Waters ACQUITY UPLC SEC Columns and Standards User Manual

[ CARE AND USE MANUAL ]

ACQUITY UPLC BEH SEC COLUMNS AND STANDARDS

CONTENTS

I.INTRODUCTION

II.CONFIGURING AN ACQUITY UPLC SYSTEM FOR USE IN SEC PROTEIN SEPARATIONS

a. Calibrators

III.GETTING STARTED

a.eCord™ Installation

b.Column Connectors

c.Column Installation

d.Column Equilibration

e.Useful Functional Tests for Benchmarking a New Column

IV. COLUMN SPECIFICATIONS AND USE

a.SEC Eluent and Needle Wash Preparation

b.Sample Preparation

c.Column Specification

V.TROUBLESHOOTING

VI. COLUMN CLEANING, REGENERATION AND STORAGE

a.Cleaning and Regeneration

b.Storage

VII. INTRODUCING eCord INTELLIGENT CHIP TECHNOLOGY

a.Introduction

b.Installation

c.Manufacturing Information

d.Column Use Information

VIII. ORDERING INFORMATION

I. INTRODUCTION

Thank you for choosing a Waters ACQUITY UPLC® BEH SEC guard, column, and/or standard mix that are integral parts of Waters ACQUITY UPLC SEC System Solution. The BEH125 SEC offering is best suited for the analysis of peptides and proteins in the molecular weight range from 1,000–80,000 Daltons that include insulin and its aggregates. The BEH200 SEC column was designed to characterize proteins ranging in molecular weight range from 10,000–450,000 Daltons that include monoclonal IgG monomers from aggregates, while our BEH450 SEC is best suited for the analyses of proteins and conjugates that range from 100,000–1.5 million Daltons

(Fig 1). All of these SEC chemistries are based on Waters Ethylene Bridged Hybrid (BEH)-based particle technology and diol-bonded surface that provide a stable chemistry with minimal non-desired secondary interactions for proteins and peptides. The columns are manufactured in a cGMP, ISO 9001 certified plant using ultra pure reagents. Each batch of Protein Separation Technology SEC packing material has been tested and qualified using a protein test mixture (that is also available for purchase), and the results are held to narrow specification ranges to ensure reproducible performance. Every column is also individually tested for efficiency. A Performance Test Chromatogram along with a Certification of

ACQUITY UPLC BEH SEC Columns

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[ CARE AND USE MANUAL ]

II. CONFIGURING AN ACQUITY UPLC SYSTEM FOR USE IN SEC PROTEIN SEPARATIONS

a. Calibrators

In order to obtain the best performance from your SEC column, it is important that your ACQUITY UPLC system is properly configured. It is recommended that only pre-cut tubing is used, and that the ID of all connecting tubing is 0.005” or less for optimal chromatographic performance.

Size-exclusion chromatography may require modifications to an existing ACQUITY UPLC system. Please refer to “Size-Exclusion and Ion-Exchange Chromatography of Proteins using the ACQUITY UPLC System” (P/N 715002147A) for specific recommendations that can be obtained at www.waters.com/chemcu

The sample loop used may affect the performance of your separation. Optimally, select the smallest volume sample loop that is required for the application. Sample loops larger than 20 µL with the ACQUITY UPLC BEH SEC column are not recommended.

III. GETTING STARTED

A Performance Test Chromatogram and a Certification of Analysis (COA) are available for each shipped ACQUITY UPLC BEH SEC column. The COA is specific to each batch of packing material and includes the batch number and chromatographic separation obtained using defined protein standards. This document is available upon request at www.waters.com/chemcu

Blue Dextran

1,000,000

ACQUITY UPLC BEH125 SEC,

1.7 µm: 1K–80K Daltons

IgM (900 kDa)

Thyroglobulin (669 kDa)

IgG (150 kDa)

ACQUITY UPLC BEH200 SEC,

100,000

Amylglucosidase (97 kDa)

1.7 µm: 10K–450K Daltons

Conalbumin (75 kDa)

Ovalbumin (44 kDa)

Myoglobin (17 kDa)

ACQUITY UPLC BEH450 SEC,

10,000

RNase A (13.7 kDa)

2.5 µm: 100K–1500K Daltons

Aprotinin (6.5 kDa)

MW

1000

Angiotensin frag 1-7 (899 Da)

Allantoin (158 Da)

Uracil (112 Da)

100

0.4

0.6

0.8

1.0

Normalized Retention Volume (Vr/VC)

Protein standards; T: 30°C; Mobile phase: 100mM sodium phosphate, pH=6.8

Figure 1. Calibration Curves on ACQUITY UPLC BEH125, BEH200, and BEH450 SEC Columns

	1	4	5	7	ACQUITY UPLC
	0.15				BEH125 SEC
			6
	2	3
AU	0.10
	0.05
	0.00

	0.12		2	4	5	7
AU	0.08	1		3
						6
	0.04
	0.00

		Thyroglobulin dimer
	0.10	(Approx. 1.4 million Daltons)	2	5
	0.08		1
AU	0.06			3
	0.04
	0.02
	0.00

