HP 241 User Manual

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HP 241 Protein Sequencer (N+C)
C-Terminal 5.0 Protein Sequencer Methods
Technical Note
The C-terminal 5.0 sequencer meth­ods implement the thiohydantoin degradation chemistry (coupling and cleavage) on the Hewlett­Packard column-based protein sequencer. Reagents R1, R2A, R4, and the PTH-Std are specific for N­terminal sequencing and are not utilized for the C-terminal 5.0 sequencer methods.
These methods are suitable for C-terminal sequence analysis of most protein samples in the low nanomole to 50 pmole range which have been applied onto a Hewlett­Packard Zitex strip. Once dry the strip is loaded into an empty RP/SAX biphasic column. No col­umn preparation is required with this method.
The methods are:
• C-Terminal 5.0
• C-Terminal 5.0 (Cycle 1)
• C-Terminal Flask 5.0
• TH Std 5.0
The column and flask methods control all of the derivatization and cleavage reactions for the thiohydantoin chemistry.
The sequence program controls all of the column and flask methods, as well as any cycle exception methods. The C-terminal 5.0 sequencer method uses a cycle 1 column exception method.
The cycle 1 method provides a longer initial drying time and a pause to provide time for proper HPLC column equilibration after switching from N-terminal sequenc­ing. There is no specialized cycle 1 method for the flask.
The TH Std 5.0 method delivers 50 pmol/100 µl TH-amino acid stan­dard solution to the on-line HPLC allowing the quantitation and iden­tification of sequenced amino acids. One may choose to run a TH-standard by using TH-std 5.0 when scheduling a sample.
S2A Neat ethyl acetate
1R Diphenylphosphorylisothiocyanate (DPP-ITC) in toluene/heptane (23:27:50) R1 Phenylisothiocyanate (PITC) in heptane (3:97) 2R Pyridine in ethylacetate (2:98) R2 Diisopropylethylamine (DIEA) in 1-propanol/water (1:6:3) S2/3 Acetonitrile/toluene (23:77) R2A Ocylamine in heptane (3:97) 3R Potassium trimethylsilanolate (0.1M) in methanol/t-butanol (50:50) S3 Acetonitrile/toluene (15:85) R3 Neat trifluoroacetic acid (TFA) L3 Acetic acid in methanol/water (1:74:25) PTH Mixture of PTH amino acids (10 pmol/100 µl) in acetonitrile with DPTU
as a marker
TH Mixture of TH amino acids (50 pmol/100 µl) in acetonitrile R4 Trifluoroacetic acid (TFA) in water (1:3) S5 Phosphate buffer (pH 2.9), 0.2% ion pairing reagent
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Method (Column): C-Terminal 5.0
Total time: 75.2 min
All metering steps deliver the speci­fied reagent or solvent to waste.
Steps 3, 25, 47, 72: The volume of R3 delivered during this step should be just enough to wet the membrane.
Steps 7, 13, 19, 22, 29, 35, 41, 44, 51, 57, 63, 66, 69,76, 79, 82, 88, 91, 109, 112: The delivery of S2/3
and S3 should completely wet the Zitex membrane. The solvent deliv­eries are held against a closed valve, enabling 1/2 to 3/4 of the membrane­containing column compartment (bottom) to remain filled with sol­vent for the duration of the step.
Steps 10, 32, 54: The delivery of 1R should completely wet the mem­brane, but not flood it. The initial delivered volume (about 1/2 the vol­ume of the column) should blow through the column wetting the Zitex strip. The 1R reagent delivery is then held against a closed valve, enabling only a small volume of 1R to sit at the bottom of the column (approxi­mately 1 slit distance as measured by the slits on the column wings).
Method Details
The steps for the C-terminal 5.0 methods are described below. The steps indicate the ranges in volume that are appropriate for the various reagents/solvent deliveries. All sequencer methods are accessed by choosing the top menu item Edit/Method in the Protein Sequencer window. The sequencer column configuration used with the C-terminal 5.0 methods consists of an empty reverse-phase (RP) sample column (top) mated with an empty strong anion exchange (SAX) column (bottom).
