Microcon-SCX Adsorptive Microconcentrator - Millipore

B I O S E P A R A T I O N S 

Microcon-SCX 

Adsorptive Microconcentrator 

User Guide 

■ For research use only, not for 

use in diagnostic procedures 

®

B I O S E P A R A T I O N S 



User Guide 

■ For research use only, not for 

use in diagnostic procedures 

99452, Rev. D, 06/00 

®

Notice 

The information in this document is subject to change without notice and should 

not be construed as a commitment by Millipore Corporation. Millipore Corporation 

assumes no responsibility for any errors that may appear in this document. 

This manual is believed to be complete and accurate at the time of publication. In 

no event shall Millipore Corporation be liable for incidental or consequential 

damages in connection with or arising from the use of this manual. 

© 1997, 1998, 2000 MILLIPORE CORPORATION. PRINTED IN THE UNITED 

STATES OF AMERICA. ALL RIGHTS RESERVED. THIS BOOK OR PARTS THEREOF 

MAY NOT BE REPRODUCED IN ANY FORM WITHOUT THE WRITTEN PERMIS- 

SION OF THE PUBLISHERS. 

Millipore is a registered trademark of Millipore Corporation or an affiliated company. 

Amicon is a registered trademark of Millipore Corporation or an affiliated company. 

Microcon is a registered trademark of Millipore Corporation or an affiliated company.

MICROCON ® 

-SCX 




Introduction 

Microcon-SCX is ideal for concentrating peptides or oligonucleotides 

and for removing low-molecular-weight contaminants from 

samples before other analytical techniques; i.e., mass spectroscopy, 

sequencing, amino acid analysis, HPLC and carbohydrate 

analysis. This device contains a strong cation exchange membrane 

which adsorbs virtually all amino acids, peptides or oligonucleotides. 

Due to fast kinetics, high extraction efficiency is achieved 

with short spin times. Recommended capacity for peptides is 2– 

400 µg/device, depending on sample and rotor type (fixed-angle 

centrifuge rotors offer less capacity than horizontal rotors). 

NOTE: Microcon-SCX is intended for concentrating and purifying 

amino acids, peptides and oligonucleotides only. The 

protocols in this instruction are not recommended for 

macromolecules larger than 10,000 MW.

2 

http://www.millipore.com 

Materials Supplied 

Three vials with attached sealing caps are included for each 

Microcon-SCX unit. The product selection guide under “Ordering 

Information” provides product numbers for various package sizes. 

Equipment Required 

Centrifuge that can accommodate 1.5 ml microcentrifuge tubes. 

Reagents ¹ 

Methanol 

Deionized water (DIW) 

Glacial acetic acid 

Ammonium hydroxide (NH4OH) 

Hydrochloric acid (HCl) 

Desorption reagents² are application-dependent. Make fresh 

daily: 

1.4 N NH4OH/MeOH (500 µl methanol, 100 µl NH4OH, 400 µl 

DIW) 

1.2 N HCl/MeOH (500 µl methanol, 100 µl HCl, 400 µl DIW) 

¹Reagents are application-specific. This is a standard protocol for sample clean-up of 

peptides; see sample elution protocols on page 4 for specific applications. 

²Use acetonitrile or isopropanol for peptides that elute from C18 columns above 50% ACN.

Microcon-SCX Adsorptive Microconcentrator User Guide 

Protocol 

For highest sample recovery or concentration, device orientation 

in a fixed-angle rotor must remain consistent with each subsequent 

centrifugation. Use reservoir’s “mold dimple” as a reference 

point to ensure consistent device orientation. 

Preparation for Sample Binding 

1. Locate “mold dimple” on brown reservoir and insert into vial, 

aligning dimple with cap strap as shown. 

Align the dimple 

with strap on vial cap. 

When placing assembled device into centrifuge rotor, always 

place cap strap toward center of rotor. Consistent orientation 

will ensure maximum sample recovery. 

2. Wet membrane by adding 100–200 µl of methanol to device; 

agitate briefly then empty methanol to waste. Repeat procedure 

with DIW. 

NOTE: When performing high-sensitivity analyses (such as 

HPLC), centrifuge (at 7,000 × g) 500 µl of methanol 

through device for 15 seconds, then 500 µl of DIW. 