ACQUITY UPLC

BEH200 SEC

ACQUITY UPLC BEH450 SEC

Note: Stds 4 and 6 above were not included in the BEH450 Column test mix

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	2	3	4	5		6	7	8 min
	Compounds:
	1. Thyroglobulin (670K)				5. Myoglobin (17K)
	2. BSA (66K)				6. Angiotensin Frag 1–7 (899)
	3. Ovalbumin (44K)				7. Uracil (112)
	4. Carbonic Anhydrase (29K)
	Columns	ACQUITY UPLC BEH125, 1.7 µm				ACQUITY UPLC BEH450, 2.5 µm
		ACQUITY UPLC BEH200 1.7 µm
	Column				4.6 x 150 mm
	Configuration
	Mobile Phase			100 mM Sodium Phosphate Buffer, pH 6.8
	Weak			100% Milli-Q® Water
	Needle Wash
	Strong				100% Milli-Q Water
	Needle Wash
	Seal wash			90/10 water/methanol
			Thyroglobulin 0.3 mg/mL
			BSA 0.3 mg/mL				Thyroglobulin 3 mg/mL
	Samples:		Ovalbumin 0.3 mg/mL				BSA 5 mg/mL
	Diluted in		Carbonic Anhydrase 0.3 mg/mL				Ovalbumin 3 mg/mL
	mobile phase		Myoglobin 0.3 mg/mL				Myoglobin 2 mg/mL
		Angiotensin Frag. 1–7 0.1 mg/mL					Uracil 0.1 mg/mL
			Uracil 0.1 mg/mL
	Injection Vol.				2 µL, Full Loop
	Flow Rate				0.3 mL/min
	Column Temp.				Ambient
	Detection		UV @ 220 nm				UV @ 280 nm
	Wavelength

Figure 2. Separation of Protein and Peptide Standards on ACQUITY UPLC BEH125, BEH200, and BEH450 SEC Columns

a. eCord Installation

The eCord button should be attached to the side of the ACQUITY UPLC column heater module. The eCord button is magnetized and does not require specific orientation.

ACQUITY UPLC BEH SEC Columns

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[ CARE AND USE MANUAL ]

b. Column Connectors

It is is recommended to use the 150 mm column stabilizer (P/N 205000365) to connect the ACQUITY UPLC BEH SEC column to the ACQUITY UPLC system. A reusable fitting is supplied with the ACQUITY UPLC column stabilizer.

c. Column Installation

1.Prior to placing the column on the system, purge the solvent delivery system of any organic or water-immiscible mobile phases. When connecting the column, orient it in the proper direction as noted by the arrow on the column inlet side which indicates the correct direction of solvent flow.

2.Flush column with 100% aqueous buffer, by pumping at a flow rate of 0.2 mL/min.

3.Ensure that the mobile phase is flowing freely from the column outlet. Attach the column outlet to the detector using .004” ID tubing (P/N 430001562). Monitor the system pressure to ensure the column is within its pressure limitations.

4.Gradually increase the flow rate, by not more than 0.1mL/min at a time, as described in Step 2.

5.Once the system pressure has stabilized, ensure that there are no leaks at either the column inlet or outlet.

d. Column Equilibration

ACQUITY UPLC BEH SEC columns are shipped in 20% methanol in water. It is important to ensure mobile-phase compatibility before changing to a different mobile-phase system. Equilibrate the column with a minimum of 10 column volumes of the buffer to be used (refer to Table 1 for column volumes).

Table 1. Empty Column Volumes in mL (multiply by 10 for flush solvent volume)

Column Dimension	Approximate Volume
4.6 x 150 mm	2.5 mL
4.6 x 300 mm	5.0 mL

e. Useful Functional Tests for Benchmarking a New Column

Waters recommends performing a benchmarking test upon receipt of your column and throughout the lifetime usage. By using a separation of common proteins with an appropriate method, you can:

Verify the performance of the column upon receipt.

Monitor the condition of the columns for extended use.

Troubleshoot separation difficulties that may arise.

The BEH125 SEC Protein Standard Mix (P/N 186006519), BEH200 SEC Protein Standard Mix (P/N 186006518), and BEH 450 SEC Protein Standard Mix (P/N 186006842) were specifically designed for this purpose with carefully chosen proteins and/or peptides to provide a good representation of the intended application. Figure 3 is an example of the Performance Test Chromatogram using

Part Number 186006519, Figure 4 is an example of the Performance Test Chromatogram using Part Number 186006518. Figure 5 is an example of the Performance Test Chromatogram using Part Number 186006842. These are typical results obtained in a waters laboratory using 4.6 x 150 mm columns on an ACQUITY UPLC system.

Buffer Preparation for Standard:

Chemicals:

-Sodium Phosphate Monobasic, Monohydrate

-Sodium Phosphate Dibasic, Anhydrous

-Milli-Q Water

Preparation of 100mM Sodium Phosphate Buffer (500 mL)

-Weigh out 500 ± 0.02 grams of H2O into a 500 mL beaker.

-Weigh out 3.55 ± 0.02 grams of Sodium Phosphate dibasic and add to the 500 mL beaker.

-Weigh out 3.45 ± 0.02 grams of Sodium Phosphate monobasic and add to the 500 mL beaker.

-Stir for a minimum of 30 minutes then filter the solution through a 0.2 µm Millipore GV filter.

-Take the pH and record the value for reference purposes (approximate pH: 6.8).

ACQUITY UPLC BEH SEC Columns

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