Step Description Primary Temperature
Time
1: Activation: Dry column DOWN 60.0 60 2: Activation: Meter R3 7.0 60 3: Activation: Deliver R3 DOWN 30.0 60 4: Wash: Flush RV2 with S3 5.0 80 5: Activation: Dry column DOWN 50.0 80 6: Wash: Meter S2/3 9.0 80 7: Wash: Deliver S2/3 DOWN (closed) 15.0 80 8: Wash: Dry column DOWN 5.0 80
9: Couple: Meter 1R 5.0 80 10: Couple: Deliver 1R UP 30.0 80 11: Couple: Dry column DOWN 10.0 80 12: Wash: Meter S2/3 and S2A 9.0 80 13: Wash: Deliver S2/3 DOWN (closed) 15.0 80 14: Wash: Dry column DOWN 5.0 80 15: Couple: Meter 2R 5.0 80 16: Couple: Deliver 2R UP 10.0 80 17: Couple: Dry column DOWN 5.0 80 18: Wash: Meter S2/3 and S2A 9.0 80 19: Wash: Deliver S2/3 DOWN (closed) 15.0 80 20: Wash: Dry column DOWN 5.0 80 21: Wash: Meter S2/3 9.0 80 22: Wash: Deliver S2/3 DOWN (closed) 15.0 80 23: Wash: Dry column DOWN 20.0 80 24: Activation: Meter R3 7.0 60 25: Activation: Deliver R3 DOWN 30.0 60 26: Wash: Flush RV2 with S3 5.0 80 27: Activation: Dry column DOWN 50.0 80 28: Wash: Meter S2/3 9.0 80 29: Wash: Deliver S2/3 DOWN (closed) 15.0 80 30: Wash: Dry column DOWN 5.0 80 31: Couple: Meter 1R 5.0 80 32: Couple: Deliver 1R UP 30.0 80 33: Couple: Dry column DOWN 10.0 80 34: Wash: Meter S2/3 and S2A 9.0 80 35: Wash: Deliver S2/3 DOWN (closed) 15.0 80 36: Wash: Dry column DOWN 5.0 80 37: Couple: Meter 2R 5.0 80 38: Couple: Deliver 2R UP 10.0 80 39: Couple: Dry column DOWN 5.0 80 40: Wash: Meter S2/3 and S2A 9.0 80 41: Wash: Deliver S2/3 DOWN (closed) 15.0 80 42: Wash: Dry column DOWN 5.0 80 43: Wash: Meter S2/3 9.0 80 44: Wash: Deliver S2/3 DOWN (closed) 15.0 80 45: Wash: Dry column DOWN 20.0 80 46: Activation: Meter R3 7.0 60 47: Activation: Deliver R3 DOWN 30.0 60 48: Wash: Flush RV2 with S3 5.0 80 49: Activation: Dry column DOWN 50.0 80 50: Wash: Meter S2/3 9.0 80 51: Wash: Deliver S2/3 DOWN (closed) 15.0 80 52: Wash: Dry column DOWN 5.0 80
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Steps 16, 38, 60: The delivery of 2R should completely wet the membrane. The initial delivered volume (about 1/2 the volume of the column) should blow through the column wetting the Zitex strip. The 2R reagent delivery is then held against a closed valve, enabling only a small volume of 2R to sit at the bottom of the column (approximately 1 slit distance as mea­sured by the slits on the column wings).
Steps 12, 18, 34, 40, 56, 62: "Meter S2/3 and S2A". The S2A flushes the delivery lines and should not wet the column. The S2/3 is involved in the column wash and is sent to the column.
Steps 85, 106: The volume of R2 delivered during this step should not wet the membrane.
Steps 86, 107: During the high pressure dry step, the volume of R2 delivered should be just enough to wet the Zitex membrane. Note: The R2 reagent does not actually "wet" the Zitex membrane like other reagents and solvents. The mem­brane will not change its appearance in the presence of R2.
Step 94: After Step 94, the volume of L3 delivered to the flask should be 50 µl (+/- 5 µl)
Steps 96, 101: The delivery of 3R should completely wet the mem­brane, but not flood it. The 3R reagent delivery is then held against a closed valve, enabling only a small volume of 3R to sit at the bottom of the column (approximately 1 slit dis­tance as measured by the slits on the column wings).