This will remove interfering substances which elute at 

25–30% acetonitrile during reverse-phase HPLC. 

3

4 


Preparation for Sample Binding, Continued 

3. Remove and empty vial. Place a clean vial on Microcon-SCX 

unit in preparation for sample binding. Save the used (empty) 

vial if you choose to do a wash step after binding and before 

elution of the sample. 

Binding Procedure 

1. Samples must contain < 0.1 M salt (if necessary, dilute 

sample). If the sample volume is greater than 500 µl after 

dilution, perform multiple bindings in one Microcon-SCX unit, 

using up to 500 µl of sample per spin. 

2. For maximum binding, samples must have a pH below the pI 

or pKa of the analyte. If necessary, add 5–10 µl of glacial 

acetic acid per 500 µl of sample before binding. 

3. Load sample into Microcon-SCX (a clean vial should be placed 

on unit after membrane washing, per step 3 of preparation 

procedure). 

4. Centrifuge at 1,200 × g for 1 minute. Be sure to maintain 

consistent orientation of device in centrifuge. If recovery is 

low, save filtrate for further analysis. 

5. A wash step (500 µl of 10 mM HCl in DIW) before elution will 

remove residual contaminates. If conducted, use vial from 

methanol/DIW wash. 

Elution Procedure 

1. Place Microcon-SCX containing bound analyte into clean vial. 

Align reservoir’s “mold dimple” with cap strap to ensure 

reservoir-vial alignment. 

2. Pipette 50 µl of desorption reagent (application-dependent) 

into unit and spin at 14,000 × g for 15 seconds. To maximize 

yields, add another 50 µl of desorption reagent and spin at 

14,000 × g for another 15 seconds. Remember to maintain 

original orientation in centrifuge.


Sample Elution Protocols for Specific Applications 

This section lists details on protocols for a variety of applications. 

Mass Spectroscopy (MS) 

Prepare 1.4 N NH 4 OH/MeOH desorption reagent, then follow 

steps 1 and 2 of elution procedure. Eluted sample may be directly 

applied to electrospray MS or neutralized with 1.2 N HCl for use 

with MALDI and/or LC/MS. Alternatively, desorption can be 

performed at low pH (see Sequence Analysis). 

NOTE: Additional analyte-bound Na + can be removed from the 

adsorbed sample by washing unit with 500 µl of 20 mM 

ammonium acetate, pH 6.0. 

Sequence Analysis 

Prepare 3 N HCl/MeOH desorption reagent, then follow steps 1 

and 2 of elution procedure. Eluted sample can be spotted onto 

glass fiber filter and sequenced. Higher concentrations of HCl (3–6 

N) in desorption reagent with 50% IPA may be necessary to 

increase sample recovery. 

Amino Acid Analysis 


steps 1 and 2 of elution procedure. If derivatizing agent (i.e., PTC) 

reacts with free amines, an additional dry down with 500 µl of 

DIW is required before hydrolysis to remove residual ammonium 

ions. Alternatively, elution can be done with 3–6 N HCl desorption 

buffer, then vacuum dried and hydrolyzed. 

HPLC Analysis 


steps 1 and 2 of elution procedure. After elution, neutralize 

eluted sample with dilute HCl (1.2 N HCl in DIW) to maximize 

sample recovery. Dilute HCl can be placed in the vial before 

elution for immediate neutralization. This is useful for samples 

which are unstable at high pH or if multiple units are run simultaneously. 

If small volumes are needed for HPLC, the final eluted 

sample will require speed vacuum lyophilization to remove ammonium 

and methanol. Optimal results with speed vacuum lyophilization 

are achieved by concentrating the sample volume to 25 

µl and diluting 1:2, before HPLC. 

5

6 


Carbohydrate Analysis 

Prepare glycoprotein for hydrolysis (2 N TFA at 100°C for 5 

hours). Following hydrolysis, dilute sample 1:1 with DIW and 

speed vac to dryness. Add 100 µl of 20 mM sodium acetate pH 5.0 

to dried samples and check pH with strip so that it is between 4 

and 5. If not, adjust pH with 0.1 N NaOH. Transfer hydrolysate 

in sodium acetate buffer to Microcon-SCX in a clean vial, centrifuge 

at 5,000 × g for 30 seconds, then increase to 14,000 × g for an 

additional 30 seconds. The filtrates, free of amino acids, are ready 

for PAD analysis. 

