha
ne
Dich
loro
met
hane
Dim
ethy
lform
amid
e
Dim
ethy
l Sul
foxid
eDi
oxan
eEt
hano
lEt
hyl A
ceta
teDi
-Eth
yl Et
her
Hept
ane
Hexa
neM
etha
nol
Met
hyl-t
-But
yl Et
her
Met
hyl E
thyl
Keto
nePe
ntan
en-
Prop
anol
Iso-P
ropa
nol
Di-Is
o-Pr
opyl
Ethe
r
Tetra
hydr
ofur
anTo
luen
e
Trich
loro
ethy
lene
Wat
erXy
lene
Table II: Solvent Miscibility Chart
PRICES AND TERMSOur prices are subject to change without notice. The price in effect when we receive your order will apply. All prices are in US Dollars and are F.O.B. Terms of payment are net 30 days. MINIMUM ORDERSWe welcome all orders, therefore, we do not have a minimum order requirement. When ordering, please include your purchase order number, complete “Ship To” and “Bill To” address, catalog number, quantity, and description of product(s). Also include your name and a phone number where you can be reached should we have any questions concerning your order. SHIPMENTSNormal processing is within 24 hours after receipt of an order. Unless special shipping requests have been made, our trained staff will send all orders by UPS Ground service. The appropriate shipping charges (freight & insurance costs) will be added to the invoice, unless otherwise instructed by the customer.
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To initiate this action, photographs of the product, including packaging and labeling of the containers, must be submitted to the UCT Representative for approval. With approval a return authorization can be initiated, and must be received within 30 days. Once the materials arrive at UCT a further inspection of the materials must be completed and accepted by our Quality Manager prior to further action of credits or replacement. UCT’s total liability is limited to the replacement cost of UCT products.
This warranty does not apply to damage resulting from misuse.
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Phone: 215.781.9255 800.385.3153Fax: 215.785.1226
UCT, Inc. 2731 Bartram Rd. Bristol, PA 19007
Email: [email protected]: www.unitedchem.com
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