Step Description Primary Temperature
Time
53: Couple: Meter 1R 5.0 80 54: Couple: Deliver 1R UP 30.0 80 55: Couple: Dry column DOWN 10.0 80 56: Wash: Meter S2/3 and S2A 9.0 80 57: Wash: Deliver S2/3 DOWN (closed) 15.0 80 58: Wash: Dry column DOWN 5.0 80 59: Couple: Meter 2R 5.0 80 60: Couple: Deliver 2R UP 10.0 80 61: Couple: Dry column DOWN 5.0 80 62: Wash: Meter S2/3 and S2A 9.0 80 63: Wash: Deliver S2/3 DOWN (closed) 15.0 80 64: Wash: Dry column DOWN 5.0 80 65: Wash: Meter S2/3 9.0 80 66: Wash: Deliver S2/3 DOWN (closed) 15.0 80 67: Wash: Dry column DOWN 5.0 80 68: Wash: Meter S3 9.0 80 69: Wash: Deliver S3 DOWN (closed) 15.0 80 70: Wash: Dry column DOWN 30.0 80 71: Cleave: Meter R3 7.0 80 72: Cleave: Deliver R3 DOWN 400.0 80 73: Wash: Flush RV2 with S3 5.0 80 74: Cleave: Dry column DOWN 160.0 80 75: !Extract: Meter S3 9.0 80 76: !Extract: Deliver S3 DOWN to flask 15.0 80 77: !Extract: Dry column UP 10.0 60 78: !Cleave: Meter S2/3 10.0 50 79: !Cleave: Deliver S2/3 DOWN 30.0 50 80: !Cleave: Dry column 10.0 50 81: !Cleave: Meter S2/3 10.0 50 82: !Cleave: Deliver S2/3 DOWN 30.0 50 83: !Cleave: Dry column 30.0 50 84: !Cleave: Meter R2 10.5 55 85: !Cleave: Deliver R2 DOWN 120.0 55 86: !Cleave: Dry column 60.0 50 87: !Cleave: Meter S2/ 3 10.0 50 88: !Cleave: Deliver S2/3 DOWN 30.0 50 89: !Cleave: Dry column 30.0 50 90: !Cleave: Meter S2/3 10.0 50 91: !Cleave: Deliver S2/3 DOWN 30.0 50 92: !Cleave: Dry column 30.0 50 93: !Cleave: Meter L3 7.0 40 94: !Cleave: Deliver L3 to transfer flask 25.0 40 95: !Cleave: Meter 3R 9.0 50 96: !Cleave: Cleave and extract to flask 30.0 50 97: !Extract: Meter S3 10.0 50 98: !Extract: Extract to flask UP 10.0 50 99: !Cleave: Dry column UP 30.0 50
100: !Cleave: Meter 3R 9.0 50 101: !Cleave: Cleave and extract to flask 30.0 50 102: !Extract: Meter S3 10.0 50 103: !Extract: Extract to flask UP 10.0 50 104: !Cleave: Dry column UP 20.0 55 105: Wash: Meter R2 10.5 55 106: Wash: Deliver R2 DOWN 120.0 55 107: Wash: Dry column DOWN 60.0 60 108: Wash: Meter S2/3 9.0 60 109: Wash: Deliver S2/3 DOWN (closed) 15.0 60 110: Wash: Dry column DOWN 15.0 60 111: Wash: Meter S2/3 9.0 60 112: Wash: Deliver S2/3 DOWN (closed) 15.0 60 113: Wash: Dry column DOWN 20.0 60
! indicates system step
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Method (Column): C-Terminal
5.0 (Cycle 1)
Total time: 88.7 min
Step 1: Cycle 1 begins with an extend­ed dry to remove any residual solvent from the sample loading process. There is also a pause to provide time for proper HPLC column equilibration after switching from N-terminal sequencing
Steps 3, 25, 47, 72: The volume of R3 delivered during this step should be just enough to wet the membrane.
Steps 7, 13, 19, 22, 29, 35, 41, 44, 51, 57, 63, 66, 69,76, 79, 82, 88, 91, 109, 112: The delivery of S2/3
and S3 should completely wet the Zitex membrane. The solvent deliv­eries are held against a closed valve, enabling 1/2 to 3/4 of the membrane­containing column compartment (bottom) to remain filled with sol­vent for the duration of the step.