NOTE: When adding sodium acetate, be sure pH does not drop to 

less than pH 4.0. Below this value, amino sugars will bind to 

the membrane, yielding low recoveries. Recommended 

controls should include: Microcon-SCX filtered monosaccharide 

standards in buffer (positive) and buffer blank 

(negative)

Microcon-SCX Adsorptive Microconcentrator User Guide 7 

Materials of Construction 

Sample reservoir: polycarbonate 

Sample vial: polypropylene 

Membrane functionality: sulfonated polystyrene divinyl benzene 

Storage 

Store Microcon-SCX devices at room temperature. 

Specifications 

Maximum sample volume: 500 µL 

Maximum relative centrifugal force: 14,000 × g 

Product Ordering Information 

This section lists the catalogue numbers for Microcon-SCX Adsorptive 

Microconcentrators. See the Technical Assistance section 

for information about contacting Millipore. You can also buy 

Millipore products on-line at www.millipore.com/purecommerce. 

Product Selection Guide 

Description 


Adsorptive Microconcentrators 

Package 

Size 

8 

24 

100 

Product 

Number 

42460 

42461 

42462

8 


Technical Assistance 

For more information, contact the Millipore office nearest you. In 

the U.S., call 1-800-MILLIPORE (1-800-645-5476). Outside the 

U.S., see your Millipore laboratory catalogue for the phone 

number of the office nearest you. You can reach us by e-mail at 

tech_service@millipore.com or visit our web site 

(www.millipore.com). 

Millipore Corporation is pleased to provide internet access to 

Material Safety Data Sheets (MSDS) for its products that contain 

hazardous materials. To obtain any MSDS documents that may be 

associated with this product, go to the MSDS page of our website 

(www.millipore.com/msds.nsf/home). 

The following is a sample of current Microcon-SCX publications 

from “Creative Solutions.” Once you are in the Millipore web site 

click on any “Solution” description as seen below for the general 

procedure and abbreviated protocol in each case. For detailed 

protocols click the word “Protocol” at the end of each “Creative 

Solution,” or use the right side of the table below. Although you 

can perform some applications with different devices, each protocol 

is written specifically for the device described. For complete 

product information for each device, see the end of each protocol. 

Solution Title Protocol For 

Solution07 DNA oligomer concentration 

and desalting 


Solution10 Glycoprotein hydrolysate 

preparation before 

carbohydrate analysis 


Solution12 Oligonucleotide recovery Micropure & Microcon 

from polyarcrylamide gel and Microcon-SCX 

Solution14 Peptide concentration and 

desalting 


Solution15 Peptide concentration and 

desalting 


Solution16 Peptide preparation before 

sequencing 

Microcon-SCX


General Limited Warranty 

Millipore Corporation ("Millipore") warrants its products will 

meet their applicable published specifications when used in 

accordance with their applicable instructions for a period of one 

year from shipment of the products. MILLIPORE MAKES NO 

OTHER WARRANTY, EXPRESSED OR IMPLIED. THERE IS NO 

WARRANTY OF MERCHANTABILITY OR FITNESS FOR A 

PARTICULAR PURPOSE. The warranty provided herein and the 

data, specifications and descriptions of Millipore products appearing 

in Millipore's published catalogues and product literature 

may not be altered except by express written agreement signed 

by an officer of Millipore. Representations, oral or written, 

which are inconsistent with this warranty or such publications are 

not authorized and if given, should not be relied upon. 

In the event of a breach of the foregoing warranty, Millipore's 

sole obligation shall be to repair or replace, at its option, the 

applicable product or part thereof, provided the customer notifies 

Millipore promptly of any such breach. If after exercising 

reasonable efforts, Millipore is unable to repair or replace the 

product or part, then Millipore shall refund to the customer all 

monies paid for such applicable product or part. MILLIPORE 

SHALL NOT BE LIABLE FOR CONSEQUENTIAL, INCIDENTAL, 

SPECIAL OR ANY OTHER INDIRECT DAMAGES RESULTING 

FROM ECONOMIC LOSS OR PROPERTY DAMAGE SUSTAINED 

BY ANY CUSTOMER FROM THE USE OF ITS PRODUCTS. 

9

Microcon-SCX Adsorptive Microconcentrator - Millipore

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