Steps 10, 32, 54: The delivery of 1R should completely wet the mem­brane, but not flood it. The initial delivered volume (about 1/2 the vol­ume of the column) should blow through the column wetting the Zitex strip. The 1R reagent delivery is then held against a closed valve, enabling only a small volume of 1R to sit at the bottom of the column (approximate­ly 1 slit distance as measured by the slits on the column wings).
Steps 16, 38, 60: The delivery of 2R should completely wet the mem­brane. The initial delivered volume (about 1/2 the volume of the column) should blow through the column wet­ting the Zitex strip. The 2R reagent delivery is then held against a closed valve, enabling only a small volume of 2R to sit at the bottom of the col­umn (approximately 1 slit distance as measured by the slits on the col­umn wings).
Step Description Primary Temperature
Time
1: Activation: Dry column DOWN and wait 120.0 60 2: Activation: Meter R3 7.0 60 3: Activation: Deliver R3 DOWN 30.0 60 4: Wash: Flush RV2 with S3 5.0 80 5: Activation: Dry column DOWN 50.0 80 6: Wash: Meter S2/3 9.0 80 7: Wash: Deliver S2/3 DOWN (closed) 15.0 80 8: Wash: Dry column DOWN 5.0 80
9: Couple: Meter 1R 5.0 80 10: Couple: Deliver 1R UP 30.0 80 11: Couple: Dry column DOWN 10.0 80 12: Wash: Meter S2/3 and S2A 9.0 80 13: Wash: Deliver S2/3 DOWN (closed) 15.0 80 14: Wash: Dry column DOWN 5.0 80 15: Couple: Meter 2R 5.0 80 16: Couple: Deliver 2R UP 10.0 80 17: Couple: Dry column DOWN 5.0 80 18: Wash: Meter S2/3 and S2A 9.0 80 19: Wash: Deliver S2/3 DOWN (closed) 15.0 80 20: Wash: Dry column DOWN 5.0 80 21: Wash: Meter S2/3 9.0 80 22: Wash: Deliver S2/3 DOWN (closed) 15.0 80 23: Wash: Dry column DOWN 20.0 80 24: Activation: Meter R3 7.0 60 25: Activation: Deliver R3 DOWN 30.0 60 26: Wash: Flush RV2 with S3 5.0 80 27: Activation: Dry column DOWN 50.0 80 28: Wash: Meter S2/3 9.0 80 29: Wash: Deliver S2/3 DOWN (closed) 15.0 80 30: Wash: Dry column DOWN 5.0 80 31: Couple: Meter 1R 5.0 80 32: Couple: Deliver 1R UP 30.0 80 33: Couple: Dry column DOWN 10.0 80 34: Wash: Meter S2/3 and S2A 9.0 80 35: Wash: Deliver S2/3 DOWN (closed) 15.0 80 36: Wash: Dry column DOWN 5.0 80 37: Couple: Meter 2R 5.0 80 38: Couple: Deliver 2R UP 10.0 80 39: Couple: Dry column DOWN 5.0 80 40: Wash: Meter S2/3 and S2A 9.0 80 41: Wash: Deliver S2/3 DOWN (closed) 15.0 80 42: Wash: Dry column DOWN 5.0 80 43: Wash: Meter S2/3 9.0 80 44: Wash: Deliver S2/3 DOWN (closed) 15.0 80 45: Wash: Dry column DOWN 20.0 80 46: Activation: Meter R3 7.0 60 47: Activation: Deliver R3 DOWN 30.0 60 48: Wash: Flush RV2 with S3 5.0 80 49: Activation: Dry column DOWN 50.0 80 50: Wash: Meter S2/3 9.0 80 51: Wash: Deliver S2/3 DOWN (closed) 15.0 80 52: Wash: Dry column DOWN 5.0 80 53: Couple: Meter 1R 5.0 80 54: Couple: Deliver 1R UP 30.0 80 55: Couple: Dry column DOWN 10.0 80 56: Wash: Meter S2/3 and S2A 9.0 80
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Steps 12, 18, 34, 40, 56, 62: "Meter S2/3 and S2A". The S2A flushes the delivery lines and should not wet the column. The S2/3 is involved in the column wash and is sent to the column.
Steps 85, 106: The volume of R2 delivered during this step should not wet the membrane.
Steps 86, 107: During the high pressure dry step, the volume of R2 delivered should be just enough to wet the Zitex membrane. Note: The R2 reagent does not actually "wet" the Zitex membrane like other reagents and solvents. The mem­brane will not change its appearance in the presence of R2.
Step 94: After Step 94, the volume of L3 delivered to the flask should be 50 µl (+/- 5 µl)
Steps 96, 101: The delivery of 3R should completely wet the mem­brane, but not flood it. The 3R reagent delivery is then held against a closed valve, enabling only a small volume of 3R to sit at the bottom of the column (approximately 1 slit dis­tance as measured by the slits on the column wings).
Step Description Primary Temperature
Time
57: Wash: Deliver S2/3 DOWN (closed) 15.0 80 58: Wash: Dry column DOWN 5.0 80 59: Couple: Meter 2R 5.0 80 60: Couple: Deliver 2R UP 10.0 80 61: Couple: Dry column DOWN 5.0 80 62: Wash: Meter S2/3 and S2A 9.0 80 63: Wash: Deliver S2/3 DOWN (closed) 15.0 80 64: Wash: Dry column DOWN 5.0 80 65: Wash: Meter S2/3 9.0 80 66: Wash: Deliver S2/3 DOWN (closed) 15.0 80 67: Wash: Dry column DOWN 5.0 80 68: Wash: Meter S3 9.0 80 69: Wash: Deliver S3 DOWN (closed) 15.0 80 70: Wash: Dry column DOWN 30.0 80 71: Cleave: Meter R3 7.0 80 72: Cleave: Deliver R3 DOWN 400.0 80 73: Wash: Flush RV2 with S3 5.0 80 74: Cleave: Dry column DOWN 160.0 80 75: !Extract: Meter S3 9.0 80 76: !Extract: Deliver S3 DOWN to flask 15.0 80 77: !Extract: Dry column UP 10.0 60 78: !Cleave: Meter S2/3 10.0 50 79: !Cleave: Deliver S2/3 DOWN 30.0 50 80: !Cleave: Dry column 10.0 50 81: !Cleave: Meter S2/3 10.0 50 82: !Cleave: Deliver S2/3 DOWN 30.0 50 83: !Cleave: Dry column 30.0 50 84: !Cleave: Meter R2 10.5 55 85: !Cleave: Deliver R2 DOWN 120.0 55 86: !Cleave: Dry column 60.0 50 87: !Cleave: Meter S2/3 10.0 50 88: !Cleave: Deliver S2/3 DOWN 30.0 50 89: !Cleave: Dry column 30.0 50 90: !Cleave: Meter S2/3 10.0 50 91: !Cleave: Deliver S2/3 DOWN 30.0 50 92: !Cleave: Dry column 30.0 50 93: !Cleave: Meter L3 7.0 40 94: !Cleave: Deliver L3 to transfer flask 25.0 40 95: !Cleave: Meter 3R 9.0 50 96: !Cleave: Cleave and extract to flask 30.0 50 97: !Extract: Meter S3 10.0 50 98: !Extract: Extract to flask UP 10.0 50
99: !Cleave: Dry column UP 30.0 50 100: !Cleave: Meter 3R 9.0 50 101: !Cleave: Cleave and extract to flask 30.0 50 102: !Extract: Meter S3 10.0 50 103: !Extract: Extract to flask UP 10.0 50 104: !Cleave: Dry column UP 20.0 55 105: Wash: Meter R2 10.5 55 106: Wash: Deliver R2 DOWN 120.0 55 107: Wash: Dry column DOWN 60.0 60 108: Wash: Meter S2/3 9.0 60 109: Wash: Deliver S2/3 DOWN (closed) 15.0 60 110: Wash: Dry column DOWN 15.0 60 111: Wash: Meter S2/3 9.0 60 112: Wash: Deliver S2/3 DOWN (closed) 15.0 60 113: Wash: Dry column DOWN 20.0 60
! indicates system step
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Method (Flask): C-Terminal Flask 5.0
Total time: 30.5 min
Step 1: During Step 1, the liquid in the flask is slowly and completely dried
Step 2: By the end of Step 2, 70 µl (+/- 5 µl) of L3 will have been deliv­ered to the flask
Step 3: By the end of Step 3, the flask will have been dry for 75-100 seconds
Step 5: After Step 5, the volume of S5 in the flask should be 75-80 µl.
TEST Delivery protocol:
To check the delivery volume of S5, copy Steps 4 and 5 to the clip­board and run from the clip­board. At the end of Step 5, remove the flask from the heating chamber. Carefully unscrew the flask cap and measure the deliv­ered volume using a syringe. Be careful that none of the delivered volume is retained on the tube sides or top of the flask.
Step 8: Before injection the injector loop should be filled, leaving the sol­vent visible in both the inlet and out­let lines of the injector loop. The time for Step 8 may have to be adjusted in order to optimize the delivery.
Step Description Primary Temperature
Time
1: Transfer: Evaporate 240.0 50 2: Transfer: Deliver L3 10.0 50 3: Transfer: Evaporate dry L3 140.0 50 4: Transfer: Meter S5 12.0 50 5: Transfer: Deliver S5 14.0 50 6: Transfer: Mix and equilibrate 10.0 50 7: Transfer: Solubilize 15.0 50 8: Transfer: Fill loop and inject 2.0 50
9: Transfer: Empty flask 60.0 50 10: Transfer: Flush with L3 60.0 50 11: Transfer: Mix 20.0 50 12: Transfer: Empty flask 30.0 50 13: Transfer: Deliver L3 to V7 15.0 50 14: Transfer: Flush with L3 60.0 50 15: Transfer: Mix 20.0 50 16: Transfer: Empty flask 30.0 50 17: Transfer: Dry waste line 100.0 50 18: Transfer: Dry vent line 100.0 50
7
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Hewlett-Packard shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing, performance or use of this material.
Information, descriptions and specifi­cations in this publication are subject to change without notice.
Copyright © 1998 Hewlett-Packard Company All rights reserved. Reproduction and adaptation is prohibited.
Printed in the U.S.A. July 1998 5967-6280E Revision 1.0
Method (Flask): TH Std 5.0
Total time: 39.8 min
Fifty picomoles of TH standard are delivered to the flask in this method.
Step 1: Cycle 1 begins with a pause to provide time for proper HPLC column equilibration after switching from N-terminal sequencing.
Step 7: Step 7 should add 100 µl (+/­2 µl) of TH-standard to the dry flask
TEST Delivery protocol:
To check the delivery volume of the TH-standards, copy the steps in the TH Std Test method to the clipboard and run from the clip­board. At the end of test pro­gram, remove the flask from the heating chamber. Carefully unscrew the flask cap and mea­sure the delivered volume using a syringe. Be careful that none of the delivered volume is retained on the tube sides or top of the flask
Step 10: After Step 10, the volume of S5 in the flask should be 75-80 µl.
TEST Delivery protocol:
To check the delivery volume of S5, copy Steps 9 and 10 to the clipboard and run from the clip­board. At the end of Step 10, remove the flask from the heating chamber. Carefully unscrew the flask cap and measure the deliv­ered volume using a syringe. Be careful that some of the delivered volume is not retained on the tube sides or top of the flask.
Step 13: Before injection the injec­tor loop should be filled, leaving the solvent visible in both the inlet and outlet lines of the injector loop. The time for Step 13 may have to be adjusted in order to optimize the delivery.
Step Description Primary Temperature
Time
1: Transfer: Empty flask 150.0 50 2: Transfer: Meter L3 60.0 50 3: Transfer: Deliver L3 25.0 50 4: Transfer: Empty flask 60.0 50 5: Transfer: Dry waste line 180.0 50 6: Transfer: Dry vent line 180.0 50 7: Transfer: Deliver TH standard 7.9 50 8: Transfer: Evaporate 300.0 50
9: Transfer: Meter S5 12.0 50 10: Transfer: Deliver S5 14.0 50 11: Transfer: Mix and equilibrate 10.0 50 12: Transfer: Solubilize 15.0 50 13: Transfer: Fill loop and inject 2.0 50 14: Transfer: Empty flask 30.0 50 15: Transfer: Meter L3 60.0 50 16: Transfer: Deliver L3 25.0 50 17: Transfer: Empty flask 60.0 50 18: Transfer: Meter L3 60.0 50 19: Transfer: Deliver L3 25.0 50 20: Transfer: Empty flask 60.0 50 21: Transfer: Dry waste line 120.0 50 22: Transfer: Dry vent line 120.0 50
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